DNA vaccine protection against SARS-CoV-2 in rhesus macaques

Abstract

The global coronavirus disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has made the development of a vaccine a top biomedical priority. In this study, we developed a series of DNA vaccine candidates expressing different forms of the SARS-CoV-2 spike (S) protein and evaluated them in 35 rhesus macaques. Vaccinated animals developed humoral and cellular immune responses, including neutralizing antibody titers at levels comparable to those found in convalescent humans and macaques infected with SARS-CoV-2. After vaccination, all animals were challenged with SARS-CoV-2, and the vaccine encoding the full-length S protein resulted in >3.1 and >3.7 log₁₀ reductions in median viral loads in bronchoalveolar lavage and nasal mucosa, respectively, as compared with viral loads in sham controls. Vaccine-elicited neutralizing antibody titers correlated with protective efficacy, suggesting an immune correlate of protection. These data demonstrate vaccine protection against SARS-CoV-2 in nonhuman primates.

Fig. 1. Construction of candidate DNA vaccines against SARS-CoV-2.

(A) Six DNA vaccines were produced expressing different SARS-CoV-2 spike (S) variants: (i) full length (S), (ii) deletion of the cytoplasmic tail (S.dCT), (iii) deletion of the transmembrane (TM) domain and cytoplasmic tail (CT) reflecting the soluble ectodomain (S.dTM), (iv) S1 domain with a foldon trimerization tag (S1), (v) receptor-binding domain with a foldon trimerization tag (RBD), and (vi) prefusion-stabilized soluble ectodomain with deletion of the furin cleavage site, two proline mutations, and a foldon trimerization tag (S.dTM.PP). Open squares depict foldon trimerization tags; red lines depict proline mutations. (B) Western blot analyses for expression from DNA vaccines encoding S (lane 1), S.dCT (lane 2), S.dTM (lane 3), and S.dTM.PP (lane 4) in cell lysates and culture supernatants using an anti-SARS polyclonal antibody (BEI Resources). (C) Western blot analyses for expression from DNA vaccines encoding S1 (lane 1) and RBD (lane 2) in cell lysates using an anti–SARS-CoV-2 RBD polyclonal antibody (Sino Biological).

Fig. 2. Humoral immune responses in vaccinated rhesus macaques.

(A to C) Humoral immune responses were assessed after immunization by (A) binding antibody ELISA, (B) pseudovirus neutralization assays, and (C) live virus neutralization assays. (D) Comparison of pseudovirus neutralization titers in vaccinated macaques (all animals as well as the S and S.dCT groups), a cohort of 9 convalescent macaques, and a cohort of 27 convalescent humans from Boston, United States, who had recovered from SARS-CoV-2 infection. NHP, nonhuman primates. (E) S- and RBD-specific antibody-dependent neutrophil phagocytosis (ADNP), antibody-dependent complement deposition (ADCD), antibody-dependent monocyte cellular phagocytosis (ADCP), and antibody-dependent NK cell activation (IFN-γ secretion, CD107a degranulation, and MIP-1β expression) are shown. Radar plots show the distribution of antibody features across the vaccine groups. The size and color intensity of the wedges indicate the median of the feature for the corresponding group (blue depicts antibody functions; red depicts antibody isotype, subclass, and FcγR binding). The principal components analysis (PCA) plot shows the multivariate antibody profiles across groups. Each dot represents an animal, the color of the dot denotes the group, and the ellipses show the distribution of the groups as 70% confidence levels assuming a multivariate normal distribution. In the dot plots above, red bars reflect median responses, and dotted lines reflect assay limits of quantitation.

Fig. 3. Cellular immune responses in vaccinated rhesus macaques.

At week 5 after immunization, cellular immune responses were assessed by (A) IFN-γ ELISPOT assays and (B) IFN-γ+ and (C) IL-4+ intracellular cytokine staining assays for CD4+ and CD8+ T cells in response to pooled S peptides. Red bars reflect median responses; dotted lines reflect assay limits of quantitation.

Fig. 4. Viral loads in rhesus macaques challenged with SARS-CoV-2 virus.

Rhesus macaques were challenged via the intranasal and intratracheal routes with 1.2 × 10⁸ VPs (1.1 × 10⁴ PFUs) of SARS-CoV-2. (A) Log₁₀ sgmRNA copies per milliliter or copies per swab (limit 50 copies) were assessed in bronchoalveolar lavage (BAL) and nasal swabs (NS) in sham controls at multiple time points after challenge. (B) Log₁₀ sgmRNA copies per milliliter in BAL and (C) log₁₀ sgmRNA copies per swab in NS in vaccinated animals at multiple time points after challenge. (D) Summary of peak viral loads in BAL and NS after challenge. Peak viral loads occurred variably on days 1 to 4 after challenge. Red lines reflect median viral loads. P values indicate two-sided Mann-Whitney tests.

Fig. 5. Immune correlates of protection.

(A and B) Correlations of (A) pseudovirus NAb titers and (B) live NAb titers before challenge with log peak sgmRNA copies per milliliter in BAL or log peak sgmRNA copies per swab in nasal swabs after challenge. Red lines reflect the best-fit relationship between these variables. P and R values reflect two-sided Spearman rank-correlation tests. (C) The heatmap (top) shows the Spearman and Pearson correlations between antibody features and log₁₀ peak sgmRNA copies per milliliter in BAL (*q < 0.05, **q < 0.01, ***q < 0.001 with Benjamini-Hochberg correction for multiple testing). The bar graph (bottom left) shows the rank of the Pearson correlation between cross-validated model predictions and data using the most predictive combination or individual antibody features for partial least squares regression (PLSR) and random forest regression (RFR). Error bars indicate SEs. The correlation heatmap (bottom right) represents pairwise Pearson correlations between features across all animals. (D) The heatmap (top) shows the difference in the means of the z-scored features between the completely protected and partially protected animals (**q < 0.01 with Benjamini-Hochberg correction for multiple testing). The dot plots show differences in log₁₀ NAb titers, RBD-specific ADCD responses, S-specific ADCD responses, and RBD-specific ADCP responses between the completely protected and partially protected animals. P values indicate two-sided Mann-Whitney tests.