ABSTRACT
Background: Currently available COVID-19 vaccination regimens in the US deliver either a homologous spike (S) mRNA prime-boost or a prime-only S DNA adenovirus-vectored antigen to elicit humoral and cell-mediated responses to confer protection against SAR-CoV-2 infection. Alternatively, heterologous vaccination using two different platforms has the potential to enhance and expand immune protection. Addition of a second SARS-CoV-2 antigen, the nucleocapsid (N) protein that is less subject to mutation and elicits vigorous T-cell responses, may also be advantageous. We report immunological responses to homologous and heterologous prime-boost vaccination regimens with a human DNA adenovirus serotype 5 S plus N (AdS+N) and/or a self-amplifying S-only mRNA vaccine (AAAH) delivered with a nanostructured lipid carrier (NLC). Methods: CD-1 mice received homologous or heterologous prime-boost combinations of AdS+N and AAAH. Priming doses were administered on Day 0, booster doses were delivered on Day 21, and mice were euthanized for blood and organ collection on Day 35. Serum was analyzed for anti-S (both wild type and variant) and anti-N IgG subtypes by ELISA. Spleen-resident CD4+ and CD8+ T cells were tested for IFN-γ, TNF-α, and IL-2 production in response to S-WT, S Delta variant and N protein overlapping peptides by intracellular cytokine staining (ICS). Splenocyte cytokine secretion upon stimulation with S-WT/N peptides was also assessed by IFN-γ and IL-4 ELISpot. Serum neutralization of the original Wuhan strain, Delta, and B.1.351 variants was assessed by a pseudovirus neutralization assay. Results: The highest humoral and T-cell responses were seen with the heterologous AAAH prime-AdS+N boost regimen, with a significant increase in T-cell responses relative to homologous vaccination. S protein-binding IgG was similar between wild type and Delta variant S proteins, with a strong/clear Th1/Th2 bias, and T cells responded to S wild type and S Delta peptides with similar levels of cytokine expression. Sera from AAAH prime-AdS+N boost mice showed the ability to neutralize Wuhan D614G, Delta, and B.1.351 (South Africa) variant pseudoviruses at high levels. Conclusion: Heterologous vaccination with the AAAH RNA vaccine prime and an AdS+N DNA boost may provide substantially improved humoral and cell-based immunity against SARS-CoV-2 variants by leveraging the advantages of each vaccine platform technology and by inclusion of immune responses to N.
ABSTRACT
Background: SARS-CoV-2 infects the host by binding of its spike receptor binding domain (S RBD) to angiotensin converting enzyme 2 (ACE2) on the surface of cells in the respiratory tract and gut;thus therapeutics that prevent this interaction are of interest because they have the potential to inhibit propagation of infection. A recombinant 'ACE2 Decoy' that competitively binds S RBD and neutralizes SARS-CoV-2 infection represents such a therapeutic approach. The impact of S RBD mutations found in the rapidly spreading UK, South African, and California SARS-CoV-2 variants on ACE2 Decoy affinity is an important factor in design. Here, we identified a high-affinity SARS-CoV-2-neutralizing ACE2 Decoy that maintains its high affinity against RBD with mutations found in emerging variants. Methods: We used Molecular Dynamic (MD) simulation of S RBD-ACE2 interactions to predict ACE2 residues that if mutated, might increase affinity for S RBD and thus successfully compete with endogenous ACE2 for binding. Recombinant ACE2-IgG1Fc and-IgAFc fusion proteins expressing mutations predicted to increase S RBD binding affinity were produced, purified, and screened for binding affinities against wild type S RBD and S RBD expressing E484K, K417N, N501Y, or L452R alone and in combinations of: K417N/N501Y;E484K/N501Y;K417N/E484K;and K417N/E484K/N501Y (found in the South African variant). The ability of the Decoy with the highest affinity to neutralize SARS-CoV-2 infection was determined by a live virus assay using Vero E6 cells. An additional mutation (H374N) to inhibit enzymatic activity of ACE2 was added to the Decoy with the highest affinity from mutation screening. Results: Eleven of the ACE2 mutations predicted to increase affinity for S RBD were tested, revealing the ACE2 Decoy with T27Y and H34A mutations to have the highest S RBD affinity and the ability to neutralize SARS-CoV-2 infection of cells. Both the ACE2 wild type (WT)-IgG1Fc and the ACE2(T27Y/H34A/H374N)-IgG1Fc Decoys showed enhanced binding affinity for S RBD with either N501Y and L452R mutations alone. Interestingly, the South African variant K417N mutation alone decreased affinity, but not in combination with the N501Y mutation. Conclusion: The potential efficacy of the ACE2 (T27Y/H34A/H374N)-IgG1FC Decoy is not decreased by the UK, South African, or California variant mutations and will undergo testing in animal models likely by expression using a human adenovirus (hAd5) construct to enhance stability.
ABSTRACT
Background: To address the need for an efficacious COVID-19 vaccine suitable for world-wide distribution, we have developed a dual-antigen vaccine incorporating genes for a modified SARS-CoV-2 spike protein (S-Fusion) and the viral nucleocapsid protein (N) with an Enhanced T-cell Stimulation Domain (N-ETSD) with the potential to increase MHC class I/II responses. The adenovirus serotype 5 platform used, hAd5 [E1-, E2b-, E3-] can be delivered in an oral formulation that overcomes cold-chain limitations. The hAd5 S-Fusion + N-ETSD vaccine was evaluated in rhesus macaques to determine both humoral and cell-mediated responses to vaccination, and protection from subsequent SARS-CoV-2 challenge. Methods: Non-human primates (NHP) received either a subcutaneous (SC) prime and two oral boosts at 2-week intervals, or one SC and one oral boost (each group n = 5). There was also a placebo group (n = 2). Humoral responses to spike (S) were determined by ELISA and T-cell responses to S and nucleocapsid (N) by ELISpot. Neutralization capability of sera was assessed by a surrogate assay and by a microneutralization assay. After SARS-CoV-2 challenge of 10e6 TCID50, genomic RNA (gRNA) and subgenomic RNA (sgRNA) were determined in nasal swab and bronchoalveolar lavage (BAL) samples by RT qPCR. Results: In response to hAd5 S-Fusion + N-ETSD vaccination, NHP generated SARS-CoV-2-neutralizing anti-spike (S) antibodies and demonstrated T-cell activation by both S and nucleocapsid (N). Both the subcutaneous (SC) prime followed by two oral boosts or an SC and oral boost protected the upper and lower respiratory tracts of non-human primates from high titer SARS-CoV-2 challenge. Notably, inhibition of viral replication began within 24 hours of challenge in both lung and nasal passages, becoming undetectable within 7 days post-challenge. Rapidly enhanced neutralization capability of sera in the two weeks after challenge suggests the presence of memory B cells that were activated by infection. Conclusion: The hAd5 S-Fusion + N-ETSD vaccine, when give as a subcutaneous prime with oral boosts, protects rhesus macaques from subsequent viral challenge. The decrease in subgenomic RNA seen at the first time point for sample collection post-challenge (24 hours) provides evidence that protection was almost immediate. The thermally-stable oral form of the vaccine has the potential to facilitate global distribution of vaccines, especially in developing nations.