Adenosine deaminase augments SARS-CoV-2 specific cellular and humoral responses in aged mouse models of immunization and challenge

Abstract

Despite numerous clinically available vaccines and therapeutics, aged patients remain at increased risk for COVID-19 morbidity. Furthermore, various patient populations, including the aged can have suboptimal responses to SARS-CoV-2 vaccine antigens. Here, we characterized vaccine-induced responses to SARS-CoV-2 synthetic DNA vaccine antigens in aged mice. Aged mice exhibited altered cellular responses, including decreased IFNγ secretion and increased TNFα and IL-4 secretion suggestive of T_H2-skewed responses. Aged mice exhibited decreased total binding and neutralizing antibodies in their serum but significantly increased T_H2-type antigen-specific IgG1 antibody compared to their young counterparts. Strategies to enhance vaccine-induced immune responses are important, especially in aged patient populations. We observed that co-immunization with plasmid-encoded adenosine deaminase (pADA)enhanced immune responses in young animals. Ageing is associated with decreases in ADA function and expression. Here, we report that co-immunization with pADA enhanced IFNγ secretion while decreasing TNFα and IL-4 secretion. pADA expanded the breadth and affinity SARS-CoV-2 spike-specific antibodies while supporting T_H1-type humoral responses in aged mice. scRNAseq analysis of aged lymph nodes revealed that pADA co-immunization supported a T_H1 gene profile and decreased FoxP3 gene expression. Upon challenge, pADA co-immunization decreased viral loads in aged mice. These data support the use of mice as a model for age-associated decreased vaccine immunogenicity and infection-mediated morbidity and mortality in the context of SARS-CoV-2 vaccines and provide support for the use of adenosine deaminase as a molecular adjuvant in immune-challenged populations.

Keywords: DNA vaccine; SARS; adenosine deaminase (ADA); adjuvant; age.

Figure 1

Aged mice display altered immune responses to SARS-CoV-2 DNA antigens. (A) 6-8 week-old “young” or 68-72 week-old “aged” C57BL/6 mice were immunized twice, separated by four weeks with 10ug of synthetic DNA plasmid encoding SARS-CoV-2 spike glycoprotein containing the D>G mutation at position 614 via electroporation and immune responses were assessed at day 14 post-2nd immunization. (B) IFNγ spot-forming units (SFU) in splenocytes as measured by ELISpot assay. SARS-CoV-2 parental spike RBD-specific endpoint titers in serum (C). Serum pseudovirus neutralization against parental (USA-WA1/2020) (D) and Omicron VOC (B.1.1.529/BA.1) spike-pseudotyped viruses (E). Serum live virus neutralization against parental (USA-WA1/2020) (F) and Delta VOC (B.1.617) spike-pseudotyped viruses (G). (H) 6-8 week-old “young” or 52-60 week-old “aged” BALB/c mice were immunized as in A, rested, transduced intranasally with a human ACE2-expressing modified AAV vector, and challenged intranasally with 1x105 PFU of parental SARS-CoV-2 (VIDO-01). Viral load was quantified in the lungs at day 4 post-challenge by qPCR (I). Bars represent the mean, symbols represent the mean of duplicate assay per animal, and error bars represent the SEM. Data are representative of two (A–G) or one independent experiment (H, I) with N= 5-10 mice per group. ns, not significant. *P<0.05, **P<0.01, and ****P<0.0001 by Kruskal-Wallis ANOVA.

Figure 2

pADA co-immunization supports enhanced antigen-specific effector function and inhibits age-associated inflammatory cytokine secretion. Mice were immunized with 10ug of spike-encoding plasmid DNA (pS) or co-immunized with 10ug od pS and 10ug of DNA plasmid encoding mouse adenosine deaminase (+pADA) twice, separated by four weeks and cellular responses were evaluated at day 14 post-second immunization (A). Interferon gamma spot-forming units in spleens (B) and lungs (C); representative IFNγ wells (D) as measured by ELISpot assay. Frequency of TNFα⁺ CD8⁺ (E) and CD4⁺ T cells in spleens (F). Frequency of IL-4+ CD4⁺ T cells (G), and the ratio of IFNγ⁺ to IL-4⁺ CD8⁺ T cells (H). Data are representative of three independent experiments with N=5 per group (B, C) or one experiment with N=5/group (E–H). Symbols represent individual animals, bars represent the mean, error bars represent the standard deviations. ns, not significant. *P<0.05, **P<0.01, and ****P<0.0001 by Mann-Whitney-U test.

Figure 3

pADA co-immunization enhances SARS-CoV-2 humoral responses and promotes long-lived antibody responses in aged mice. Mice were immunized as in Figure 2 and antibody responses were evaluated 12-14 days post-2^nd immunization (A). (B) Serum binding, (C) pseudovirus neutralization, and (D) live virus neutralization against SARS-CoV-2 parental (USA-WA1/2020). Serum binding IgG1 (E), IgG2c (F), and the ratio of IgG2c:IgG1 (G). Serum antibody affinity as measured by surface plasmon resonance against parental SARS-CoV-2 S1 (H), RBD (I), and S2 (J) spike subunits. Omicron (B.1.529/BA.1) (K), BA.2 (L) and BA.4/5 (M) RBD Serum binding endpoint titers. Mice were immunized twice separated by four weeks with pS alone or co-immunized with pS and pADA as in A, and antibody responses were evaluated 72 days post-2nd immunization (N). (O) Serum binding, (P) pseudovirus neutralization, and (Q) live virus neutralization against SARS-CoV-2 parental (USA-WA1/2020) at 6072 days post second immunization. Data are representative of one experiment. Symbols represent the mean of duplicate assays for individual animals, bars represent the geometric mean, error bars represent the geometric standard deviation. *P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001 by Mann-Whitney-U test.

Figure 4

pADA enhances protection from mouse-adapted SARS-CoV-2 challenge in aged mice. Young (6-12 week-old, black circles), and adult (24-30 week-old, gray-filled circles) C57BL/6 mice were immunized twice as in Figure 2 and challenged with 1x10⁵ plaque-forming units of mouse-adapted SARS-CoV-2 (MA-SAR2) on day 19 post-challenge and morbidity and mortality were monitored until day 7 post-challenge (A). Parental SARS-CoV-2 (USA-WA1/2020) receptor-binding domain (RBD)-specific IgG endpoint titers at day 10 post-2^nd immunization (B). Survival curves for all animals (C), and weight loss among empty plasmid immunized (pVax) (D), pS-only immunized (E), and pADA co-immunized (F) mice. SARS-CoV-2 nucleoprotein N1 (G) and N2 (H) RNA in the lungs of animals at day 7 post-challenge. Data are representative of a single experiment with N=5 (12 and 24 wk-old) and N=10 (66-68wk old) mice per group. Symbols represent the mean of duplicate (B) or triplicate (G, H) assays for individual animals, bars represent group mean and error bars represent the SEM. Symbols represent individual animals (C–F). ns, not significant. **P<0.01 and ****P<0.0001 by Kruskall-Wallis ANOVA (C) or Mann-Whitney-U test between indicated groups (B, D–H).

Figure 5

pADA co-immunization supports protection in a human ACE2tg aging mouse SARS-CoV-2 challenge model. Young (6-12 week-old) or aged (52-68 week-old) human ACE2 transgenic mice were immunized twice as in Figure 2 and challenged with 1x10⁵ plaque-forming units of SARS-CoV-2 (VIDO-01) on day 19 post-challenge and morbidity and mortality were monitored until day 15 post-challenge (A). SARS-CoV-2 lung viral loads as measured by TCID50 assay among young (B), aged (C), and (D) all mice including survivors(triangles) and non-survivors (circles). Weight loss among empty-plasmid (pVax) (E), pS-only (F), and pADA co-immunized (G) young animals, and overall survival among young animals (H). Weight loss among empty-plasmid (pVax) (I), pS-only (J), and pADA co-immunized (K) aged animals, and overall survival among aged animals (L). Symbols represent the mean of triplicate assays for individual animals (B–D) or weights of individual animals (E–G, I–K). Lines represent the group probability of survival (H, L). ns, not significant. *P<0.05 and **P<0.01, by Kruskal-Walis ANOVA.

Figure 6

pADA co-immunization corrects age-associated increases in proinflammatory and pro-regulatory gene signatures. Mice were immunized twice with pS alone or co-immunized with pADA as in Figure 2 and popliteal and inguinal lymph nodes from 10 mice per immunization condition were pooled and subjected to single-cell RNA sequencing (scRNAseq) and ingenuity pathway analysis (IPA). IPA analysis of differentially regulated pathways in aged lymph nodes as compared to young lymph nodes following pS-only immunization among CD8⁺ T cells (A) and CD4⁺ T cells (B). IPA analysis of differentially regulated pathways in aged lymph nodes as compared to young lymph nodes following pS and pADA co-immunization among CD8⁺ T cells (C) and CD4⁺ T cells (D). (E) Select differentially expressed genes in aged animals compared to young animals among follicular helper T (T_FH) cells. (F) Select differentially expressed genes in aged animals compared to young animals among T regulatory (T_Reg) cells. (G) Select differentially expressed genes in the dendritic cell (DC) compartment of aged animals among immunization groups.