Complete Protection of Nasal and Lung Airways Against SARS-CoV-2 Challenge by Antibody Plus Th1 Dominant N- and S-Specific T-Cell Responses to Subcutaneous Prime and Thermally-Stable Oral Boost Bivalent hAd5 Vaccination in an NHP Study

Abstract

BackgroundTo address the dire need for a safe and effective vaccine to protect individuals from and reduce transmission of SARS-CoV-2, we developed a COVID-19 vaccine that elicits not only robust humoral responses but also activates T cells. Our bivalent vaccine expresses both an optimized viral spike (S) protein (S-Fusion) and the viral nucleocapsid (N) protein with an Enhanced T-cell Stimulation Domain (N-ETSD) that directs N to the endo/lysosomal subcellular compartment to enhance MHC class II responses. The vaccine antigens are delivered by the second-generation adenovirus serotype 5 [E1-, E2b-, E3-] platform (hAd5) that has been safely administered and found to be effective in generating tumor-specific T cells even in the presence of pre-existing adenovirus immunity. Here, we report our findings on the safety and efficacy of our hAd5 S-Fusion + N-ETSD subcutaneous (SC) prime and thermally-stable oral boost vaccine in generating SARS-CoV-2-neutralizing antibodies, eliciting N- and S-specific T-cell responses, and providing complete protection with the clearing of virus after challenge in Non-Human Primates (NHP). A key objective of the study was to explore the efficacy of a novel thermally-stable oral hAd5 S-Fusion + N-ETSD to serve as a booster dose following an SC prime.MethodsGroup 1 NHP received the hAd5 S-Fusion + N-ETSD vaccine on Days 0 and 14 by SC injection (1011 VP), and on Day 28 by a single oral boost (1010 VP); Group 2 received vaccination on the same schedule, but with an SC prime and two oral boosts. Group 3 placebo NHP were dosed with vehicle-only SC-oral-oral. Blood for the isolation of sera and PBMCs was collected throughout the study. ELISA was used for determination of anti-S IgG levels, cPass™ for presence of neutralizing antibodies, and ELISpot for interferon-γ(IFN-γ) and interleukin-4 (IL-4) secretion by T cells.On Study Day 56, all NHP were challenged with intratracheal/intranasal 1 × 106 TCID50/mL SARS-CoV-2. Bronchoalveolar lavage (BAL) samples were collected on Day 42 pre-challenge and at several time points post-challenge; nasal swabs were collected daily post-challenge. NHP were euthanized on Study Day 70 and tissue collected for histopathological analyses. Viral load and active viral replication were determined in BAL and nasal swab specimens by RT qPCR of genomic and subgenomic RNA, respectively. Safety was determined by cage-side observations such as weight as well as hematology and clinical chemistry analyses of blood.ResultsThe hAd5 S-Fusion + N-ETSD vaccine, both SC and oral, elicited no apparent toxicity seen in clinical chemistry, hematology, or cage-side observations. Neutralizing antibodies were induced in 9 of 10 vaccinated NHP and anti-S IgG positive titers in 10 out of 10. Th1 dominant T-cell responses were elicited by both S and N antigens, with responses being greater for N. Viral replication was inhibited from Day 1 post-SARS-CoV-2 challenge with complete protection in all (10/10) primates within 7 days of challenge from both nasal passages and lung. Replicating SARS-CoV-2 dropped immediately and was undetectable as soon 3 days post-challenge. There was a rapid decline in lymphocytes from the periphery on Day 1 post-challenge and a rebound within 3 days following challenge that was significantly higher by Day 14 post-challenge in Group 1 as compared to Group 3 placebo NHP.ConclusionsIn the rhesus macaque NHP model, the bivalent hAd5 S-Fusion + N-ETSD subcutaneous and oral vaccine provided complete protection of nasal passages and lung against SARS-CoV-2 challenge by eliciting neutralizing antibodies plus Th1 dominant N- and S-specific T-cell responses. Inhibition of viral replication within the first 24 hours post-challenge, in vaccinated NHP compared to placebo NHP, suggests the presence of SARS-CoV-2-specific cytotoxic T cells that rapidly cleared infected cells. The rapidity of clearance implies that shedding of live viruses may be attenuated as a result of vaccination and thus the vaccine has the potential to prevent transmission of virus by infected individuals. Clinical trials of hAd5 S-Fusion + N-ETSD are ongoing. The hAd5 S-Fusion + N-ETSD subcutaneous prime/boost vaccine has completed Phase 1 clinical trials and Phase 2/3 trials are actively recruiting. The thermally-stable oral vaccine will enter Phase 1 trials as a prime and boost, as well as explored to provide a boost to subcutaneous vaccination.

Molecular dynamic simulation reveals E484K mutation enhances spike RBD-ACE2 affinity and the combination of E484K, K417N and N501Y mutations (501Y.V2 variant) induces conformational change greater than N501Y mutant alone, potentially resulting in an escape mutant

“…Although not the subject of the investigation described here, we are developing a dual-antigen human adenovirus serotype 5 (Ad5) platform-based vaccine that delivers both a spike protein with a linker to increase cell surface expression and humoral responses (S-Fusion) and the highly antigenic and conserved nucleocapsid (N) protein with a signal sequence (an Enhanced T-cell Stimulation Domain, ETSD) to direct it to subcellular compartments that enhance MHC I and II responses 18 . It is our belief that the vaccine, hAd5 S-Fusion + N-ETSD, due to its ability to elicit cell-mediated in addition to humoral immune responses, as shown in both a rodent model 19 and non-human primates 20 , offers hope to those regions such as South Africa wherein dangerous variants of SARS-CoV-2 have swept the country.…”

A recombinant ‘ACE2 Triple Decoy’ that traps and neutralizes SARS-CoV-2 shows enhanced affinity for highly transmissible SARS-CoV-2 variants

“…16,41–43 We anticipate going forward into our next studies using the dimeric IgA 44 fusion protein decoy expressed by the human adenovirus serotype 5 E1, E2b, E3 deleted (hAd5 [E1-, E2b-, E3-]) platform that we have used successfully in our vaccine development. 45,46 This platform can readily be used to generate oral and/or intranasal formulations to further facilitate delivery. Our ACE2 Triple Decoy delivered in vivo using the hAd5 platform is anticipated to overcome barriers to successful delivery and will be tested in animal models of SARS-CoV-2 infection in future studies.…”

Prime hAd5 Spike + Nucleocapsid Vaccination Induces Ten-Fold Increases in Mean T-Cell Responses in Phase 1 Subjects that are Sustained Against Spike Variants

“…Here we show that our strategy of including N-ETSD, confirmed to be directed to the endo-/lysosomal compartment with S optimized for cell-surface display 1,47 results in recognition of the…”