Patent Application
20220193225
ACE2 Angiotensin-converting enzyme-2 ADE Antibody-Dependent Enhancement APC Antigen-Presenting Cell ARDS Acute Respiratory Distress Syndrome BCG Bacillus-Calmette-Guerin BCR B-cell receptor BHK Baby hamster kidney BSL Biologic Safety Level C Capsid SARS-2/COVID-19 Novel Coronavirus-19 CTL Cytotoxic T Lymphocyte DC Dendritic Cell E Envelope EMEM Eagles Minimal Essential Media EV71 Enterovirus 71 GOI Gene of Interest GP Glycoprotein HA Hemagglutinin HAI Hemagglutinin Inhibition IFA Influenza A IgA Immunoglobin A IgG Immunoglobin G IgM Immunoglobin M IL Interleukin IN Intranasal IRES Internal Ribosome Entry Site M Matrix M1 Macrophage type 1 M2 Macrophage type 1 MCP-1 Macrophage Chemokine Protein-1 MERS Middle East Respiratory Syndrome mRNA messenger RNA NAB Neutralizing antibody NK Natural Killer NP Nucleocapsid Protein NTD N-Terminal Domain PRR Pattern Recognition Receptor RBD Receptor Binding Domain SARS Severe Acute Respiratory Syndrome S1 Spike 1 S2 Spike 2 TCID Tissue Culture Infectious Dose TLR Toll Like Receptor UTR Untranslated Region VEE Venezuelan Equine Encephalitis Virus VLP Virus-Like Particle Variant of Concern VOC VRP Viral Replicative Particle
This invention is in the field of preventative vaccines for viral diseases. Severe Acute Respiratory Syndrome Coronavirus (SARS-2/COVID-19), appeared on 12/12/19 in the city of Wuhan, China. SARS-2/COVID-19 represents a threat to public health and society of a magnitude similar to the Spanish Flu pandemic of 1918-1920 which killed an estimated 50 million people out of 500 million infections. There are several factors which contribute to the threat posed by SARS-2/COVID-19:
SARS-2 Phylogeny
SARS-2/COVID-19 belongs to family Coronaviridae, subfamily Betacoronaviridae. SARS-2/COVID-19 is a large, enveloped, positive-strand RNA virus of approx. 120 nm in diameter. Its 31.6 kb genome is large for RNA viruses, and codes for the major structural protein products Nucleoprotein (NP), Matrix (M), Spike (S), and Envelope Protein (E). There are also several non-structural genes coding for enzymes including RNA-dependent RNA polymerase. Unique among RNA viruses, Coronaviruses carry a gene for a proofreading enzyme which limits mutations. Many types of Coronaviruses are implicated in cases of the common cold. The structural locations and purpose of the described proteins are:
Phylogenetically, SARS-2/COVID-19 is related to several other coronaviruses, some of which cause human disease. SARS-1 appeared in China in 2004, and caused severe upper and lower respiratory syndrome, with a mortality of 10-12%. SARS-2/COVID-19 shares 79.5% sequence homology with SARS-1, and the discovery of the ten-fold stronger binding of SARS-2/COVID-19 to the main receptor Angiotensin-converting enzyme 2 (ACE2), compared with SARS-1, may explain its rapid transmissibility. Other coronaviruses similar to SARS-2/COVID-19 include Middle Eastern Respiratory Syndrome (MERS) virus, which apparently crossed over from camels to humans in 2012, and had a mortality of 34.2%. SARS-2/COVID-19 shares 96.2% sequence homology with the bat virus CovRaTG13, and the discovery of SARS-2/COVID-19 RNA at the Wuhan market where wild-caught animals, including bats, are sold for food, provides a plausible link for the virus spread.
There are no specific treatments approved for SARS-2/COVID-19, although various drugs approved for other viral and non-viral diseases are being tested. The efficacy record for RNA antivirals, unless targeting unique enzymes such as those expressed by HIV, is low compared to antibiotics. Undoubtedly, vigorous exploration in these areas will continue, but success is by no means assured.
The most efficacious means of reducing or eliminating the spread of viruses is through the use of prophylactic (preventative) vaccine. Vaccines have not been successful in many cases, including HIV, but have virtually eliminated such diseases as smallpox and polio. A successful vaccine provokes protective immune responses to viral antigens (proteins of immunological significance). While no vaccine has proven 100% safe and effective, the vaccines in use today prevent millions of illnesses and countless deaths from infectious diseases each year. These responses of the adaptive immune system include specific antibodies which recognize, and bind to short amino acid (peptide) sequences on the exposed portions of the virus. They are proteins consisting of both heavy and light amino acid chains. One end is termed Fab or antigen-binding, the other terminus is called Fc, which binds complement or the Fc receptor of macrophages or other effector cells. Antibodies are termed either neutralizing or non-neutralizing. Neutralizing antibody (NAB), can block virion-host cell receptor binding or fix complement for virion destruction. Antibodies are produced by mature B lymphocytes (plasma cells), after picking up antigen presented by Dendritic Cells through their B-cell receptor (BCR).
Emergence of Mutant Variants of Concern (VOC)
The SARS-2 Spike 1 contains a Receptor-Binding Domain (RBD), which binds to human ACE2 receptors. Mutant V.O.C. such as the Delta strain have been identified that combine antibody evasion with higher transmission capacity. These mutants are capable of causing breakthrough infections and illness even inf fully-vaccinated persons.
Antibody Types:
The “gold standard” of protection against challenge with Influenza A is to achieve a titer of specific IgG3 to the HA antigen of the virion of at least 1:40 for 50% protection. While IgG3 antibodies to SARS-2/COVID-19 will definitely play a role in vaccines efficacy, there are significant challenges to a successful vaccine using IgG3 against the Spike (S1 and S2), protein antigens.
The first obstacle is the evidence that specific IgG3 recognizing SARS-2/COVID-19 declines rapidly. A study found that of 59 patients who recovered from SARS-2/COVID-19, only 16.7% had high (>ID50 of 2000), levels of NAB after 60 days. A recent study of 243 health care workers in Japan given two shots of a mRNA vaccine found that levels of neutralizing antibody dropped to undetectable levels after six months. A vaccine which relies solely on NaB to spike proteins of SARS-2/COVID-19, like Influenza A, may fail to provide adequate protection.
Significant Factors in SARS-2/COVID-19 Vaccine Design
The second obstacle facing a vaccine design with a foundation based on NAB to SARS-2/COVID-19 Spike antigens (S-IgG), is safety. Antibodies, especially those in blood (serum IgG), can be protective or cause harm. Antibodies carry two receptors, the Fab end attaches to the antigen, and the Fc end fixes complement for destruction of the virus, or engulfment by a macrophage. Many patients who die from SARS-2/COVID-19 infection present with Acute Lung Injury (ALI). Immunopathology appears to play in important role, with elevated levels of IL-6 and IL-8 creating a cytokine storm, damaging delicate alveoli and impairing oxygen uptake.
In 1967, researchers were surprised to find that 80% of children vaccinated with Respiratory Syncytial Virus (RSV), were hospitalized compared with 5% of unvaccinated children. A review of human patients with SARS-2/COVID-19 revealed that recovering patients developed NAB after 20 days, but patients who died developed them sooner at 14.7 days. This points to an aggravating effect of antibodies in ARDS cases. Evidence points to a damaging effect of serum IgG antibodies against S1/S2.
A Chinese macaque model of SARS-1 showed evidence that the presence of S-IgG from a vaccine increased the likelihood of ALI and lack of protection. Macrophages can exist as either M1 activated or M2 regulatory. The S-Ig attached to the virus attracted M1 macrophages through the Fc receptor. This is known as antibody-dependent enhancement (ADE), and is well-characterized in the viral infection caused by dengue.
This ADE induced high levels of the inflammatory MCP-1 chemokine and cytokines, including TNFα, IL-6, and IL-8. This activation abrogated the macrophages M2 wound-healing properties, so damage accumulated in the pulmonary spaces without the capacity for repair. This could explain the 20-25% of patients who recover from SARS-2/COVID-19 showing evidence of “ground glass” opacities seen on CT images.
In summary, it appears that the major mechanism conferring protection against Influenza A, serum IgG, could have both a protective and a detrimental role in SARS-2/COVID-19. A vaccine designed only to raise serum IgG titers against S1/S2 would likely fail to confer full protection, or even aggravate disease. An alternative to using full-length S1/S2 in a vaccine design is to use only the receptor-binding domain (RBD), of S1. Studies have shown that NAB against the RBD are very effective and do not carry the risk of ADE. For the intranasal component, there is value to using the full-length S1/S2 construct as mucosal IgA is not implicated in ADE. Clearly, a novel vaccine design is required to deal with this unusual phenomenon.
Another significant obstacle to a SARS-2/COVID-19 vaccine is the presence of >60 polysaccharide chains attached to the S1 complex. These can hinder antibody binding, and the high degree of glycosylation (although lower than SARS-2/COVID), contributes to the failure of vaccines against HIV.
An ideal vaccine design for SARS-2/COVID-19 would include:
Current SARS-2/COVID Vaccine Technologies
Currently, there are at least six vaccines for SARS-2/COVID-19 in clinical trials, and many more in pre-clinical development. A live, attenuated version of SARS-2/COVID-19 would most likely have a high-risk potential for causing disease, and killed vaccines are often sub-optimal inducers of protective CTL responses. Given these factors, the six designs represent emergent technologies in the field of vaccine design. These can be classified as:
Each of these systems have strengths and weaknesses. Proper vaccine design involves an intensive review and analysis of the known properties of the pathogen and the correlates of immunity (Neutralizing antibody titers, CTL type and numbers, etc.). After this exercise, a review of the available new vaccine and adjuvant technologies can be performed, and promising candidates for testing identified.
Vector Design Strengths:
Vector Design Drawbacks:
mRNA Vaccine Strengths:
mRNA Vaccine Drawbacks:
Sub-Unit Vaccine Strengths:
Sub-Unit Vaccine Drawbacks:
Cytotoxic T Lymphocytes in Vaccine Design
T lymphocytes originate from c-kit+ Sca1+ hematopoietic stem cells (HSC) in the bone marrow. The cells migrate to the thymus where they undergo further selection into CD4+ and CD8+ cells. CD4+ cells are termed helper-inducer T cells, though they also have regulatory and cytolytic functions. CD4+ T cells recognize longer peptides of 12-24 amino acids presented by Class II MHC complexes on dendritic cells.
These cells act as critical accessory cells between Dendritic Cells, which process and present antigen, and effector cells CD8+ cytotoxic T cells (CTL) and B cells producing antibodies. This occurs mainly through contact-mediated activation in lymph tissue, and the secretion of cytokines: IL-2, IL-4, IL-7, IL-10, IL-12, IL-17, IL-17, IL-21, etc. When a CD4+ T cell received both antigen and activation signals, it develops cytolytic capacity and secretes IL-2, IL-7, IL-12, IL-15, and IL-21. When a CD4+ cell receives an antigenic signal in the absence of activation, it develops regulatory capacity, down-grading immune responses through secretion of IL-4 and IL-10. In this manner immune responses are tightly regulated.
CD8+ T cells (CTL), are sometime suppressor/regulatory, but most often assume a cytolytic function, recognizing short, 8-12 amino acid chains presented by Class I MHC present on most somatic cells. When a viral peptide is presented, the cytolytic machinery composed of perforin and Granzyme B destroys the target cell by perforation of the membrane and induction of apoptosis.
Together, CD4+ and CD8+ cells can provide protective immunity from viruses, either in the context of recovery from disease or through vaccination. The best-know model for CTL importance in vaccination is Influenza A. Studies show the importance of CD4+ cells as they recruit early innate system effectors into the lung to blunt virus infection, and promote strong, effective protection mediated by B and CD8+ T cells. These cells, which predominantly recognize peptides from the conserved NP and M proteins, reside in the memory pool after vaccination or infection. The recall response is noted for its strength and speed compared with primary challenge.
Models appear to prove the importance of CD4+ And CD8+ cells. A single immunodominant peptide (S525) from the SARS spike protein, protected 80% of C57/BL6 from SARS viral challenge. Humans with higher precursor frequency of CTL recognizing internal Flu virus proteins have greater protection than those making responses against the HA spike protein, which is not heavily conserved and subject to high mutation rates. Although internal proteins can mutate, these mutations are often fatal as they impair assembly of the core and adjacent proteins.
Types of Vaccines Currently Licensed for Use
The original vaccine was Vaccinia or cowpox virus. Dr. William Jenner made the astute observation that milkmaids were often immune to smallpox. Inoculation of non-pathogenic cowpox virus into humans raised Ab and CTL responses that were cross-protective against the more lethal smallpox.
The Types of Vaccines Currently in Use are Summarized Below:
A live, attenuated vaccine against SARS-2/COVID-19 appears unlikely to be approved for testing due to safety concerns. A killed vaccine may have a higher margin of safety, but the lack of CTL protection is an obstacle, and the rapidly declining IgG titers to the virus make this approach problematic. Sub-unit vaccines may offer a degree of protection with a high margin of safety, but will require combination with an approach designed to induce strong CTL responses to have a chance at >50% protection of subjects. DNA and mRNA designs hold promise, but may provoke auto-immune responses. Vector designs are safer than live or killed vaccines, but pre-existing antibodies to the vector and the inefficiency of GOI translation can reduce the desired immune effect.
In summary, it appears that current vaccine technologies in approved use have serious deficiencies when applied to SARS-2/COVID-19. A novel and improved approach is needed, and is described herein.
The invention is comprised of two components, a viral replicative vector carrying a SARS-2 transgene administered by hypodermic syringe, and a mixture of SARS-2 variant spike proteins in a surfactant adjuvant administered by intranasal inhalation. The invention is designed to provide a safe method of inducing powerful blood antibody (IgG), mucosal antibody (IgA), and T cell responses against mutant strains of SARS-2. Currently, mRNA and vector vaccine designs can provide IgG protection which is diminished after several months, limited T cell responses, and poor IgA responses.
Vaccine Component #1
VEE VRP Expressing SARS-2/COVID-19 Spike 1 RBD (Prime)
Construction of VRP Consisting of VEE3000/3526 with SARS-2/COVID-19 RBD Gene Insertions.
A new form of vaccine offering the protection of a live attenuated vaccine and the safety of a killed vaccine is the recombinant viral vector design. Recombinant viral vectors use genetic engineering to insert foreign transgenes into the vector genome. The transgenes are then produced by the host cell as viral proteins capable of inducing an immune response. Alphaviruses are small, enveloped RNA viruses of family Togaviridae, subfamily Alphaviridae. Examples include Sindbis, Venezuelan Equine Encephalitis (VEE), and Semliki Forest Virus. Of these, attenuated strains of VEE transformed into recombinant vectors have been tested in human volunteers with an acceptable safety record in cancer immunotherapy trials.
VEE has some unique attributes for use as a vaccine vector. First, existing Neutralizing antibody (NaB), to VEE is very rare outside the NE region of South America. Second, VEE has a cell tropism for Dendritic Cells (DC), which act as central regulators of the immune system. DC of the CD11b infected with VEE-VRP migrate to lymph nodes to prime powerful CTL and antibody responses through interactions with CD4+ helper/inducer subsets and CD4+ follicular helper cells which sustain strong, long-lasting antibody responses to viral pathogens.
Some more advantages of VEE-VRP are that the use of a bipartite helper-plasmid construction allows for in vitro assembly of infectious VEE particles. These particles, when injected into humans, are capable of infecting DC, but the progeny particles are antigenic/infectious but replication-incompetent. This induces a powerful yet safer immune response than a replication-competent vector. Another advantage is the use of Internal Ribosome Entry Sites (IRES) from a virus such as the human Enterovirus EV71. This allows for more efficient translation of the foreign gene, increasing the antigenicity and resulting immune response.
There are Three Main Types of Vector Systems in Use:
The Venezuelan Equine Encephalitis (VEE3000/3526) Virus Recombinant Particle (VRP)
Venezuelan Equine Encephalitis (VEE) is a medium size (70 nm), enveloped RNA virus of family Togaviridae, subfamily Alpphaviridae. The virus is transmitted by the bite of an infected Aedes mosquito, and causes potentially fatal encephalitis in horses and humans. Bovine, Canine and Porcine species can be infected, but do not show symptoms. There are six different serologically distinct subtypes and numerous strains of VEE. The largest recent outbreak occurred in Colombia in 1995, with over 14,000 cases and 26 reported deaths.
VEE has been the subject of research for both vaccine and biowarfare projects. The TC-83 strain, developed by USAMRID at Ft. Detrick, Md., is available to military and other persons serving in high-risk areas. The TC-83 strain is manufactured by 83 serial passages in guinea pig cardiac cells.
Several members of Alphaviridae, including VEE, are preferred platforms for recombinant vector systems to express foreign viral antigens in a VRP particle. These can have the advantages of high immunogenicity and safety as they are replication-restricted. The vectors can be constructed using the parent sequence of VEE3000 to produce the VEE3526 VRP platform. The advantages of the VEE3526 platform is that while the original VEE3000 strain is highly immunogenic, it can only by assembled in Biosafety Level-3 (BSL-3), facilities. The VEE3526 strain is prepared by deletion of the furin cleavage site in the Envelope 3 (E3) gene [Δ56RKRR59], and a 2nd site resuscitation in E1.
The recombinant VRP offer significant advantages to Naked RNA and Layered systems for viral vaccines, as they mimic pathogenic virus strains to a certain degree (glycosylation of envelope glycoproteins when expressed in mammalian cell lines, one round of replication). Among VRP, the VEE3000/3526 platform represents a wild-type strain with robust vaccine capabilities due to its superior DC-infecting capabilities, among other advantages. These include the generation of recombinant proteins, production of VLP, and in vivo efficacy as a vaccine against emergent viruses. It also can be constructed in BSL-2 conditions, increasing scale-up capability for production.
Construction of Split Helper VEE3000/3526 VRP Vectors
In a split-helper vector design, a second copy of the 265 promoter is inserted into the genome either immediately upstream of the authentic promoter or between the E1 gene and the beginning of the 3′ untranslated region. A foreign gene of interest (GOD, is then inserted into the genome just downstream of the second 26S promoter such that a second sub-genomic mRNA containing the foreign gene is transcribed. For added translation of the GOI, an IRES sequence cloned from Enterovirus 71 (EV71), can be inserted between the 265 promoter and the GOI.
The EV71 IRES element (strain 7423/M5/87) can be PCR amplified from pdc/MS DNA using primers dc/M5 (EcoRI) F and dc/MS (BamHI) R. The EV71 IRES PCR product is then digested with EcoRI and BamHI restriction enzymes and ligated into the VEE3000 VRP-RBD and plasmids downstream of the 265 promoters and upstream of the SARS-2/COVID gene sequences.
These VEE vectors replicate in infected cells under GMP conditions and assemble into infectious particles. These particles, when injected into humans, can infect DC, but progeny particles are replication incompetent as they lack the two helper plasmids for complete VRP construction. When such vectors are based on vaccine strains of alphaviruses, they can be utilized in vivo for immunization against both the alpha-virus vector and the pathogen from which the heterologous gene was derived. The use of the VEE capsid and the VEE glycoprotein on two separate helper RNAs reduce the probability of recombination events by a factor of 10 e4.
To construct a VEE3000/3526 vector that can be manufactured in BSL-2 conditions, deletion of the entire furin cleavage site between VEE E3 and E2 can be performed, with a secondary site resuscitation mutation in E1 that allows production in a mammalian cell line such as Vero or BHK-21. These modifications prevent possible reversions-to-virulence in the mammalian cell. This new system uses sequences of the wild-type VEE strain, including the 5′ and 3′ untranslated regions (UTR).
The viral capsid and glycoprotein genes are inserted into separate helper plasmid constructs between the 26S subgenomic promoter and the start of the 3′ UTR. After linear alignment of the three plasmid constructs are tied by ligase, the RNA transcripts are electroporated or transfected into BHK-21 cells or another suitable cell line. Cell culture supernatants are then harvested by pipetting, then filtered by ultra-centrifugation through 60 nm Millipore filters. Filtered VRP particles are then measured for titer by plaque assay on Vero E6 cells using serial ten-fold dilutions and calculation of viral plaques after 48 hours and 72 hours.
The following contains the materials and methodology used to construct and test the VEE 3526 VRP clones (VEE3000/3526 VRP-SARS-2/COVID-RBD), containing the sequences of the SARS-2/COVID-19 RBD sequence.
Plasmid Construction and Insertion of SARS-2/COVID-19 Genes
An example of construction of the two recombinant VEE VRP particles, each carrying a structural gene from SARS-2/COVID-19, is described below.
In order to insert the desired gene (Spike 1-RBD for SARS-2/COVID-19, the complete genomes of VEE 3000 must be cloned. The parent VEE 3000 is derived from the Trinidad Donkey strain of VEE (GenBank L01442.2 Genebake VEE TDS). The VEE cDNA is downstream from a T7 RNA polymerase promoter so that linearization of the clone downstream of the VEE sequences, and subsequent in vitro transcription with T7 polymerase, yields infectious VEE genomic replicas. Plasmid SARS-2/COVID-19-RBD is constructed using a T7 promoter, containing the complete RBD sequence of the Delta strain of SARS-2/COVID-19 Spike-1 RBD (parent sequence Genbank 01K504), and is used to produce VEE3526-SARS-2/COVID-19-RBD. This sequence is located from nt #21481 to 25325 and is listed in the accompanying ASCII text file “B.1.617.2 Delta Spike Sequence Text File”.
The VEE replicon is prepared from a plasmid carrying a complete cDNA copy of the VEE genome modified to contain a second 26S promoter followed by a multiple cloning site from Cla12 adaptor plasmid. The insertion of EV71 IRES sequences downstream of the 26S promoter and upstream of the SARS-2/COVID transgene allows for more efficient translation. The double promoter clone is digested with ApaI, which cleaves within the 265 promoters bracketing the structural protein genes. Re-ligation reconstitutes a single 265 promoter followed by a multiple cloning site, which is used to insert the heterologous SARS-2/COVID-19 gene fragment. For insertion of these plasmids, a shuttle vector is used.
The helper constructs are derived from the pVEE3000 clone by partial deletion of the genes encoding the VEE nonstructural proteins. When necessary, incompatible 5′ and 3′ overhanging ends are made blunt by treatment with T4 DNA polymerase prior to re-ligation of the plasmid.
The bipartite helper system consisted of individual Capsid (C)- and glycoprotein (GP)-helper RNAs which are constructed from VEE3000/3526 520±7505. In the C-helper, nt 8495±11229 are deleted by digestion of VE3000 A 520±7505 with HpaI and religation of the 3.8-kb DNA fragment. In the GP-helper, nt 7565±8386 are deleted by digestion of VEE3000 520±7505 with Tth111I and SpeI followed by ligation of the 5.7-kb DNA fragment with the synthetic double-stranded oligonucleotide 5′-TAGTCTAGTCCGCCAAGATGTCA-3′. This oligonucleotide contained Tth111I and SpeI overhanging ends at the 5′ and 3′ ends, respectively, and reconstituted the 265 promoter downstream from the Tth111I site, the initiation codon normally used for the capsid protein, and the first codon of E3.
Transcription and Transfection
Plasmid templates are linearized by digestion with NotI at a unique site downstream from the VEE3000 cDNA sequence, and capped run-off transcripts were prepared in vitro with the RiboMAX T7 RNA polymerase kit. BHK cells are transfected by electroporation and incubated in 75-cm2 flasks at 37° C. in 5% CO2. For the preparation of VRP, transcripts of both the replicon and the helper plasmids were co-electroporated into BHK cells, and the culture supernatants were harvested at 30 hrs. after transfection.
Analysis by Western Blot of fractionated VRP harvested from transfected culture supernatants can be performed to confirm expression of the SARS-2/COVID-19 genes. Alternatively, monoclonal antibodies with GFP-tags can be utilized on whole VRP for the spike protein, and on sonically fractionated VRP for the nucleocapsid protein.
Scale-Up and Purification
For large-scale production of VRP, BHK or other suitable cell lines (Vero E6, e.g.), can be expanded by serial culture passage into Master and Working Cell Bank systems after appropriate tests confirm absence of pathogens. Cells from the Working Bank can then be expanded in successively larger flasks, then transferred to roller bottles with supplemented EMEM media. When 80-90% confluent, these roller bottles can be inoculated with the VRP for production.
Cells and supernatant are then removed and purified by standard means (Benzonase treatment, DNAase, Tangential Flow Filtration sucrose density gradient centrifugation), to remove unwanted cell debris. The final VRP particles can then be titered by plaque assay and have antigens confirmed by ELISA and Western Blot. The two VRP types are then combined in a 50/50 mixture for final fill and finish.
Storage and Administration
After titer has been determined by plaque assay, the VRP clones can be stored at −20° C. after lyophilization for reconstitution with EMEM and sterile water prior to administration. Alternatively, the VRP can be stored in a preservative (15% Trehalose sugar, 2% F127 surfactant, and 2% Human Serum Albumin, e.g.), and stored cold at 2-4° C.T. The titer of virus equal to the appropriate dose determined by animal studies then can be administered by hypodermic injection to the optimum site for maximum immune activation and safety. These dosages and site of injection will be determined by results of applicable animal models using young, healthy, and aged or immunosuppressed animals.
Vaccine Component #2 SARS-2/COVID-19 Spike1 Glycoprotein+SF10 Adjuvant Intranasal Administration (Boost)
Review
While the prime component of the novel vaccine design may be successful in generation of high titers of neutralizing IgG antibodies and CTL against SARS-2/COVID-19 virus, a strong mucosal immune response is critical to providing the first line of defense against respiratory viruses. In order to achieve this goal, the “boost” strategy seeks to elevate the immune response induced by the prime phase. To achieve multiple layers of protection in both the Upper and Lower respiratory tracts, intranasal administration of SARS-2/COVID-19 antigens follows the “prime” injection.
Intranasal administration has been used with success against the Influenza A virus. Several preclinical studies on adjuvant-combined, nasal-inactivated vaccines revealed that nasal S-IgA Abs, a major immune component in the upper respiratory tract, reacted with homologous virus hemagglutinin (HA) and were highly cross-reactive with viral HA variants, resulting in protection and cross-protection. Serum-derived IgG Abs, which are present mainly in the lower respiratory tract, are less cross-reactive and cross-protective.
Inactivated influenza vaccines induce both S-IgA and IgG Ab responses in the respiratory tract when administered intranasally with an appropriate adjuvant. In addition, one clinical study demonstrated that the ability of human S-IgA Abs to neutralize influenza viruses increased with increasing polymerization of IgA (IgA Abs can form dimers, trimers, tetramers, and larger polymers). This suggests that polymeric S-IgA plays a crucial role in protecting against both homologous and variant influenza viruses.
The literature suggests that mucosal IgA Abs are secreted actively, whereas mucosal IgG Abs diffuse from the serum to the mucus. These results also support previous findings that S-IgA Abs play a primary role in preventing influenza virus infection in the upper respiratory tract, whereas serum IgG Abs play a predominant role in preventing the progression to lethal influenza-induced pneumonia in the lower respiratory tract.
Thus, the ability of S-IgA Abs to provide cross-protection depends on polymeric structures, which displayed increasing neutralization activity with increasing polymerization. These results suggest that the presence of large polymeric S-IgA Abs with higher neutralization activity in the respiratory tract play a crucial role in providing protection against homologous and variant influenza viruses.
While the “prime” component of the described novel vaccine should be capable of inducing mucosal immune responses, a “boost” regimen may be required in order to increase the levels of large, polymeric S-IgA antibodies for more complete protection against SARS-2/COVID-19. The equivalent antigen to Influenza A HA is the SARS-2/COVID-19 spike glycoprotein.
The SARS-2/COVID-19 S gp is a trimeric protein with two sub-components, S1 and 52. These have multiple glycosylation sites branching from Asparagine residues. As the binding of amino acids to sugars is less favorable than other proteins, these sites represent a further obstacle to vaccine developers. S1 is expressed in the free virion, and upon binding of the Receptor-Binding Domain (RBD), a conformational change is triggered from a “down” to an “up” position. This allows S2 to be exposed, increasing the binding force to the cellular Angiotensin-converting enzyme-2 (ACE2) receptor. This has implications for vaccine developers, as antibodies directed against S2 may not be able to access their binding site when in the “down” position.
The use of various cells to produce viral gp for vaccine subunits has been employed, but mammalian cells are preferred as they can attach the saccharide chains required to duplicate the viral gp structure and antigenicity. The following described a method of production and purification of SARS-2/COVID-19 Spike 1/2 glycoproteins using a Vaccinia (Cowpox), VRB12 and pRB21 plasmid viral vector system expressed in a CV-1 (green monkey kidney) cell line.
Materials Required
Transfection Process Steps
Step #1 of the procedure is to grow stocks of the parental mutant virus vRB12.
The SARS-2 COVID-19 Spike 1 glycoproteins from selected mutant strains can be cloned into plasmid pRB21 and purified by commercial providers using an appropriate standard transfection technique for the size gene. An example is given below.
Step #2 involves plating CV-1 cells for generation of the viruses in supplemented DMEM (5% FBS+1× penicillin/streptomycin).
Step #3 involves transfection of vRB12 infected CV-1 cells with pRB21 plasmids
Step #4 involves plaque purification of rVV
Production Scale-Up and Purification of Final Glycoprotein
The desired protein expression can be confirmed by Western Blot technique. After confirmation, one mini-stock of this can be used for seed virus stock using standard seeding and culture techniques. After harvesting, save working stock in aliquots of 1 mL and store at −20° until ready to infect CV-1 cells.
Step #4 is the production of large amounts of COVID-19 gp using Vero E6 cells in Roller bottles.
Vaccine Adjuvant Review
Viral subunit proteins are antigenic, but unless combined with other compounds to stimulate the immune response (adjuvants), they are poorly immunogenic. In order to stimulate innate immunity, higher titers of both serum IgG and mucosal IgA, and induce clones of CD4+ and CD8+ CTL with receptors specific for viral proteins, an adjuvant must be employed.
The original adjuvants were aluminum salts. (Freund's adjuvants, complete or incomplete). These act to stabilize and preserve the antigens from premature degradation, and attract macrophages and DC to the injection site. Over the years, new adjuvants became available as advances in immunology and molecular biology gave rise to new forms of immune stimulants.
An ideal adjuvant should protect the antigens without interfering with their structure, attract antigen-presenting cells (APC), and stimulate immune response without undue toxicity. Bacterial toxins were developed as potent adjuvants, but toxic effects, including partial paralysis, led to their being discarded. Bacterial flagellin protein lacks the toxic effect of many bacterial cell wall extracts, and has been proven safe for vaccine injected via parenteral route, but its safety by the intranasal route is unproved. Chitosan, a mucopolysaccharide from crustaceans, can cause reactions in persons allergic to shellfish. Allergic reactions to substances delivered to the respiratory tract are especially hazardous.
Another category of adjuvants with many properties suitable for intranasal administration are Liposome bodies. These are self-assembling lipid vesicles with aqueous cores in sizes from 10-300 nm, similar to viruses. They can be linked to Toll-Like-Receptor (TLR) ligands for added adjuvant power. TLR ligands activate both innate and adaptive immune mechanisms, leading to cytokine release and effector cells up-regulating cytolytic activity (Perforin and Granzyme B genes).
SF-10 is a compound of synthetic human pulmonary surfactant or its bovine equivalent, Surfacten™, a phospho-lipoprotein made by type 2 alveolar cells, with a carboxy vinyl polymer as a viscosity improver. SF-10 effectively induces Flu A anti-HA neutralizing IgA antibodies in nasal and lung washes as well as IgG in sera. SF-10 effectively delivers antigen to DC and promotes cross-presentation to CTL, yielding high numbers of effector CD4+ and CD8+ cells specific for the viral antigen. SF-10+HA up-regulated perforin and Granzyme B in splenic cells after intranasal administration. This was proven experimentally after depletion of CTL deprived animals of protection after SF-10+Flu A HA i.n. vaccination. CD4+ cells were more critical than CD8+ cells in this protection, so the addition of SF-10 to a suspension of SARS-2/COVID-19 Spike antigens may impart critical advantages for a SARS-2/COVID-19 prime-boost vaccine strategy.
Advantages of Using SF-10 as Intranasal Adjuvant for SARS-2/COVID-19
SF-10 Preparation
SF-10 is made using commercially available Surfacten™ (Mitsubishi Pharma, Tokyo, Japan), plus 1,2 Dipalmitoyl1-phoshphtidylcholine (DPPC), and palmitate (PA). Synthetic human surfactant (SSF), is available for Nippon Fine Chemical (Osaka, Japan). Synthetic SP-related peptides (>80% grade), are available from Greiner (Frickenhausen, Germany). Carboxy vinyl polymer is available from Sigma-Aldrich (St. Louis, Mo.).
SSF is prepared by mixing the three lipids, DPPC, PG, and PA, plus various peptides (below) at a molar ratio of 75:25:30:0-6, respectively. SSF samples at 4 mg/mL can then be lyophilized for storage.
Amino Acid Sequence of Peptides Comprising SF-10
Mixing of SARS-2/COVID-19 Spike Glycoprotein and SF-10
SARS-2/COVID gp is treated for 3 minutes with a sonic oscillator followed by upside-down mixing every 30 minutes for 2 hours at RT, then stored at 4° C.
A mixture of SSF and an appropriate dose (75 ug), of SARS-2/COVID-Spike 1/2 gp is incubated at 42° C., the critical temperature of Surfacten lipids, for 10 minutes with gentle mixing, followed by freezing at −75° C., and then lyophilized. Lyophilized SARS-2/COVID-19 Spike ½ gp+SSF is dissolved in sterile saline and added to an atomizer unit and stored at 4° C. before use.
Administration of SARS-2/COVID-19 Spike Glycoprotein and SF-10
The atomizer is removed from cold storage, shaken, and the cap is removed. The subject places the atomizer at one nostril and depressed the plunger while inhaling through the nose.
Prime-Boost Strategy of Novel SARS-2/COVID-19 Vaccine
Prime-boost refers to the administration of multiple antigens, in sometimes varying formulations and routes of administration, in order to increase protection against viral challenge. For the invention described herein, the plan is:
The goal of the strategy is to stimulate a strong, multi-layered immune response in both the Upper and Lower Respiratory Tracts. Further Investigations with animal subjects may require some modification of the described dosing or administration plan.
Addressing Mutations and Emergent Strains of SARS-2/COVID-19
While DNA viruses are inherently stable, RNA viruses exhibit a high level of mutations. This is due to the relative accuracy of DNA polymerase (error rate of 1 in 10{circumflex over ( )}e7 base pairs) to RNA polymerase (error rate 1 in 10{right arrow over ( )}e4 base pairs). While many of these errors are fatal to the virion, some, especially those occurring in envelope gp antigens, can allow escape by inhibiting binding of neutralizing antibody, or CTL recognition of an epitope.
IFA exhibits considerable antigenic drift (moderate changes in genome) and also antigenic shift (dramatic changes in genome leading to impact on immune responses). Antigenic drift mutations may or may not impact immunity, but antigenic shift mutations lead to emergent strains, requiring new vaccine formulations for protection. The 2017 IFA vaccine had an estimated protective rate of 36%
SARS-2/COVID-19 exhibits moderate mutation rates compared with IFA, but this could be due to its relatively recent emergence, and lack of selective pressure from vaccine or natural immunity. There are several strains circulating, with unknown impact on disease severity or future vaccine development.
The traditional method of overcoming IF mutations is through new annual vaccine formulations to induce a new range of NAB against HA targets. However, this approach has several drawbacks:
Serum NAB may not be the ultimate goal of a vaccine protective against emergent strains. There is a consensus among thought leaders of respiratory virus vaccine development that serum NAB is only part of an overall protective strategy. Critical elements of this strategy include:
A Multi-Part Prime-Boost Strategy as Described Herein could be Capable of Achieving these Goals:
| Number | Date | Country | |
|---|---|---|---|
| 63103910 | Aug 2020 | US |
