TW202311282A - Compositions and vaccines for treating and/or preventing coronavirus variant infections and methods of using the same - Google Patents

Abstract

The present disclosure is directed to compositions and methods useful for treating, as well as vaccinating against, SARS-CoV-2 viral infections, including SARS-CoV-2 variant viral infections.

Description

Compositions and vaccines for treating and/or preventing coronavirus variant infection, and methods of use thereof

In the last two decades, outbreaks of Severe Acute Respiratory Syndrome (SARS, 2002-2004 (Ksiazek et al., 2003; Drosten et al., 2003)) and Middle East Respiratory Syndrome (MERS, 2012-present (Zaki et al., 2012)) is a major threat to global public health.

Respiratory syndromes caused by coronaviruses (CoVs) that are transmitted from person to person through close contact result in high morbidity and mortality in infected individuals. Although SARS and MERS initially presented with a mild, influenza-like illness with fever, dyspnea, and cough, progression to more severe symptoms was characterized by atypical interstitial pneumonia and diffuse alveolar damage. Both SARS-CoV and MERS-CoV can cause acute respiratory distress syndrome (ARDS), the most severe form of acute lung injury, in which alveolar inflammation, pneumonia, and hypoxic lung conditions lead to respiratory failure, multiple Organ disease and death (Lew et al., 2003).

Vaccines have been considered the gold standard for preventing and eradicating infectious diseases against the human population and conferring long-term immune protection benefits on individuals. Although several SARS-CoV-2 vaccines have been developed, they are less effective against the numerous SAR-CoV-2 variants that have emerged in the past year.

Therefore, new compositions and methods are needed to effectively stimulate antiviral immunity especially against SARS-CoV-2 variants. The present invention fulfills these needs.

In one aspect, compositions are described herein comprising: (a) a vector comprising a plastid encoding at least one viral antigen, wherein the viral antigen is from a variant of SARS-CoV-2; (b) a vector comprising comprising an antigen recognized by CD1d; and (c) at least one pharmaceutically acceptable carrier, wherein at least one of carrier (a) and carrier (b) is an intact, bacterial-derived pocket cell or a killed bacterial cell .

In another aspect, the SARS-CoV-2 variant is selected from the group consisting of: (a) UK SARS-CoV-2 variant (B.1.1.7/VOC-202012/01); (b) has B.1.1.7 variant of E484K; (c) B.1.617.2 (δ) variant; (d) B.1.617 variant; (e) B.1.617.1 (κ) variant; (f) B.1.617.3 variant; (g) South African B.1.351 (β) variant; (h) P.1 (γ) variant; (i) B.1.525 (η) variant; (j) B. 1.526 (ι) variant; (k) λ (lineage C.37) variant; (l) ε (lineage B.1.429) variant; (m) ε (lineage B.1.427) variant; (n) ε (lineage CAL.20C) variant; (o) ζ (lineage P.2) variant; (p) θ (lineage P.3) variant; (q) R.1 variant; (r) lineage B. 1.1.207 variant; and (s) lineage B.1.620 variant.

In another aspect, the SARS-CoV-2 variant is selected from the group consisting of: a SARS-CoV-2 variant comprising: (a) a L452R spinin substitution; (b) an E484K spinin substitution; (c ) K417N spinin substitution; (d) E484K spinin substitution; (e) N501Y spinin substitution; (f) K417T spinin substitution; (g) E484K spinin substitution; (h) N501Y spinin substitution; and (i) SARS-CoV-2 mutant strains, which have one or more of the following missense mutations: N440, L452R, S477G/N, E484Q, E484K, N501Y, D614G, P681H, P681R and A701V.

In one aspect, the vaccine composition may comprise a carrier (a) which additionally comprises at least one viral antigen from a SARS-CoV-2 strain (eg, a non-mutant strain). For example, the SARS-CoV-2 strains can be selected from the group consisting of L strains, S strains, V strains, G strains, GR strains and GH strains. In another aspect, the SARS-CoV-2 viral antigen can be composed of a polynucleotide comprising the sequence of SARS-CoV-2 or a polynucleotide having at least 80% sequence identity to a polynucleotide comprising the sequence of SARS-CoV-2 nucleotide code.

In one aspect of the compositions described herein, the plastid encodes the spine (S) protein, nucleic acid protein shell (N) protein, membrane (M) protein, and sleeve protein of SARS-CoV-2 or SARS-CoV-2 variants. At least one of membrane (E) proteins. In addition, plastids can encode spine (S) protein, nucleic acid protein shell (N) protein, membrane (M) protein and envelope (E) protein or any combination thereof (for example, spinin from mutant strains and envelope protein from non-mutant strains).

In another aspect, the plastid can encode the receptor binding domain (RBD) of the spike protein of SARS-CoV-2 or a variant of SARS-CoV-2.

In one aspect of the compositions described herein, the carrier (a) is a first whole, bacteria-derived pocket cell or a killed bacterial cell, and the carrier (b) is a second whole, bacteria-derived pocket cell or killed bacterial cells. In another aspect, vector (a) and vector (b) are the same whole, bacterial-derived pocket cell or killed bacterial cell comprising an antigen recognized by CD1d and encoding at least one virus SARS-CoV-2 The plastid of the mutant virus antigen. In another aspect, one of vector (a) and vector (b) is not an intact, bacterial-derived pocket cell or a killed bacterial cell, and the other of vector (a) or vector (b) One is intact, bacteria-derived pocket cells or killed bacterial cells.

In one embodiment, the antigen recognized by CDld comprises glycosphingolipids. For example, the antigen recognized by CDld can be selected from the group consisting of α-galactosylceramide (α-GalCer), the C-glycosidic form of α-galactosylceramide (α-C-GalCer), half The 12-carbon acyl form of lactosylceramide (β-GalCer), β-D-glucopyranosylceramide (β-GlcCer), l,2-diacyl-3-O-galactosyl-sn - Glycerol (BbGL-II), glycolipid containing diacylglycerol (Glc-DAG-s2), ganglioside (GD3), neurotrihexosylceramide (Gg3Cer), glycoside phosphatidylinositol ( GPI), α-glucuronyl ceramide (GSL-1 or GSL-4), isoglucotrihexosyl ceramide (iGb3), lipophosphoglycan (LPG), lysophosphatidyl choline ( LPC), α-galactosylceramide analogs (OCH), threitol ceramide, and derivatives of any of them.

In another aspect, the antigen recognized by CDld comprises α-GalCer. Alternatively, antigens recognized by CDld may include synthetic α-GalCer analogs. For example, the antigen recognized by CDld may comprise a synthetic α-GalCer analog selected from the group consisting of 6′-deoxy-6′-acetamide α-GalCer (PBS57), naphthaleneurea α-GalCer (NU-α- GC), NC-α-GalCer, 4ClPhC-α-GalCer, PyrC-α-GalCer, α-carba-GalCer, carba-α-D-galactose α-GalCer analog (RCAI-56), 1-deoxy - Neo-inositol α-GalCer analog (RCAI-59), 1-O-methylated α-GalCer analog (RCAI-92) and HS44 aminocyclitol ceramide.

In one aspect, the antigen recognized by CDld is an IFNy agonist.

The compositions described herein may be formulated for any pharmaceutically acceptable use. Examples of pharmaceutically acceptable formulations include, but are not limited to, oral administration, injection, nasal administration, pulmonary administration or topical administration.

In another aspect, vaccine compositions comprising at least one whole, bacterially-derived miniature cell or killed bacterial cell, and comprising within the miniature cell or cells: (a) Plastids of spiky proteins in multiples: SARS-CoV-2 variant α (B.1.1.7.UK), SARS-CoV-2 variant β (B.1.351. SA), SARS-CoV-2 variant Strain δ (B.1.617.2 India (India)) and/or SARS-CoV-2 variant γ (P.1 Brazil (Brazil)); and (b) α-galactosylceramide. Alternatively, the vaccine composition may comprise (a) and (b) within a single pocket cell. In addition, the plastids of the vaccine composition may encode spike proteins from each of: SARS-CoV-2 variant alpha (B.1.1.7.UK), SARS-CoV-2 variant beta (B.1.351 . SA), SARS-CoV-2 variant δ (B.1.617.2 India) and SARS-CoV-2 variant γ (P.1 Brazil).

The disclosure also encompasses methods of treating viral infections and/or vaccinating against viral infections comprising administering to an individual in need thereof a composition described herein.

In one aspect, the individual suffers from or is at risk of developing lymphopenia. In another aspect, the individual is considered to be at risk of serious disease and/or serious complications from viral infection. For example, an "elderly" individual who is at higher risk of serious illness and/or serious complications from a viral infection is about 50 years or older, about 55 years or older, about 60 years or older, or about 65 years old or above.

In another aspect of the methods described herein, the individual suffers from one or more pre-existing conditions selected from the group consisting of diabetes, asthma, respiratory disorder, hypertension, and heart disease. In yet another aspect, the individual is immunocompromised. For example, an individual may be immunocompromised due to AIDS, cancer, cancer treatment, hepatitis, autoimmune disease, steroid reception, immunosenescence, or any combination thereof.

In one embodiment, administration of a composition described herein increases the chance of survival after exposure to a coronavirus. For example, the chance of survival can be increased by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%, if using Measured by any clinically recognized technique.

In yet another aspect, administration of the compositions described herein reduces the risk of transmission of the coronavirus. For example, the reduction in risk of transmission can be about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%, As measured using any clinically recognized technique.

In all methods described herein, the administering step can be performed by any pharmaceutically acceptable method.

In another aspect, an individual may be exposed or expected to be exposed to an individual with coronavirus infectivity. Additionally, an individual with coronavirus infectivity may have one or more symptoms selected from the group consisting of fever, cough, shortness of breath, diarrhea, sneezing, runny nose, and sore throat.

In one embodiment, individuals of the methods described herein are health care workers, 60 years of age or older, frequent travelers, military personnel, caregivers, or individuals with pre-existing conditions that increase the risk of death from infection.

In another aspect, the method further comprises administering one or more antiviral drugs. For example, one or more antiviral drugs may be selected from the group consisting of: chloroquine, darunavir, galidesivir, interferon beta, lopinavir , ritonavir, remdesivir and triazavirin.

In the methods of the present disclosure, an antigen recognized by CDld induces a Th1 interleukin response in an individual. For example, interleukins can include IFNγ.

In another aspect, a first pocket-sized cell comprising an antigen recognized by CDld and a second pocket-sized cell comprising a plastid encoding at least one SARS-CoV-2 variant viral antigen are administered to the individual simultaneously. In yet another aspect, a first pocket-sized cell comprising an antigen recognized by CDld and a second pocket-sized cell comprising a plastid encoding at least one viral antigen are sequentially administered to the individual. Alternatively, the present disclosure encompasses methods in which a first pocket-sized cell comprising an antigen recognized by CDld and a second pocket-sized cell comprising a plastid encoding at least one viral antigen are repeatedly administered to an individual.

In the methods described herein, the first pocket-sized cells comprising an antigen recognized by CDld and the second pocket-sized cells comprising a plastid encoding at least one viral antigen can be administered at least once a week, twice a week, three times a week, or Administered to individual four times per week.

The above summary of the invention and the following brief description and implementation are exemplary and explanatory. It is intended to provide further details of the invention but should not be construed as limiting. Other objects, advantages and novel features will be apparent to those skilled in the art from the following description of the invention.

Cross References to Related Applications

This application claims priority to U.S. Provisional Patent Application No. 63/224,838, filed July 22, 2021; and U.S. Patent Application No. 17/480,073, filed September 20, 2021, in their entirety The disclosure is incorporated herein by reference in its entirety. I. Overview

This disclosure relates to novel compositions useful for treating and/or vaccinating against coronavirus infection viruses, and particularly in patient populations that are elderly, immunocompromised (e.g., from cancer, HIV, hepatitis, autologous immune diseases, organ transplant patients receiving immunosuppressive therapy) and/or with comorbidities. These patient populations are unlikely to mount a robust anti-COVID immune response from any of the existing COVID-19 vaccines. COVID-19 vaccines currently in use in at least one region of the world include Pfizer/BioNTech Comirnaty ^® COVID-19 Vaccine, Moderna COVID-19 Vaccine (mRNA 1273), Janssen/Ad26.COV 2.S, SII developed by Johnson & Johnson /Covishield and AstraZeneca/AZD1222 vaccines (developed by AstraZeneca/Oxford and manufactured by State Institute of India and SK Bio, respectively), Sinopharm manufactured by Beijing Bio-Institute of Biological Products Co Ltd (subsidiary of China National Biotec Group (CNBG)) COVID-19 vaccine and Sinovac Biotech Ltd. CoronaVac COVID-19 vaccine.

SARS-CoV-2 (Severe Acute Respiratory Syndrome-Coronavirus Type 2) is the causative agent of the COVID-19 pandemic and despite global vaccination efforts, the pandemic has not abated, especially with the high concern variant (variants of concern, VOC) continued to appear. Structurally, SARS-CoV-2 has 4 proteins; spine (S), mantle, membrane, and nucleic acid protein shell (Chan et al., 2020). The S-protein receptor binding domain (RBD) binds to the human vasoconstrictor peptide-converting enzyme 2 (hACE2) receptor on host cells (Song et al., 2018) and is responsible for cell attachment and fusion during viral infection. The S-protein is 1273 amino acids (aa) in length and consists of an N-terminal signal peptide (1-13 aa), including an N-terminal domain (14-305 aa) and an RBD (319-541 aa) The composition of the S1 subunit (14 - 685 aa) and the S2 subunit (residues 686-1273 aa) of the 2020 (Lan et al., 2020). RBD is a key neutralizing target and current vaccines are mainly aimed at eliciting RBD-specific neutralizing antibodies and T cell responses (Brouwer et al., 2020).

According to the US Centers for Disease Control and Prevention (CDC), a variant of SARS-CoV-2 has one or more characteristics that distinguish it from other variants in circulation. As expected, multiple variants of SARS-CoV-2 have been documented in the United States and globally throughout the pandemic. To inform local outbreak investigations and understand national trends, scientists compare genetic differences between viruses to identify variants and how they are related to each other. The US Department of Health and Human Services (HHS) established the SARS-CoV-2 Interagency Group (SIG) to improve CDC, National Institutes of Health (NIH), Coordination between Food and Drug Administration (FDA), Biomedical Advanced Research and Development Authority (BARDA) and Department of Defense (DoD). This interagency group is focused on the rapid characterization of emerging variants and actively monitoring their potential effects on key SARS-CoV-2 countermeasures, including vaccines, therapeutics and diagnostics. https://www.cdc.gov/coronavirus/2019-ncov/variants/variant-info.html (accessed July 21, 2021).

Throughout the COVID-19 pandemic, genetic variants of SARS-CoV-2 have emerged and spread around the world. Viral mutations and variant strains are routinely monitored in the United States by means of sequence-based surveillance, laboratory studies, and epidemiological investigations.

Immunologists and health authorities warn that not everyone develops a robust immune response to Covid-19, especially the elderly or those with weakened immune systems. The immune response can also depend on how much virus a person is exposed to or the severity of their illness. Vaccines appear to provide longer-lasting protection than infections. Wall Street Journal , "COVID-19 Immune Response Could be Long Lasting, but Variants Present Risks" (July 16, 2021). Many immunocompromised persons fail to elicit a strong immune response even after full vaccination. As the number of cases caused by the delta variant increases in Israel, it has begun booster shots for those with weakened immune systems. Ditto. A CDC advisory panel is scheduled to discuss the possibility of additional doses for immunocompromised individuals next week. Ditto. Therefore, there is a particular need for a vaccine that will be effective in this high-risk patient population.

For COVID-19 vaccines, high-affinity antigen-specific antibodies, CD8 ⁺ T-cell, and memory B-cell responses are critical for maximal protection against variant strains of high concern (VOC). This paper presents the results of immunization of mice and human volunteers with bacterially-derived non-viable nanocells (EDV ^TM ) packaged with SARS-CoV-2 expressing spinin and the IFNγ-stimulating adjuvant α - Bacterial plastids of galactosylceramide (EDV-COVID-αGC). EDV-COVID-αGC was shown to trigger iNKT-permissive dendritic cell activation/maturation, and follicular helper T cell homology helps B cells undergo germinal center-based somatic hypermutation and produce cells capable of neutralizing α, β, γ, δ and ο High affinity antibodies to VOCs, including memory B cell responses. Type I and Type II interferon stimulation and S-specific CD8 ⁺ T cells were also achieved. EDV-COVID-αGC was lyophilized, stored and shipped at room temperature.

For a vaccine to be successful, the host needs a robust immune system, which is sub-optimal in immunocompromised patients (e.g. cancer, HIV), and thus these patients remain susceptible to infection with SARS-CoV-2 and VOCs (Haidar et al., 2021 ; Liang et al., 2020; Pegu et al., 2021; Uriu et al., 2021). In addition, since the currently approved vaccines need to be stored and transported at a temperature of -20°C to -70°C, the storage life is only 3 to 6 months, so there are logistics problems.

In one aspect, the present disclosure sets forth a novel vaccine (designated EDV-COVID-αGC) comprising submicron diameter, non-live, chromosomal nanocell EDV ^™ (EnGeneIC Dream Vector) packaged with (i) Type I interferons carrying S-protein coding sequences stimulate bacterial gene expression recombinant plastids, (ii) plastids expressing S-proteins produced in the cytoplasm of nanocells, and (iii) type II interferons stimulate glycolipid adjuvant Agent αGC ( Figure 11A ). The EDV line was derived from a mutant non-pathogenic Salmonella typhimurium bacterium that germinated from the bacterium during its normal replication due to chromosomal mutation-induced asymmetric cell division (MacDiarmid et al., 2009; MacDiarmid et al., 2007 ). Single-chain Fv bispecific (scFv) antibodies targeting EDV have been used to deliver cytotoxic payloads and small molecules to solid cancers in several Phase I and Phase IIa clinical trials in solid tumors. Tumor stabilization/regression, prolonged overall survival, and minimal to no toxicity despite repeated dosing have been achieved in these patients who have exhausted all treatment options (Kao et al., 2015; van Zandwijk et al., 2017; Whittle et al. , 2015). A. Clinical Data

The data presented here show, for example, that EDV-COVID-αGC can deliver the SARS-CoV-2 S protein and αGC to dendritic cells (DCs), thereby eliciting S-specific humoral and cellular responses, where wild-type, αGC , β, γ and δ mutant strains have a broad-spectrum neutralization rate greater than 90%, and the broad-spectrum neutralization rate of the ο mutant strain is greater than 70%. In addition, the results of 4 volunteers prior to the phase I clinical trial of EDV-COVID-αGC are presented, which echo the preclinical data to date.

Despite more than two years of unprecedented global efforts to contain the COVID-19 pandemic, this effort has been thwarted by the persistent emergence of VOCs that has led to varying degrees of decline in vaccine protective efficacy. In addition, current vaccines display limited protective efficacy in immunocompromised populations (eg, those with cancer, HIV, organ transplants, and autoimmune diseases). Logistical issues such as the need to store and transport vaccines at -20°C to -70°C and a shelf life of only 3 to 6 months make it difficult to provide these vaccines to rural populations, especially in Africa. A novel COVID-19 vaccine that readily overcomes these limitations is described herein.

The vaccine comprises submicron (eg 400 nm in one aspect) non-viable, chromosomal nanocells (EDV™; EnGeneIC Dream Vector) derived from a non-pathogenic strain of S. typhimurium. Bacterial strains carry mutations that lead to asymmetric cell division during normal bacterial cell division, where EDV germinates at the poles of mutant strains (MacDiarmid et al., 2007). Purified EDV was prepackaged with bacterial gene expression recombinant plastids carrying the SARS-CoV-2 S protein-encoding gene (or the other SARS-CoV proteins disclosed). Plastids express the S-protein in the bacterial cytoplasm during normal bacterial growth, and when EDV is formed, significant concentrations of the S-protein segregate into the EDV cytoplasm. In addition, EDV-COVID nanocells were further packaged with αGC (EDV-COVID-αGC). 109 EDVs showed about 16 ng of S-protein, about 30 ng of αGC and about 100 plastid copies per EDV.

Previous studies have shown that after systemic administration, EDV is phagocytosed by professional phagocytes such as macrophages and DCs and degraded in lysosomes to release drug, nucleic acid or adjuvant payloads into the cytoplasm (MacDiarmid et al. 2007). Flow cytometry studies showed that EDV-COVID-αGC efficiently delivered both S-polypeptide and αGC to murine bone marrow-derived JAWSII DC and αGC glycolipid antigen presented MHC class I-like molecule CD1d with similar efficiency as free αGC presented on the DC surface ( Figure 5B ). The same DC can also present the S-polypeptide on the cell surface via MHC class II molecules ( FIG . 11C ).

Display of αGC:CD1d on the DC cell surface recruits iNKT cells carrying an invariant TCR known to bind to CD1d-associated αGC on DCs, which leads to rapid secretion of IFNγ (Bricard and Porcelli, 2007), as seen only in EDV-COVID-αGC mouse group seen ( Figure 11E ). Vaccination with EDV-COVID-αGC resulted in significant serum IFNγ release by day 28 in 6 human volunteers ( FIG . 14B ), suggesting activation of iNKT cells via αGC:CDld display on APCs ( FIG. 12G ). In contrast, the currently licensed mRNA vaccine (BNT162b2) showed transient serum IFNγ release that waned by day 8 (Bergamaschi et al., 2021). This is not surprising since the mRNA vaccine did not elicit antigen-specific antibodies via the iNKT/DC pathway. This iNKT cell activation and IFNγ secretion is critical for the activation of the high affinity antibody production pathway depicted in Figure 12G .

DCs that engulf EDV are further activated by pathogen-associated molecular patterns (PAMPs) such as EDV-associated LPS (Sagnella et al., 2020). This activation released TNF[alpha], which was evident in all four EDV-containing groups ( FIG. 11H ).

Activated iNKT cells have been shown to promote DC maturation through CD40/40L signaling and the cytokines IFNγ and TNFα (Hermans et al., 2003). It was also established that DC express the co-stimulatory molecule CD80/86, but upon activation by iNKT cells, the expression of these molecules was rapidly upregulated, as shown in a cancer study by EnGeneIC (data not shown). Upregulation of CD40L on the surface of DCs induced their maturation and secretion of IL-12 ( Fig . 1 IF ). Again, the secretion of IL-12 was only observed in the EDV-COVID-αGC group. This promotes the cytolytic function of cytotoxic CD8+ T cells and the priming of CD4+ T cells (Vinuesa et al., 2016) to provide cognate help for antibody production by B cells.

Splenic CD8+ cytotoxic T cells from mice immunized with EDV-COVID-αGC exhibited the highest number of CD3+/CD69+ cytotoxic T cells compared to all other groups ( FIG. 13C , D ). T cell responses are important for early viral clearance and long-term protection of memory S-specific T cells (Sattler et al., 2020). This data indicated that EDV carrying the S protein was able to induce CD8+ T cell specificity, which was further enhanced by the incorporation of αGC.

It is well established that B cells bearing MHC class II-presented protein antigens first interact homologously with TFH cells at the junction between T cell-rich regions and B-cell follicles of secondary lymphoid tissue (Eertwegh et al., 1993 ; Garside et al., 1998; Toellner et al., 1996). Engagement of the MHC class II/antigen complex on these B cells with the TCR on the TFH cell surface results in the cognate co-stimulatory molecule CD40L (Ma and Deenick, 2014), the inducible T-cell co-stimulator ICOS (Beier et al., 2000 ) and rapid upregulation of PD-1 ( FIG. 12G ).

Binding of ICOS ligand expressed on naive B cells (Hu et al., 2011 ) to ICOS on TFH cells is a necessary condition for the development of pre-TFH to fully differentiated TFH cells. ICOS/ICOSL signaling also results in the release of several cytokines, including IFNγ, IL-4, IL-10, IL-17, IL-2, IL-6, and IL-21 (Bauquet et al., 2009; Bonhagen et al. , 2003; Crotty, 2014; Löhning et al., 2003).

IL-6 has been shown to promote the differentiation of activated CD4+ T cells into TFH cells during immune responses. IL-6 has been shown to be elevated in the EDV-COVID-αGC injected group compared to all controls ( Fig. 11I ). The secretion of TNF[alpha] and IL-6 ( Fig. 11H , I ) was short-term, self-limited and none of the mice experienced any significant side effects. IL-10 also has anti-inflammatory properties, which play a key role in limiting the host response to inflammatory interleukin cells such as TNFα. IL-10 is part of the innate immune response to EDV - associated LPS, and thus was present to the same extent in all EDV-containing groups ( Fig. 11K ). When stimulated with S protein trimer, spleen cells from EDV-immunized mice showed that CD4+ T cells from EDV-COVID and EDV-COVID-αGC mice produced IFNγ but not IL-4, but not IL-4 from other This was not the case for the group ( FIG. 13E ), indicating that CD4+ T cells elicited antigen -specific Th1-type responses after vaccination.

IL-21 is mainly expressed by TFH cells and stimulates the proliferation of B cells and their differentiation into plasma cells. Class switching of B cells interacting with CD-40L to IgG and IgA is also promoted by IL-21 (Avery et al., 2008). A highly significant increase in IL-21 was also observed only in EDV-COVID-αGC-treated mice ( FIG . 11J ), suggesting that αGC in EDV-COVID is critical for activating the CD4+ TFH cell line, possibly due to DC activation of TFH cells licensed by iNKT. In terms of S-specific antibody responses, IL-21 plays a role in T cell-dependent B cell activation, differentiation, germinal center (GC) responses (Avery et al., 2010) and selection of B cells that secrete antigen-specific affinity antibodies Key role. There are no reports on IL-21 secretion after mRNA vaccines.

B cell activation leads to the extrafollicular proliferation of long-lived antibody-producing B cells as plasmablasts, or their entry into the GC for subsequent development of memory or plasma cells (MacLennan et al., 2003). Following homologous interaction of TFH cells in follicles, proliferating B cells generate GCs and undergo somatic hypermutation (SHM) and affinity maturation in their immunoglobulin V region genes, which generate higher affinity plasma cells and memory cells (Ansel et al., 1999; Breitfeld et al., 2000; Gunn et al., 1998; Jacob et al., 1991; Kim et al., 2001; MacLennan, 1994; Schaerli et al., 2000). In GC, TFH cells provide further B cell help mainly through the secretion of IL-21 and CD40L co-stimulation, which are the two main factors of B cell activation and differentiation. IL-21 also induces class switching to IgGl and IgG3 by human naive B cells and increases secretion of these Ig isotypes by human memory B cells (Pène et al., 2004).

B cells isolated from mouse bone marrow at day 28 after the initial injection, when co-incubated with S-protein, showed that B cells from EDV-COVID-αGC-immunized mice produced significantly higher levels of S compared to all other groups Specific IgM ( Figure 13A ) and IgG ( Figure 13B ). This indicates that EDV-COVID-αGC treatment induces SARS-CoV-2-specific memory B cells that can respond rapidly to S protein re-exposure.

The presence of antigen-specific memory B cells was detected in PBMCs of 6 healthy volunteers 28 days after EDV-COVID - αGC vaccination ( Fig . , K ).

At both dose levels, 28 days after the initial dose and a booster dose was given on the 21st day, high levels of anti-S protein IgM were detected in the serum of most EDV-COVID-αGC immunized i mice ( Fig . 12 A ) and IgG ( FIG. 12B ) antibody titers. Mice immunized with EDV-COVID also elicited IgM and IgG antibodies, but IgG responses were lower ( Fig. 12A , B ). Incorporation of αGC into EDV-COVID resulted in a significant and sustained increase in S-specific IgG titers. The S-specific IgG responses of EDV-COVID and EDV-COVID-αGC were similar on day 7 and 21 ( Fig. 12D , E , F ), however, the potentiation effect was only observed on day 28 in EDV-COVID-αGC Significantly in mice of αGC, suggesting that the incorporation of αGC may direct EDV-COVID-αGC to the iNKT-permissive DC pathway, which is known to lead to germinal center B cell activation/maturation and high-titer antibody secretion.

Interestingly, EDV-COVID is not expected to elicit S-specific antibodies via the iNKT/DC/TFH pathway, and thus is not expected to elicit via the iNKT/DC/TFH pathway elicited by EDV-COVID-αGC, Immunoglobulin class switching from an initial IgM response to IgG is initiated.

Surrogate virus neutralization tests showed that 100% of mice immunized with EDV-COVID-αGC were positive for neutralizing antibodies to wild-type SARS-CoV-2 virus ( Figure 13F and 13G ). In contrast, neutralizing antibodies were only detected in 50% ( Fig. 13F ) and 75% ( Fig. 13G ) of EDV-COVID-immunized mice, highlighting the importance of αGC in this vaccine. When tested against 4 common SARS-CoV-2 variant strains α (B.1.1.7), β (B.1.351), γ (P.1) and δ (B.1.617.2), the results were 3 × 10 ⁹ EDV-COVID-αGC-immunized mice strongly neutralized α (100%; Fig. 13 H ), β (80%; Fig. 13 I ), γ (90%; Fig. 13 J ) and δ (90%; Fig . 13K ) S-protein binding. In contrast, EDV-COVID insufficiently neutralized α (50%; FIG. 13F ), β (0% ; FIG. 13I ), γ (40 % ; FIG. 13J ) and δ (50%; FIG. 13J ). K ) mutant strains.

Similar results were seen in 4 healthy human volunteers, where PRNT90 (90% reduction in binding of the respective RBD to hACE2) virus neutralization by serum antibodies at day 28 showed Wuhan (100%), α (100%) Strong neutralization of RBD of , β (100%), γ (100%), δ (100%) and ο (75%). In contrast, data from the mRNA vaccine (BNT162b2) was only available for PRNT50 analysis (only 50% neutralization of the RBD was required) and these results required 2-fold (α), 5-10-fold (β), 2-5 Fold (γ), 2- to 10-fold (delta) serum dilutions are reduced to achieve PRNT50 (https://covid19.who.int/, 2021), and more than 22-fold for o (Cele et al., 2021).

With the help of T cells, B cells that react with protein antigens can differentiate into extrafollicular plasma cells, producing a rapid wave of low-affinity antibodies, or grow and differentiate in follicles, producing germinal centers. Data from sVNT showed that antibodies elicited by EDV-COVID-αGC could provide a high level of neutralization of RBD from VOC, suggesting that these are high-affinity antibodies, possibly due to B cells entering GC and undergoing SHM resulting in high affinity Antibody. In contrast, antibodies generated by immunization with EDV-COVID did not confer protection against the VOC RBD, suggesting that these B cells did not receive help from the fully cognate TFH, resulting in the formation of released plasma cells, resulting in a low-lying effect against the VOC RBD. Affinity antibodies.

This data suggests that even in highly immunocompromised populations, the EDV-COVID-αGC vaccine may have the potential to early activate the immune system with a broad antiviral immune response, and if these patients are infected with SARS-CoV-2 or its variants strains, may help to protect against the occurrence of severe lymphopenia in such patients. It is also interesting to note that, contrary to the results obtained in the mouse study, no early elevation of IL6 and TNFα after EDV vaccine administration was observed in human volunteers.

Serum sVNT from 6 healthy volunteers of the Covid vaccine Phase I clinical trial showed strong neutralizing activity (> PRNT90 equivalent) to the wild type, delta and o variants of SARS-CoV2 virus by day 28. Neutralization results for the o variant from 5 volunteers who had received at least 2 doses of Pfizer vaccine are also shown for comparison ( FIG. 14A ). These results demonstrate the effectiveness of the vaccines described herein compared to currently approved products, especially against the o variant, the current major variant of the SARS-CoV2 virus.

In addition, a significant increase in CD4+ and CD8+ central memory T cells was observed by day 28, followed by an increase in the number of antigen-specific B cells as well as memory B cells, indicating successful B cell activation, as shown in Figure 12G .

IFNγ could be detected in the supernatant of ex vivo PBMCs stimulated with SARS-CoV-2 spinin ( FIG. 14F ), confirming the proposed pathway ( FIG. 12G ). Furthermore, the number of CD69+ T cells in ex vivo PBMCs further increased upon S-protein stimulation, which was not observed at day 1 ( FIG. 14G ). This indicates that circulating T cell populations are specific for SARS-CoV-2 antigens, as observed with other vaccines (Ewer et al., 2021).

Successful vaccination is currently believed to be dependent on antibody and T cell-mediated immunity, and although at least type I and type II interferons have been recognized to elicit broad antiviral immunity, due to the diverse effects these interferons exhibit, few Likely to contain current and future viral pandemics, a combination of broad specific and non-specific antiviral immunity and specific memory B and T cell responses may be required.

Even more important are the types of antibodies elicited by the vaccines described herein. Most vaccines do not elicit the iNKT-licensed DC pathway and thus B cells rapidly release low-affinity antibodies, which are then unable to neutralize the VOC RBD. In contrast, as B cells undergo affinity maturation and SHM in GC, the DC pathway permissive by iNKT elicits high-affinity antibodies and these high-affinity antibodies can be highly effective in neutralizing VOC RBDs.

EDV based cancer therapeutics or COVID vaccines can be lyophilized after manufacture and can be stored and shipped around the world at room temperature. The shelf life of EDV cancer therapeutics has now been shown to exceed 3 years and the EDV-COVID-αGC vaccine has a stability of more than 1 year. B. The state of this disclosure

The SARS-CoV-2 vaccine compositions described herein comprise at least one antigen from a variant of SARS-CoV-2, and in other aspects may comprise at least one antigen from a plurality of variants of SARS-CoV-2 (e.g., α, β, γ, δ) antigens. The vaccine composition may additionally comprise a SARS-CoV-2 antigen from a non-mutant SARS-CoV-2 strain.

The mature SARS-CoV-2 virus has four structural proteins, namely, mantle, membrane, nucleic acid protein shell, and spines. It is believed that all of these proteins can be used as antigens to stimulate neutralizing antibodies and increase CD4+/CD8+ T cell responses. In one aspect, the spinin antigen from a variant strain of SARS-CoV-2 is utilized in the compositions described herein. As noted above, the composition may additionally comprise viral antigens from a non-mutant strain of SARS-CoV-2.

In another aspect, the SARS-CoV-2 antigen from the mutant or non-mutant strain can be the receptor binding domain (RBD) of spinin, the site involved in binding to the human ACE2 receptor.

In one aspect, the vaccine compositions described herein comprise, within a single pocket-sized cell, a bacterial gene-expressed plastid encoding at least one SARS-CoV-2 antigen (e.g., spinin), the spinin expressed by the plastid ( or other SARS-CoV-2 antigens) and α-galactosylceramide as an adjuvant to trigger IFNγ responses. In other aspects, plastids may encode more than one SARS-CoV-2 antigen, for example from SARS-CoV-2 variants (e.g., α, β, γ, δ, or other variants described herein or not yet identified) ) and the spike protein of the SARS-CoV-2 strain.

In other aspects, the present disclosure encompasses compositions comprising a first pocket-sized cell that elicits an IFNγ response comprising α-galactosylceramide as an adjuvant and comprising a protein encoding at least one SARS-CoV-2 antigen (e.g., spinin). ) of the bacterial gene expression plastid of the second pocket cell and the spike protein (or other SARS-CoV-2 antigen) expressed by the plastid. Again, in other aspects, plastids may encode more than one SARS-CoV-2 antigen, e.g., from SARS-CoV-2 variants (e.g., α, β, γ, δ, or those described herein or not yet identified). other mutant strains) and the spike protein of SARS-CoV-2 strains.

Unlike current COVID-19 vaccines, bacterial pocket-sized coronavirus vaccines are expected to be effective against COVID-19 variants, both existing and emerging variants. This is due to, as described herein, the design of the bacterial pocket cell coronavirus vaccine resulting in broad antiviral efficacy, in contrast to all currently used COVID-19 vaccines. Effectively combating mutant strains is critical to the long-term success and management of the COVID-19 pandemic.

Exemplary advantages of the vaccine compositions of the invention described herein over other COVID-19 vaccines are detailed in the table below. EDV-COVID-19 therapeutic vaccine Other vaccines such as Pfizer-BioNTech , Moderna , Regeneron IgG and IgM reactions of anti-spillin serum yes yes Nasal and oral mucosal secretory IgA responses against spinin delivered intranasally yes no Antiviral IFN-α and IFN-β responses yes Only a portion, such as those carrying nucleic acids such as mRNA Antiviral IFN- γ response yes no Alleviate lymphopenia, especially in the elderly yes no Activate WBC to fight viral infection yes only a part CD8+ cytotoxic T cells and iNKT cells specifically respond to SARS-CoV-2 yes no. Some were able to generate a CD8+ T cell response. Is it effective in immunocompromised patients ( such as cancer ) yes no Effective in older adults with comorbidities yes only a part toxicity none Severe toxicity in some patients Effective in patients with autoantibodies against IFNα yes. This is because it also induces IFNβ and IFNγ antiviral responses no. This is because it does not trigger other antiviral interferon responses Can the vaccine be easily altered to cover emerging mutant strains of the SARS-CoV-2 virus yes. A new plastid carrying the gene sequence for the mutant protein is simply added to EDV. The same plastid can carry multiple sequences no. Completely new vaccines had to be designed. Whether it acts as both a vaccine as well as a therapeutic yes No - one or the other storage and transport issues no problem. Can be stored and transported at room temperature. Serious Problem. Store and transport at -20°C to -70°C Storage life more than 3 years 2 months to 6 months Product Cost Cheap to make. globally affordable Some are quite expensive and banned in many countries.

1-3 depict various exemplary vaccine constructs according to the present disclosure. The first construct ( FIG. 2A ) shows a typical EDV-COVID-19 vaccine composition comprising bacterial pocket-sized cells containing a combination of: (i) a bacterial gene expression plasmid encoding spinin, (ii) composed of The plastid expresses spinin, and (iii) glycolipid α-galactosylceramide as an adjuvant to elicit an IFNγ response, and the second construct ( FIG . 3 ) shows the EDV-COVID-19 vaccine composition comprising Bacterial pocket cells containing combinations of: (i) bacterial gene expression plasmids encoding the spike protein of SARS-CoV-2 and spike proteins encoded by multiple variant strains, (ii) multiple spike proteins, including spike proteins produced by The spike protein encoded by the plasmid, and (iii) glycolipid α-galactosylceramide as an adjuvant. Both plastids are bacterial expression plastids, so spinin is produced in the cytoplasm of EDV.

A key point of these exemplary constructs is that the plastid has the bacterial expression of the bacterial origin of replication, and thus it does not replicate in human cells and does not integrate into the chromosome. Plastids remain episomal and degrade when the cell completes its life cycle.

In another aspect herein, the vaccine compositions described herein comprise one or more pocket-sized cells comprising a plastid with a bacterial gene expression promoter produced in the parental bacterial strain SARS-CoV-2 antigens (such as spinin or other SARS-CoV-2 antigens), which are then isolated into recombinant pocket cells. Thus, in one aspect, the composition carries plastids, spinin (or other SARS-CoV-2 antigens), and α-galactosylceramide in the same pocket cell or in multiple pocket cells.

In another variant, plastids can carry mammalian gene expression promoters, so spinin is expressed in human professional phagocytes only when the pocket cells are phagocytized, the plasmid is released, and the mRNA is expressed in the mammalian nucleus. Therefore, this composition differs from the ones described above in that the pocket-sized cell composition carries recombinant plastids with mammalian gene expression promoters and mutants or variations from SARS-CoV-2 and colonized downstream of the promoters Spiny protein gene (or other SARS-CoV-2 antigen) and α-galactosylceramide of strain SARS-CoV-2. Thus, in this vaccine composition, spinin is absent from the pocket cells.

In another aspect, the gene sequence from SARS-CoV-2 and mutant/mutant SARS-CoV-2 viruses selected in plastids may include the entire spinin-encoding gene or only the human ACE2 receptor binding ( RBD) gene sequence, this is because it is expected that the antibody response is directed against the RBD region of these viral spike proteins (or other SARS-CoV-2 antigens).

1-3 depict various exemplary vaccine constructs according to the present disclosure. The first construct ( FIG. 2A ) shows a typical EDV-COVID-19 vaccine composition comprising bacterial pocket-sized cells containing a combination of: (i) a bacterial gene expression plasmid encoding spinin, (ii) composed of The plastid expresses spinin, and (iii) glycolipid α-galactosylceramide as an adjuvant to elicit an IFNγ response, and the second construct ( FIG . 3 ) shows the EDV-COVID-19 vaccine composition comprising Bacterial pocket cells containing combinations of: (i) bacterial gene expression plasmids encoding the spike protein of SARS-CoV-2 and spike proteins encoded by multiple variant strains, (ii) multiple spike proteins, including spike proteins produced by The spike protein encoded by the plasmid, and (iii) glycolipid α-galactosylceramide as an adjuvant. Both plastids are bacterial expression plastids, so spinin is produced in the cytoplasm of EDV.

Furthermore, bacterial pocket cells or EDVs are only engulfed by professional phagocytes such as macrophages, dendritic cells and NK cells. It cannot enter normal cells. Finally, more than 2,400 doses of EDV (bacterial pocket cells) carrying various drugs, nucleic acids, and sugar esters have been administered to more than 170 terminal cancer patients in Australia and the United States, despite repeated dosing (in many patients 15 to 50 repeated doses), but with minimal to no toxic side effects.

The plastid can also be a mammalian expressing plastid, wherein the gene expressing promoter can be a mammalian expressing promoter. Therefore, spinin is not produced in the cytoplasm of EDV. Conversely, when EDV is decomposed in the lysosomes of professional phagocytes such as macrophages, dendritic cells, and NK cells, the plastids are released, exported to the nucleus, and express spines after expressing mRNA from mammalian gene expression promoters. protein.

The EDV-COVID-19 vaccine can be administered intramuscularly, intranasally, or orally. In general, intramuscular administration is preferred. However, the vaccine can be administered intranasally or orally to induce secretory IgA responses in the mucosal tract and lungs. Furthermore, this will trigger innate and adaptive immune responses in the lung and oral passages.

Vaccines can be mixed and matched because the same vaccine can be given intramuscularly and intranasally to elicit robust systemic and mucosal immune responses.

Specifically, Figure 1A shows a scanning electron microscope image showing the generation of EnGeneIC Dream Vector (EDV) nanocells from a safe strain of bacteria Salmonella typhimurium, and Figure 1B shows a transmission electron micrograph image, It shows the structure of empty EDV bacterial nanocells, where the diameter is about 400 nm. Figure 2A is a graphical representation of an EDV-COVID-19 vaccine composition comprising a bacterial expression plasmid ("EDV"), such as that shown in Figure 1B, wherein the EDV first expresses the spine of SARS-CoV-2 in the EDV cytoplasm protein and additionally carries the glycolipid α-galactosylceramide IFN-γ stimulator. The expressed spinins encoded by SARS-CoV-2 are designated by the stars on Figure 2A. Figure 2B shows exemplary vials containing lyophilized EDV-COVID-19 vaccine compositions.

Figure 3 is a graphical representation of an EDV-COVID-19 vaccine composition comprising a bacterial expression plasmid ("EDV") (such as that shown in Figure 1B), wherein the EDV contains (i) a plasmid expressed from the original SARS- Selected spike proteins of CoV-2 and multiple genetic variants (e.g., delta variant and Brazilian variant), (ii) a gene expression promoter that expresses all proteins as a single mRNA and individual proteins in the EDV cytoplasm, (iii) Multiple spinins, including spinins produced by SARS-CoV-2, Brazilian variant spinins, and delta variant spinins, and (iv) CD1d-restricted forms as adjuvants or IFN-γ stimulators iNKT cell antigen glycolipid α-galactosylceramide (α-GalCer). The encoded expressed spinins are designated by the stars on FIG. 3 .

The EDV-COVID-19 vaccine composition can be easily lyophilized and stored at room temperature with a shelf life of more than 3 years. The EDV-COVID-19 vaccine composition can be shipped by courier anywhere in the world and stocked eg in a hospital pharmacy. Delivery and storage can also be performed at room temperature. Furthermore, its low manufacturing cost means that the EDV-COVID-19 vaccine composition is affordable worldwide.

The genome sequences of many different SARS-CoV-2 strains and variants, including the spike proteins of these viruses, are known. See eg Figure 9 . A new virus strain arises when a virus undergoes one or more mutations that change its behavior in some way, but a mutant strain occurs when a virus undergoes any type of mutation. Examples of SARS-CoV-2 strains include L strains, S strains, V strains, G strains, GR strains and GH strains. www.sciencedaily.com/releases/2020/08/200803105246.htm.

Examples of known SARS-CoV-2 variants include (but are not limited to) (1) UK SARS-CoV-2 variant (B.1.1.7/VOC-202012/01), also known as alpha variant (B .1.1.7 (α)); (2) B.1.1.7 variant with E484K; (3) B.1.617.2 (δ) variant; (4) B.1.351 (β) variant, also Known as the South African variant; (5) P.1 (γ) variant; (6) B.1.525 (η) variant; (7) B.1.526 (ι) variant; (8) B.1.617 (κ , δ) mutant strain; (9) B.1.617.1 (κ) mutant strain; (10) B.1.617.2 mutant strain; (11) B.1.617.3 mutant strain; (12) λ (lineage C. 37) mutant strains; (13) ε (lineage B.1.429, B.1.427, CAL.20C) mutant strains; (14) ζ (lineage P.2) mutant strains; (15) θ (lineage P.3) mutation strain; (16) R.1 variant; (17) lineage B.1.1.207 variant; and (18) lineage B.1.620 variant.

Other SARs-CoV-2 variants include (1) L452R spike protein substitution, (2) E484K spike protein substitution, (3) K417N, E484K, N501Y spike protein substitution, (4) K417T, E484K, N501Y spike protein substitution SARS-CoV-2 mutant strains, and (5) SARS-CoV-2 mutant strains with one or more of the following erroneous mutations: N440, L452R, S477G/N, E484Q, E484K, N501Y, D614G, P681H, P681R and A701V.

See also (1) Lu et al., "Genomic Characterization and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding," The Lancet , 395 :565-574 (2020-02-22); (2) Galloway et al. People, "Emergence of SARS-CoV-2 B.1.1.7 Lineage - United States, December 29, 2020 - January 12, 2021," Centers for Disease Control and Prevention , 70 (3):95-99 (January 22, 2021) (SARS-CoV-2 UK variant sequencing, lineage B.1.1.7 (20I/501Y.V1)); (3) Toovey et al., “Introduction of Brazilian SARS-CoV- 2 484K.V2 related variants s into the UK," J. Infect. , 82 (5):e23-e24 (February 3, 2021) (describes two Brazilian variants); (4) Sah et al., " Complete Genome Sequence of a 2019 Novel Coronavirus (SARS-CoV-2) Strain Isolated in Nepal," ASM Journals, Microbiology Resource Announcements , 9 (11) (March 11, 2020); and (5) "Variants of SARS- CoV-2”, www.wikipedia.org/wiki/Variants_of_SARS-CoV-2#Notable_missense_mutations .

Surprisingly and in contrast to at least some COVID-19 vaccines currently in use, the vaccine compositions described herein generate immunity as measured by IgG titers against various SARS-CoV-2 variants. Specifically, Figures 10A-D show the results of IgG titers following administration to five different groups of mice ( n =6/group; ELISA samples were run in triplicate): Group 1 = saline; Group 2 = EDV (bacterial pocket cells without payload); Group 3 = EDV _control (EDV carrying a plastid without an insert expressing spinin (i.e., only the plastid backbone)); Group 4 = EDV _Covid ( Bacterial pocket cells comprising plastids and encoded SARS-CoV-2 spike protein) and group 5 = EDV _{Covid +} α _GC (construct shown in Figure 2A ). Mice were given 3 × 10 ⁹ EDV. The results shown in Figures 10A-D detailing S1 unit-specific IgG titers at day 28 after split-dose IM demonstrate that serum IgG titers obtained from mice treated with EDV-COVID-GC correlated well with all four (1) SARS-CoV-2 variant α (B.1.1.7.UK) ( Fig. 10A ); (2) SARS-CoV-2 variant β (B.1.351. SA) (Fig. 10B); (3) SARS-CoV-2 variant δ (B.1.617.2 India); and (4) SARS-CoV-2 variant γ (P.1 Brazil). II. Composition

The composition comprises a combination of: (a) a vector comprising a plasmid encoding at least one viral antigen from a variant of the SARS-CoV-2 virus and optionally additional viral antigens from SARS-CoV-2; and (b) a vector comprising an antigen recognized by CD1d, wherein at least one of the two vectors is an intact, bacterial-derived pocket cell or a killed bacterial cell, and wherein the two vectors are present on at least one pharmaceutical in an acceptable carrier. An exemplary antigen recognized by CDld is α-galactosylceramide (α-GalCer), which stimulates IFNγ, which is critical for viral immunity. In another aspect, both vectors are whole, bacterial-derived pocket cells or killed bacterial cells, including two separate bacterial-derived pocket cells or killed bacterial cells or together in a single bacterial source Sexual pocket cells or killed bacterial cells.

In another aspect, the vector or intact, bacterial-derived pocket cells may comprise one or more of the four major structural proteins of the SARS-CoV-2 virus or variant strain or antigenic fragments thereof, such as spine (S ) protein, nucleic acid protein shell (N) protein, membrane (M) protein and envelope (E) protein.

In another aspect, as described above, one or the other (but not both) of the plastid payload and the CDld-recognized payload can be passed through cells that are not intact, bacteria-derived miniature cells, or killed bacteria. Vector administration of cells. Examples of such non-pocket-sized cellular carriers are liposomes, polymeric carriers, recombinant viral envelopes (virions), and immunostimulatory complexes (ISCOMs). See, eg, Bungener et al. (2002), Kersten et al. (2003), Daemen et al. (2005), Chen et al. (2012) and Yue et al. (2013). Available at https://www.ncbi.nlm.nih.gov/pubmed/12428908 (Bungener); https://www.meta.org/papers/liposomes-and-iscoms/12547602 (Kersten); https://www .ncbi.nlm.nih.gov/pubmed/15560951 (Daemen); https://journals.plos.org/plosone/article?id=10.1371/journal.pone.0039039 (Chen); https://pubs.rsc .org/en/content/articlelanding/2013/bm/c2bm00030j#!divAbstract (Yue).

The compositions can be administered via any pharmaceutically acceptable method, such as, but not limited to, injection (parenteral, intramuscular, intravenous, intraportal, intrahepatic, peritoneal, subcutaneous, intratumoral, or intradermal administration), via Oral administration, application of the formulation to a body cavity, inhalation, insufflation, nasal administration, pulmonary administration, or any combination can also be used.

The composition can be administered as a vaccine to individuals at risk of infection with a variant of SARS-CoV-2, or the composition can be administered as a therapeutic agent to an individual suffering from infection with a variant of SARS-CoV-2 infection.

In highly immunocompromised end-stage cancer patients, intact bacterial pocket cells (also known as "EnGeneIC Dream Vector™" or EDV™) have been shown to lead to: (1) CD8+ T cells, macrophages, NK cells , activation and proliferation of dendritic cells and iNKT cells. This result is exactly what would be expected for a CoV-2 therapeutic/vaccine.

According to one aspect, the present disclosure provides the use of recombinant, whole bacterial pocket cells comprising plastids encoding viral proteins for infection by administering the composition to the virus in the manufacture of compositions or in a method for treating and/or preventing disease in a person who is at risk of viral infection. The disease to be treated in this context is infection with the SARS-CoV-2 virus or variant strains.

The main areas of treatment/vaccine against SARS-CoV2 currently being explored include: (1) antiviral drugs (eg Gilead Sciences; the nucleotide analog Remdesivir); (2) cocktail monoclonal antibodies ( such as Regeneron); and (3) an attenuated virus used as a vaccine to stimulate a potent antibody response to viral proteins. Each of these strategies faces difficulties, but most importantly, none of these approaches can solve the problem of lymphopenia in elderly and immunocompromised patients to be able to overcome viral infections. In the absence of a robust immune system, this patient population remains the most vulnerable and most likely to die from the disease.

In previous EnGeneIC disclosures, the use of plastid-packaged pocket cells in the treatment of neoplastic diseases has been demonstrated, where the main function of the plastid is to encode siRNA or miRNA to deactivate genes responsible for cell proliferation or drug resistance in cancer cells silence.

In the present disclosure, the function of the plastid-packaged minicells component of the complete composition, which includes CDld-recognized antigens, such as α-GC-packaged minicells, has novel functions not previously shown or described. Function. Specifically, plastids are used to encode viral proteins in the parent bacterial cell, and these proteins segregate into pocket-sized cells upon asymmetric cell division. These viral proteins are delivered to the lysosomes of antigen processing and presentation cells (APCs) such as macrophages and dendritic cells. Following antigen processing, viral protein epitopes are displayed on the surface of APCs via MHC class I and II molecules, which is expected to result in a potent antibody response to the viral protein. In addition, plastids, which are themselves double-stranded nucleic acids, are recognized by nucleic acid sensing proteins in APCs, and this then triggers the secretion of type I interferons (IFNα and IFNβ).

This unique dual triggering of antibody responses to viral proteins and type I interferon responses not only results in clearance of viral particles released from infected cells, but also enables cells of the immune system to recognize virus-infected cells and kill them. This dual trigger has not been described previously, in particular the ability of type I interferons to trigger a hitherto uncharacterized mechanism by which they can recognize and kill virus-infected cells. In the present disclosure, the triggering of IFNγ after presentation of α-GC/CDld to iNKT cell receptors is key to enhance antiviral immunity. The exact mechanism of action is unknown, but IFNγ is critical in identifying and destroying virus-infected cells. In addition, IFNγ has never been reported to enhance serum IgG antibody responses to any antigen. The present invention demonstrates this phenomenon for the first time. This finding is clearly visible in Figures 6B-6D and Figures 7A-7D, where EDV compositions carrying plastids and spinin did not elicit high levels of IgG antibodies against spinin. In contrast, EDV compositions carrying the same COVID plasmid and spinin, and additionally carrying αGC, provided a significant increase in IgG antibody titers to spinin. The sole function of αGC is to rapidly trigger the secretion of IFNγ, which then stimulates a massive antiviral immune response. This is the first discovery showing that one or more effectors of IFNγ enhance antiviral serum IgG antibody titers.

In the United States, several clinical trials have been conducted in which intact, bacterial-derived pocket cells loaded with anticancer agents and intact, bacterial-derived pocket cells loaded with microRNA mimics have been administered to humans in methods for treating cancer . See, eg, ClinicalTrials.gov Identifier Nos. NCT02766699, NCT02687386, and NCT02369198. Also, in Australia, bacterial pocket cells loaded with α-GC are being administered to patients in a phase IIa clinical trial in terminal cancer patients. The results have shown that the intact, bacterial-derived miniature cell line loaded with α-GC is a potent stimulator of IFN-γ. See Trial ID No. ACTRN12619000385145. Thus, the in vivo efficacy of intact, bacterial-derived pocket cells loaded with CD1d-recognized antigens has been shown in humans, and has additionally been shown that intact, bacterial cells loaded with target compounds (e.g., anticancer compounds, rather than viral antigens) Efficacy of derived miniature cells in humans.

Additionally, the disclosed compositions have the additional benefit of restoring elderly and immunocompromised patients from lymphopenia (rapid depletion of lymphocytes including macrophages, dendritic cells, NK cells, and CD8+ T cells). Key function, lymphopenia is the main reason why viruses like SARS-CoV-2 predominate in these patients and eventually cause respiratory distress syndrome and eventually death. In particular, the pocket cells of the composition themselves activate macrophages via recognition of pathogen-associated molecular patterns (PAMPs) such as LPS. This provides activation, maturation and proliferation signals to quiescent monocytes in the bone marrow, resulting in a marked increase in activated macrophages and dendritic cells. In addition, pocket cell-associated PAMPs also activate NK cells and also stimulate proliferation of these cells. Furthermore, activated macrophages and dendritic cells enter the infected area and engulf the virus-infected apoptotic cells. They then migrate into the draining lymph nodes and activate naive CD8+ T cells, which are then activated and proliferate.

Therefore, the pocket-sized cell component of the composition can overcome lymphopenia in these elderly and immunocompromised patients by means of PAMP signaling, and the activation of these lymphocytes helps to overcome viral infection and prevent patients from falling into respiratory distress and die. A. Background on SARS - CoV - 2 variants

A growing body of research shows that both infection and vaccination trigger an immune response to Covid-19 that lasts for months or even years, but the vaccine's ability to fight known variants makes vaccination critical to containing the virus. Recent studies have shown that mutant strain variants, including the delta strain currently dominant in the United States, can partially evade immune responses from previous infections and vaccinations. Comprehensive vaccination still appears to provide solid protection against it. As the delta variant takes hold in the United States, a combination of immunity from infection and vaccination will likely act as a buffer, epidemiologists say. But the virus still has a chance to spread. Wall Street Journal , "COVID-19 Immune Response Could be Long Lasting, but Variants Present Risks" (July 16, 2021).

The U.S. Government SARS-CoV-2 Interagency Group (SIG) developed a variant classification scheme that defines three types of SARS-CoV-2 variants: (1) SARS-CoV-2 Variant of Interest ; (2) Variant of Concern of SARS-CoV-2; and (3) Variant of High Consequence of SARS-CoV-2.

SARS-CoV-2 "mutants to watch out for" are defined by the CDC as variants with specific genetic markers, changes in receptor binding, reduced neutralization of antibodies against previous infection or vaccination, reduced therapeutic efficacy , potential diagnostic impact, or projected increases in transmissibility or disease severity.

Possible attributes of a VOI include, for example, specific genetic markers predicted to affect transmission, diagnosis, treatment, or immune escape, and/or evidence that it is the cause of an increased proportion of cases or a unique cluster of outbreaks.

SARS-CoV-2 variants of note (VOI) include B.1.427 (Pango lineage), which has spinin substitutions: L452R, D614G, and has been named "E". It was first identified in the United States (California). Notable attributes include approximately 20% increase in transmission and a slight decrease in sensitivity to the combination of bamlanivimab and etesevimab; however, the clinical significance of this decrease is unclear . Alternative monoclonal antibody therapy is available, and mutant strains exhibit reduced serum neutralization during convalescence and post-vaccination. On June 29, 2021, this variant was downgraded from VOC due to the significant decrease in the proportion of B.1.427 lineage viruses circulating nationwide and available data indicating that vaccines and treatments are effective against this variant.

The second SARS-CoV-2 VOI line, B.1.429 (Pango lineage), has spinin substitutions: S13I, W152C, L452R, D614G, and has been designated "E". Notable attributes include an approximately 20% increase in transmission and a decrease in sensitivity to the combination of banivirumab and eltersuumab; however, the clinical significance of this decrease is unclear. Alternative monoclonal antibody therapy is available, and mutant strains exhibit reduced serum neutralization during convalescence and post-vaccination. On June 29, 2021, this variant was downgraded from VOC due to the significant decrease in the proportion of B.1.429 lineage viruses circulating nationwide and available data indicating that vaccines and treatments are effective against this variant.

A third SARS-CoV-2 VOI line, B.1.525 (Pango lineage), has spinin substitutions: A67V, 69del, 70del, 144del, E484K, D614G, Q677H, F888L, designated "n". The variant was first identified in United Kingdom/Nigeria - December 2020. Notable attributes include a potential reduction in therapeutic neutralization for some Emergency Use Authorization (EUA) monoclonal antibodies and a reduction in serum neutralization potential during recovery and post-vaccination.

A fourth SARS-CoV-2 VOI lineage B.1.526 (Pango lineage) with spinin substitutions: L5F, (D80G*), T95I, (Y144-*), (F157S*), D253G, (L452R*), (S477N*), E484K, D614G, A701V, (T859N*), (D950H*), (Q957R*), and named "ι". The variant strain was first identified in the United States (New York) - December 2020. Notable attributes include decreased sensitivity to the combination of banivirumab and etesivirumab monoclonal antibody therapy; however, the clinical significance of this decrease is unclear. Alternative monoclonal antibody therapy is available, and mutant strains exhibit reduced serum neutralization during convalescence and post-vaccination. The B.1.526.1 subpedigree has been merged with this parental pedigree.

The fifth SARS-CoV-2 VOI lineage B.1.617.1 (Pango lineage) has spinin substitutions: (T95I), G142D, E154K, L452R, E484Q, D614G, P681R, Q1071H, and is named "κ". Variant strain identified for the first time in India - December 2020. Notable attributes include the potential for treatment neutralization of some EUA monoclonal antibodies and the potential for decreased serum neutralization after vaccination.

The sixth SARS-CoV-2 VOI line B.1.617.3 (Pango lineage) has spinin substitutions: T19R, G142D, L452R, E484Q, D614G, P681R, D950N, and is named "20A". The mutant strain was first identified in India - October 2020. Notable attributes include the potential for treatment neutralization of some EUA monoclonal antibodies and the potential for decreased serum neutralization after vaccination.

A SARS-CoV-2 "variant of very high concern" (VOC) is defined by the CDC as having increased transmissibility, more severe disease (such as increased hospitalization or death), neutralizing antibodies developed during previous infection or vaccination Variant strains with evidence of significantly reduced, reduced effectiveness of treatments or vaccines, or failure of diagnostic tests.

Potential attributes of a VOC, in addition to those of a VOI, include: (a) evidence of diagnostic, therapeutic, or vaccine impact; (b) broad interference with diagnostic test targets; (c) materially reduced sensitivity to one or more classes of therapy (d) evidence of a neutral and significant reduction in antibodies produced during prior infection or vaccination; (e) evidence of reduced vaccine-induced protection from severe disease; (f) evidence of increased transmissibility; and ( g) Evidence of increased disease severity.

The first VOC line B.1.1.7 (Pango lineage) with spinin substitutions: 69del, 70del, 144del, (E484K*), (S494P*), N501Y, A570D, D614G, P681H, T716I, S982A, D1118H ( K1191N*), and named "α". The variant was first identified in the UK and notable attributes include (1) approximately 50% increased transmission, (2) possible increased severity based on hospitalization and case fatality rates, (3) limited response to EUA monoclonal antibody therapy No effect on susceptibility, and (4) minimal effect on convalescence and post-vaccination seroneutralization.

The second VOC line, B.1.351 (Pango lineage), has spinin substitutions: D80A, D215G, 241del, 242del, 243del, K417N, E484K, N501Y, D614G, A701V, and is named "β". The variant was identified for the first time in South Africa, and notable attributes include (1) an approximately 50% increase in transmission, (2) a marked sensitivity to the combination of banivirumab and eltersuvirumab monoclonal antibody therapy decreased, but other EUA monoclonal antibody treatments are available, and (3) serum neutralization decreased during convalescence and post-vaccination.

A third VOC line B.1.617.2 (Pango lineage) with spinin substitutions: T19R, (V70F*), T95I, G142D, E156-, F157-, R158G, (A222V*), (W258L*), ( K417N*), L452R, T478K, D614G, P681R, D950N, and named "δ". Variant strains were first identified in India, and noteworthy properties include (1) increased infectivity, (2) potential decreased treatment neutralization of some EUA monoclonal antibodies, and (3) post-vaccination serum neutralization possibly lower. AY.1, AY.2, and AY.3 are currently clustered with B.1.617.2.

The third VOC line, P.1 (Pango lineage), has spinin substitutions: L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, D614G, H655Y, T1027I, and is named "γ". The variant was first identified in Japan/Brazil, and notable properties include (1) significantly reduced sensitivity to the combination of banivirumab and eltersuvir monoclonal antibody treatment, but other EUA monoclonal antibodies Antibody therapy is available, and (2) serum neutralization decreases during recovery and post-vaccination.

Finally, a SARS-CoV-2 "variant of high consequence" (VHC) is defined by the CDC as one with clear evidence that preventive measures or medical countermeasures (MCM) are significantly less effective relative to previously circulating variants. mutant strain. In addition to possible attributes of VOC, possible attributes of VHC include the following effects on medical countermeasures (MCM): (1) exhibiting diagnostic failure, (2) significantly reduced vaccine effectiveness, a higher proportion of vaccine breakthrough cases, or Evidence of minimal protection against severe disease induced, (3) markedly reduced susceptibility to multiple Emergency Use Authorization (EUA) or approved therapeutic agents, and (4) increased clinical illness and hospitalization for more severe disease. VHCs will be required to notify the WHO under the International Health Regulations, report to the CDC, announce strategies to prevent or contain transmission, and recommend updated treatments and vaccines. Currently, there are no variants of SARS-CoV-2 that have risen to high shock levels.

As of June 28, 2021, four notable variants of SARS-CoV-2 have been reported in the United States: B.1.1.7 (α), B.1.351 (β), P.1 (γ), and B. .1.617.2 (δ). “About Variants of the Virus that Causes COVID-19,” www.cdc.gov/coronavirus/2019-ncov/variants/variant.html (June 28, 2021).

The B.1.1.7 (α) mutant strain was first detected in December 2020 in the United States. It was initially detected in the UK. α. (B.1.1.7) Variant strains of COVID-19 appear to be more transmissible, with an approximately 50% increase in transmission compared to previously circulating variant strains. This variant strain may also have an increased risk of hospitalization and death. www.mayoclinic.org/diseases-conditions/coronavirus/expert-answers/covid-variant/faq-20505779 (accessed 16 July 2021).

The B.1.351 (β) mutant strain was first detected in the United States at the end of January 2021. Its initial strain was detected in South Africa in December 2020. The β (B.1.351) variant appears to be more transmissible, with approximately a 50% increase in transmission compared to previously circulating variants. It also reduces the effectiveness of some monoclonal antibody medicines and antibodies produced from previous COVID-19 infections or COVID-19 vaccines. www.mayoclinic.org/diseases-conditions/coronavirus/expert-answers/covid-variant/faq-20505779 (accessed 16 July 2021).

The P.1 (γ) variant was first detected in January 2021 in the United States. P.1 was initially identified from travelers from Brazil who were tested during routine screening at Japanese airports in early January. Gamma (P.1) variants reduce the effectiveness of some monoclonal antibody medicines and antibodies generated from previous COVID-19 infection or COVID-19 vaccines. www.mayoclinic.org/diseases-conditions/coronavirus/expert-answers/covid-variant/faq-20505779 (accessed 16 July 2021).

Finally, the B.1.617.2 (δ) variant was detected for the first time in March 2021 in the US lineage. Its original strain was identified in India in December 2020. Based on current data, variant B.1.1.7 (δ) is the most common variant in the United States. Ditto. B.1.1.7 (δ) mutant strains are potentially more transmissible than other mutant strains. Studies have shown that it spreads easily in indoor sports facilities and in households. This variant may also reduce the effectiveness of some monoclonal antibody treatments and antibodies produced by the COVID-19 vaccine. www.mayoclinic.org/diseases-conditions/coronavirus/expert-answers/covid-variant/faq-20505779 (accessed 16 July 2021).

These variants appear to spread more easily and faster than other variants, which may lead to more cases of COVID-19. Ditto. An increase in the number of cases will put more pressure on medical resources, lead to more hospitalizations, and possibly more deaths. “About Variants of the Virus that Causes COVID-19,” www.cdc.gov/coronavirus/2019-ncov/variants/variant.html (June 28, 2021).

Different variants have emerged in Brazil, California, and elsewhere. A variant known as B.1.351, which first appeared in South Africa, may have the ability to reinfect people who have recovered from earlier versions of the coronavirus. It may also have some resistance to some coronavirus vaccines being developed. Nonetheless, other vaccines currently being tested appear to protect people infected with B.1.351 from severe disease. www.hopkinsmedicine.org/health/conditions-and-diseases/coronavirus/a-new-strain-of-coronavirus-what-you-should-know (accessed July 16, 2021).

Patients infected with SARS-CoV or MERS-CoV initially present with mild influenza-like illness with fever, dyspnea, and cough. Most patients recover from this disease. However, the most vulnerable groups are patients over 65 years of age and patients with comorbidities (such as cancer, HIV, etc.) that lead to immunosuppression, where the disease progresses to more severe symptoms and is characterized by atypical interstitial pneumonia and diffuse alveolar damage. Both SARS-CoV and MERS-CoV can cause acute respiratory distress syndrome (ARDS), the most severe form of acute lung injury, in which alveolar inflammation, pneumonia, and lung hypoxia lead to respiratory failure, multi-organ disease and 50% ARDS patients died. As the disease progresses, lymphopenia is usually observed. Most deaths due to CoV-2 infection are the result of severe lymphopenia in immunocompromised patients, and the disease predominates, leading to ARDS.

Coronavirus (SARS-CoV-2; COVID-19) causes atypical pneumonia in infected individuals and symptoms include fever, dry cough, and fatigue. Most patients have lymphopenia (decreased white blood cell count, especially T cells, B cells and NK cells). Current observations indicate that patients most likely to die from the disease are those who are immunocompromised (the elderly and those with immunosuppressive diseases such as cancer) and those with diabetes and other underlying health problems such as hypertension , heart disease and respiratory disease) patients. Patients in the former group are most likely to be exacerbated by lymphopenia and thus uncontrolled viral replication and infection in both lungs, resulting in acute respiratory distress syndrome (ARDS).

Once the key cells of the immune system (eg, T cells, B cells, macrophages and NK cells) are depleted, viral proliferation prevails. In older patients, immune function is not as robust as in younger patients. Research has shown that, in most people, their immune function is good in their 60s and even into their 70s. Immune function declines rapidly after age 75 or 80.

COVID-19 spreads rapidly through human-to-human transmission, with a median incubation period of 3.0 days (range 0 to 24.0) and a time from symptom onset to development of pneumonia of 4.0 days (range 2.0 to 7.0) (Guan et al., 2020) . Fever, dry cough, and fatigue are common symptoms at the onset of COVID-19 (Huang et al., 2020). Most patients had lymphopenia and bilateral ground-glass opacity changes on chest CT scans (Huang et al., 2020; Duan and Qin, 2020). No specific antiviral treatment or vaccine is available. There is an urgent need to develop a vaccine based on SARS-CoV-2.

Vaccine preparations based on whole virions, including inactivated and attenuated virus vaccines, are considered desirable as they are based on previous studies on the prevention and control of seasonal influenza vaccines (Grohskopf et al., 2018). The genome sequence of the first SARS-CoV-2 (Wuhan-Hu-1) has been completed (GenBank accession number MN908947.3; Wu et al., 2020). Large-scale cultivation of SARS-CoV-2 has been performed, and inactivated virus vaccines were prepared by using established physical and chemical methods such as UV light, formaldehyde and β-propiolactone (Jiang et al., 2005). The development of attenuated virus vaccines can also be achieved by screening for continuously transmitted SARS- CoV-2 to achieve (Regla-Nava et al., 2015). Both inactivated and attenuated virus vaccines have their own disadvantages and side effects (Table 1; reproduced from Shang et al., 2020). Table 1. Advantages and disadvantages of different vaccine strategies. vaccine strategy advantage shortcoming references inactivated virus vaccine Easy to prepare; safe; high titer neutralizing antibody May not be suitable for highly immunosuppressed individuals 25 Attenuated virus vaccine Rapid development; induces high immune response Potential for phenotypic or genotypic reversion; can still cause some disease 25 subunit vaccine High safety; consistent production; can induce cellular and humoral immune response; high titer neutralizing antibody High cost; low immunogenicity; requires repeated doses and adjuvants 12,14 viral vector vaccine Safety; induces high cellular and humoral immune responses Pre-existing immunity may be present 12 DNA vaccine Easier to design; high safety; high titer neutralizing antibody Low immune response in humans; repeated doses can cause toxicity twenty three mRNA vaccine Easier to design; high fitness; induce strong immune response Highly unstable under physiological conditions twenty three

All of the new therapies being developed are either (i) antiviral drugs used systemically to prevent viral multiplication, or (ii) attenuated viruses used as vaccines to stimulate a strong antibody response to viral proteins.

None of these therapies is likely to prevent the death of infected immunocompromised patients as is currently seen in the case of influenza virus infected patients. The largest number of deaths due to influenza infection each year are immunocompromised patients and the elderly.

Effective immunotherapy strategies for the treatment of diseases such as cancer depend on the activation of innate and adaptive immune responses. Cells of the innate immune system interact with pathogens through conserved pattern recognition receptors, while cells of the adaptive immune system recognize pathogens through various antigen-specific receptors generated by somatic DNA rearrangement. Invariant natural killer T (iNKT) cell lines bridge a subset of lymphocytes of the innate and adaptive immune systems (type I NKT). iNKT cells express an invariant α-chain T-cell receptor (Vα24-Jα18 in humans and Vα14-Jα18 in mice) mediated by a certain protein present in the context of the non-pleomorphic MHC class I-like protein CD1d. Some glycolipids are specifically activated. CDld binds to various dialkyl lipids and glycolipids, such as the glycosphingolipid alpha-galactosylceramide (α-GalCer). iNKT cell TCR recognition of CDld-lipid complexes leads to the release of pro-inflammatory and regulatory cytokines, including the Th1 cytokine interferon gamma (IFNγ). The release of cytokines in turn activates adaptive cells (such as T and B cells) and innate cells (such as dendritic cells and NK cells).

α-GalCer (also known as KRN7000, chemical formula C ₅₀ H ₉₉ NO ₉ ) is a synthetic glycolipid derived from the structure-activity relationship study of galactosylceramide isolated from the sponge Agelas mauritianus . α-GalCer is a potent immunostimulant and exhibits potent antitumor activity in a number of in vivo models. A major challenge of using α-GalCer for immunotherapy is that it induces anergy in iNKT cells as it can be presented by other CDld expressing cells in peripheral blood such as B cells. Delivery of α-GalCer was also shown to induce hepatotoxicity. B. SARS-CoV-2

The coronavirus genome encodes four major structural proteins: the spine (S) protein, the nucleic acid protein shell (N) protein, the membrane (M) protein, and the envelope (E) protein, all of which are required to produce structurally complete virus particles. Some CoVs do not require full integration of structural proteins to form complete, infectious virions, suggesting that some structural proteins may be dispensable, or that these CoVs may encode other proteins with overlapping compensatory functions. Individually, each protein plays a major role in the structure of the virus particle, but it also participates in other aspects of the replication cycle. The S protein mediates attachment of the virus to host cell surface receptors and subsequent fusion between the virus and host cell membranes to facilitate virus entry into the host cell. In some CoVs, expression of S at the cell membrane can also mediate cell-cell fusion between infected cells and adjacent uninfected cells. This formation of giant, multinucleated cells, or syncytia, is thought to be a strategy to allow direct transmission of the virus between cells, which destroys virus neutralizing antibodies.

The SARS-CoV-2 spine (S) glycoprotein has been shown to bind to the cell membrane protein vasoconstrictor-converting enzyme 2 (ACE2) for entry into human cells. COVID-19 has been shown to bind to ACE2 via the S protein on its surface. During infection, the S protein splits into subunits S1 and S2. S1 contains a receptor binding domain (RBD) that allows the coronavirus to bind directly to the peptidase domain (PD) of ACE2. S2 thus likely plays a role in membrane fusion.

Unlike other major structural proteins, N is the only protein mainly used to combine with the CoV RNA genome to form the nucleic acid protein coat. Although N is largely involved in processes associated with the viral genome, it is also involved in other aspects of the CoV replication cycle and the host cell's response to viral infection. Transient expression of N was shown to substantially increase the production of virus-like particles (VLPs) in some CoVs, suggesting that it may not be required for envelope formation, but for full virion formation.

The M protein is the most abundant structural protein and defines the shape of the viral envelope. Think of it as the central organizer of CoV assembly, interacting with all other major coronavirus structural proteins. Homotypic interactions between M proteins are the main driving force behind virion envelope formation, but alone are not sufficient for virion formation. The combination of M and N stabilizes the nucleic acid protein shell (N protein-RNA complex) and core of the virus particle, and finally promotes the completion of virus assembly. Together, M and E constitute the viral envelope and their interaction is sufficient to generate and release VLPs.

The CoV envelope (E) protein is the smallest of the major structural proteins. Its mesangial body protein, which is involved in multiple aspects of the virus life cycle, such as assembly, budding, mantle formation, and pathogenicity. During the replication cycle, E is expressed in large quantities within infected cells, but only a small fraction is incorporated into the virion envelope. Most of the proteins are located in intracellular trafficking sites, where they participate in CoV assembly and budding. Recombinant CoVs lacking E displayed significantly reduced virus titers, severely impaired virus maturation, or produced reproductively incompetent progeny, demonstrating the importance of E in virus production and maturation.

The coronavirus is a virus in which the gene system is single-stranded mRNA, with a 3'-UTR and a poly-A tail. In a subset of coronaviruses including 2019-nCoV, SARS and MERS, the 3'-UTR contains a highly conserved sequence (among an otherwise quite variable message) that folds into a unique structure called s2m( stem two motif). Although s2m appears to be extremely conserved in sequence and is required for viral survival, its exact function is unknown. The 2019 Wuhan novel coronavirus (COVID-19, formerly 2019-nCoV) has an almost identical s2m sequence (and thus structure) to SARS.

The SARS-CoV-2 genome sequence has been released and published at https://www.ncbi.nlm.nih.gov/genbank/sars-cov-2-seqs/ ( downloaded on March 24 , 2020 ) , including Multiple complete nucleotide sequences of viruses from all over the world and sequences of specific viral genes, such as S gene, N gene, M gene, etc. Examples include GenBank Accession Nos. MN908947.3, MN975262.1, NC_045512.2, MN997409.1, MN985325.1, MN988669.1, MN988668.1, MN994468.1, MN994467.1, MN988713.1, and MN138384.1. SARS-CoV-2 is an enveloped single- and positive-sense RNA virus whose genome consists of 29,891 nucleotides encoding 12 putative open reading frames responsible for the synthesis of viral structural and nonstructural proteins (Wu et al., 2020; Chen et al., 2020). Mature SARS-CoV-2 has four structural proteins, namely, mantle, membrane, nucleic acid protein shell, and spine (Chen et al., 2020). All of these proteins can be used as antigens to stimulate neutralizing antibodies and increase CD4+/CD8+ T cell responses (Jiang et al., 2015). However, subunit vaccines require multiple booster injections and appropriate adjuvants to be effective, and some subunit vaccines (such as hepatitis B surface antigen, PreS1 and PreS2) may not produce protective responses in clinical trials. DNA and mRNA vaccines, which are easier to design and can enter clinical trials very quickly, are still in the experimental phase. Vaccines based on viral vectors can also be rapidly constructed and administered without adjuvants. However, the development of such vaccines may not begin until antigens containing neutralizing epitopes are identified. The E and M proteins have important functions in the viral assembly of coronaviruses, and the N protein is necessary for the synthesis of viral RNA. Deletion of the E protein abolishes the virulence of CoVs, and several studies have explored the potential of recombinant SARS-CoV or MERS-CoV with mutant E proteins as live attenuated vaccines. The M protein can enhance the immune response against SARS-CoV induced by the N protein DNA vaccine; however, the conserved N protein in the CoV family means that it is not a suitable candidate for vaccine development, and the N protein against SARS-CoV-2 Antibodies do not provide immunity against infection. The key glycoprotein S of SARS-CoV-2 is responsible for virus binding and entry. The S precursor protein of SARS-CoV-2 can be proteolytically split into S1 (685 aa) and S2 (588 aa) subunits. The S2 protein is extremely conserved among SARS-CoV-2 viruses and shares 99% identity with bat SARS-CoV. Vaccine design based on S2 protein can enhance broad-spectrum antiviral effect and is worthy of testing in animal models. Antibodies directed against the conserved stem region of influenza hemagglutinin have been found to exhibit broadly cross-reactive immunity but are less potent at neutralizing influenza A virus. In contrast, the S1 subunit consists of a receptor-binding domain (RBD) that mediates viral entry into sensitive cells via the host vasoconstrictor-converting enzyme 2 (ACE2) receptor. The S1 protein of 2019-nCoV shares about 70% identity with human SARS-CoV. The highest number of amino acid changes in the RBD are located in the external subdomain, which is responsible for the direct interaction between the virus and the host receptor. C. Summary of How the Disclosed Compositions Work to Treat Viral Infections and / or Vaccine Against Viral Infections

The present invention aims to intervene in the early stages before or after infection of a virus, such as a coronavirus (eg SARS-CoV or MERS-CoV). The compositions and methods address problems including: (i) overcoming lymphopenia to prevent viral infection/disease from overtaking the patient's own immune defenses, (ii) stimulating high titer systemic antibodies against proteins exposed on the surface of the virus, to rapidly clear viral particles released from infected cells and thereby limit infection of other healthy cells, (iii) stimulate potent type I and type II interferon responses, which are known to be specific by virtue of multiple effects (e.g., antiviral immunity) sexual stimulation and elimination of virus-infected cells) rapidly combats a range of different viral infections, and (iv) elicits virus-specific CD8+ T cell responses that rapidly identify and kill virus-infected cells.

To address these and other needs, according to one aspect, the present invention provides compositions comprising combinations of: (i) vectors, which may be whole pocket cells of bacterial origin, which are optionally reconstituted and packaged There are plasmids encoding viral proteins that stimulate antibody responses to viral proteins and stimulate type I interferons; (ii) vectors, which may be whole pocket cells of bacterial origin, which are optionally reconstituted and packaged There is an antigen recognized by CD1d, and (iii) at least one pharmaceutically acceptable carrier. Carriers packaged with antigens recognized by CDld (such as α-GalCer) act to stimulate type II interferons. The role of the pocket cell carrier itself is to stimulate the activation, maturation and proliferation of immune system cells. In another aspect, whole bacterial-derived pocket cells can also be replaced with killed bacterial cells.

Thus, in certain embodiments, described herein are compositions comprising an immunogenic effective amount of a combination of: (a) a vector or whole, bacterial-derived pocket cells or killed bacterial cells that encapsulate One or more viral antigens and plastids, and (b) vectors or intact, bacterial-derived pocket cells or killed bacterial cells that encapsulate antigens recognized by CD1d, such as α-galactosylceramide (α-GalCer ). In some embodiments, the antigen recognized by the encapsulated CDld can be taken up by phagocytes (eg, dendritic cells or macrophages). After uptake, CDld-recognized cellular antigens form complexes with CDld in lysosomes of phagocytic cells and are subsequently transported to the surface of phagocytes, where CDld-recognized antigens bound to CDld are presented for recognition by iNKT cells. In some embodiments, the cell antigen recognized by CD1d is induced by iNKT cells to respond to Th1 cytokines, specifically IFNγ, and the iNKT cells recognize the cell antigen recognized by CD1d bound to CD1d on the surface of phagocytic cells. IFNγ is also known to trigger a potent antiviral immune response. The ability of CDld-restricted NKT cells to activate innate and adaptive immune responses led to the idea that these cells could modulate immunity to infectious agents. In addition, CDld-restricted iNKT cells can directly contribute to host resistance due to the expression of various effector molecules that can mediate antimicrobial effects. The CD1 protein is an antigen-presenting molecule that presents lipid antigens to T cells.

In one aspect, the purpose of administering the compositions described herein to an individual in need is to rapidly free the individual from lymphopenia and at the same time activate key cells of the immune system to fight viral infections, especially the elderly and immune impaired patients. This will prevent the progression of viral infection and resulting death in these patients. As a result, infected individuals will suffer from mild flu-like symptoms and recover more quickly as the body's own immune system turns the balance back.

In one aspect of the disclosure, genes encoding all four SARS-CoV-2 structural proteins (mantle, membrane, nucleic acid protein shell, and spines) were colonized in plastids carrying bacterial origins of replication and bacterial genes were used for the genes The expression promoter is transcribed so that the protein is expressed only in EDV™ producing bacterial cells and sequestered into the EDV™ cytoplasm. Thus, all four SARS-CoV-2 proteins can be expressed from a single bacterially expressed promoter. Alternatively, the gene can be transcribed under a mammalian gene expression promoter such that the protein is expressed only by mammalian cells. Recombinant plastids can be transformed in S. typhimurium strains that produce pocket-sized cells. This recombinant intact, bacteria-derived pocket-sized cell therapeutic is expected to elicit potent antibody responses to all four CoV-2 proteins.

In addition, when recombinant intact, bacteria-derived pocket cells were systemically administered to patients infected with the CoV-2 virus, the intact, bacteria-derived pocket cells were rapidly engulfed by professional phagocytes such as macrophages and dendritic cells and Intact, bacterial-derived pocket cells disintegrate in lysosomes, releasing plastid DNA. This DNA is then recognized by intracellular DNA sensors such as cGAS, AIM2, IFI16 and others and this will trigger a type I interferon (IFNα and IFNβ) response. These interferons are known to be potent inducers of antiviral defense.

It is well known that early in infection, IFN stimulation leads to changes in the cellular transcriptional program leading to an antiviral state characterized by the activation of a large number of host genes with partially defined antiviral functions (Schoggins et al., 2011).

In some embodiments, the antigen recognized by CDld is a glycosphingolipid. In some embodiments, the glycosphingolipid is selected from the group consisting of α-galactosylceramide (α-GalCer), C-glycosidic form of α-galactosylceramide (α-C-GalCer), galactosylceramide 12-carbon acyl form (β-GalCer), β-D-glucopyranosylceramide (β-GlcCer), l,2-diacyl-3-O-galactosyl-sn-glycerol (BbGL- II), glycolipid containing diacylglycerol (Glc-DAG-s2), ganglioside (GD3), neurotrihexosylceramide (Gg3Cer), glycoside phosphatidylinositol (GPI), α- Glucuronylceramide (GSL-1 or GSL-4), isotrihexosylceramide (iGb3), lipophosphoglycan (LPG), lysophosphatidylcholine (LPC), α- Galactosylceramide analogs (OCH), threitolceramide and derivatives of any of them. In some embodiments, the glycosphingolipid is α-GalCer. In some embodiments, the glycosphingolipids are synthetic α-GalCer analogs. In some embodiments, the synthetic α-GalCer analog is selected from 6′-deoxy-6′-acetamide α-GalCer (PBS57), naphthaleneurea α-GalCer (NU-α-GC), NC-α- GalCer, 4ClPhC-α-GalCer, PyrC-α-GalCer, α-carba-GalCer, carba-α-D-galactose α-GalCer analog (RCAI-56), 1-deoxy-neo-inositol α- GalCer analogs (RCAI-59), 1-O-methylated α-GalCer analogs (RCAI-92), and HS44 aminocyclylceramide. In some embodiments, the antigen recognized by CDld is derived from a bacterial antigen, a fungal antigen, or a protozoan antigen.

In some embodiments, the immune response generated in the target cells comprises the production of type I interferons, including interferon-alpha and/or interferon-beta.

This bacterial pocket cell therapy will reduce the severity and duration of the disease in almost all patients, making it more like the common cold.

Alternatively, the treatment can be administered as a vaccine to healthy individuals to prevent infection by viruses carrying proteins encoded by recombinant plastids carried by the pocket cells.

In one embodiment, the adjuvant composition comprises (a) an immunogenically effective amount of an antigen recognized by encapsulated CDld, and (b) a pocket-sized cell carrying a recombinant plastid encoding one or more viral antigens.

In one embodiment, the CDld-recognized antigen and the recombinant plasmid are packaged in two intact bacteria-derived miniature cells or killed bacterial cells.

The antigen recognized by CDld is contained within the first whole bacterium-derived miniature cell or killed bacterial cell, and the recombinant plasmid encoding the viral antigen is contained within the second whole bacterium-derived miniature cell or killed bacterial cell.

In some embodiments, the encapsulated CDld-recognized antigen (eg, α-GalCer) and a pocket cell line carrying a recombinant plasmid encoding at least one viral antigen are administered simultaneously. In some embodiments, pocket cell lines encapsulating CDld-recognized antigens (eg, α-GalCer) and carrying recombinant plastids encoding viral antigens are administered sequentially. In some embodiments, a pocket cell line encapsulating an antigen recognized by CDld (eg, α-GalCer) and carrying a recombinant plasmid encoding a viral antigen is administered multiple times. In some embodiments, at least once a week or twice a week or three times a week or every Thursday the pocket cell line encapsulates the antigen recognized by CD1d (such as α-GalCer) and carries the recombinant plasmid encoding the viral antigen administration until the disease subsides.

After infection with SARS-CoV-2, the purpose of this therapy will be to achieve the following: (1) Stimulate innate and adaptive immunity by recruiting fresh monocytes and dendritic cells from bone marrow and activating NK cells. This will keep the patient's immune status high as the disease progresses and prevent the development of lymphopenia. (2) Physiologically fully tolerate the secretion of type I (IFNα and IFNβ) and type II (IFNγ) interferons. It is well known that early in viral infection, IFN stimulation results in altered cellular transcriptional programs, resulting in an antiviral state characterized by the activation of a large number of host genes with partially defined antiviral functions. This activation will enable rapid elimination of virus-infected cells and reduction of viral replication. (3) Secretion of antibodies against the four structural proteins of the virus (envelope, membrane, spine and nucleic acid protein shell) and this is aimed at clearing the bulk of virus particles released from infected cells. All of the above are expected to have minimal to no toxicity. III. Whole Bacteria-Derived Pocket Cells

The term "pocket cell" is used herein to denote a derivative of a bacterial cell that lacks chromosomes ("chromosome-free") and is produced by disrupting the coordination of cell division and DNA segregation during binary fission. Pocket cells differ from other small vesicles, such as so-called "membrane vesicles" (approximately 0.2 µm or smaller in size), which are spontaneously produced and released under certain conditions, but not due to specific genetic reprogramming. row or episomal gene expression. Bacterial-derived miniature cell lines as used in the present disclosure are completely intact and are distinguished from other derivatives of bacterial cells that do not contain chromosome-free forms characterized by disruption or degradation, or even removal, of the outer or delimiting membrane. The intact membranes of the pocket cells that characterize the present disclosure allow retention of therapeutic payloads within the pocket cells until release of the payload.

Intact, bacterial-derived minicells, or EDV™, are non-nucleated, non-viable nanoparticles produced by inactivating genes that control normal bacterial cell division, thereby derepressing the cell's polar sites. In addition, compared with, for example, current stealth liposomal drug carriers (e.g., DOXIL) (liposomal doxorubicin) that can only pack about 14,000 molecules/particle or "armed antibodies" that can carry less than 5 drug molecules In contrast, bacterial pocket cells can easily accommodate a payload of up to 1 million drug molecules.

Pocket cells used in the present disclosure can be prepared from bacterial cells such as E. coli and S. typhimurium . Prokaryotic chromosome duplication is associated with normal binary fission involving the formation of the mid-cell septum. For example, in E. coli, mutations in min genes such as minCD abrogate the inhibition of septum formation at the cell poles during cell division, resulting in normal daughter cells and pocket-sized cells with fewer chromosomes.

In addition to minimal operon mutations, pocket-sized cells with fewer chromosomes are also generated following a series of other gene rearrangements or mutations affecting septum formation, eg, in divIVB1 in B. subtilis . Pocket cells can also be formed following perturbations in the expression levels of genes involved in cell division/chromosomal segregation. For example, overexpression of minE leads to division and generation of polar bodies of pocket cells. Similarly, pocket-sized cells with fewer chromosomes can result from defects in chromosome segregation, such as the smc mutation in B. subtilis, the spoOJ deletion in B. subtilis, the mukB mutation in E. coli, and the parC mutation in E. coli. In addition, CafA can enhance the rate of cell division and/or inhibit chromosome partitioning after replication, which leads to the formation of chained cells and pocket-sized cells with fewer chromosomes.

Thus, due to the conserved nature of bacterial cell division among these bacteria, the pocket cells of the disclosure can be prepared from any bacterial cell, whether of Gram-positive or Gram-negative origin. In addition, as noted above, the miniature cells used in this disclosure should have intact cell walls (i.e., be "intact miniature cells") and should be isolated from other vesicles not caused by specific gene rearrangements or episomal gene expression. Vesicles (eg, membrane vesicles) are differentiated and separated.

In a given embodiment, the parental (source) bacterium of the pocket-sized cells may be Gram-positive, or it may be Gram-negative. In one aspect, the parental bacterium is selected from one or more of the following: Terra-/Glidobacteria (BV1), Proteobacteria (BV2), BV4 (including Spirochaete, Sphingobacteria Class (Sphingobacteria) and Planctobacteria-Verrucobacteria/Chlamydia (Planctobacteria)). According to another aspect, the bacterial line is selected from one or more of the following: Firmicutes (BV3) (for example Bacilli, Clostridia) or Softicutes/Mollicutes Class Tenericute/Mollicute or Actinobacteria (BV5) (eg Actinomycetales or Bifidobacteriales).

According to the present disclosure, the bacterial cells killed are non-viable prokaryotic cells of bacteria, cyanobacteria, eubacteria and archaebacteria, as defined in Bergey's Manual Of Systematic Biology, 2nd edition. Such cells are considered "intact" if they have intact cell walls and/or membranes and contain genetic material (nucleic acid) endogenous to the bacterial species. Methods for preparing killed bacterial cells are described, for example, in US 2008/0038296.

In still other aspects, the bacteria are selected from one or more of the following: Eubacteria (Chloroflexi, Deinococcus-Thermus), Cyanobacteria, Thermodesulfobacteria, thermophiles (Aquificae, Thermotogae), alpha, beta, gamma (Enterobacteriaceae), delta or epsilon Proteobacteria, Spirochetes, Fibrobacteres, Chlorobi/Bacteroidetes, Chlamydiae/Verrucomicrobia, Planctomycetes, Acidobacteria (Acidobacteria), Chrysiogenetes, Deferribacteres, Fusobacteria, Gemmatimonadetes, Nitrospirae, Synergistetes ), Dictyoglomi, Lentisphaerae, Bacillales, Bacillaceae, Listeriaceae, Staphylococcaceae, Milk Lactobacillales, Enterococcaceae, Lactobacillaceae, Leuconostocaceae, Streptococcaceae, Clostridiales, Halanaerobiales , Thermoanaerobacterales, Mycoplasmatales, Entomoplasmatales, Anaeroplasmatales, Acholeplasmatales, Halophiles ( Haloplasmatales), Actinomycineae, Actinomycetaceae, Corynebacterineae, Mycobacteriaceae, Nocardiaceae, Corynebacteriaceae , Frankinae, Frankiaceae, Micrococcineae, Brevibacteriaceae and Bifidobacteriaceae.

For pharmaceutical use, compositions of the present disclosure should contain miniature cells or killed bacterial cells that are separated as fully as possible from immunogenic components and other toxic contaminants. Methods for purifying bacterial-derived miniature cells to remove free endotoxin and parental bacterial cells are described, for example, in WO 2004/113507. Briefly, purification achieves the removal of (a) smaller vesicles, such as membrane vesicles, typically less than 0.2 µm in size, (b) free endotoxin released from cell membranes, and (c) parental bacterial (whether living or dead) and their fragments, which are also sources of free endotoxin. This removal is especially useful with 0.2 µm filters to remove smaller vesicles and cell debris, 0.45 µm filters to remove parental cells after they are induced to form filaments, to kill live bacteria Cell antibiotics and antibodies against free endotoxin are performed.

Following the purification procedure, the present inventors discovered that despite their different bacterial origins, all intact pocket cells were approximately 400 nm in size, ie larger than membrane vesicles and other smaller vesicles, but still smaller than the parent bacteria. Size determination of pocket cells can be performed by using solid-state techniques such as electron microscopy or liquid-based techniques such as dynamic light scattering. The magnitude values produced by each such technique may have a margin of error, and such values may vary between techniques. Thus, pocket cells in a dry state can be measured by electron microscopy to be approximately 400 nm ± 50 nm. Dynamic light scattering can measure the size of the same pocket-sized cells to be about 500 nm ± 50 nm. In addition, drug-packaged, ligand-targeted pocket cells can again be measured using dynamic light scattering to approximately 400 nm to 600 nm ± 50 nm.

Another structural element of killed bacterial cells or pocket cells derived from Gram-negative bacteria is the O-polysaccharide component of lipopolysaccharide (LPS), which is embedded in the outer membrane via lipid A anchors. This component is a chain of repeating carbohydrate residue units, each chain of repeating units having as many as 70 to 100 repeating units of 4 to 5 sugars. Since the chains are not rigid, in liquid environments, such as in vivo, they can adopt a wavy, flexible structure to produce the overall appearance of algae in Coral Sea environments; that is, the chains move with the liquid while Remains anchored to the pocket cell membrane.

As mentioned above, dynamic light scattering can provide pocket cell size values from about 500 nm to about 600 nm, influenced by the O-glycan fraction. However, pocket cells from Gram-negative and Gram-positive bacteria equally readily pass through 0.45 µm filters, demonstrating an effective pocket cell size of 400 nm ± 50 nm. The above-mentioned size spreads are encompassed by the present invention, and are in particular indicated by the modifier "about" and the like in the phrase "about 400 nm in size".

With regard to toxic contaminants, compositions of the present disclosure preferably contain less than about 350 EU of free endotoxin. Exemplary in this regard are the following free endotoxin levels: about 250 EU or less, about 200 EU or less, about 150 EU or less, about 100 EU or less, about 90 EU or less, about 80 EU or less, about 70 EU or less, about 60 EU or less, about 50 EU or less, about 40 EU or less, about 30 EU or less, about 20 EU or less, about 15 EU or less, about 10 EU or less, about 9 EU or less, about 8 EU or less, about 7 EU or less, about 6 EU or less, about 5 EU or less, about 4 EU or less, about 3 EU or less, about 2 EU or less, about 1 EU or less , about 0.9 EU or less, about 0.8 EU or less, about 0.7 EU or less, about 0.6 EU or less, about 0.5 EU or less, about 0.4 EU or less, about 0.3 EU or less, about 0.2 EU or less, about 0.1 EU or less, about 0.05 EU or less, or about 0.01 EU or less.

Compositions of the present disclosure may also comprise at least about 10 ⁹ pocket cells or killed bacterial cells, such as at least about 1 x 10 ⁹ , at least about 2 x 10 ⁹ , at least about 5 x 10 ⁹ , or at least 8 x 10 ⁹ . In some embodiments, the composition comprises no more than about ¹⁰ pocket cells or killed bacterial cells, such as no more than about 1 x ¹⁰ or no more than about 9 x ¹⁰ or no more than about 8 × 10 ¹⁰ . IV. Antigen recognized by CD1d

The compositions and methods of the invention include vectors, which may be whole bacteria-derived pocket cells containing antigens recognized by CDld. These antigens result in increased levels (eg, active or expressed levels) of type II interferons (eg, IFN-γ (gamma)). IFN-γ is involved in the regulation of immune and inflammatory responses; in humans, only one type of interferon-γ exists. It is produced in activated T cells and natural killer cells. IFN-γ enhances the effects of type I IFN. IFN-γ released by Th1 cells recruits leukocytes to the site of infection, leading to increased inflammation. It also stimulates macrophages to kill engulfed bacteria. IIFN-γ released by Th1 cells is also important in regulating Th2 responses.

The IFNy cytokine is released by innate natural killer (NK) cells upon binding natural antigens, but glycosphingolipid compounds can act as potent activators of both innate and adaptive immune responses. Exposure to glycosphingolipids induces potent interleukin responses in innate natural killer T (iNKT) cells, including type II interferons, IFN-γ, and many interleukins (Th1-, Th2- and/or Th17-type interleukins ). iNKT cells then induce DCs to mature and exhibit T cell helper-like functions, which lead to the development of cytotoxic T cell responses.

Examples of glycosphingolipids that can be used to induce an IFN type II response are described herein and include the C-glycosidic form of α-galactosylceramide (α-C-GalCer), α-galactosylceramide (α-GalCer ), the 12-carbon acyl form of galactosylceramide (β-GalCer), β-D-glucopyranosylceramide (β-GlcCer), l,2-diacyl-3-O-galactose Glycerol-sn-glycerol (BbGL-II), glycolipid containing diacylglycerol (Glc-DAG-s2), ganglioside (GD3), neurotrihexosylceramide (Gg3Cer), glycoside phosphatidyl Inositol (GPI), α-glucuronylceramide (GSL-1 or GSL-4), isoglotrihexosylceramide (iGb3), lipophosphoglycan (LPG), lysophosphatidylceramide Choline (LPC), α-galactosylceramide analog (OCH) and threitol ceramide. In certain embodiments, the pocket cells disclosed herein comprise α-galactosylceramide (α-GalCer) as a type II IFN agonist.

α-GC (INF type II agonist) is known to stimulate the immune system by activating a type of white blood cell called natural killer T cells (NKT cells).

Pocket cells can deliver type II IFN agonists directly to cells of the immune system in order to enhance iNKT cell activation and type II interferon IFN-γ production in vivo. Non-targeted intact, bacteria-derived pocket cells are engulfed by phagocytes of the immune system, where they are broken down in endosomes, and αGC is presented to iNKT cells for immune activation. Thus, in some embodiments, the pocket cells provide targeted delivery of Type II interferon agonists. In other embodiments, the compositions disclosed herein comprise non-targeted pocket cells comprising a Type II interferon agonist.

IFN-γ production is controlled by antigen presenting cells (APC) secreted cytokines, most notably interleukin (IL)-12 and IL-18. These cytokines act as a bridge in the innate immune response to link infection to IFN-γ production. Macrophage recognition of many pathogens induces the secretion of IL-12 and chemokines. The chemokines attract NK cells to sites of inflammation, and IL-12 promotes IFN-γ synthesis in these cells. The combination of IL-12 and IL-18 stimulation further increased IFN-γ production in macrophages, natural killer cells and T cells. Accordingly, any one of these proteins, or a combination thereof, is a suitable agent for the purposes of this disclosure.

Negative regulators of IFN-γ production include IL-4, IL-10, transforming growth factor beta, and glucocorticoids. Proteins or nucleic acids that inhibit these factors will be able to stimulate IFN-γ production.

Also suitable in this context are polynucleotides encoding IFN-γ or genes that activate the production and/or secretion of IFN-γ.

Agents that increase IFN-gamma levels can also be viral vaccines. A number of viral vaccines are available that induce IFN-[gamma] production without causing infection or other types of adverse effects. An exemplary such viral vaccine agent is an influenza (influenza) vaccine.

Serum concentrations of IFN-γ required to effectively activate the host immune response were lower when patients also received administration of drug-loaded, bispecific antibody-targeted pocket cells, or killed bacterial cells. Thus, in one aspect, the methods of the invention result in an increase in serum IFN-γ concentration of no greater than about 30,000 pg/mL. In another aspect, the serum IFN-γ concentration does not increase by more than about 5000 pg/mL, 1000 pg/mL, 900 pg/mL, 800 pg/mL, 700 pg/mL, 600 pg/mL, 500 pg/mL mL, 400 pg/mL, 300 pg/mL, 200 pg/mL, or 100 pg/mL. In another aspect, the resulting serum IFN-γ concentration is at least about 10 pg/mL, or at least about 20 pg/mL, 30 pg/mL, 40 pg/mL, 50 pg/mL, 60 pg/mL, 70 pg/mL pg/mL, 80 pg/mL, 90 pg/mL, 100 pg/mL, 150 pg/mL, 200 pg/mL, 300 pg/mL, 400 pg/mL, or 500 pg/mL.

According to some aspects, the agent is an IFN-γ protein or an engineered protein or an analog. In some aspects, administration achieves about 0.02 ng to 1 microgram of IFN-γ/ml host blood. In one aspect, the achieved IFN-γ concentration in the blood of the host is about 0.1 ng to about 500 ng/ml, about 0.2 ng to about 200 ng/ml, about 0.5 ng to about 100 ng/ml, about 1 ng to about 50 ng/ml or about 2 ng to about 20 ng/ml. V. Loading of SARS - CoV -2 Virus and Variant Virus Antigens and Antigens Recognized by CD 1 d into Pocket Cells or Killed Bacterial Cells

Viral antigens and antigens recognized by CDld can be directly packaged in the miniature cells or killed bacterial cells by co-incubating a plurality of whole miniature cells or killed bacterial cells with the antigen in a buffer. Buffer composition can be varied according to conditions well known in the art to optimize antigen loading in intact miniature cells. Buffers can also vary depending on the antigen (eg, in the case of nucleic acid payloads, depending on the nucleotide sequence or the length of the nucleic acid to be loaded into the pocket cells). Exemplary buffers suitable for loading include, but are not limited to, phosphate buffered saline (PBS). Once packaged, the antigen remains inside the pocket cells and is protected from degradation. Extended incubation studies using siRNA-packaged minicells incubated in sterile saline showed, for example, no leakage of siRNA.

An antigen (eg, a protein that can be encoded by a nucleic acid) can be introduced into a pocket-sized cell by converting an antigen-encoding vector (eg, a plastid) into a parent bacterial cell. When a miniature cell is formed from a parental bacterial cell, the miniature cell retains some copies of the plastid and/or the expressed product (eg, antigen). Further details of packaging and expression products into pocket-sized cells are provided in WO 03/033519.

For example, data presented in WO 03/033519 demonstrate that recombinant pocket cells carrying mammalian gene expression plasmids can be delivered to phagocytic and non-phagocytic cells. WO 03/033519 also describes the genetic transformation of a parental bacterial strain producing pocket-sized cells using heterologous nucleic acid carried on episomally replicating plastid DNA. When the parental bacteria separate from the pocket cells, some episomal DNA segregates into the pocket cells. The resulting recombinant pocket cells are readily engulfed by mammalian phagocytes and degraded by intracellular phagolysosomes. In addition, some recombinant DNA escapes the phagolysosomal membrane and is transported to the nucleus of mammalian cells, where the recombinant gene is expressed. In other embodiments, multiple antigens can be packaged in the same pocket cell.

Antigens can be packaged in the miniature cells by creating a concentration gradient of the antigen between the extracellular medium comprising the miniature cells and the cytoplasm of the miniature cells. When the extracellular medium contains a higher concentration of antigen than the cytoplasm of the miniature cells, the antigen naturally moves down this concentration gradient into the cytoplasm of the miniature cells. However, when the concentration gradient was reversed, the antigen did not move out of the pocket cells. Further details of the active agent loading process and its surprising properties are found, for example, in US Patent Application Publication No. 2008/0051469. Ⅵ. Preparation

The present disclosure includes within its scope combinations comprising: (a) vectors, whole bacterial pocket cells or killed bacterial cells comprising at least one viral antigen as payload; and (b) vectors, whole bacterial cells Miniature cells or killed bacterial cells comprising at least one CDld-recognized antigen as a payload, both present in at least one pharmaceutically acceptable carrier. The at least one viral antigen and the at least one CDld-recognized antigen may be in the same or different vectors, whole bacterial miniature cells or killed bacterial cells. At least one of the viral antigen and the antigen recognized by CDld is present in intact bacterial pocket cells.

In another aspect, one of the viral antigen and at least one CDld-recognized antigen is present in a non-bacterial cellular vehicle, such as a liposomal vehicle.

In some aspects, the antigen recognized by CDld is the interferon type II agonist alpha-galactosylceramide.

Compositions of the disclosure may be presented in unit dosage form, eg, in ampoules or vials, or in multi-dose containers, with or without added preservatives. The compositions may be solutions, suspensions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Suitable solutions are isotonic with the blood of the recipient and are exemplified by, for example, saline, Ringer's solution and dextrose solution. Alternatively, the formulation may be in the form of a lyophilized powder for reconstitution with a suitable vehicle such as sterile, pyrogen-free water or physiological saline. The formulations may also be in the form of a depot preparation. Such long-acting formulations can be administered by implantation (eg, subcutaneously or intramuscularly) or by intramuscular injection. In some embodiments, administering comprises enteral or parenteral administration. In some embodiments, administering comprises administration selected from the group consisting of oral, buccal, sublingual, intranasal, rectal, vaginal, intravenous, intramuscular, and subcutaneous injection.

In some aspects, compositions comprising pocket cells are provided that include a therapeutically effective amount of a viral antigen and a therapeutically effective amount of a CDld-recognized antigen. A "therapeutically effective" amount of an antigen is an amount that elicits a pharmacological response when administered to an individual in accordance with the present disclosure.

Thus, in the context of the present disclosure, when administering pocket cells carrying a therapeutic payload, a therapeutically effective amount can be determined with reference to the prevention or amelioration of viral infection in animal models or human subjects, as further set forth below. An amount that proves to be a "therapeutically effective amount" for a particular individual in a given situation may not be effective in 100% of individuals similarly used to treat a viral infection, even if that amount is considered "therapeutically effective" by a skilled practitioner. In this regard, appropriate dosages will also vary depending on, for example, the stage and severity of the viral infection, and whether the individual has any underlying adverse medical conditions, age 60+, or immunocompromised. A. Investment route

The formulations of the present disclosure can be administered by various routes and to various sites in the body of a mammal to achieve a desired local or systemic therapeutic effect. Delivery can be accomplished by any pharmaceutically acceptable route, such as oral administration, application of the formulation to a body cavity, inhalation, nasal administration, pulmonary administration, insufflation, or by injection (e.g., parenteral, intramuscular , intravenous, portal vein, intrahepatic, peritoneal, subcutaneous, intratumoral, or intradermal administration). Combinations of approaches may also be used. B. Purity

The bacterial pocket cells are substantially free of contaminating parental bacterial cells. Accordingly, formulations free of miniature cells preferably comprise less than about 1 contaminating parent bacterial cell per ¹⁰ miniature cells, less than about 1 contaminating parent bacterial cell per ¹⁰ miniature cells, less than In about 1 contaminating parent bacterial cell/10 ⁹ pocket cells, in less than about 1 contaminating parent bacterial cell/10 ¹⁰ pocket cells, or in less than about 1 contaminating parent bacterial cell/10 ¹¹ pocket cells type cells.

Methods of purifying pocket cells are known in the art and described in PCT/IB02/04632. One such method combines cross-flow filtration (feed flow parallel to the membrane surface; Forbes, 1987) with dead-end filtration (feed flow perpendicular to the membrane surface). Optionally, the filter pack may be preceded by differential centrifugation at low centrifugal force to remove a portion of the bacterial cells and thereby enrich the supernatant for miniature cells.

A particularly effective purification method utilizes bacterial filamentation to increase the purity of the pocket cells. Accordingly, a method of purifying miniature cells may comprise the steps of (a) subjecting a sample containing miniature cells to conditions that induce the parental bacterial cells to adopt a filamentous form, followed by (b) filtering the sample to obtain purified miniature cells preparation.

Combinations of known mini-cell purification methods are also possible. A highly efficient combination of methods is as follows: Step A: Differential centrifugation of the bacterial cell culture producing pocket-sized cells. This step can be performed at 2,000 g for about 20 minutes, which removes most of the parental bacterial cells while leaving pocket-sized cells in the supernatant; Step B: Density gradient centrifugation using an isotonic and non-toxic density gradient medium. This step separates the miniature cells from many contaminants, including parental bacterial cells, with minimal loss of the miniature cells. Preferably, this step is repeated within the purification method; Step C: Cross-flow filtration with a 0.45 μm filter to further reduce parental bacterial cell contamination. Step D: Stress-Induced Filamentization of Residual Parental Bacterial Cells. This can be done by subjecting the minicell suspension to any of several stress-inducing environmental conditions; Step E: Antibiotic treatment to kill parental bacterial cells; Step F: Cross-flow filtration to remove small contaminants (such as membrane vesicles, membrane fragments, bacterial fragments, nucleic acids, media components, etc.) and enriched pocket cells. A 0.2 μm filter can be used to separate the miniature cells from small contaminants and a 0.1 μm filter can be used to concentrate the miniature cells; Step G: Dead-end filtration to eliminate filamentous dead bacterial cells. This step may use a 0.45 um filter; and Step H: Removal of endotoxin from the minicell preparation. Anti-lipid A coated magnetic beads can be used for this step. C. Investment schedule

In general, the formulations disclosed herein can be used at appropriate dosages, as defined by routine testing, to achieve optimal physiological effect while minimizing any potential toxicity. Dosage regimens can be selected based on various factors, including age, weight, sex, medical condition of the patient; severity of the condition to be treated, route of administration, and renal and hepatic functions of the patient.

Achieving optimal precision in the concentration of miniature cells and drug within a range for maximum efficacy and minimal side effects may require a regimen based on the kinetics of miniature cells and the availability of antigen to the target site and target cells. The distribution, balance and elimination of pocket cells or antigens may be considered when determining the optimal concentration for a treatment regimen. When pocket cells and antigens are used in combination, their doses can be adjusted to achieve the desired effect.

In addition, dosage administration of formulations can be optimized using pharmacokinetic/pharmacodynamic modeling systems. For example, one or more dosage regimens can be selected and a pharmacokinetic/pharmacodynamic model can be used to determine the pharmacokinetic/pharmacodynamic profile of the one or more dosage regimens. A dosage regimen that achieves the desired pharmacokinetic/pharmacodynamic response can then be selected for administration based on the particular pharmacokinetic/pharmacodynamic profile. See, eg, WO 00/67776.

Specifically, the formulations can be administered at least once daily for several days (three to four), or until symptoms of viral infection resolve. In one embodiment, the formulation is administered at least once daily until resolution of the viral disease.

More specifically, the formulations can be administered at least once per day for up to about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, About 14, About 15, About 16, About 17, About 18, About 19, About 20, About 21, About 22, About 23, About 24, About 25, About 26, About 27, About 28, About 29, About 30 or about 31 days. Alternatively, the formulation can be administered about once per day, every about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 11, about 12, about 13, about 14 , about 15, about 16, about 17, about 18, about 19, about 20, about 21, about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30 or about Administer approximately once for 31 days or more.

The compositions can be administered in a single daily dose, or the total daily dosage can be administered in divided doses of two, three or four times daily. VII. Definition

Unless defined otherwise, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art.

For convenience, the meanings of certain terms and phrases used in the specification, examples, and appended claims are provided below. Other terms and phrases are defined throughout the specification.

The singular forms "a, an" and "the" include plural referents unless the context clearly dictates otherwise.

As used herein, "about" will be understood by those skilled in the art and will vary to some extent depending on the context in which it is used. "About" will mean up to ±10% of the particular term if the use of the term is unclear to those skilled in the art in the context in which the term is used.

As used herein, the term "comprise" and variations of that term (eg, "comprising, comprises, comprised") are not intended to exclude other additives, components, integers or steps, unless the context requires otherwise.

The phrases "biologically active" and "biological activity" are used to qualify or denote (as the case may be) the effect of a compound or composition on living matter. Thus, a material is biologically active or has biological activity if it interacts with or has an effect on any cellular tissue in the human or animal body, for example by reacting with proteins, nucleic acids or other molecules in the cell.

The terms "individual", "subject", "host" and "patient" used interchangeably in this specification refer to any mammalian individual for whom diagnosis, treatment or therapy is desired. An individual, subject, host or patient can be a human or a non-human animal. Thus, suitable subjects may include, but are not limited to, non-human primates, cows, horses, dogs, cats, guinea pigs, rabbits, rats, and mice.

The terms "treatment, treating, treat" and the like mean achieving a desired pharmacological and/or physiological effect in a patient. The effect may be prophylactic in terms of completely or partially preventing viral infection and/or therapeutic in terms of effect in viral infection. Alternatively or additionally, the desired effect of treatment may be an increase in overall patient survival, progression-free survival, or a decrease in adverse effects.

The phrase "pharmaceutical grade" means that it is devoid of parental cell contamination, cell debris, free endotoxin, and other pyrogens to adequately meet safety requirements for intravenous administration in humans. See, eg, "Guidance for Industry - Pyrogen and Endotoxins Testing", U.S. Food and Drug Administration (June 2012).

"Payload" in this specification identifies or defines a biologically active substance that is to be loaded or has been loaded in a pocket-sized cell for delivery to a targeted host cell.

The term "substantially" generally means at least 90% similarity. In some embodiments, "substantially" means ± 0.2° in the context of the first X-ray powder diffraction pattern being substantially as shown in the second X-ray powder diffraction pattern. In some embodiments, "substantially" means ±0.4°C in the context of the first differential scanning calorimetry thermogram being substantially as shown in the second differential scanning calorimetry thermogram. In some embodiments, "substantially" in the context of the first thermogravimetric analysis being substantially as shown in the second thermogravimetric analysis refers to ±0.4% by weight. In some embodiments, "substantially purified" means at least 95% pure. This includes at least 96, 97, 98 or 99% purity. In other embodiments, "substantially purified" refers to about 95, 96, 97, 98, 99, 99.5, or 99.9% pure, including increments therein.

As used herein, "therapeutic activity" or "activity" may refer to an activity whose effects are consistent with a desired therapeutic outcome in humans or in non-human mammals or other species or organisms. Therapeutic activity can be measured in vivo or in vitro. For example, desired effects can be assayed in cell culture.

As used herein, the phrase "therapeutically effective amount" shall mean a dose of a drug that provides a specific pharmacological response when administered to a large number of individuals in need of such treatment. It is important to emphasize that a therapeutically effective amount of an antigen administered to a particular individual in a particular situation will not always be effective in treating the viral infections described herein, even if such a dose is considered a therapeutically effective amount by those skilled in the art.

The present technology thus broadly set forth will be more readily understood by reference to the following examples, which are provided by way of illustration and not intended to limit the present technology. Example Example 1

The purpose of this example is to illustrate the preparation of a SARS-CoV-2 vaccine comprising antigens from variants of the SARS-CoV-2 vaccine.

Figure 1A depicts scanning electron microscope images showing the production of EnGeneIC Dream Vector (EDV™) nanocells (i.e., intact, bacteria- derived pocket cells) from a safe strain of the bacterium Salmonella typhimurium, and Figure 1B Depicted is a transmission electron micrograph image showing the structure of an empty EDV bacterial nanocell with a diameter of approximately 400 nm. Vectors or bacterial pocket cells are used as vehicles for SARS-CoV-2 variant antigens, SARS-CoV-2 antigens and adjuvants described herein.

Figure 2A is a graphical representation of an EDV-COVID-19 vaccine composition comprising a bacterial expression plasmid ("EDV"), such as that shown in Figure 1 B , where EDV first expresses the expression of SARS-CoV-2 in the EDV cytoplasm Spinin and additionally carried or loaded with CD1d-restricted iNKT cell antigen glycolipid α-galactosylceramide (α-GalCer) IFN-γ as an adjuvant or stimulator. The expressed spinins encoded by SARS-CoV-2 are designated by the stars on Figure 2A. Figure 2B shows exemplary vials containing lyophilized EDV-COVID-19 vaccine compositions.

Figure 3 is a graphical representation of an EDV-COVID-19 vaccine composition comprising an intact bacterial pocket cell containing an expression plastid (such as shown in Figure 1B ), wherein the bacterial pocket cell comprises (i) a plastid, which Expression of the selected spike protein from the original SARS-CoV-2, SARS-CoV-2 delta variant, and SARS-CoV-2 Brazilian variant, (ii) a gene expression promoter that expresses all proteins in the cytoplasm of EDV as single mRNA and individual proteins, (iii) multiple spinins including spinin produced by SARS-CoV-2, Brazilian variant spinin and delta variant spinin, and (iv) as adjuvants or stimulators The CD1d-restricted iNKT cell antigen glycolipid α-galactosylceramide (α-GalCer) IFN-γ. The encoded expressed spinins are designated by the stars on Figure 3 . After administration to an individual in need thereof, the vaccine composition acts to stimulate an antibody response to viral proteins. Plastid dsDNA is recognized by intracellular nucleic acid sensors and triggers IFNα and IFNβ responses.

The product can be lyophilized. Products based on whole bacteria-derived pocket cells are extremely stable and when lyophilized vials with anticancer compounds and whole bacteria-derived pocket cells loaded with α-GC are simply placed in a normal refrigerator at a hospital pharmacy at 4°C On storage, the vials have shown stability for more than 3 years. It can be shipped anywhere in the world by courier, as previously demonstrated in US cancer trials using EDV (eg bacterial pocket cells).

Patient Administration: When a patient wishes to administer, the vial can be reconstituted in 1 ml sterile saline and injected i.v. as a bolus injection.

Plasmids encoding proteins of the SARS-CoV-2 virus and virus variants can be transformed into strains that generate intact bacteria-derived pocket cells that will express viral proteins in the bacterial cytoplasm. Many proteins are segregated in the intact bacterium-derived pocket cell cytoplasm when they are generated during asymmetric bacterial division. This has been demonstrated in several studies in which heterologous exogenous proteins have been expressed in bacterial cells that give rise to intact bacterium-derived miniature cells, and the proteins are segregated in the cytoplasm of the intact bacterium-derived miniature cells.

The expected outcome of plastid-packaged bacteria-derived minicells is an antibody response to all viral proteins plus a type I interferon response.

The injected whole bacteria-derived pocket cells will be engulfed by cells of the immune system (macrophages, NK cells and dendritic cells) in the lymph nodes, liver and spleen. Intact bacteria-derived pocket cells typically enter endosomes and are disassembled in lysosomes and release plastids, which escape into the cytoplasm.

The cytoplasmic DNA sensor that will recognize plastid DNA is a class of pattern recognition receptors (PRRs), which induce the production of type I interferons (IFNα and IFNβ) and trigger the induction of a rapid and potent innate immune response. It is well known that type I interferons have potent antiviral effects.

SARS-CoV-2 virus and virus variant proteins are released from intact bacteria-derived pocket cells that are disassembled in lysosomes and undergo antigen processing and presentation on the cell surface via MHC class II. This triggers a potent antibody response to viral antigenic epitopes. This further induces a CD4+/CD8+ T cell response against virus infected cells and this will enhance the antiviral response.

Activation, maturation and proliferation of fresh bone marrow-derived monocytes, together with activation and proliferation of macrophages, dendritic cells, NK cells, B cells, and T cells, is expected to overcome that observed in elderly and immunocompromised SARS-CoV2 patients Lymphopenia.

Expected outcome of α-galactosylceramide-packaged intact bacterial-derived pocket cells - induction of IFN-γ responses: EDV™ α-GC is also produced by cells of the immune system (macrophages, NK cells and dendritic cells). Pocket cells of complete bacterial origin disassemble and release α-GC in intracellular lysosomes, which are taken up by lysosome-associated CD1d (MHC class I, such as molecules involved in the presentation of exogenous glycolipids) and transported to the cell surface . This α-GC/CDld complex is recognized by the invariant T cell receptor on invariant NKT cells and this results in a rapid release of IFN-γ. IFN-γ is known to be a potent stimulator of specific antiviral immune responses and is thus expected to enhance rejection of viral infection.

Whole bacterium-derived pocket cell therapeutics have been shown to be safe in human cancer patients where over 1,500 doses have been administered in over 140 patients with minimal to no side effects despite repeated dosing. Example 2

This example is directed to a study evaluating the feasibility of using bacterial pocket cells loaded with EDV _Covid-αGC (EDV _Covid-αGC ) as a vaccine against SARS-CoV-2.

α-GC and spinin, as well as a plastid encoding spinin DNA sequence, can be successfully incorporated into a single EDV (EDV _Covid-αGC ). The EDVs are then administered by subcutaneous (SC), intravenous (IV) and intramuscular (IM) injection. It was found that administration by intramuscular injection produced the strongest initial interferon response at 8 h post injection and the highest spinin-specific IgG titers at 1 week post injection compared to all other strategies tested.

EDV _Covid-αGC and corresponding controls were then administered by intramuscular injection and incorporation of αGC in EDV resulted in a significant increase in IFNα, TNFα, IFNγ, IL12 and IL6 production 8 h after treatment. This was accompanied by an increase in the amount of cytotoxic T cells in the spleens of EDV _{Covid -αGC-} treated mice. These T cells responded to stimulation with echinin ex vivo and expressed CD69+CD137+.

Figure 4 A - C shows the administration of bacterial pocket cells containing α-galactosylceramide (α-GalCer) to three pancreatic cancer patients (CB03, CB17 and CB41) over a period of 39 days or over a period of 46 days Results of administration to 4 pancreatic cancer patients (CB11, CB14, CB18 and CB41). Measurements of serum IFN-α (pg/mL) ( FIG . 4A ) and serum IFN- γ ( FIG. 4B ) are shown on the Y-axis of the graphs plotted in FIGS . 4A and 4B . The data showed that EDV-αGC elicited a Th1 response and increased lymphocyte content in pancreatic cancer patients. Figure 4A shows a sustained increase in serum IFNα levels from all 3 patients after 2 doses of EDV-αGC , and Figure 4B shows a sustained increase in serum IFNγ levels from all 3 patients after 2 doses of EDV-αGC. IFN levels were measured by ELISA from patient serum samples obtained throughout the treatment period. Figure 4C shows the results of measuring lymphocyte counts ( ^xlO9 /L) in four pancreatic cancer patients (CB11, CB14, CB18 and CB41) over a period of 46 days after 2 doses of EDV-αGC. The results depicted in Figure 4C show that lymphocyte counts were elevated within the normal range (1.0-4.0) in four pancreatic cancer patients. Lymphocyte levels were measured from patient blood samples throughout the treatment period by the pathology service.

Four weeks after initial treatment, mice injected with EDV _Covid-αGC contained the highest amount of spinin-specific IgG and IgM compared to all tested controls. B cells extracted from these mice were able to produce IgG and IgM in response to spikin stimulation in vitro. In addition, splenocytes from EDVCovid-αGC-treated mice contained the highest amount of antiviral CD69+ CD137+ cytotoxic T cells and ex vivo stimulation of these splenocytes using spinin produced an increase in viral antigen-specific CD69+ cytotoxic T cells. Furthermore, sera from EDV _Covid-αGC- injected mice exhibited the strongest inhibition of spinin binding to hACE receptors in vitro, indicating neutralization of the antibodies produced. Interestingly, sera from mice receiving any form of αGC also exhibited measurable but non-antigen-specific antiviral effects.

In conclusion, incorporation of αGC into EDV _Covid is extremely important for maximal SARS-CoV-2 spike protein potency. The results of this study indicate that IM administration of EDV _Covid-αGC is a viable strategy to combat the current Covid-19 pandemic.

Materials and methods

SARS - CoV -2 Spike Protein Bacterial Expression Plasmid Design : The Expression Cassette is prepared by placing the coding nucleotide sequence of SARS-CoV-2 (Covid-19) Spike Protein (Genebank MN908947.3) in the modified β- Generation of the 3'-end of the amidase promoter, which had previously been tested for expression in a strain of Salmonella typhimurium (Su, Brahmbhatt et al., Infection and Immunity , 60 (8): 3345-3359 (1992) ). The expression cassette was then inserted between the Kpn 5' and Sal I 3' sites of the M13 multiple selection site of the PUC57-Kan backbone plastid to generate P-Blac-Cov2S. The control plasmid P-Blac was generated by removing the Cov2S sequence from P-Blac-Cov2S.

Figures 5A - H show the construct design of the EDV-SARs-CoV-2 vaccine ( Figure 5A ). The expression cassette is generated by placing the coding nucleotide sequence of the SARS-Cov-2 (Covid-19) spike protein (Genebank MN908947.3) at the 3'-end of the modified β-lactamase promoter, which The promoter was previously tested for expression in a S. typhimurium strain (Su et al., Infection and Immunity , 60 (8):3345-3359 (1992)). The expression cassette was then inserted between the Kpn 5' and Sal I 3' sites of the M13 multiple selection site of the PUC57-Kan backbone plastid to generate P-Blac-Cov.

P - Blac - Cov 2 S and P - Blac - Cov 2 S were selected and colonized in the EDV - producing strain of Salmonella typhimurium and then P - Lac - Cov 2 S and spinin were incorporated into EDV : P- Blac-Cov2S and P-Blac-Cov2S were electroporated on the chemically competent Salmonella typhimurium intermediate strain (4004) (which lacks plasmid body restriction mechanism). Transformants were recovered in TSB medium at 37°C for 1.5 hours, and then plated on TSB agar plates containing 75 µg/ml kanamycin (#K4000, Sigma-Aldrich, St. Louis, Missouri). Isolates were picked into TSB broth with 75 µg/ml kanamycin and plastid DNA was extracted using a Qiagen miniprep kit (#27104, Qiagen, Hilden, Germany) according to the manufacturer's instructions. Subsequently, the extracted plastid DNA from 4004 was electroporated into an EDV-producing S. typhimurium strain (ENSm001 ) of EnGeneIC Pty. Ltd. as described above. Bacteria harboring P-Blac-Cov2S will produce the encoded Covid2 spike protein which is incorporated into EDV along with plastid DNA to produce EDV _COVID . EDV (EDVCONT) containing P-Blac will be used as a control.

To determine the plastid content of EDV _COVID and EDV _CONT , plastids were extracted from ^2x109 EDV using the Qiaprep Spin miniprep kit (Qiagen) according to the manufacturer's instructions. Empty EDVs were treated in the same manner and served as controls. The number of DNA plasmids was then measured by absorbance at 260 nm using a biophotometer (Eppendorf). The copy number of the plastid was calculated using the following:

Western Blot : Use 100 µL of B-PER™ (Thermo Fisher) bacterial protein supplemented with 10% (v/v) lysozyme (Sigma-Aldrich) and 1% (v/v) DNaseI (Qiagen) Extraction Reagents Proteins were extracted from ^2x1010 EDVCOVID. The extracted samples were then centrifuged at 12,000 g for 10 min and the supernatant was collected. The remaining pellet was also collected and resuspended in 100 μl PBS. Incubate 23 µl supernatant and precipitated protein samples with 5 µl loading buffer and 2 µl DTT (Sigma-Aldrich) at 80°C for 20 min, then load the entire content of each sample on NuPAGE 4-12 % Bis-Tris mini gel (Life Technologies) and run at 190 V for about 80 min. Samples were then transferred using an iBlot 2 machine and the membrane was blocked using Superblock blocking buffer (Thermo Fisher), and then blocked with a 1:2000 rabbit polyclonal SARS-CoV2 spike antibody (also cross-reactive with the S1 subunit, Sino Biological, Beijing , China) and incubated overnight at 4°C. The membrane was then washed with PBST and incubated with HRP-conjugated anti-rabbit secondary antibody (1:5000) (Abcam) for 1 h at room temperature. Blots were developed using Lumi-Light western blot substrate (Roche) and visualized using Chemidoc MP (Biorad).

α - Galactosylceramide loading into EDVCOVID and cell culture: α-Galactosylceramide glycolipid adjuvant (α-GC) was loaded into EDVCOVID using a proprietary method developed by EnGeneIC to generate EDVCOVID-αGC.

JAWSII cells (ATCC) were treated with EDVCOVID-αGC at 1×10 ⁴ EDV-COVID-αGC/cell in a 96-well Perfecta 3D hanging drop plate (Sigma). JAWSII cells treated with 4 µg/mL α-GC were used as positive control. Cultures were then incubated at 37°C and 5% CO ₂ for 24 h, and cells were harvested and stained with CDld-αGC antibody (ThermoFisher) and analyzed using a Gallios flow cytometer (Beckman). Results were analyzed using Kaluza Analysis software (Beckman).

Animal studies : 6-7 week old female Balb/c mice were obtained from Animal Resources Company, Western Australia. Mice were acclimatized for one week before the start of the experiment. Mice were injected with appropriate therapeutic agents via SC and IM injections, and serum was collected via tail vein at 8 h, 1 week, and 4 weeks after injection, and spleen and bone marrow were collected.

Figure 5 D -5 H shows the results after intramuscular (IM) injection of 2 × 10 ⁹ EDV-COVID-α-GC into five groups of BALB/c mice, in which the concentration of IFNα (pg/mL) (Figure 5D), IFNγ concentration (pg/mL) (Fig. 5E), IL12p40 concentration (pg/mL) (Fig. 5F), IL6 concentration (pg/mL) (Fig. 5G) and TNFα concentration (pg/mL) (Fig. 5H) are shown in Fig. on the Y axis. The results showed that administration resulted in a strong type I interferon response within 8 h after injection. Five groups of mice (n = 6/group) were: Group 1 = saline; Group 2 = EDV (bacterial miniature cells without payload); Group 3 = EDV _control (carrying inserts that did not express spinin plastid (i.e. only plastid backbone) of EDV); Group 4 = EDV _Covid (bacterial pocket cells containing SARS-CoV-2 spike protein), and Group 5 = EDV _{Covid + α GC} ( Fig. 2A structure shown in).

Figures 6A-6F show Balb/c mice ^{( n} = 8 rats/group). 28 days after the initial dose (with booster administration on day 21), high levels of anti-S protein IgM ( FIG. 6 A ) and IgG ( Figure 6 B ) Antibody titers. Figure 6C shows the results after B cells were isolated from mouse bone marrow and co - cultured with SARS-CoV-2 S protein ex vivo 28 days after the initial injection. It was found that B cells isolated from EDV-COVID-α-GC immunized mice produced significantly higher amounts of S protein-specific IgG in response to the presence of S protein compared to all other groups tested. Figure 6D shows the results of neutralizing antibody assays showing that 100% of the sera from mice immunized with EDV-COVID-α-GC resulted in inhibition of the binding of SARS-CoV-2 RBD to the hACE2 receptor. Inhibition of RBD binding to the hACE2 receptor was assessed using the cPASSTM SARS-CoV-2 Neutralizing Antibody Assay (FDA-approved; Tan et al., Nature Biotech , 2020) for detection in various species.

ELISA : According to the manufacturer's instructions, IL-12p40, IFN-γ, TNFα, IL-6, IL2, IFNα and The content of IFNβ. The concentration of protein present was determined by calculating the absorbance of the samples against a standard curve constructed in the same assay using purified protein.

For the analysis of anti-RBD specific IgG and IgM antibodies, 96-well plates (Immulon 4 HBX; Thermo Fisher Scientific) were suspended at 4°C with 50 µl/well of 2 µg/ml anti-covid spine RBD protein (Genetex) in PBS (GIBCO) The solution in it is coated. The next day, the capping protein solution was removed and the samples in each well were blocked with 100 μl/well of 3% skim milk prepared in PBS with 0.1% Tween 20 (PBST) for 1 h at room temperature. During this time, serial dilutions of mouse sera were prepared in 1% skim milk prepared in PBST. The blocking solution was then removed and 100 μl of each serially diluted serum sample was added to the plate and incubated for 2 h at room temperature. At the end of the incubation period, the plate was washed three times with 250 µl/well of 0.1% PBST, and then 100 µl of goat anti-mouse IgG/IgM-horseradish peroxidase (HRP) conjugate prepared in 0.1% PBST was added. 1:3,000 dilution of linked secondary antibody (ThermoFisher). Samples were incubated for 1 h at room temperature and then washed again three times with 0.1% PBST. Once completely dry, samples were visualized by incubation with TMD. Reactions were then stopped and samples were read at 490 nm using a KC Junior plate reader (BioTek Instruments).

Antibody titers were determined using ELISA by generating 1 :3 serial dilutions of treated mouse serum samples and expressed as the reciprocal of the highest dilution with a positive result.

Statistical analysis : Student's T-tests and one-way ANOVA were implemented using Prism 8 (GraphPad). A P value < 0.05 was considered statistically significant.

result

To achieve effective and efficient delivery of the vaccine with a single injection, αGC was coloaded in EDV _Covid to generate EDV _Covid-αGC . The function of co-loaded αGC was tested by examining its presentation on JAWSII cells by CDld ligand after EDVCovid-αGC treatment. A high percentage of JAWII cells expressing CDld-αGC after treatment was found to be comparable or higher than their levels treated with 3 μg/mL free αGC. Western blot analysis was performed to ensure that spinin incorporated into EDVCovid-αGC was not affected by the secondary incorporation of αGC.

The effect of different delivery methods on EDV _Covid-αGC was then evaluated in vivo. Mouse serum samples were collected from treatments administered by subcutaneous (SC), intravenous (IV) and intramuscular (IM) injections and analyzed by ELISA for IFNα ( FIG. 7C ; serum IFNα 8 h after injection concentration), IFNγ ( Fig. 7D ; serum IFNγ concentration of 8h after injection), IL12 ( Fig. 7E ; IL12p40 serum concentration of 8h after injection), IL6 ( Fig. 7F ; IL6 serum concentration of 8h after injection) and TNFα ( Fig. 7G ; Concentration of serum TNFα 8h after injection). It was found that EDV _Covid-αGC administered by IM injection was excellent in inducing the production of all the tested interkines in mice 8 h after injection.

Differences between the different administration methods of EDV _Covid-αGC were further confirmed when spikin-specific antibodies were analyzed 1 week after the initial injection. High spinin-specific IgG titers were detected in the sera of EDVCovid-αGC-treated mice by IM injection compared to by SC injection. It was concluded that administration of EDV Covid-αGC by IM injection is a preferred delivery strategy due to the high level of initial interferon response and subsequent high IgG titers.

Detailed analysis of the initial interferon response after IM injection of EDV, EDV _αGC , EDV _Control , EDV _Control-αGC , EDV _Covid , EDV _Covid-αGC revealed that the early interferon response in mice is mainly caused by the administration of αGC carried by EDV (with or without concomitant antigen-specific plastids) induction. See Figure 12 A (the serum IFNα concentration of 8h after IM injection); Figure 12 B (the serum IFNγ concentration of 8h after IM injection); Figure 12 C (the IL6 serum concentration of 8h after IM injection); Figure 12 D (the serum concentration of IL6 after IM injection 8h serum TNFα concentration); and Figure 12E ( IL12p40 serum concentration 8h after IM injection).

FACS analysis of splenocytes from mice 1 week after injection showed an increased number of CD3+CD8+ cytotoxic T cells in mice injected with EDV _Covid-αGC compared with saline group. AIMS analysis was performed on isolated splenocytes and it was found that when stimulated with spinin, the viral antigen-specific CD69+CD137+ population within the cytotoxic T cell population was increased to a higher level than the PHA-stimulated positive control.

The highest levels of spinin-specific IgG and IgM were observed in the serum of EDV _Covid-αGC- treated mice administered by IM injection 4 weeks after the initial injection. Interestingly, it was also found that the sera of mice treated with EDV _Control-αGC also contained spinin "specific" antibodies. This finding was confirmed by neutralizing antibody analysis. While serum from mice treated with EDV _Covid-αGC contained the highest amount of neutralizing antibodies, serum from mice treated with EDV _Control-αGC , EDV _Covid , and EDV _αGC also resulted in measurable levels of spinosin binding to hACE receptors suppression. This appears to suggest that αGC alone has antiviral properties, where administration of the compound may result in inhibition of virus binding to cells in vivo. In contrast, injection of EDV _Covid alone without the addition of αGC was able to generate neutralizing antibodies in serum, although at much lower levels compared with treatment with EDV _Covid-αGC . This demonstrates the importance of incorporating αGC as an immune adjuvant into this system as an important part of a functional vaccine.

To further demonstrate the specificity of the antibody response, B cells were extracted from the bone marrow of treated mice 4 weeks after the initial injection and stimulated with spinin for 48 hours in vitro. B cells from mice treated with EDV _Covid-αGC produced the highest levels of spinin-specific IgG and IgM compared to all other treatment groups. Figure 5B shows the results of a mouse experiment in which four groups of mice were evaluated (Group 1 = untreated; Group 2 = EDV without payload; Group 3 = administered free αGC; and Group 4 = administered Bacterial pocket cell vaccine comprising a combination of SARS-CoV-2 spike protein and αGC (depicted in Figure 2A )). The data presented in Figure 5B demonstrate that EDV ^™ -COVID-α-GC is capable of efficient delivery of α- GC into murine bone marrow-derived JAWII cells and is presented by CD1d-ligand with similar efficiency as free α-GC . Figure 5C shows Western blot analysis using polyclonal antibodies against RBD and S1 subunit, ^{where the results demonstrate the presence of the EDV™} -COVID -GC spike protein. The incorporation of bacterially expressed proteins into EDV ^™ occurs during cell division and segregation of cytoplasmic proteins.

Figure 6 E shows the results of E-FACS analysis of CD8+ cytotoxic T cells in mouse splenocytes, which show that mice immunized with EDV _COVID-α-GC were 4 weeks after the initial injection (1 booster on day 21). ) had the highest amount of antigen-specific memory CD137+CD69+ cytotoxic T cells, for example, there was a significantly higher number of CD137+ CD69+ cytotoxic T cells in EDV _Covid-αGC- treated mice compared to all other treatment groups group. CD137+ signaling is required for CD8+ T cell antiviral responses. Figure 6F shows the results of ex vivo AIMS analysis showing spine antigen - specific CD8+ T cell responses. CD69+/CD8+ T cell numbers increased after stimulation with Covid spike protein in the EDV-Covid and EDV-Covid-aGC groups, but not in any other group. PHA was used as a positive control. These results indicated that both the plastid and the protein contained within EDV reacted specifically.

Thus, FACS analysis of isolated splenocytes from treated mice showed that EDV _Covid-αGC treatment resulted in an increase in CD69+ CD137+ cytotoxic T cells compared to all other treatment conditions. It was also observed that when ex vivo spheroids were stimulated with spinin, viral antigen-specific CD69+ CD137- cells within the cytotoxic T cell population were positive for PHA stimulation similar to those from EDV _Covid-αGC and EDV _Covid- treated mice Control speed increased. This was not observed in all other treatment groups. This indicates that, unlike the antiviral response triggered by EDV _Covid-αGC treatment, the antiviral properties of αGC may be broad-spectrum rather than antigen-specific. Example 3

The purpose of this example is to demonstrate the immunity generated against a variant strain of SARS-CoV-2 using the vaccine compositions described herein.

Two SARS-CoV-2 variant strains of high concern UK (B.1.1.7) variant and South African (B.1.351) variant. The UK (B.1.1.7) variant (also known as the α SARS-CoV-2 variant) has been reported to have a 71% higher transmission rate than other variants ( BMJ , "Covid-19: What have we learned about the new variant in the UK?" December 23, 2020). One report indicated that UK variants appeared to gain dominance by competing with existing circulating variant populations, strongly suggesting natural selection of viruses that are more transmissible at the population level (Lauring et al., "Genetic Variants of SARS- CoV-2-What do They Mean?” JAMA , 325 (6):529-531 (February 9, 2021). The South African (B.1.351) variant was associated with increased infectivity, higher viral load, and was defined by an unusually high number of mutations. ( www . thermofisher . com / blog / ask - a - scientist / what - you - need - to - know - about - the -501 y - v 2 - b -1-351 - south - african - variant - of - sars - cov -2/ , access July 16, 2021).

Figure 7A - 7D is shown by analyzing the specificity and cross-reactivity of RBD and S1 subunits of UK (B.1.1.7) and South Africa (B.1.351) mutant strains of the virus from serum IgG from immunized mice Robustness of immunity generated by EDV-COVID-α-GC. The results showed that although UK variant strain RBD-specific IgG was produced in some EDV-COVID-α-GC immunized mice ( Figure 7A ), higher S1-specific IgG antibody titers were observed ( Figure 7B ), This indicates that the binding of the S protein-specific antibody is mainly outside the RBD. A similar trend was observed for the SA variant strains ( Fig. 7 C and D ).

This data demonstrates the surprisingly broad effectiveness of the vaccine compositions of the disclosure against SARS-CoV-2 variants. Example 4

The purpose of this example is to demonstrate the immunity generated against a variant strain of SARS-CoV-2 using the vaccine compositions described herein.

Five groups of mice were administered the COVID variant vaccines described herein and various controls. Five groups of mice ( n = 6/group; ELISA samples were run in triplicate): Group 1 = saline; Group 2 = EDV (bacterial pocket cells without payload); Group 3 = EDV _control (EDV carrying a plastid without an insert expressing the spinin (i.e., only the plastid backbone)); Group 4 = EDV _Covid (bacterial pocket cells comprising plastids and encoded SARS-CoV-2 spinin) and Group 5 = EDV _{Covid + α GC} (the construct shown in Figure 2A ).

Figures 10A - D show the results of IgG titers after administration of 3 x 10 ⁹ EDV to five different groups of mice on days 1 and 21, where serum analysis was performed on day 28. The results shown in Figures 10A-D detailing the S1 subunit-specific IgG titers at day 28 demonstrate that serum IgG titers obtained from mice treated with EDV-COVID-GC correlate with all four mutant virus spikes. Proteins strongly bound to: (1) SARS-CoV-2 variant α (B.1.1.7.UK) ( Fig. 10A ); (2) SARS-CoV-2 variant β (B.1.351. SA) (Fig. 10B ); (3) SARS-CoV-2 variant δ (B.1.617.2 India); and (4) SARS-CoV-2 variant γ (P.1 Brazil).

This data demonstrates the surprisingly broad effectiveness of the vaccine compositions of the disclosure against SARS-CoV-2 variants. Example 5

The purpose of this example is to illustrate a proposed clinical trial using the COVID-19 vaccine described herein.

The clinical trial in healthy volunteers will include intramuscular injection of 8 × 10 ⁹ EDV-COVID-GC on day 1 and day 21 ( Figure 2A ), and serum analysis will be performed on day 28 and month 3. The results are expected to demonstrate immunity generated against the SARS-CoV-2 variant, as measured by IgG analysis. Example 6

This example illustrates a phase I/IIa, open-label study to identify plastids encoding the SARS-CoV-2 spike protein and glycolipid a-galactose nerves packaged in EDV in non-COVID-19-infected volunteers aged 18 years and older Safety of amide-based EDV nanocells (EDV-plastid-spine-GC).

All participants were otherwise healthy and did not report any history of chronic health conditions. Individuals were identified by PCR testing as naive to SARS-CoV-2 and naïve to previous COVID-19 vaccines. All subjects received a dose of 9 x ¹⁰⁹ EDV-COVID-αGC with the same booster dose on day 21. All doses were administered in the clinic, and safety monitoring was performed for 3 hours on the day of dosing, including vital signs, laboratory tests, and monitoring of adverse events.

Samples were collected at 4 time points: pre-vaccine baseline (time point 1), before the day 21 booster vaccination (time point 2) and one week after the day 28 booster (time point 3). Subjects are also scheduled to return at the 3-month and 6-month time points. Each study visit included the collection of 20 mL of peripheral blood. The study began in September 2021, and by the time the study was submitted, a number of volunteers had volunteered to participate in the study. Complete data (all time points) for six volunteers are presented here.

Animal studies : 6-8 week old female BALB/C mice were obtained from the Animal Resource Center in Western Australia. Mice were acclimatized for one week before the start of the experiment. Treatment groups (n = 4-10, depending on the trial) included EDV-COVID-αGC and a control group consisting of saline, EDV, EDV-αGC, EDV-CONTROL (control plastids) and EDV-COVID. The initial experiment involved a 2 x ¹⁰⁹ im pellet dose to unilateral flank on day 0, followed by a 1 x ¹⁰⁹ boost on day 21. Subsequent experiments applied higher im, but due to the limitation of acceptable particle volume/concentration per flank, the dose of 3 × 10 ⁹ particles was divided into 1.5 × 10 ⁹ particles per dorsal flank, with the same amount on day 21 Dose and delivery regimens were boosted. Depending on the experiment, serum and tissue were collected 8 h, 7 days, 21 days and 28 days after the initial injection. Blood was collected via cardiac puncture immediately after euthanasia, or tail bled for ongoing analysis. Other tissues harvested included spleen, lymph nodes, and bone marrow from the femur.

method details

Recombinant CoV proteins and antibodies: SARS-CoV-2 spike protein was purchased from ACRObiosystems Inc. SARS-CoV-2 (Cov-19) Spike protein, His tag, super stable trimer (MALS & NS-EM verified) (Cat. No. SPN-C52H9) was used to analyze IgG and IgM responses in early experiments, and For the assay of activated immune markers (AIM). Subsequently, as new high-concern variants emerged and the availability of recombinant proteins increased, the following products were purchased: SARS-CoV-2 UK αS1 protein (HV69-70del, Y144del, N501Y, A570D, D614G, P681H, T716I, S982A , D1118H), His tag (Cat. No. SPN-C52H6); SARS-CoV-2 S UK α protein RBD (N501Y), His tag (Cat. No. SPD-C52Hn); SARS-CoV-2 SA β S protein ( L18F, D80A, D215G, 242-244del, R246I, K417N, E484K, N501Y, D614G, A701V) trimer, 50ug Cat. #SPN-C52Hk; SARS-CoV-2 SA β S protein RBD (K417N, E484K, N501Y ), His tag (MALS verification) (Cat. No. SPD-C52Hp); SARS-CoV-2 Brazil γ S1 protein (L18F, T20N, P26S, D138Y, R190S, K417T, E484K, N501Y, D614G, H655Y, T1027I, V11 (Cat. #SPN-C52Hg); SARS-CoV-2 Indian delta spine S1 (T95I, G142D, E154K, L452R, E484Q, D614G, P681R), His tag (Cat. No. S1N-C52Ht); SARS-CoV-2 o Spipin HRP (RBD, G339D, S371L, S373P, S375F, K417N, N440K, G446S, S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, His tag)-HRP (Cat No. Z03730).

The SARS-CoV-2 (COVID-19) spike-antibody system against S1 and S2 subunits was purchased from Genetex (Cat. No. GTX135356 and No. GTX632604), and was used to confirm the S protein in EDV ^TM by Western blot method. Antigen affinity purified SARS-CoV-2 (2019-nCoV) spine RBD rabbit PAb (Cat. No. 40592-T62, Sino Biological) was used to quantify the S protein in EDV using ELISA.

Cell line: JAWSII mouse bone marrow-derived dendritic cells (ATCC ^® CRL-11904™) were cultured in α-minimal essential medium with ribonucleosides and deoxyribonucleosides (4 mM L-glut, 1 mM sodium pyruvate , 5 ng/ml GMCSF and 20% FBS) at 37°C, 5% CO ₂ .

Generation of plastids expressing the SARS-CoV-2 S protein under a bacterial promoter : the expression cassette is obtained by incorporating the encoding nucleotides of the SARS-Cov-2 (Covid-19) spike protein (Wuhan sequence; GeneBank MN908947.3) The acid sequence was placed at the 3'-end of a modified β-lactamase promoter that was previously used for expression in S. typhimurium strains (Su et al., 1992). The expression cassette was then inserted between the KpnI 5' and SalI 3' sites of the M13 multiple selection site of the pUC57-Kan backbone plastid to generate pLac-CoV2. Sequences were optimized for S. typhimurium codon usage prior to fabrication by the Genscript service. Negative control plastid pLac-control was generated by removing the CoV2 sequence from pLac-CoV2 as described above ( FIGS. 5C and D and 11A-K ).

Colonization of pLac-CoV2 and pLac in EDV-producing strains of Salmonella typhimurium and evaluation of plastids and S proteins in EDV

Colonization: PLac-Cov2 and pLac-CoV2-control were electroporated into a chemically competent S. typhimurium intermediate strain (which lacks the plastid restriction mechanism) using Gene Pulser Xcell™ (Bio-Rad, Hercules CA). Transformants were recovered in TSB medium at 37°C for 1.5 hours, and then plated on TSB agar plates containing 75 µg/ml kamycin (Sigma-Aldrich, St. Louis, Missouri). Isolates were picked into TSB broth with 75 µg/ml kanamycin and plastid DNA was extracted using a Qiagen miniprep kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. Subsequently, extracted plastid DNA from the 4004 strain was electroporated into an EDV ^™ producing S. typhimurium strain (ENSm001 ) as described above. A clone containing pLac-CoV2 produces an encoded Covid-19 spike protein that, along with plastid DNA, is incorporated into EDV during cell division to generate EDV-COVID. EDV containing pLac (EDV-CONTROL) was generated in the same manner to serve as a negative control.

To determine the plastid content of EDV-COVID and EDV-CONTROL, plastids were extracted from ^2x109 EDV using the Qiaprep Spin miniprep kit (Qiagen) according to the manufacturer's instructions. Empty EDVs were treated in the same manner and served as controls. The number of DNA plasmids was then measured by absorbance at OD _{260 nm} using a biophotometer (Eppendorf). The copy number of the plastid was calculated using the following:

Western blotting: using 100 µL of B-PER™ (Bacterial Protein Extraction Reagent; Thermo Fisher) supplemented with 10% (v/v) lysozyme (Sigma-Aldrich) and 1% (v/v) DNaseI (Qiagen) Proteins were extracted from 2 × 10 ¹⁰ EDV-COVID. The extracted samples were then centrifuged at 12000 g for 10 min and the supernatant was collected. Pellets were also harvested and resuspended in 100 µl PBS. Incubate 23 µl of supernatant and precipitated protein samples with 5 µl of loading buffer and 2 µl of DTT (Sigma-Aldrich) at 80°C for 20 min, then load the entire contents of each sample on NuPAGE 4- 12% Bis-Tris mini Protein Gel (ThermoFisher) and run at 190 V for about 80 min. The gel was then transferred using the iBlot 2 system (ThermoFisher), after which the membrane was blocked using SuperBlock™ blocking buffer (ThermoFisher), and subsequently blocked with a 1:1000 rabbit polyclonal anti-SARS-CoV-2 spike (S1 ) antibody (Genetex) Or 1:1000 mouse monoclonal anti-SARS-CoV-2 spine (S2) antibody (Genetex) stained and incubated overnight at 4°C. The membrane was then washed with PBST and incubated with HRP-conjugated anti-rabbit (1:5000) (Abcam) or anti-mouse (1:5000) (ThermoFisher) IgG secondary antibody at RT for 1 h. Blots were developed using Lumi-Light western blot substrate (Roche) and visualized using Chemidoc MP (Bio-rad).

Evaluation of EDV S protein by ELISA : 4×10 ⁹ EDV-COVID particles were pelleted by centrifugation at 13000 g for 8 min. 100 µL of B-Per™ Bacterial Lysis Reagent supplemented with 100 µg/reaction of lysozyme (Sigma) and 5 U/reaction of rDNase I (Macherey-Nagel) was added to each sample and incubated at RT on a vortex shaker Incubate at 600 rpm for 2 h. The samples were then mixed with 1 :5 dithiothreitol (ThermoFisher) and placed on an 80°C heat block (Eppendorf) with agitation at 600 rpm for an additional 20 min. Protein quantities were analyzed using the DC protein assay kit (Bio-rad) following the manufacturer's instructions.

Standards were generated by serial dilution of spinin (ACRObiosystems) to achieve the following concentrations: 2000, 1000, 500, 250, 125, 62.5, 31.3 pg/mL. The EDV-COVID S protein sample was diluted 1:1000 in PBS. Standards and EDV spinin samples were then coated on ELISA plates, sealed and incubated O/N at 4°C. Plates were then washed 3 times with 300 µL PBST using a plate washer. 200 µL of protein-free blocking buffer (Astral Scientific) was added to the plate, the plate was sealed and incubated for 1 h at RT.

The spiny RBD rabbit PAb detection antibody (Sino Biological) was diluted 1:10000 in 10 mL PBST and added at 100 µL/well and incubated at RT for 1 h. Plates were washed in PBST as described above, then 100 µL of sheep anti-rabbit IgG (H+L)-peroxidase (Merck, 1:10000) in 10 mL of PBST was added. Incubate the sealed plate for 30 min at RT in the dark. Plates were washed again as above and 100 µL per well of TMB solution (ThermoFisher) was added. The reaction was stopped by adding 50 µL per well of 2 _MH2SO4 within minutes _of TMB addition. Samples were analyzed at OD _450nm using a µQuant plate reader (Bio-TEK Instruments, Inc.) and KC junior software.

Loading α - galactosylceramide into EDV-COVID : As described above, the EDV-COVID nanoparticles carrying the S protein were purified in large quantities by the biofermentation of the parent bacterium Salmonella typhimurium, and then tangentially Flow filtration (TFF) was used to purify EDV-COVID particles from their parents (MacDiarmid et al. , 2007). EDV-COVID particles were then buffer exchanged from the medium into PBS pH 7.4 (Dulbecco's; ThermoFisher) supplemented with 0.5% tyloxapol (Sigma-Aldrich) based on the protocol described in Singh et al. (2014) , and then load αGC.

Alpha-galactosylceramide glycolipid adjuvant (αGC; Advanced Molecular Technologies, Melbourne) depot was formulated in 100% DMSO (Sigma). Stock αGC was added to the EDV-COVID solution in PBS at a final concentration of 10 µM (equivalent to 8.58 µg/mL). Co-cultivation of EDV-COVID particles with αGC was performed overnight at 37°C with stirring. Unloaded αGC was removed by washing the particles in PBS pH 7.4 (Dulbecco's; ThermoFisher) with a 0.2 µm TFF system. EDV-COVID-αGC particles were then concentrated in PBS pH 7.4 followed by buffer exchange into 200 mM trehalose (Cat. No. T9531, Sigma) ready for vial filling and lyophilization.

Prior to use in animal experiments, batch vials of EDV-COVID-αGC underwent quality control testing, including particle count, homogeneity, sterility, S protein concentration, plastid copy number, and αGC concentration per 10 ⁹ EDV particles. The uptake of loaded αGC by dendritic cells (DC) and presentation by CDld T cell receptor was carried out as follows.

αGC uptake and presentation by murine DC : JAWII cells (ATCC) were treated with EDV-COVID-αGC in 96-well Perfecta3D hanging drop plate (Sigma-Aldrich) with 1×10 ⁹ EDV-COVID-αGC/well. JAWSII cells treated with 2 µg/mL αGC (Advanced Molecular Technologies) were used as a positive control. Cultures were then incubated at 37°C and 5% CO ₂ for 24 h, and cells were harvested and stained with PE-conjugated CDld:αGC complex antibody (ThermoFisher, 1:2000) and analyzed using Gallios flow cytometry (Beckman). Results were analyzed using Kaluza Analysis software (V.2.1, Beckman).

Detection of spinin and CD1d- related αGC in murine DCs after EDV-COVID-αGC co-cultivation : JAWII cells (ATCC) were seeded on 96-well hanging drop plates (Sigma-Aldrich) at 5 × ¹⁰⁴ cells/well . EDV alone, EDV-αGC, EDV-CONTROL, EDV-COVID and EDV-COVID-αGC were co-incubated with cells at 1×10 ⁹ EDV/well. Untreated JAWSII cells were used as controls. The samples were incubated at 37°C and 5% CO ₂ for 48 h, then collected and treated with PE anti-mouse αGC:CD1d complex antibody (ThermoFisher, 1:2000) and SARS-CoV-2 S1 protein polyclonal primary antibody (Genetex , 1:2000) were co-stained for 30 min at room temperature in the dark. Samples were then restained with Alexa Fluor 647 goat anti-rabbit IgG (H+L) highly cross-adsorbed secondary antibody (ThermoFisher, 1:1000) at 4°C for 20 min and analyzed using a Gallios flow cytometer (Beckman Coulter) . Mouse IgG2a and rabbit IgG (Biolegend) were used as isotype controls. DAPI was used to differentiate live/dead cells, and single stained samples were used to generate compensation. The samples were analyzed using Kaluza analysis software (V2.1, Beckman Coulter).

Extraction of αGC from EDV-COVID-αGC for quantification: The αGC extraction method was adapted from previous similar studies (Sartorius et al., 2018; von Gerichten et al., 2017). Obtain the necessary number of EDV vials to achieve a total of 4 x ¹⁰¹⁰ EDV per sample for extraction of αGC. A sample of EDV alone was used as a negative control.

All lyophilized vials were resuspended in 400 µL of PBS (Dulbecco's, Ca ²⁺ Mg ²⁺ free, ThermoFisher). Each sample was aliquoted to give approximately 2 x ¹⁰¹⁰ EDV/sample in Eppendorf tubes (ie, two tubes per sample), and all samples were centrifuged at 13200 rpm for 7.5 min. The supernatant was removed from each sample and the EDV pellet was resuspended in 800 µL PBS for each 2 x ¹⁰¹⁰ and centrifuged again as above. The supernatant was removed again, and all samples were resuspended in 500 µL of UltraPure™ H ₂ O (ThermoFisher).

For α-GC extraction, each 500 µL sample was transferred to a conical bottom 2 mL microtube (Axygen). One stainless steel bead (5 mm) was added to each sample and the samples were then homogenized using agitation on a Qiagen TissueLyser II Homogenizer (Qiagen). Homogenization was performed with two rounds of 2 min stirring at 25 Hz, with short pauses between sets. The lysates were then transferred to new tubes and 500 µL aliquots from each sample were combined to yield 1 mL samples (beads were left).

Lipids were then extracted from each 1 mL sample by adding 1 mL of chloroform/methanol (2:1 CHCl3:MeOH ratio), manual vigorous shaking, and incubating at 37°C for 15 min with sonication for 1 min every 5 min. After 15 min, the samples were centrifuged at 2000 g for 10 min in a tabletop microcentrifuge. Transfer the organic layer (bottom) to a new tube. The samples were dried and then analyzed.

Quantitative LC-MS/MS analysis of αGC : standard α-galactosylceramide (αGC) and internal standard (IS) D-galactosyl-β-1,1' N-palmityl-D- Erythro-sphingosine was dissolved in DMSO to 1 mM or 2 mM, and if necessary, heated at 60-80°C for 5 min to dissolve. Prior to data acquisition, stock dilutions were prepared in MeOH:H ₂ O (95:5) using standard stocks. Prepare a working IS solution with a final concentration of 200 ng/mL.

Standards were prepared by using five calibration points (62.5, 125, 250, 500 and 1000 ng/mL) using αGC (STD) spiked with 200 ng/mL IS (Sartorius et al., 2018). Unknown samples were quantified using standard:IS area ratios as calibration curve (CC) points or linearity. Samples were dried and rehydrated in 1 ml of working IS diluent.

Samples together with freshly prepared CC standards were acquired on a TSQ Altis (ThermoFisher) triple quadrupole mass spectrometer (MS) interfaced with a Vanquish (ThermoFisher) UHPLC (ultra high performance liquid chromatography) (LC). The LC-MS instrumental method employed for data acquisition was optimized according to Sartorius et al. (2017) (Sartorius et al., 2018). Xcalibur and TraceFinder software were used for data acquisition and analysis (ThermoFisher), respectively.

Chromatography (LC) was performed on an Acquity BEH Phenyl column (Waters, 100 × 2.1 mm, 1.7 μm) using a short gradient program from 95:5 MeOH/H2O to 100% MeOH in 1 min, followed by 100 Dissolution was carried out by isocratic elution under % MeOH for 4 min. The flow rate was set at 0.4 mL/min and the column temperature was 40 °C. αGC was eluted at RT of 1.53 min, and IS was eluted at 1.07 min. For quantitative purposes and to confirm analytical identity, MRM transitions were monitored for both STD and IS. The strongest transition for each compound (ie, m/z 856.7 > 178.9 for STD and m/z 698.5 > 89.2 for IS) was used for the analytical reaction.

Separation of serum : Coagulate whole blood samples in SST vacuum blood collection tubes (VACUETTE®) for 1 h at RT. After centrifugation at 800 g for 10 min, the serum layer was aliquoted and stored at -80°C for detection of SARS-CoV-2-specific antibodies by ELISA and neutralizing antibody assays.

Murine splenocyte isolation : Tissue suspensions were isolated from dissected spleens of treated BALB/C mice using a Dounce homogenizer and resuspended in RPMI-1640 medium (Sigma-Aldrich). The homogenized tissue was then filtered through sterile 70 µm MACS SmartStrainers (Miltenyi Biotec) and incubated with erythrocyte lysis buffer Hybri-Max™ (Sigma-Aldrich) as recommended by the manufacturer. Cells were then resuspended in 2.5 mL of autoMACS running buffer (Miltenyi Biotec) and passed through a 70 µm MACS SmartStrainer to obtain a single cell suspension.

Interleukin ELISA : IFNγ, TNFα, IL-6, IFNα, IL-12p40, IL-10, IL-2, and IL-4 from mouse serum were measured using the ^DuoSet® ELISA kit from R&D Systems according to the manufacturer’s instructions Measurement. Serum levels of IL-21 were analyzed using the LEGEND MAX mouse IL-21 ELISA kit (Biolegend) according to the manufacturer's instructions. Interleukin concentrations were determined by calculating the absorbance of the samples against a standard curve constructed in the same assay using purified material.

S - protein RBD and S 1 IgG / IgM serum titer ELISA : To analyze anti-RBD specific IgG and IgM antibodies, 96-well plates (Immulon 4 HBX; Thermo Fisher Scientific) were used at 4°C with 50 µL/well of 2 µg /mL solution of RBD or S1 protein of the corresponding variant tested (ACRObiosystems) suspended in PBS (GIBCO) was applied. The next day, the coating protein solution was removed and 100 µL of 3% skim milk blocking solution or protein-free blocking solution (G-Biosciences) in PBS/0.1% Tween 20 (PBST) was added and incubated for 1 h at RT. Serial dilutions of mouse sera were prepared in 1% skim milk/PBST or protein-free blocking solution. The blocking solution was removed and 100 μL of each serum sample was added to the plate and incubated for 2 h at RT. After incubation, the cells were washed 3 times with 250 µL of 0.1% PBST, and then 100 µL of goat anti-mouse IgG (H+L) or IgM (Heavy)-horseradish peroxidase ( HRP) conjugated secondary antibody (ThermoFisher, 1:3000). Samples were incubated for 1 h at RT and washed three times with 0.1% PBST. Once completely dry, samples were visualized by incubation with TMB. The reaction was then terminated and samples were read at OD _{490 nm} using a KC Junior plate reader (BioTek Instruments).

Antibody titers were determined using ELISA by generating 1 :3 serial dilutions of treated mouse serum samples and expressed as the reciprocal of the highest dilution with a positive result.

Extraction of B cells from murine bone marrow : A 0.5 mL microcentrifuge tube was pierced at the bottom with a 21 gauge needle and then placed into a 2 mL tube. Isolated mouse tibias and femurs were placed in the 1 mL tubes with the cut side of the bone at the bottom. Bone marrow cells were extracted from tibia and femur by centrifugation at ≥10000 g for 30 s. The pelleted cells were resuspended in 1 mL of RPMI-1640 medium (Sigma-Aldrich) and incubated with Hybri-Max™ Erythrocyte Lysis Buffer (Sigma-Aldrich) for 5 min. The lysis buffer was neutralized with 15 mL of RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) (Interpath), and centrifuged at 300 g for 10 min. Cells were resuspended in RPMI-1640 medium in a final volume of 10 mL for final counts. B cell lines were isolated using the Pan B Cell Isolation Kit (Miltenyi Biotec) according to the manufacturer's instructions.

B cell stimulation and ELISA : ELISA microplates were coated with 2 µg/mL SARS-CoV-2 spinin trimer (ACRObiosystems) and incubated overnight at 4°C. The microplate was washed 3x with phosphate-buffered saline (PBS) and blocked with 200 μl/well protein-free blocking buffer PBST (G-Biosciences) for 2 h at RT.

Mouse splenocytes were isolated from treated mice and ^1x105 cells were seeded per well in 200 µL AIMV medium and incubated at 37°C for 48 h.

At the end of the incubation period, cells were removed from each well, and each microplate was washed 5 times with 200 µL/well of 0.05% Tween 20 in PBST. Samples were then incubated with 100 µL/well of 1:5000 mouse IgG-HRP in PBST for 2 h at RT in the dark, then washed 3x in 250 µL PBST. The presence of Covid-specific IgG was detected by adding 100 µL/well of TMB substrate system and incubating for up to 20 min or until a chromogenic solution was formed. The enzyme reaction was stopped by adding 50 µL/well of 2N H2SO4 stop solution. Samples were analyzed using a CLARIOstar microplate reader (BMG LABTECH) at OD _{450 nm} with OD _{540 nm} as a reference wavelength and analyzed using MARS software.

Activation-induced marker (AIM) analysis: The isolated splenocytes were seeded in AIMV (Life Technologies) serum-free medium in 96-well U-bottom plates at 1×10 ⁶ cells/200 µL/well. Cells were stimulated with 1 µg/mL SARS-CoV-2 trimer (ACRObiosystems) for 24 h at 37°C, 5% CO ₂ . 1 µg/mL DMSO was used as negative control and 10 µg/mL PHA (Sigma) as positive control. After 24 h of stimulation, samples were collected in 1.5 mL microcentrifuge tubes, cells were collected by pipetting up and down, and centrifuged at 300 g for 10 min. Supernatants were collected and frozen for processing of IFNγ by ELISA (DuoSet, R&D Systems).

For T cell activation staining, cell pellets from above were washed twice in 500 µL FACS buffer and centrifuged as above. The final cell pellet was resuspended in 500 µL of FACS buffer and stained with the appropriate antibody (included in the kit) for 30 minutes at RT in the dark. Cells were then centrifuged at 300 g for 5 min and washed twice with 500 µL FACS buffer. They will then be fixed in 1% paraformaldehyde for 10 min at 4°C and then centrifuged again at 300 g for 5 min. Finally resuspended in 300 µL of FACS buffer and analyzed on a Gallios flow cytometer (Beckman). A single stained sample and a mouse IgG isotype control were used to generate staining compensation.

Th1/Th2 phenotype analysis: Th1/Th2 phenotype analysis was performed using mouse Th1/Th2/Th17 phenotype analysis kit (BD). First, according to AIM analysis, isolated splenocytes were seeded in AIMV (Life Technologies) serum-free medium in 96-well U-bottom plates at 1 × 10 ⁶ cells/200 µL/well. Cells were stimulated with 1 µg/mL SARS-CoV-2 trimer (ACRObiosystems) for 24 h at 37°C, 5% CO ₂ . 1 µg/mL DMSO was used as a negative control. After 24 h of stimulation, add 1 μL of BD GolgiStop™ (protein transport inhibitor; BD) per 200 μL/well of cell culture, mix well and incubate at 37°C for another 2 h. Cells were then centrifuged at 250 g for 10 min and washed twice with staining buffer (FBS) (BD). Cells were counted and approximately 1 million cells were transferred to each flow tube for immunofluorescent staining according to the manufacturer's instructions. Cells were protected from light throughout the staining procedure. First, cells were fixed by spinning at 250 g for 10 min at RT and thoroughly resuspended in 1 mL of cold BD Cytofix™ Buffer (BD) and incubated for 10-20 min at RT. After fixation, cells were pelleted at 250 g for 10 min at RT and washed twice in staining buffer (FBS) at RT. Staining buffer was removed by spinning and the cell pellet was resuspended in 1X BD Perm/Wash™ buffer (BD) diluted in distilled water and incubated at RT for 15 min. Spin cells at 250 g for 10 min at RT and remove supernatant. For staining, the fixed/permeabilized cells were thoroughly resuspended in 50 μL of BD Perm/Wash™ Buffer with 20 μL/tube of the mix included in the kit (mouse CD4 PerCP-Cy5 .5 (clonal: RM4-5), mouse IL-17A PE (clonal: TC11-18H10.1), mouse IFN-GMA FITC (clonal: XMG1.2), mouse IL-4 APC (clonal: 11B11 )) or an appropriate negative control incubation. Samples were incubated in the dark at RT for 30 min before FACs analysis on a Gallios flow cytometer (Beckman). Manual compensation was performed for each channel using a single staining control.

SARS-CoV-2 Surrogate Virus Neutralization Test ( Mice and Human Samples ) : The evaluation of neutralizing antibodies was performed using the FDA-approved "cPASS SARS-CoV-2 Surrogate Virus Neutralization Test Kit" (Genscript) (Tan et al. People, 2020) implementation. The kit is a blocking ELISA detection tool that simulates the process of virus neutralization, and is suitable for use with sera from mice and other species. Capture plates are pre-coated with hACE2 protein. The desired hACE2-coated plate strips were plated and the remainder stored at 2-8°C. According to the protocol, HRP-RBD (Wuhan, Genscript) was diluted 1:1000 in HRP dilution buffer to give a total of 10 mL. Mouse and human serum samples, PBMC supernatants, and positive and negative controls were diluted 1:10 (10 µL + 90 µL sample dilution buffer) and HRP-RBD was diluted 1:1 (60 µL + 60 µL) to allow neutralizing Ab to bind to HRP-RBD. The mixture was incubated at 37°C for 30 min. Add 100 µL of sample or control to appropriate wells. Plates were covered with plate sealant and incubated at 37°C for 15 min. Remove plate sealant and wash 4 times with 260 µL of 1X wash solution. Plates were pat dried after washing. 100 µL of TMB solution was then added to each well and the plate was incubated for up to 15 min at RT in the dark. Add 50 µL of stop solution to terminate the reaction. Absorbance was immediately analyzed at OD _450nm using a CLARIOstar microplate reader. HACE2 receptor binding inhibition was calculated using the formula provided by the manufacturer (% inhibition = 1-(OD value of sample/OD value of negative control) × 100%. According to the specification sheet, positive values are interpreted as > 30% and negative values Interpreted as <30%.

To evaluate neutralizing antibodies against mutant SARS-CoV-2 strains, the following HRP-RBD proteins were purchased from Genscript to replace the cPASS kit: SARS-CoV-2 α-spike proteins (RBD, E484K, K417N, N501Y, Avi and His tag)-HRP, SARS-CoV-2 β-spike protein (RBD, N501Y, Avi and His tag)-HRP, SARS-CoV-2 γ-spike protein (RBD, E484K, K417T, N501Y, Avi and His tag)- HRP, SARS-CoV-2 δ spike protein (RBD, L452R, T478K, Avi and His tag)-HRP, SARS-CoV-2 δ spike protein HRP (RBD, G339D, S371L, S373P, S375F, K417N, N440K, G446S , S477N, T478K, E484A, Q493R, G496S, Q498R, N501Y, Y505H, His tag)-HRP.

Neutralization titer analysis: Dilute serum samples at 1:1, 1:10, 1:20, 1:40, 1:80, 1:160, 1:320 and 1:640 and use FDA-approved "cPASS The SARS-CoVv-2 Surrogate Virus Neutralization Test Kit" was assayed against wild-type SARS-CoV-2 virus as previously described. Neutralizing titers were determined as final serum dilutions resulting in greater than or equal to 30% inhibition of RBD binding to hACE2.

FACS Analysis of T and B Cells in Human Samples : T cell analysis was performed using DuraClone IM T cell subpopulation tubes (Beckman Coulter). Add 1 x ¹⁰⁶ purified PBMCs directly to a 100 µL tube and incubate for 30 min at RT in the dark. Samples were then pelleted at 300 g for 5 min and washed once in 3 ml PBS. Resuspend the final sample in 500 µL of PBS containing 0.1% formaldehyde. Compensation for the assay was generated using purified PBMC using the compensation kit provided in the IM DuraClone T cell subset tube.

Volunteer PBMC lines were analyzed using the Human SARS-CoV-2 Spike B Cell Assay Kit (Miltenyi Biotec). Briefly, PBMCs were stained with SARS-CoV2 spinin-biotin and then co-labeled with streptavidin PE and streptavidin PE-Vio 770 to eliminate the chance of non-specific binding. Cells were then analyzed using 7AAD, CD19, CD27, IgG and IgM, and then using FACS. All compensations were performed using UltraComp eBeads™ Plus compensation beads (ThermoFisher). Samples were analyzed using a Gallios flow cytometer (Beckman Coulter) and analyzed using Kaluza software (Beckman Coulter).

Samples were processed using a Gallios flow cytometer (Beckman) and results were analyzed using Kaluza Analysis software (ver 2.1, Beckman).

Activation-induced marker (AIM) analysis in human samples: PBMC from volunteers were seeded in AIMV (Life Technologies) serum-free medium in 96-well U-bottom plates at 1 × 10 ⁶ cells/200 µL/well. Cells were stimulated with 2 µg/mL SARS-CoV-2 trimer (ACRObiosystems) for 24 h at 37°C, 5% CO ₂ . 2 µg/mL DMSO was used as negative control and PHA (eBiosciences) as positive control. After 24 h of stimulation, samples were collected in 1.5 mL microcentrifuge tubes, cells were collected by pipetting up and down, and centrifuged at 300 g for 10 min. Supernatants were collected and frozen for IFNγ treatment by ELISA (DuoSet, R&D Systems) and for SARS-CoV-2 wild-type surrogate virus neutralization testing using the cPASS kit (Genscript). A negative control of the samples was also used for IL-21 analysis using the IL-21 human ELISA kit (ThermoFisher) according to the manufacturer's instructions.

Results : SARS - CoV -2 EDV formulation and dual antigen presentation: cancer therapeutic EDV was co-packaged with cytotoxic payloads via scFv bispecific antibodies specific for EDV lipopolysaccharide and cancer cell receptors (e.g. EGFR) Targets cancer cells (MacDiarmid et al., 2007). In this example , EDV-COVID-αGC, a double-packaged nanocell carrying the SARS-CoV-2 spike protein and the glycolipid adjuvant α-galactosylceramide, was generated ( FIG . 11A ). pLac-CoV2 bacterial recombinant plasmids expressing the SARS-CoV-2 S-protein under the modified β-lactamase promoter ( Fig . 11B ) were transformed into EDV-producing S. typhimurium and purified EDV- COVID Nanocells were shown to contain two subunits of the S-protein by Western blotting using polyclonal antibodies against S1 and monoclonal antibodies against the S2 subunit ( Fig. 5C ) . EDV plastid extraction and quantification gave a pLac-CoV2/EDV plastid copy number of about 100 copies, while protein quantification showed about 16ng spinin/10 ⁹ EDV.

Purified EDV-COVID was loaded on αGC to generate EDV-COVID-αGC and LC-MS/MS measurement of lipid extracted EDV-COVID-αGC showed about 30 ng αGC/10 ⁹ EDV. Flow cytometric analysis of murine JAWS II cells treated with EDV-COVID-αGC and stained with anti-CDld:αGC revealed uptake of αGC and CDld-mediated surface presentation ( FIG . 5B ). Furthermore, co-staining of JAWS II cells with anti-Spine S1 and anti-CDld:αGC confirmed the presentation of both S-protein and αGC on the surface of DCs after co-incubation with EDV-COVID-αGC ( FIG . 11C ).

Early cytokine responses in mice treated with EDV-COVID-αGC: BALB / c mice intramuscularly (im) inoculated with a single first dose of 2 × 10 ⁹ or 3 × 10 ⁹ EDV compared to controls -COVID-αGC caused 8 h serum samples to show elevated Th1 cell immune response cytokines. As shown in Figure 11 ( D - I ) , IFNα, IFNγ, IL-12p40, IL -2. TNFα and IL-6 increased to significantly higher levels in the EDV-COVID-αGC group, which shows the influence of αGC. IL-21, a Th2 cytokine factor critical for antiviral activity, was significantly elevated at 8 h in mice treated with EDV-COVID-αGC ( FIG . 11 J ). IL-10 (also a Th2 cytokine) was relatively elevated in all groups ( Fig. 11K ) .

S- protein-specific antibodies in mice treated with EDV -COVID-αGC : analysis on day 28 using S-protein-specific ELISA im administered with 2 × 10 ⁹ or 3 × 10 ⁹ EDV and on day 21 Serum IgM and IgG antibody titers of equal boosted mice. Both dose levels of EDV-COVID and EDV-COVID-αGC gave elevated IgM ( Fig. 12 A ) and IgG ( Fig . 12 B ) S-protein specificity compared to saline, EDV and EDV-CONTROL groups Antibody titer. Compared with EDV-COVID, the antibody titer of EDV-COVID-αGC is higher. For both EDV-COVID and EDV-COVID-αGC, IgM ( FIG. 12C ) and IgG ( FIG. 12D ) levels showed an increase to comparable levels at day 7. Before the booster on the 21st day, the IgM titers of both EDV-COVID and EDV-COVID-αGC groups decreased ( Fig . 12E ), but IgG still increased, especially in the EDV-COVID-αGC group ( Fig. 12F ).

S - protein-specific B and T cell responses: In order to study B cell responses after immunization of mice at two levels of 2 × 10 ⁹ and 3 × 10 ⁹ , bone marrow-derived B cells were isolated from SARS-CoV-2 S-protein stimulation and measurement of S-specific IgM and IgG titers secreted by B cells. A dose of 2 × 10 ⁹ EDV-COVID-αGC resulted in significantly increased IgM and IgG content compared to all other groups dosed at 2 × 10 ⁹ ( p = 0.0081, p < 0.0001), and similarly, with 3×10 ⁹ EDV-COVID-αGC resulted in significantly increased IgM and IgG content compared to all other groups dosed at 3×10 ⁹ ( p <0.0001, p =0.0175) ( FIG . 13 A and B ).

When the activation marker CD69 was analyzed by flow cytometry as a percentage of CD3/CD69 within the isolated splenic CD8 ⁺ T cell population, a dose of 2 × 10 ⁹ EDV-COVID-αGC was significantly more effective in utilizing S-protein compared to DMSO stimulation. Stimulation resulted in a higher T cell response ( p = 0.0159) ( Fig. 13C ) . This higher % CD3/CD69 ratio was also observed at a dose of 3×10 ⁹ ( p =0.0185) ( FIG . 13D ). In Figure 13E , the Th1/ Th2 phenotype analysis of isolated splenocytes stimulated by S-protein showed that CD4+ T cells from EDV-COVID and EDV-COVID-αGC mice were significantly higher in IFNγ was produced within 24 h, but IL-4 was not produced.

Multi-strain neutralization of EDV-COVID-αGC measured using the Surrogate Virus Neutralization Test ( sVNT ) : The FDA-approved cPASS™ sVNT kit was used to assess 2 × 10 ⁹ and 3 × 10 ⁹ (for wild-type Wuhan strain) and for α, β, γ and δ strains 3 × 10 ⁹ levels of neutralizing antibodies in mouse serum on day 28. According to the instructions of the kit, the samples were considered to be neutralized as positive at the 30% inhibition level (Jung et al., 2021; Tan et al., 2020). According to these guidelines, at two doses of 2 × 10 ⁹ and 3 × 10 ⁹ , 100% of mice treated with EDV-COVID-αGC neutralized the RBD of wild-type SARS-CoV-2, while the corresponding dose of EDV- COVID showed RBD neutralization in 50% and 75%, respectively ( Fig. 13 F and G ). 100% of mice treated with 3×10 ⁹ EDV-COVID-αGC neutralized the respective RBDs from the α strain, 80% for β and 90% for γ and δ ( FIG. 13H - K ) .

Clinical results : Healthy volunteers from the Phase I study cohort who received 9x10 ⁹ EDV-COVID-αGC showed strong neutralizing activity (>PRNT ₉₀ equivalent). For comparison, the neutralization results also included the o variant of 5 volunteers who received at least 2 doses of Pfizer vaccine ( FIG. 14A ). Serum IFNγ ( FIG. 14B ) and IFNα ( FIG. 14C ) levels of these volunteers persisted. Total PBMC analysis showed an increase in CD4 ⁺ ( FIG. 14D ) and CD8 ⁺ ( FIG. 14E ) circulating memory B cells in vaccinated volunteers from day 1 to day 28. Ex vivo PBMCs isolated on day 28 produced elevated levels of IFNγ in response to SARS-CoV2 spike protein stimulation ( FIG. 14F ). Similarly, CD69 ⁺ T cells in PBMCs increased after echinin stimulation ( FIG. 14G ). Two months after the initial injection, the percentage of spinin-specific B cells increased in PBMCs ( Figure 14H ), and a similar trend was observed in spinin-specific memory B cells ( Figure 14I ). B cell class switching was observed 2 months after the initial injection, as evidenced by a general trend towards decreased numbers of IgM ⁺ B cells ( Figure 14J ) and increased numbers of IgG ⁺ B cells ( Figure 14K ).

While certain embodiments have been illustrated and described, it should be understood that changes and modifications can be made thereto according to ordinary skill in the art without departing from the technology in broader aspects as defined in the appended claims .

The embodiments illustratively set forth herein may be suitably practiced in the absence of any element or elements, limitation or limitations, not specifically disclosed herein. Thus, for example, the terms "comprising", "including", "containing", etc. should be read broadly and without limitation. Furthermore, the terms and expressions employed herein are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions to exclude any equivalents or parts thereof of the features shown and described, but It should be recognized that various modifications may be made within the scope of the claimed technology. Additionally, the phrase "consisting essentially of" will be understood to include both those specifically listed elements as well as additional elements which do not materially affect the basic and novel characteristics of the claimed technology. The phrase "consisting of" excludes any unspecified element.

The disclosure is not limited to the particular embodiments set forth in this application. Many modifications and changes can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. Functionally equivalent methods and compositions within the scope of the disclosure, in addition to those enumerated herein, will be apparent to those skilled in the art from the foregoing descriptions. Such modifications and variations are intended to be within the scope of the appended patent applications. The disclosure should be limited only by the terms of the accompanying claims and the full scope of equivalents to which such claims are entitled. It is to be understood that this disclosure is not limited to particular methods, reagents, compounds or compositions, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

Furthermore, when features or aspects of the invention are described in terms of the Markush group, those skilled in the art will recognize that the disclosure is also thereby described in terms of any individual member or subgroup of members of the Markush group .

As will be understood by those skilled in the art, for any and all purposes, particularly in terms of providing a written description, all ranges disclosed herein also encompass any and all possible subranges and combinations of subranges thereof, including endpoints. Any listed range is readily identifiable as adequately describing and enabling the same range to be divided into at least equal halves, thirds, quarters, fifths, tenths, etc. As a non-limiting example, each range discussed herein can be easily divided into a lower third, a middle third, an upper third, etc. Those skilled in the art will also appreciate that all language such as "highest," "at least," "greater than," "less than," and the like include the recited numbers and refer to ranges that may then be divided into the subcategories discussed above. scope. Finally, as those skilled in the art will understand, the scope includes each individual member.

All publications, patent applications, issued patents, and other documents mentioned in this specification are hereby incorporated by reference as if each individual publication, patent application, issued patent, or other document were specifically and are individually indicated to be incorporated herein by reference in their entirety. Definitions incorporated by reference contained in the text are excluded to the extent they contradict definitions in this disclosure.

Other embodiments are set forth in the following claims. references

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Figure 1A depicts a scanning electron microscope image showing the generation of EnGeneIC Dream Vector (EDV™) nanocells ( i.e., intact, pocket-sized cells of bacterial origin) from a safe strain of the bacterium Salmonella typhimurium , and FIG. 1 B shows a transmission electron micrograph image showing the structure of an empty EDV bacterial nanocell with a diameter of about 400 nm.

Figure 2A is a graphical representation of an EDV-COVID-19 vaccine composition comprising a bacterial expression plasmid ("EDV") , such as that shown in Figure 1B, where EDV first expresses the expression of SARS-CoV-2 in the EDV cytoplasm Spinin and additionally carried or loaded with CD1d-restricted iNKT cell antigen glycolipid α-galactosylceramide (α-GalCer) IFN-γ as an adjuvant or stimulator. The expressed spinins encoded by SARS-CoV-2 are designated by the stars on Figure 2A. Figure 2B shows an exemplary vial containing a lyophilized EDV- COVID -19 vaccine composition.

Figure 3 is a graphical representation of an EDV-COVID-19 vaccine composition comprising a bacterial expression plasmid ("EDV") (such as that shown in Figure 1B), wherein the EDV contains (i) a plasmid expressed from the original SARS- Selected spike proteins of CoV-2 and multiple genetic variants (e.g., delta variant and Brazilian variant), (ii) a gene expression promoter that expresses all proteins as a single mRNA and individual proteins in the EDV cytoplasm, (iii) Multiple spinins including spinin produced by SARS-CoV-2, Brazilian variant spinin and delta variant spinin, and (iv) CD1d-restricted iNKT cell antigens as adjuvants or stimulators Glycolipid α-galactosylceramide (α-GalCer) IFN-γ. The encoded expressed spinins are designated by the stars on FIG. 3 .

Figure 4 A - C shows the administration of bacterial pocket cells containing α-galactosylceramide (α-GalCer) to three pancreatic cancer patients (CB03, CB17 and CB41) over a period of 39 days or over a period of 46 days Results of administration to 4 pancreatic cancer patients (CB11, CB14, CB18 and CB41). Measurements of serum IFN-α (pg/mL) ( FIG . 4A ) and serum IFN- γ ( FIG. 4B ) are shown on the Y-axis of the graphs plotted in FIGS . 4A and 4B . The data showed that EDV-αGC elicited a Th1 response and increased lymphocyte content in pancreatic cancer patients. Figure 4A shows that serum IFNα levels from all 3 patients continued to increase after 2 doses of EDV-αGC, and Figure 4B shows serum IFNγ levels from all 3 patients after 2 doses (one week apart) of EDV - αGC Continued to increase. IFN levels were measured by ELISA from patient serum samples obtained throughout the treatment period. Figure 4C shows the results of measuring lymphocyte counts ( ^x109 /L) in four pancreatic cancer patients (CB11, CB14, CB18 and CB41) over a 46-day period after 2 doses (one week apart) of EDV-αGC. The results depicted in Figure 4C show that lymphocyte counts were elevated within the normal range (1.0-4.0) in four pancreatic cancer patients. Lymphocyte levels were measured from patient blood samples throughout the treatment period by the pathology service.

Figures 5A - H show the construct design of the EDV- SARS -CoV - 2 vaccine ( Figure 5A ). The expression cassette is generated by placing the coding nucleotide sequence of the SARS-Cov-2 (Covid-19) spike protein (Genebank MN908947.3) at the 3'-end of the modified β-lactamase promoter, which The promoter has previously been used for expression in S. typhimurium strains (Su et al., Infection and Immunity , 60 (8):3345-3359 (1992)). The expression cassette was then inserted between the Kpn 5' and Sal I 3' sites of the M13 multiple selection site of the PUC57-Kan backbone plastid to generate P-Blac-Cov. Figure 5B shows the results of a mouse experiment in which four groups of mice were evaluated (Group 1 = untreated; Group 2 = EDV without payload; Group 3 = administered free αGC; and Group 4 = administered Bacterial pocket cell vaccine comprising a combination of SARS-CoV-2 spike protein and αGC (depicted in Figure 2A)). The data presented in Figure 5B using FACS analysis demonstrated that EDV ^™ -COVID-α-GC was able to efficiently deliver α-GC to murine bone marrow-derived JAWII cells with CD1d-ligand in a manner similar to free α-GC . Efficiency presents. Figure 5 D -5 H shows the results of intramuscular (IM) injection of 2 × 10 ⁹ EDV-COVID-α-GC into five groups of BALB/c mice, in which the concentration of IFNα (pg/mL) ( Figure 5 D ), IFNγ concentration (pg/mL) ( Fig. 5 E ), IL12p40 concentration (pg/mL) ( Fig. 5 F ), IL6 concentration (pg/mL) ( Fig. 5 G ) and TNFα concentration (pg/mL) ( Fig. 5H ) is shown on the Y - axis of the graph. The results showed that administration resulted in a strong type I interferon response within 8 h after injection. Five groups of mice ( n = 6/group; ELISA samples run in triplicate) were: Group 1 = saline; Group 2 = EDV (bacterial pocket cells without payload); Group 3 = EDV _control (EDV carrying a plastid without an insert expressing the spinin (i.e., only the plastid backbone)); Group 4 = EDV _Covid (bacterial pocket cells comprising plastids and encoded SARS-CoV-2 spinin) and Group 5 = EDV _{Covid + α GC} (the construct shown in Figure 2A ).

Figure 6 A ^- 6 F shows that ^Balb /c mice ( n = 8 responses in each group). 28 days after the initial dose (with booster administration on day 21), high levels of anti-S protein IgM ( FIG. 6 A ) and IgG ( Figure 6 B ) Antibody titers. Figure 6C shows the results after B cells were isolated from mouse bone marrow and co - cultured with SARS-CoV-2 S protein ex vivo 28 days after the initial injection. It was found that B cells isolated from EDV-COVID-α-GC immunized mice produced significantly higher amounts of S protein-specific IgG in response to the presence of S protein compared to all other groups tested. Figure 6D shows the results of neutralizing antibody assays showing that 100% of the sera from mice immunized with EDV-COVID-α-GC resulted in inhibition of the binding of SARS-CoV-2 RBD to the hACE2 receptor. Inhibition of RBD binding to the hACE2 receptor was assessed using the cPASS ^™ SARS-CoV-2 Neutralizing Antibody Assay (FDA approved; Tan et al., Nature Biotech , 2020) for detection in various species. Figure 6E shows the results of FACS analysis of CD8+ cytotoxic T cells in mouse splenocytes, which show that mice immunized with EDV _COVID-α-GC had the highest expression at 4 weeks after the initial injection (1 boost on day 21). Quantities of antigen-specific memory CD137+CD69+ cytotoxic T cells, eg, in EDV _Covid-αGC treated mice there was a significantly higher number of CD137+CD69+ populations within the cytotoxic T cell population compared to all other treatment groups. CD137+ signaling is required for CD8+ T cell antiviral responses.

Figure 6F shows the results of ex vivo AIMS analysis showing spine antigen - specific CD8+ T cell responses. The number of CD69+ CD8+ T cells increased after stimulation with Covid-spike protein in the EDV-Covid and EDV-Covid-aGC groups, but not in any other group. PHA was used as a positive control. These results indicate that both the plastid and the protein contained within EDV give rise to S protein-specific responses.

Figure 7A - 7D is shown by analyzing the specificity and cross - reactivity of RBD and S1 subunits of UK (B.1.1.7) and South Africa (B.1.351) mutant strains of the virus from serum IgG from immunized mice Robustness of immunity generated by EDV-COVID-α-GC. The results showed that although UK variant strain RBD-specific IgG was produced in some EDV-COVID-α-GC immunized mice ( Figure 7A ), higher S1-specific IgG antibody titers were observed ( Figure 7B ), This indicates that the binding of the S protein-specific antibody is mainly outside the RBD. A similar trend was observed for the SA variant strains ( Fig. 7 C and D ).

Figure 8 shows the genome of the SARS-CoV-2 virus, identifying transcription sites and protein coding domains. www.viralzone.espasy.ort/resources/nCoV_genome_bis.png.

Figure 9 shows a representative phylogenetic tree of SARS-CoV-2 virus and known variants. Whole-genome SARS-CoV-2 sequence was downloaded from GISAID (https://www.gisaid .org/) on January 19, 2021, using MAFFT: https://mafft.cbrc.jp/alignment/software/ Aligned and manually edited using BioEdit v7.2.5. The phylogenetic tree construction system was implemented using FastTree v2.1.11 with Shimodaira-Hasegawa class local clade support and displayed using FigTree v1.4.4. Excerpted from Toovey et al., J. Infect . , 82 (5):e23-324 (3 February 2021) .

Figure 10 A - D shows the results of IgG titers after administration to five different groups of mice ( n =6/group; ELISA samples were run in triplicate): Group 1 = saline; Group 2 = EDV (without Payloaded Bacterial Pocket Cells); Group 3 = EDV _control (EDV carrying plastids (i.e., plastid backbone only) without inserts expressing spinin); Group 4 = EDV _Covid (comprising plastids and Bacterial pocket cells encoding the SARS-CoV-2 spike protein) and group 5 = EDV _{Covid + α GC} (construct shown in Figure 2A ). The mice were given 3 x 10 ⁹ EDV. The results shown in Figure 10 A - D detailing the S1 subunit-specific IgG titers at day 28 after split dose IM demonstrate that serum IgG titers obtained from mice treated with EDV-COVID-GC were correlated with all Spike proteins of four mutant strains strongly bound: (1) SARS-CoV-2 variant α (B.1.1.7.UK) ( Fig. 10 A ); (2) SARS-CoV-2 variant β (B. 1.351. SA) ( Fig. 10 B ); (3) SARS-CoV-2 variant δ (B.1.617.2 India); and (4) SARS-CoV-2 variant γ (P.1 Brazil).

Figure 11 A - K depicts SARS-CoV-2 S-protein construct design, antigen processing and presentation to DC cells and ability to elicit Th1 and Th2 responses. Figure 11 A shows an image of EDV-COVID-αGC depicting LPS, membrane and nanocellular contents including plac-CoV-2 plastids, S-protein and αGC. Figure 11 B shows the construct: SARS-CoV-2 spike protein nucleotide sequence (Genbank MN908947.3), which is located at the 3'-end of the modified constitutively expressed β-lactamase promoter and inserted into the PUC57- Between the KpnI 5' and SalI 3' sites of the M13 multi-selection site of the Kan backbone plastid to generate plac-CoV2. Figure 11E shows co-staining of JAWSII cells with anti-CDld:αGC and anti-Spin Ab, which demonstrates delivery of αGC and S - protein by EDV, where EDV-COVID-αGC delivers both S-protein and αGC to the same cells On the surface. Figure 11D-F : IM injection of 2 and 3 (x 10 ⁹ ) EDV-COVID-αGC in BALB/c mice resulted in increased IFNα, IFNγ, IL-12-p40 levels 8 h after the first dose: Figure 11G : IL-2 content in 2 × 10 ⁹ and 3 × 10 ⁹ EDV-CoV2-αGC particle doses 8 h after the first dose. Figures 11H and 11I : TNFα and IL-6 levels in 2 and 3 (x ¹⁰⁹ ) EDV-CoV2-αGC particle doses 8 h after the 1st dose. FIG. 11 J : IL-21 content of 3×10 ⁹ doses measured on day 28. Fig. 11 K : IL-10 content in 3 × 10 ⁹ EDV-CoV2-αGC particle doses 8 h after the first dose.

Figure 12 A - G . S - Specific IgM and IgG titers. ( FIG. 12A ) Day 28 IgM S - protein specific titers for 2×10 ⁹ and 3×10 ⁹ dose levels. ( FIG. 12B ) Day 28 IgG S-protein specific titers for 2×10 ⁹ and 3×10 ⁹ dose levels. ( FIG. 12C-F ) IgM and IgG S-protein specific titers at 3×10 ⁹ doses at day 7 ( FIG. 12C , D ) and at day 21 ( FIG. 12E , F ). ( FIG. 12G) Schematic diagram of dendritic cell activation permitted by iNKT; (1) EDV-COVID-αGC im injected into mice, (2) phagocytized by dendritic cells (DC), degraded in lysosomes, ( 3.1) αGC is released from EDV, (3.2) CD1d binds to αGC, (3.3) CD1d:αGC complex is displayed on the surface of DC cells, (4.1) spine polypeptide is also released from EDV, (4.2) MHC class II binds to S- Peptides, (4.3) These were displayed on the surface of the same DC cells. (5) binding of the iNKT semi-invariant T cell receptor to the CD1d/αGC complex, (6) rapid secretion of IFNγ, which triggers the upregulation of CD40 ligands in DCs, which, via CD80, CD86, CCR7, MHC class I molecules, Upregulation of the inflammatory cytokine IL-12 and the chemokine CCL17 induces DC maturation/activation and increased co-stimulatory capacity. (7) Binding of the CDld:αGC complex to the iNKT TCR triggers the release of perforin, which kills the CDld/αGC complex of displaying DCs. (8) S-polypeptide is released from dying DC, (9) is endocytosed by activated CD11c+ DC and (10) naive B cells via B cell surface receptors, and (11, 12) is displayed on the surface of each cell via MHC class II superior. (13) MHC class II/spine binding on the DC surface to CD4+ TCRβ on CD4+ T follicular helper ( _TFH ) cells, and (14.1) these signals induce _TFH cell differentiation and the chemokine receptor CXCR5 Up-regulation and down-regulation of CCR7, which allows the cells to migrate to the T/B border. (14.2) B cells activated by BCR S-peptide engagement increase CCR7 expression and migrate to the T/B follicle border to seek cognate CD4+ T cells. (15) TCRβ recognizes the S-peptide/MHC II complex on B cells so that TFH cells (16) can express CD40 ligand and ICOS, and (17) secrete cytokines IL-21, IFNγ, IL-4 , IL-2 and IL-10. _TFH cells were strongly enriched for cells expressing the highest levels of IL-21. (18) This homology helps stimulate B cells to undergo robust proliferation, induce Ig class switching, and differentiate into plasma-like cells capable of secreting all major Ig isotypes. (19) Within GC, B cells undergo somatic hypermutation and only B cells with high affinity antibodies are selected. (20) These plasma cells secrete high-affinity S-specific antibodies that neutralize various S-mutants. (21) These B cells differentiate into long-lived memory B cells. Throughout this process, IL-21 induces the expression of CD25, which enables B cells to respond to IL-2, also derived from _TFH cells, which promotes the effects of IL-21. Similarly, IL-21 induces the expression of IL-6R on PCs, which allows these cells to integrate survival signals through IL-6. (22) DCs displaying S-peptide via MHC class II also elicited S-specific CD8+ T cell responses.

Figure 13A-K . Ex vivo AIM analysis of murine bone marrow-derived B cells and splenocytes and surrogate virus neutralization test (sVNT) of mouse sera. IgM ( FIG. ^13A ) and ^IgG ( Figure 13 B ) S-protein specific titers. #: Significant difference compared with all 2 × 10 ⁹ injected mice; *: Significant difference compared with all 3 × 10 ⁹ groups. After stimulating isolated splenocytes with SARS-CoV-2 S-protein, S in CD8 ⁺ cytotoxic T cell populations in 2 × 10 ⁹ ( Figure 13C ) and 3 × 10 ⁹ ( Figure 13D ) EDV-immunized mice Specific CD69 expression. *: Significant difference compared to DMSO (-ve) stimulated control. Data are presented as mean ± SEM. Expression of IFNγ (Th1) and IL-4 (Th2) in CD3 ⁺ CD4 ⁺ T cell populations in isolated splenocytes stimulated by SARS-CoV-2 S-protein ( FIG. 13E ). ( FIG. 13 F-K ) The virus neutralization test (VNT) using the cPASS ^™ SARS-CoV-2 neutralizing antibody assay (FDA-approved) detected in various species was used to evaluate the inhibition of RBD binding to the hACE2 receptor. ( FIG. 13F ) Sera from mice immunized with 2×10 ⁹ and ( FIG. 13G ) 3×10 ⁹ EDV were directed against SARS - CoV-2 RBD Wuhan wild-type VNT. Subsequent VNT was carried out against α ( Fig. 13 H ), β ( Fig . 13 I ), γ ( Fig . 13 J ) and δ ( Fig . 13 K ) mutant RBD using serum from 3 × 10 ⁹ EDV-immunized mice.

Figures 14A-K . Data from the first 4 EDV-COVID clinical trial volunteers 28 days after initial injection. Data are from cohort 2 of a phase 1 clinical trial . ( FIG. 14A) SVNT analysis of volunteer sera against wild-type (WT), delta, o and o BA2 variants of SARS-CoV2 RBD at days 1, 21, 28 and 3 months after initial injection. Results from 5 volunteers who received at least 2 doses of Pfizer vaccine were used for comparison. ( FIG. 14B) Serum IFNy levels on days 1, 21 and 28 after initial injection. ( FIG. 14C) Serum IFN[alpha] levels on days 1, 21 and 28 after the initial injection. ( FIG. 14D ) Analysis of CD4 ⁺ central memory T cells (CD45RA ⁻ CD27 ⁺ CCR7 ⁺ CD3 ⁺ CD4 ⁺ ) at days 1 and 28. ( FIG. 14E ) Analysis of CD8 ⁺ central memory T cells (CD45RA ⁻ CD27 ⁺ CCR7 ⁺ CD3 ⁺ CD8 ⁺ ) at days 1 and 28. ( FIG. 14F) Ex vivo PBMC production of IFNγ following SARS-CoV2 spiken stimulation at days 1 and 28. ( FIG. 14G ) CD69 expression in T cells (CD45 + ^{CD3 +} CD69 ⁺ ) in ex vivo PBMCs after SARS-CoV2 spinin stimulation at days 1 and 28. ( FIG. 14H ) Amount of spinin-specific CD19 ⁺ B cells in PBMCs at 1, 28 days, 2 months and 3 months after initial injection. ( FIG. 14I ) Amount of spinin - specific CD19 ⁺ CD27 ⁺ memory B cells in PBMCs at 1, 28 days, 2 months and 3 months after initial injection. ( FIG. 14J ) Amounts of IgM ⁺ CD19 ⁺ CD27 + memory B cells in PBMCs at 1, 28 days, 2 months ^and 3 months after initial injection. ( FIG. 14K ) Amount of IgG ⁺ CD19 ⁺ CD27 + memory B cells in PBMCs at 1, 28 days, 2 months ^and 3 months after initial injection. Data are presented as mean ± SEM.

Claims (23)

A composition comprising: (a) a vector comprising a plasmid encoding at least one viral antigen derived from a variant of SARS-CoV-2; and (b) a vector comprising an antigen recognized by CD1d; and (c) at least one pharmaceutically acceptable carrier, Wherein at least one of the vector (a) and the vector (b) is an intact, bacterial-derived pocket cell or a killed bacterial cell. The composition of claim 1, wherein the mutant strain of SARS-CoV-2 is selected from the group consisting of: (a) UK variant of SARS-CoV-2 (B.1.1.7/VOC-202012/01); (b) B.1.1.7 mutant strain with E484K; (c) B.1.617.2 (δ) mutant strain; (d) B.1.617 mutant strain; (e) B.1.617.1 (κ) mutant strain; (f) B.1.617.3 mutant strain; (g) South African B.1.351 (β) mutant strain; (h) P.1 (γ) mutant strain; (i) B.1.525 (η) mutant strain; (j) B.1.526 (ι) mutant strains; (k) λ (lineage C.37) mutant strain; (l) ε (pedigree B.1.429) mutant strain; (m) ε (lineage B.1.427) mutant strain; (n) ε (lineage CAL.20C) mutant strain; (o) ζ (lineage P.2) mutant strains; (p) θ (lineage P.3) mutant strains; (q) R.1 mutant strain; (r) Lineage B.1.1.207 variants; and (s) Pedigree B.1.620 mutant strain. The composition of claim 1, wherein the SARS-CoV-2 variant strain is selected from the group consisting of: SARS-CoV-2 variant strain, which comprises: (a) L452R spinin substitution; (b) E484K spinin substitution; (c) K417N spinin substitution; (d) E484K spinin substitution; (e) N501Y spinin substitution; (f) K417T spinin substitution; (g) E484K spinin substitution; (h) N501Y spinin substitution; and (i) SARS-CoV-2 mutant strains, which have one or more of the following missense mutations: N440, L452R, S477G/N, E484Q, E484K, N501Y, D614G, P681H, P681R and A701V. As the composition of claim 1, wherein: (a) the vector (a) additionally comprises at least one viral antigen from a strain of SARS-CoV-2; and/or (b) The vector (a) additionally comprises at least one viral antigen from a SARS-CoV-2 strain, and wherein the SARS-CoV-2 strain is selected from the group consisting of: L strain, S strain, V strain, G strain , GR strain and GH strain; and/or (c) The vector (a) additionally comprises at least one viral antigen from a SARS-CoV-2 strain, wherein the viral antigen is encoded by a polynucleotide comprising a sequence of SARS-CoV-2, or is combined with a polynucleotide comprising a SARS-CoV- The polynucleotide encoding the sequence of 2 has at least 80% sequence identity. As the composition of claim 1, wherein the plasmid codes: (a) At least one of spine (S) protein, nucleic acid protein shell (N) protein, membrane (M) protein and envelope (E) protein of SARS-CoV-2 or SARS-CoV-2 variant strain; and /or (b) the spine (S) protein, nucleic acid protein shell (N) protein, membrane (M) protein and the envelope (E) protein; and/or (c) The spine (S) protein of SARS-CoV-2 or SARS-CoV-2 mutant strain; and/or (d) The receptor binding domain (RBD) of the spike protein of SARS-CoV-2 or SARS-CoV-2 variants. As the composition of claim 1, wherein: (a) the vector (a) is a first intact, bacteria-derived pocket cell or killed bacterial cell, and the vector (b) is a second intact, bacteria-derived pocket cell or killed bacterial cell; and/ or (b) vector (a) and vector (b) are the same whole, bacterial-derived pocket cell or killed bacterial cell comprising the antigen recognized by the CD1d and a plasmid encoding at least one viral antigen; and/or (c) One of vector (a) and vector (b) is not intact, pocket cells of bacterial origin or killed bacterial cells and the other of vector (a) or vector (b) is intact , pocket cells of bacterial origin or killed bacterial cells. The composition as claimed in item 1, wherein the antigen recognized by the CD1d: (a) contain glycosphingolipids; and/or (b) selected from the group consisting of α-galactosylceramide (α-GalCer), the C-glycosidic form of α-galactosylceramide (α-C-GalCer), 12 of galactosylceramide Carbonyl form (β-GalCer), β-D-glucopyranosylceramide (β-GlcCer), l,2-diacyl-3-O-galactosyl-sn-glycerol (BbGL-II ), glycolipid containing diacylglycerol (Glc-DAG-s2), ganglioside (GD3), neurotriohexosylceramide (gangliotriaosylceramide) (Gg3Cer), glycoside phosphatidylinositol (GPI), α-glucuronylceramide (GSL-1 or GSL-4), isoglobotrihexosylceramide (iGb3), lipophosphoglycan (LPG), lysophosphatidylcholine ( LPC), α-galactosylceramide analogs (OCH), threitol ceramide, and derivatives of any of them; and/or (c) contains α-GalCer; and/or (d) Contains synthetic α-GalCer analogs; and/or (e) Comprising synthetic α-GalCer analogs selected from the group consisting of: 6′-deoxy-6′-acetamide α-GalCer (PBS57), naphthaleneurea α-GalCer (NU-α-GC), NC-α -GalCer, 4ClPhC-α-GalCer, PyrC-α-GalCer, α-carba-GalCer, carba-α-D-galactose α-GalCer analog (RCAI-56), 1-deoxy-neo-inositol α - GalCer analogs (RCAI-59), 1-O-methylated α-GalCer analogs (RCAI-92), and HS44 aminocyclitol ceramides; and/or (f) is an IFNγ agonist. A vaccine composition comprising at least one whole, bacterial-derived pocket cell or killed bacterial cell, and comprising within the pocket cell or cells: (a) A plastid encoding a spike protein from one or more of: SARS-CoV-2 variant alpha (B.1.1.7.UK), SARS-CoV-2 variant beta (B.1.351 .SA), SARS-CoV-2 variant delta (B.1.617.2 India) and/or SARS-CoV-2 variant gamma (P.1 Brazil); and (b) α-Galactosylceramide. The vaccine composition according to claim 8, wherein (a) and (b) are contained in a single pocket cell. As the vaccine composition of claim 8, wherein the plastid code is from each SARS-CoV-2 variant α (B.1.1.7.UK), SARS-CoV-2 variant β (B.1.351.SA), Spike proteins of SARS-CoV-2 variant δ (B.1.617.2 India) and SARS-CoV-2 variant γ (P.1 Brazil). The composition according to claim 1, wherein the composition is formulated for oral administration, injection, nasal administration, pulmonary administration or local administration. A composition according to any one of claims 1 to 11 for use in a method of treating viral infection and/or vaccination against viral infection. The composition used in claim 12, wherein the individual: (a) suffer from or are at risk of developing lymphopenia; and/or (b) considered to be at risk of serious illness and/or serious complications from viral infection; and/or (c) about 50 years of age or over, about 55 years of age or over, about 60 years of age or over, or about 65 years of age or over; and/or (d) have one or more pre-existing conditions selected from the group consisting of: diabetes, asthma, respiratory disorders, hypertension and heart disease; and/or (e) Immunocompromised; and/or (f) Immunocompromised due to AIDS, cancer, cancer treatment, hepatitis, autoimmune disease, receiving steroids, immunosenescence, or any combination thereof. The composition used as claimed in item 12, wherein: (a) increase chances of survival after exposure to coronavirus; and/or (b) Reduce the risk of transmission of the coronavirus. As the composition used in claim 14, wherein: (a) the chance of survival is increased by about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%, if any Measured by clinically recognized techniques; and/or (b) the reduction in the risk of transmission is about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%, if using Measured by any clinically recognized technique. The composition for use according to claim 12, wherein the administration is by any pharmaceutically acceptable method. The composition for use as claimed in claim 12, wherein the individual is exposed or expected to be exposed to an individual with coronavirus infectivity. The composition used in claim 17, wherein the individual with coronavirus infectivity has one or more symptoms selected from the group consisting of fever, cough, shortness of breath, diarrhea, sneezing, runny nose and sore throat. The composition for use according to claim 12, wherein the individual is a health care worker, 60 years of age or older, frequent traveler, military personnel, caregiver, or an individual with a pre-existing condition at increased risk of death from infection. As the composition used in claim 12, it (a) further comprising administering one or more antiviral drugs; and/or (b) further comprising administering one or more antiviral drugs, wherein the one or more antiviral drugs are selected from the group consisting of: chloroquine, darunavir, galidesivir, Interferon beta, lopinavir, ritonavir, remdesivir, and triazavirin. The composition for use according to claim 12, wherein the antigen recognized by CD1d induces a Th1 interleukin response in the individual, and optionally wherein the interleukin comprises IFNγ. As the composition used in claim 12, wherein: (a) a first pocket-sized cell line comprising an antigen recognized by CD1d and a second pocket-sized cell line comprising a plastid encoding at least one viral antigen are administered simultaneously to the individual; and/or (b) a first pocket-sized cell line comprising an antigen recognized by CD1d and a second pocket-sized cell line comprising a plastid encoding at least one viral antigen are sequentially administered to the individual; and/or (c) repeated administration to the individual of a first pocket cell line comprising the antigen recognized by CD1d and a second pocket cell line comprising the plastid encoding at least one viral antigen; and/or (d) a first pocket cell line comprising the antigen recognized by the CD1d and a second pocket cell line comprising the plastid encoding at least one viral antigen at least once a week, twice a week, three times a week or on Thursdays administered to the individual. A use of the composition according to any one of claims 1 to 11 for the manufacture of a medicament, wherein the medicament can be used for treating viral infection and/or vaccination against viral infection.

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