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Novavax NVX-2373 Insect Transgene founded from Ebola technology

Novavax NVX-CoV2373 - Phase 1u20132 Trial of a SARS-CoV-2 Recombinant Spike Protein Nanoparticle Vaccine<br>

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Novavax NVX-2373 Insect Transgene founded from Ebola technology

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  1. Novavax NVX-CoV2373 - Phase 1–2 Trial of a SARS-CoV-2 Recombinant Spike Protein Nanoparticle Vaccine Commenced: May 2020 - Reported: September 2, 2020 (Funded by the Coalition for Epidemic Preparedness Innovations; ClinicalTrials.gov number, NCT04368988) METHODS We initiated a randomized, placebo-controlled, phase 1–2 trial to evaluate the safety and immunogenicity of the rSARS-CoV-2 vaccine (in 5-μg and 25-μg doses, with or without Matrix-M1 adjuvant, and with observers unaware of trial-group assignments) in 131 healthy adults. In phase 1, vaccination comprised two intramuscular injections, 21 days apart RESULTS After randomization, 83 participants were assigned to receive the vaccine with adjuvant and 25 without adjuvant, and 23 participants were assigned to receive placebo. No serious adverse events were noted. Reactogenicity was absent or mild in the majority of participants, more common with adjuvant, and of short duration (mean, ≤2 days). One participant had mild fever that lasted 1 day. Unsolicited adverse events were mild in most participants; there were no severe adverse events. The addition of adjuvant resulted in enhanced immune responses, was antigen dose– sparing, and induced a T helper 1 (Th1) response CONCLUSIONS At 35days, NVX-CoV2373appeared to be safe, and it elicited immune responses that exceeded levels in Covid-19 convalescent serum. The Matrix-M1 adjuvant induced CD4+ T-cell responses that were biased toward a Th1 phenotype. NVX-CoV2373 contains MatrixM1 adjuvant6 and a recombinant SARS-CoV-2 (rSARS-CoV-2) nanoparticle vaccine, constructed from the full-length (i.e., including the transmembrane domain), wild-type SARS-CoV-2 spike glycoprotein, which mediates attachment of the virus to the human angiotensin-converting enzyme 2 (hACE2) receptor of host cells for cellular entry and serves as a key target for development of antibodies and vaccines.7,8 In rodent and nonhuman primate challenge models, NVX-CoV2373 induced high titers of antibodies measured against anti-spike protein that blocked hACE2 receptorbinding and achieved neutralization of wild-type virusthat exceeded the magnitude of responses measured in human convalescent serumand that provided protection against SARS-CoV-2 challenge.9,10

  2. As a safety measure, 6 participants (Sentinels) were initially randomly assigned in a 1:1 ratio to the 5-μg and 25-μg rSARS-CoV-2 plus Matrix-M1groups (groups C and D), vaccinated in an open-label manner, and observed for reactogenicity for 48 hours. rSARS-CoV-2, manufacturedatEmergent Biosolutions, is a recombinant nanoparticle vaccine constructed from the full-length(i.e., including the transmembrane domain), wild-type SARS-CoV-2 spike glycoprotein (GenBank accession number, MN908947; nucleotides 21563–25384) optimized in the established baculovirusSpodoptera frugiperda (Sf9) insect cell expression system.9rSARS-CoV-2is generated with 682-QQAQ-685 mutations at the S1/S2 cleavage sitesto confer protease resistanceand two proline substitutions at residues K986P and V987P at the top of the heptad repeat 1/central helix in theS2 subunit to stabilize the construct in a prefusion conformation. rSARS-CoV-2 isresistant to proteolytic cleavage, binds with high affinity to the hACE2 receptor, and demonstrates thermostability.9,11 Matrix-M1, a saponin-based adjuvant,12wasmanufactured by Novavax Of the 131 participants who received injections, 23 received placebo (group A), 25 received 25-μg doses of rSARS-CoV-2 (group B), 29 received 5-μg doses of rSARS-CoV-2 plus Matrix-M1, including three sentinels (group C), 28 received 25-μg doses of rSARS-CoV-2 plus Matrix-M1, including three sentinels (group D), and 26 received a single 25-μg dose of rSARS-CoV-2 plus Matrix-M1followed by a single dose of placebo (group E). All 131 participants received their first vaccination on day 0, and all but 3 received their second vaccination at least 21 days later; exceptions include 2 in the placebo group (group A) who withdrew consent (unrelated to any adverse event) and 1 in the 25-μg rSARS-CoV-2 + Matrix-M1 group (group D) who had an unsolicited adverse event (mild cellulitis) Of note, the mean duration of reactogenicity events was 2 days or less for both the first vaccination and second vaccination periods. T-cell responses in 16 participants who were randomly selected from groups A through D, 4 participants per group, showed that adjuvanted regimens induced antigen-specific polyfunctional CD4+ T-cell responses that were reflected in IFN-γ, IL-2, and TNF-αproduction on spike protein stimulation. A strong bias toward this Th1 phenotype was noted; Th2 responses (as measured by IL-5 and IL-13 cytokines) were minimal(Fig. 5). Although the effector mechanisms that might lead to protection with a Covid-19 vaccine are yet not known, it is presumedthat neutralizing antibodies will be associated with protection; this has led to the use of microneutralizationtesting in all recent human Covid-19 vaccine trials.16 The theoretical concernfor vaccine-induced enhanced disease is in part addressed in this trialby the use of an adjuvant, which stimulates both high neutralizing antibody responses and T cells with a predominant Th1 phenotype, both of which are suggestedto be important in vaccine candidate selection.23,24

  3. Interleukins, from 1 to 37, and interferon-y: Receptors, functions, and roles in diseases

  4. The theoretical concern for vaccine-induced enhanced disease is in part addressed in this trialby the use of an adjuvant, which stimulates both high neutralizing antibody responses and T cells with a predominant Th1 phenotype, both of which are suggested to be important in vaccine candidate selection.23,24

  5. Ten thousand UK volunteers will be invited to join a leading phase 3COVID-19 NVX-CoV2373 vaccine trial - 25 September 2020 The Phase 3 study will test the safety and effectiveness of a promising new vaccine, developed by US biotechnology company Novavax, across a broad spectrum of people, including those from a variety age groups and backgrounds. Calling on some of the thousands of volunteers who have joined the fight against coronavirus through the NHS Vaccine Registry, the phase 3 trials are the second to commence in the UK and will be undertaken at a number of National Institute for Health Research (NIHR) regional sites across the UK, including Lancashire, the Midlands, GreaterManchester, London, Glasgow and Belfast. The Registrywas launched in July to help create a database of people who consent to be contacted by the NHS to take part in clinical studies, to help speed up the development of a safe and effective vaccine. The UK government has secured 60 million doses of the Novavax vaccine, which will be manufactured using FUJIFILM Diosynth Biotechnologies’ facilities in Stockton-on-Tees, north east England. Professor Paul Heath, NovavaxPhase 3 trial Chief Investigator and Professor of Paediatric Infectious Diseases at St George’s University Hospitals NHS Foundation Trust said: This is only the second Phase 3 vaccine trial to be initiated in the UK, and the first Phase 3 trial with the Novavax vaccine anywhere in the world…The vaccinehas successfully gone through its early safety trialsand we’re extremelyencouraged by its performance so far. Chair of the government’s Vaccines TaskforceKate Bingham said: “Finding a safe and effectivevaccinethat works for the majority of the UK population is the best way to tackle this devastating disease. Whilst social distancing, testing and other measures can help reduce the impact of coronavirus, the only long-term solution to beating it will be finding a vaccine.” The Novavax vaccine comprises a recombinant nanoparticle technologycontaining an engineered coronavirus spike protein and the saponin-based adjuvant Matrix-M designed to enhance the immune response and stimulate high levels of neutralising antibodies. Half the study participants will receive the trial coronavirus vaccine, delivered in 2 doses, and half will receive a saline placebo, also delivered in 2 doses - a so called ‘blinded trial’ - Study participants can expect to make around 6 visits to their local trial centre over 13 months.

  6. [1] The UK has secured access to a total of 6 different candidates, across 4 different vaccine types, reflecting the government’s strategy to ensure the UK has a supply of vaccines should any of these prove safe and effective through clinical trial research. This is in addition to the University of Oxford’s vaccine being developed with AstraZeneca, and includes agreements with the BioNTech/Pfizer alliance, Valnevaand GSK/Sanofi Pasteur. The 4 different vaccine classes that the governmenthas secured to date for the UK are: adenoviral vaccines (Oxford/AstraZeneca, Janssen) mRNA vaccines (BioNTech/Pfizer, Imperial) inactivated whole virus vaccines (Valneva) protein adjuvant vaccines (GSK/Sanofi, Novavax) The UKis actively working with the vaccine alliance GAVI, The Coalition for Epidemic Preparedness Innovations(CEPI), the World Health Organisation(WHO) and a group of other countries to help buy vaccines as well as to ensure equitable distribution of vaccines to low-income countries. A new NHS service was launched in July 2020 to enable people across the UK to sign up for information on COVID-19 vaccine trials. The NHS COVID-19 vaccine research registry, developed in partnership with NHS Digital, The service was commissioned as part of the UK government’s Vaccine Taskforce inconjunction with the National Institute for Health Research (NIHR) The Vaccine Taskforce (VTF) was set up under the Department for Business, Energy and Industrial Strategy (BEIS) in May 2020. It is chaired by biotech and life sciencesexpert Kate Bingham, who was appointed by the Prime Minister Boris Johnson. [1[ https://www.gov.uk/government/news/10000-uk-volunteers-to-take-part-in-new-covid-19-vaccine-trials

  7. NCT04583995

  8. NVX-CoV2373 vaccine protects cynomolgus macaque upper and lower airways against SARS-CoV-2 challenge - 19 October, 2020 Funding for certain studies was provided by the Coalition for Epidemic Preparedness Innovations (CEPI), Following intranasaland intra-tracheal challengewith SARS-CoV-2, immunized macaques were protected against upper and lower infection and pulmonary disease. These results support on-going phase 1/2 clinical studiesof the safety and immunogenicityof NVX-CoV2327 vaccine (NCT04368988). We have developed a recombinant nanoparticle vaccine constructed from the full–length, wild-type SARS–CoV-2 spike glycoprotein (GenBankgene sequence MN908947, nucleotides 21563–25384) optimized for the baculovirus-Spodoptera frugiperda (Sf9) insect cell expression system [1]. NVX-CoV2373 vaccine also induces multifunctional CD4+ T-cell responses of IFN-y, IL-2, andTNF-a biased towards a Th1 phenotype, and generates antigen-specific germinal center B cellsin the spleen [1]. 2. Materials and methods 2.1. Cell lines, virus, antibody reagents, and receptors Vero E6 cells (ATCC, CRL-1586) were maintainedin Minimal Eagles Medium (MEM) supplemented with 10% fetal bovine serum, 1% glutamine and 1% penicillin and streptomycin. The SARS-CoV-2 (*WA-1, 2020) isolated was obtained from the Center for Disease Control and stock virus prepared in Vero E6 cells. Histidine-tagged hACE2 receptorwas purchased from Sino Biologics (Beijing, CN). Rabbit anti-SARS-CoV spike protein was purchased form Biodefense and Emerging Infections Research Resources Repository 2.2. Recombinant SARS-CoV-2 spike protein NVX-CoV2327 was codon optimized synthetically produced from the full-length S glycoprotein gene sequence (GenBank MN908947 nucleotides 21563–25384 - see slide 20) for expression in Spodoptera frugiperda(Sf9) cells (GenScript Piscataway, NJ, USA) as described [1]. Briefly, the S1/S2 furin cleavage site 682-RRAR-685 was modified 682-QQAQ-685 and two proline substitutions introduced at positions K986P and V987P (2P) to stabilize the full length SARS-CoV-2 S [3]. 2.6. SARS-CoV-2 challenge procedure The virus challenge study was done at BIOQUAL, Inc. within a BSL-3 containment facility. SARS-CoV-2 generated from isolate 2019-nCoV/USA-WA1/2020was received from BEI Resources (NR-52281; lot # 70033175) and expanded in Vero E6 cellsfor challenge stock generation. *2019-nCoV-/USA-WA1/2020 was also used in the German study for Pfizer BTN162b2 in April 2020. The USA/WA1/2020 from June, 2020also has the D614G northern European mutation.

  9. Thefall armyworm, Spodoptera frugiperda, is a lepidopteran pest that feeds in large numbers on the leaves, stems and reproductive parts of more than 350 plant species, causing major damage to economically important cultivated grasses such as maize, rice, sorghum, sugarcane and wheatbut also other vegetable crops and cotton. Native to the Americas, it has been repeatedly intercepted at quarantinein Europe and was first reported from Africa in 2016 where it caused significant damage to maize crops

  10. December 28, 2020 Phase 3 trial of Novavax investigational COVID-19 vaccine opens. NIH- and BARDA-fundedtrial will enrol up to 30,000 volunteers. 2 doses of 5 μg - 1 dose each on Days 0 and 21. SARS-CoV-2 Recombinant Spike Protein Nanoparticle Vaccine (SARS-CoV-2 rS) With Matrix-M1™ Adjuvant . Completes: March 21, 2021. Estimated study completion date: December 30, 2022 The National Institute of Allergy and Infectious Diseases (NIAID), part of the National Institutes of Health, and the Biomedical Advanced Research and Development Authority (BARDA), part of the U.S. Department of Health and Human ServicesOffice of the Assistant Secretary for Preparedness and Response, are funding the trial. Department of Health and Human Services(sponsor)

  11. “BARDAmanagesProject Bioshield, which involves the procurement and advanced development of medical countermeasures for Chemical, Biological, Radiologicaland Nuclearagents (CBRN), as well as the advanced development of procurement of medical countermeasures for pandemic Influenza and other emerging infectious diseases that fall outside the auspices of Project Bioshield. In addition, BARDA manages the Public Health Emergency Medical Countermeasures Enterprise (PHEMCE) and is a key partner in implementing PMEMCE strategies”

  12. About Matrix-M™ Novavax’ patented saponin-based Matrix-M™ adjuvant has demonstrated a potent and well-tolerated effect by stimulating the entry of antigen presenting cells into the injection site and enhancing antigen presentation in local lymph nodes, boosting immune response. Information about the trial and how to enroll in PREVENT-19 is available on clinicaltrials.gov under trial identifier NCT04611802.

  13. NCT04611802

  14. Press release - Novavaxpublishes positive efficacy data for its COVID-19 vaccine- 28 January, 2021 Novavaxtoday published positive data from the UK phase 3 studyof its COVID-19 vaccine candidate, showing it to be 89.3% effective in preventing coronavirus in participants. The study was conducted during the period the new COVID-19 variantwas first observed in Kentand began to circulate widely, with today’s results showing it was effective against the variantduring the phase 3 trial. Novavax’s candidate differs from those currently being used in the UK, combining an engineered protein from the virus that causes COVID-19 with a plant-based ingredient (Saponin) to help generate a stronger immune response. The data published today come from more than 15,000 people (NCT04583995 Trial ID: 2019nCoV-302 – see slide 12) who were recruited through the National Institute of Health Research vaccine registry, which was launched in July 2020 to support the UK’s efforts to deliver vaccines for COVID-19. Nearly 4,000 people in the study were over the age of 65. Intotal, more than 7.4 million people across the UK have now had a least one dose of the vaccine. the UK government has invested £127 million to fund a state-of-the-art manufacturing innovation centre in Braintree, Essex, in collaboration with the Cell and Gene Therapy Catapult, to accelerate the mass production of a successful Covid-19 vaccine in the UK due to open in December 2021, The Governmenthas also provided £4.7 million funding to the Catapultto ensure that the UK has the best skills and expertise in vaccine manufacturing and advanced therapies. The government has also created the UK’s first dedicated Vaccine Manufacturing and Innovation Centre (VMIC) and accelerated its development with £93 million of investment The government has established a Rapid Deployment Facility with £8.75 million of investment which is manufacturing vaccines at scale. Through the Vaccines Taskforce, the UK has secured early access to 367 Million doses of 7 of the most promising vaccines so far. To date, the UK government has invested over £230 million into manufacturing a successful vaccine.

  15. 1A Vaccine Trial for COVID-19 - Trial ID: 2019nCoV-301 January 30, 2021

  16. 1SARS-CoV-2 spike glycoprotein vaccinecandidateNVX-CoV2373 immunogenicity in baboons and protection in mice - January 14,2021 Unique to SARS CoV-2 is the insertion of a polybasic RRAR furin-like cleavage motif in the S1/S2 cleavage site10. Proteolytic cleavage of the S protein generates the S2 stalk that is conserved across human coronaviruses and the less conserved S1 cap11. The N-terminal domain (NTD) and the receptor-binding domain (RBD) are located in the S1 subunit. The fusion peptide (FP), two heptad repeats (HR1 and HR2), central helix (CH), transmembrane (TM) domain, and cytoplasmic tail (CT) are located in the S2 subunit. Three S1/S2protomers non-covalently associate to form the functional S-trimer. We have developed a SARS-CoV-2 S subunit vaccine (NVXCoV2373) constructedfrom the full-length S-protein and produced in the established Sf9 insect cell expression system. Here, we describe a stable prefusion S-protein structure generated by mutating the furin cleavage site to be resistant to cleavage and utilization of two proline substitutions at the apex of the CH11 In mice, the vaccineelicits protection against SARS-CoV-2 infection with no evidence of vaccine associated enhanced respiratory disease (VAERD). These results support the clinical development of the NVX-CoV2373 vaccine for prevention of COVID-19 (NCT04368988). Results SARS-CoV-2 S glycoproteins. The SARS-CoV-2 S-gene (MN908947.3, nucleotides 21563–25384) encoding the full length 1273 amino acid spike protein was used as a backbone to produce spike protein variants. The BV2365 single mutant was generated by mutating the putative furin cleavage site682-RRAR-685 to 682-QQAQ-685, and the NVX-CoV2373 double mutant was generated with 682-QQAQ-685 and two proline substitutionsat residues K986P and V987P (Fig. 1a). Synthetic full-length wild-type (WT), the single mutant BV2365, and double mutant NVX-CoV2373 genes were codon optimized for insect cells and cloned into recombinant baculovirus for expression in Sf9 cells. SARS-CoV-2 S stability under stressed conditions. The stability of a COVID-19 vaccine forglobal distribution is critical. Only oxidizing conditions with hydrogen peroxide reduced the bindingof NVX-CoV2373 by eightfold(IC50= 120 ng mL−1) [See Ascorbic Acid studies] 1SARS-CoV-2 spike glycoprotein vaccine candidate NVX-CoV2373 elicits 1 immunogenicity in baboons and protection in mice, PRE-PRINT, June 29, 2020

  17. NVX-CoV2373 protection against SARS-CoV-2 in Ad/hACE2 mice. Mice were vaccinated with NVX-CoV2373 to evaluate the induction of protective immunity against challenge with SARS CoV-2 by comparing single-dose or prime/boost vaccination strategies in a live virus challenge model. Mice were immunized with a single priming doseor a prime/boost regimen with NVX-CoV2373/ Matrix-M as described above. Since mice do not support replication of WT SARS-CoV-2 virus, BALB/c mice were [intranasally] transduced with adenovirus encoding human ACE2 receptor(Ad/hACE2) which renders them permissive to infection with SARS-CoV-2(refs. 21,22). At 4 days post transduction, micewere challenged with 105 plaque forming units (pfu)/mouseof SARSCoV-2 (WA1 strain). Following challenge, mice were weighed daily and pulmonary histology and viral load were analysed at 4 and 7 days post challenge. Weight loss during the experiment paralleled the viral load findings, with animals receiving single dose of 10 μg, 1 μg, and 0.1 μg NVX-CoV2373/Matrix-M showing marked protection from weight loss compared to the unvaccinated placebo animals. These results confirmed that NVX-CoV2373 confers protection against SARS-CoV-2 and that low doses of the vaccineassociated with lower serologic responsesdo not exacerbate weight loss or induce exaggerated illness. The NVX-CoV2373-immunized mice showed a significant reduction in lung pathology at both 4 and 7dpiin a dose dependent manner. The prime only group displayed reduced inflammation at the 10 μg and 1 μg doseswith a reduction in inflammation surrounding the bronchi and arteriolescompared to placebo mice. Multifunctional cytokine analysis of CD4+ and CD8+ T cells in mice. To determine the role of Matrix-M in generating T cell responses, we immunized groups of mice (N = 6/group) with 10 μg NVX-CoV2373 alone or with 5 μg Matrix-Min a two-dose regimen spaced 21 days apart. The number of IFN-γ-secreting cells after ex vivo stimulation increased sevenfoldin spleensof miceimmunized with NVX-CoV2373/Matrix-M compared to NVX-CoV2373 alone as measured by the ELISpotassay. Importantly, we found the frequency of IFN-γ+, TNF-α+, and IL-2+ cytokine-secreting CD4+ and CD8+ T cells was significantly higher (p < 0.0001) in spleensfrom the NVX-CoV2373/Matrix-M-immunized mice compared to miceimmunized without adjuvant Type 2 cytokines IL-4 and IL-5 secretion from CD4+ T cells was also determined by ICCS and ELISpot, respectively. We found that immunization with NVX-CoV2373/Matrix-M also increased type 2 cytokines IL-4 and IL-5 secretion (twofold) compared to immunization with NVX-CoV2373 alone, but to a lesser degree than enhancement of type 1 cytokine production (e.g. IFN-γincreased 20-fold).

  18. These results indicate that administration of the Matrix-M adjuvant led to an antigen specific CD4+ T cell development, which was at least balanced betweenTh1 and Th2 phenotypes or, in most animals, was Th1-dominant We next evaluated the immunogenicity of the vaccinein adult baboons. In this study, adult olive baboons were immunized with a dose range (1 μg, 5 μg, and 25 μg) of NVX-CoV2373 with 50 μg Matrix-M adjuvant administered by IM injection in two doses spaced 21 days apart To assess the adjuvant activity of Matrix-M in nonhuman primates, an additional group of animals (ten adult baboons 10–16 years of age) was immunized with 25 μg of NVX-CoV2373without the adjuvant. Anti-S protein IgGtiterswere detected within 21 days of a singlepriming immunization in animals immunized with NVX-CoV2373/Matrix-M across all the dose levels (GMT=1249–19,000). Importantly, animals immunized with NVXCoV2373 without adjuvant had minimal or no detected anti-S IgG titer(GMT <125) after one immunization, which was not boosted by a second immunization PBMCs (peripheral blood mononuclear cells)were collected 7 days after the secondimmunization(day 28) and T cell response was measured by ELISpot assay. PBMCs from animals immunized with 5 μg or 25 μg NVXCoV2373/Matrix-M had the highest number of IFN-γ secreting cells, which was fivefold greater on averagecompared to animals immunized with 25 μg NVX-CoV2373 alone or 1 μg NVXCoV2373/Matrix-M. Type 2 cytokine IL-4 levels were too low to be detected in baboons by ELISpot analysis. The mean anti-S IgG levels were sevenfold higher in immunizedbaboons (EC50=152,060, 95%CI, 60,767–243,354) compared to convalescent serum (EC50=21,136, 95%CI, 11,473–30,799). Baboons receiving the vaccinehad eightfold higher bindingand receptor inhibiting antibodies(50% RI=478, 95%CI, 161.1–794.4) compared to levels in COVID-19 convalescent serum(50% RI =61, 95%CI, 35.7–85.5) (Fig. 9). Therefore, NVX-CoV2373vaccine induced bindingand functional antibodies in a nonhuman primate at levels comparableorhigher than individuals recovered from COVID-19. The functional immunity induced by this nanoparticle vaccine and Matrix-M adjuvant clearly depends on both the adjuvant and antigen componentsand mirrorsthe human experience with influenza hemagglutinin vaccine23and a naïve population with Ebola recombinant protein nanoparticle vaccines24 Subcutaneous injection of Matrix-M has been shown to promote the recruitment of leukocytes to local draining lymph nodesand the spleenin mice, along with an increase in activation marker CD69 on monocytes and T-, B-, natural killer, and dendritic cells in the draining lymph nodes. It is hypothesizedthat Matrix-M induces a local transient pro-inflammatory response with recruitment, activation, and maturation of immune cells, specifically stimulating dendritic cell uptakeand processing of antigens with enhanced presentation25–27. There was no evidence of VAERD in challenged mice immunized with low doses of NVXCoV2373

  19. While multiple animal models have been developed for infection with human coronaviruses, including SARS-CoV, MERS-CoV, and now SARS-CoV-2, to date none of them fully represent the pathology or clinical symptoms of human infection. A murinehACE2 *transduced challenge model with WT virus is considered robustand was recently used to evaluate protection against SARSCoV-2 *Transduction, a process of genetic recombination in bacteria in which genes from a host cell(a bacterium) are incorporated into the genome of a bacterial virus(bacteriophage) and then carried to another host cell when the bacteriophage initiates another cycle of infection. In generaltransduction, any of the genes of the host cell may be involved in the process; in special transduction, however, only a few specific genes are transduced. It has been exploited as a remarkable molecular biological technique for altering the genetic construction of bacteria, for locating bacterial genes, and for many other genetic experiments. *https://www.britannica.com/science/transduction-microbiology Of note, the adenovirus-based hACE-2 transduction itself (Ad5/hACE2) gives rise to some background inflammatory changeswhich are present in all animals and are, of course, not responsive to prophylaxis targeting of SARS-CoV-2. The safety, immunogenicity, and efficacy of NVX-CoV2373 with Matrix-M adjuvant are currently being evaluated in multiple nonhuman primate models and a phase 1/2 human clinical trial (NCT04368988)30 Methods Cell lines, virus, antibody reagents, and receptors. Vero E6 cells(ATCC, CRL-1586) were maintained in Minimal Eagles Medium (MEM) supplemented with 10% fetal bovine serum, 1% glutamine, and 1% penicillin and streptomycin. The SARS-CoV-2 (WA-1) isolated was obtained from the Center for Disease Control (WA-1 strain) and stock virus prepared in Vero E6 cells. Rabbit anti-SARS-CoV S protein was purchased from Biodefense and Emerging Infections Research Resources Repository Recombinant baculovirus constructs were plaque purified and master seed stocks prepared and used to produce the working virus stocks. Recombinant baculovirus stocks were prepared by infecting Sf9 cells with a multiplicity of infection (MOI) of ≤0.01 pfu per cell31–33 Expression and purification. SARS-CoV-2 S proteins were produced in Sf9 cells expanded in serum-free medium to a density of 2–3 × 106 cells mL−1 and infected with recombinant baculovirus at MOIof ≤0.1 pfu per cell. Cells were cultured at 27 ± 2 °C and harvested at 68–72 hpost-infection by centrifugation. The mouseSARS-CoV-2 challenge study was conducted at the University of Maryland ABSL-3 containment facility. Animals were fed certified chow that had been analysed for ensure no contaminants were in the feed. Waterwas available ad libitum throughout the studies. Mice used: FemaleBALB/c mice (7–9 weeks old) Refer also to Research paper: Adjuvanted SARS-CoV-2 spike proteinelicits neutralizing antibodies and CD4 T cell responses after a single immunization inmice, 2021

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