Heterologous Vaccination with SARS-CoV-2 Spike saRNA Prime followed by DNA Dual-Antigen Boost Induces Robust Antibody and T-Cell Immunogenicity against both Wild Type and Delta Spike as well as Nucleocapsid Antigens

We assessed if immune responses are enhanced in CD-1 mice by heterologous vaccination with two different nucleic acid-based COVID-19 vaccines: a next-generation human adenovirus serotype 5 (hAd5)-vectored dual-antigen spike (S) and nucleocapsid (N) vaccine (AdS+N) and a self-amplifying and -adjuvanted S RNA vaccine (SASA S) delivered by a nano-lipid carrier. The AdS+N vaccine encodes S modified with a fusion motif to increase cell-surface expression. The N antigen is modified with an Enhanced T-cell Stimulation Domain (N-ETSD) to direct N to the endosomal/lysosomal compartment and increase MHC class I and II stimulation potential. The S sequence in the SASA S vaccine comprises the D614G mutation, two prolines to stabilize S in the prefusion conformation, and 3 glutamines in the furin cleavage region to increase cross-reactivity across variants. CD-1 mice received vaccination by homologous and heterologous prime > boost combinations. Humoral responses to S were the highest with any regimen including the SASA S vaccine, and IgG bound to wild type and Delta (B.1.617.2) variant S1 at similar levels. An AdS+N boost of an SASA S prime particularly enhanced both CD4+ and CD8+ T-cell responses to both wild type and Delta S peptides relative to all other vaccine regimens. Sera from mice receiving SASA S homologous or heterologous vaccination were found to be highly neutralizing of all pseudovirus strains tested: Wuhan, Beta, Delta, and Omicron strain. The findings here support the clinical testing of heterologous vaccination by an SASA S > AdS+N regimen to provide increased protection against emerging SARS-CoV-2 variants.

Rapid Identification of Neutralizing Antibodies against SARS-CoV-2 Variants by mRNA Display

The increasing prevalence of SARS-CoV-2 variants with the ability to escape existing humoral protection conferred by previous infection and/or immunization necessitates the discovery of broadly-reactive neutralizing antibodies (nAbs). Utilizing mRNA display, we identified a set of antibodies against SARS-CoV-2 spike (S) proteins and characterized the structures of nAbs that recognized epitopes in the S1 subunit of the S glycoprotein. These structural studies revealed distinct binding modes for several antibodies, including targeting of rare cryptic epitopes in the receptor-binding domain (RBD) of S that interacts with angiotensin- converting enzyme 2 (ACE2) to initiate infection, as well as the S1 subdomain 1. A potent ACE2-blocking nAb was further engineered to sustain binding to S RBD with the E484K and L452R substitutions found in multiple SARS-CoV-2 variants. We demonstrate that mRNA display is a promising approach for the rapid identification of nAbs that can be used in combination to combat emerging SARS-CoV-2 variants.

Single Prime hAd5 Spike (S) + Nucleocapsid (N) Dual Antigen Vaccination of Healthy Volunteers Induces a Ten-Fold Increase in Mean S- and N- T-Cell Responses Equivalent to T-Cell Responses from Patients Previously Infected with SARS-CoV-2

In response to the need for a safe, efficacious vaccine that elicits vigorous T cell as well as humoral protection against SARS-CoV-2 infection, we have developed a dual-antigen COVID-19 vaccine comprising both the viral spike (S) protein modified to increase cell-surface expression (S-Fusion) and nucleocapsid (N) protein with an Enhanced T-cell Stimulation Domain (N-ETSD) to enhance MHC class I and II presentation and T-cell responses. The antigens are delivered using a human adenovirus serotype 5 (hAd5) platform with E1, E2b, and E3 regions deleted that has been shown previously in cancer vaccine studies to be safe and effective in the presence of pre-existing hAd5 immunity. The findings reported here are focused on human T-cell responses due to the likelihood that such responses will sustain efficacy against emerging variants, a hypothesis supported by our in silico prediction of T-cell epitope HLA binding for both the first-wave SARS-CoV-2 A strain and the B.1.351 strain K417N, E484K, and N501Y spike and T201I N variants. We demonstrate the hAd5 S-Fusion + N-ETSD vaccine antigens expressed by previously SARS-CoV-2-infected patient dendritic cells elicit Th1 dominant activation of autologous patient T cells, indicating the vaccine antigens have the potential to elicit immune responses in previously infected patients. For participants in our open-label Phase 1b study of the vaccine (NCT04591717; https://clinicaltrials.gov/ct2/show/NCT04591717), the magnitude of Th-1 dominant S- and N-specific T-cell responses after a single prime subcutaneous injection were comparable to T-cell responses from previously infected patients. Furthermore, vaccinated participant T-cell responses to S were similar for A strain S and a series of spike variant peptides, including S variants in the B.1.1.7 and B.1.351 strains. The findings that this dual-antigen vaccine elicits SARS-CoV-2-relevant T-cell responses and that such cell-mediated protection is likely to be sustained against emerging variants supports the testing of this vaccine as a universal booster that would enhance and broaden existing immune protection conferred by currently approved S-based vaccines.

The Dual-Antigen Ad5 COVID-19 Vaccine Delivered as an Intranasal Plus Subcutaneous Prime Elicits Th1 Dominant T-Cell and Humoral Responses in CD-1 Mice

In response to the need for an efficacious, thermally-stable COVID-19 vaccine that can elicit both humoral and cell-mediated T-cell responses, we have developed a dual-antigen human adenovirus serotype 5 (hAd5) COVID-19 vaccine in formulations suitable for subcutaneous (SC), intranasal (IN), or oral delivery. The vaccine expresses both the SARS-CoV-2 spike (S) and nucleocapsid (N) proteins using an hAd5 platform with E1, E2b, and E3 sequences deleted (hAd5[E1-, E2b-, E3-]) that is effective even in the presence of hAd5 immunity. In the vaccine, S is modified (S-Fusion) for enhanced cell-surface display to elicit humoral responses and N is modified with an Enhanced T-cell Stimulation Domain (N-ETSD) to direct N to the endosomal/lysosomal pathway to increase MHC I and II presentation. Initial studies using subcutaneous (SC) prime and SC boost vaccination of CD-1 mice demonstrated that the hAd5 S-Fusion + N-ETSD vaccine elicits T-helper cell 1 (Th1) dominant T-cell and humoral responses to both S and N. We then compared SC to IN prime vaccination with either an SC or IN boost post-SC prime and an IN boost after IN prime. These studies reveal that IN prime/IN boost is as effective at generating Th1 dominant humoral responses to both S and N as the other combinations, but that the SC prime with either an IN or SC boost elicits greater T cell responses. In a third study to assess the power of the two routes of delivery when used together, we used a combined SC plus IN prime with or without a boost and found the combined prime alone to be as effective as the combined prime with either an SC or IN boost in generating both humoral and T-cell responses. The findings here in CD-1 mice demonstrate that combined SC and IN prime-only delivery has the potential to provide broad immunity - including mucosal immunity - against SARS-CoV-2 and supports further testing of this delivery approach in additional animal models and clinical trials.

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

The highly-transmissible SARS-CoV-2 variants now replacing the first wave strain pose an increased threat to human health by their ability, in some instances, to escape existing humoral protection conferred by previous infection, neutralizing antibodies, and possibly vaccination. Thus, other therapeutic options are necessary. One such therapeutic option that leverages SARS-CoV-2 initiation of infection by binding of its spike receptor binding domain (S RBD) to surface-expressed host cell angiotensin-converting enzyme 2 (ACE2) is an ACE2 decoy that would trap the virus by competitive binding and thus inhibit propagation of infection. Here, we used Molecular Dynamic (MD) simulations to predict ACE2 mutations that might increase its affinity for S RBD and screened these candidates for binding affinity in vitro. A double mutant ACE2(T27Y/H34A)-IgG1FC fusion protein was found to have very high affinity for S RBD and to show greater neutralization of SARS-CoV-2 in a live virus assay as compared to wild type ACE2. We further modified the double mutant ACE2 decoy by addition of an H374N mutation to inhibit ACE2 enzymatic activity while maintaining high S RBD affinity. We then confirmed the potential efficacy of our ACE2(T27Y/H34A/H374N)-IgG1FC Triple Decoy against S RBD expressing variant-associated E484K, K417N, N501Y, and L452R mutations and found that our ACE2 Triple Decoy not only maintains its high affinity for S RBD expressing these mutations, but shows enhanced affinity for S RBD expressing the N501Y or L452R mutations and the highest affinity for S RBD expressing both the E484K and N501Y mutations. The ACE2 Triple Decoy also demonstrates the ability to compete with wild type ACE2 in the cPass surrogate virus neutralization in the presence of S RBD with these mutations. Additional MD simulation of ACE2 WT and decoy interactions with S RBD WT or B.1.351 variant sequence S RBD provides insight into the enhanced affinity of the ACE2 decoy for S RBD and reveals its potential as a tool to predict affinity and inform therapeutic design. The ACE2 Triple Decoy is now undergoing continued assessment, including expression by a human adenovirus serotype 5 (hAd5) construct to facilitate delivery in vivo. Summary sentenceAn ACE2(N27Y/H34A/H374N)-IgG1FC fusion protein decoy sustains high affinity to all SARS-CoV-2 spike receptor binding domain (RBD) protein variants tested, shows enhanced affinity for the N501Y and L452R variants, and the highest affinity for combined N501Y and E484K variants.