Low pre-existing endemic human coronavirus (HCoV-NL63)-specific T cell frequencies are associated with impaired SARS-CoV-2-specific T cell responses in people living with HIV

Abstract

Background: Understanding how HIV affects SARS-CoV-2 immunity is crucial for managing COVID-19 in sub-Saharan populations due to frequent coinfections. Our previous research showed that unsuppressed HIV is associated with weaker immune responses to SARS-CoV-2, but the underlying mechanisms are unclear. We investigated how pre-existing T cell immunity against an endemic human coronavirus HCoV-NL63 impacts SARS-CoV-2 T cell responses in people living with HIV (PLWH) compared to uninfected individuals, and how HIV-related T cell dysfunction influences responses to SARS-CoV-2 variants.

Methods: We used flow cytometry to measure T cell responses following PBMC stimulation with peptide pools representing beta, delta, wild-type, and HCoV-NL63 spike proteins. Luminex bead assay was used to measure circulating plasma chemokine and cytokine levels. ELISA and MSD V-PLEX COVID-19 Serology and ACE2 Neutralization assays were used to measure humoral responses.

Results: Regardless of HIV status, we found a strong positive correlation between responses to HCoV-NL63 and SARS-CoV-2. However, PLWH exhibited weaker CD4⁺ T cell responses to both HCoV-NL63 and SARS-CoV-2 than HIV-uninfected individuals. PLWH also had higher proportions of functionally exhausted (PD-1high) CD4⁺ T cells producing fewer proinflammatory cytokines (IFNγ and TNFα) and had elevated plasma IL-2 and IL-12(p70) levels compared to HIV-uninfected individuals. HIV status didn't significantly affect IgG antibody levels against SARS-CoV-2 antigens or ACE2 binding inhibition activity.

Conclusion: Our results indicate that the decrease in SARS-CoV-2 specific T cell responses in PLWH may be attributable to reduced frequencies of pre-existing cross-reactive responses. However, HIV infection minimally affected the quality and magnitude of humoral responses, and this could explain why the risk of severe COVID-19 in PLWH is highly heterogeneous.

Keywords: COVID-19; HCoV-NL63; HIV; SARS-CoV-2; T-cell response; antibody response.

Figure 1

Comparison of HCoV-specific T cells in convalescent HIV-infected and HIV-uninfected individuals and healthy controls. Blood samples collected between 1- and 28-days post infection during the second (beta) and third (delta) waves were used. Intracellular cytokine staining (ICS) was performed to detect cytokine-producing T cells to HCoV overlapping peptide pools in HIV-uninfected (HIV-/SARS-CoV-2+, n = 47) and PLWH (HIV+/SARS-CoV-2+, n = 20) individuals and healthy controls (HIV-/SARS-CoV-2-; HC, n = 11). (A) Representative flow cytometry plots for the identification of antigen-specific CD4⁺ and CD8⁺ T cells based on expression IFNγ and TNF-α, following 18-h stimulation with HCoV peptides pools. (B) Summary plots showing the frequency of HCoV-specific CD4⁺ and CD8⁺ T cells (IFNγ⁺ and TNF-α⁺). (C) Representative flow cytometry plots for the identification of antigen-specific CD4⁺ and CD8⁺ T cells based on expression of IFNγ and TNF-α, following 18-h stimulation with SARS-CoV-2 peptides pools. (D) Summary plots showing the frequency of SARS-CoV-2-specific CD4⁺ and CD8⁺ T cells (IFNγ⁺ and TNF-α⁺). (E) Correlation of HCoV-specific and SARS-CoV-2-specific CD4⁺ T cells in HIV-infected and HIV-uninfected individuals based on expression of IFNγ and TNF-α. (F) Correlation of HCoV-specific and SARS-CoV-2-specific CD8⁺ T cells in HIV-infected and HIV-uninfected individuals based on expression of IFNγ and TNF-α. Significance was determined by two-tailed Mann-Whitney test and the two-tailed nonparametric Spearman test was used for correlation analysis, p< 0.05 was considered statistically significant. *p< 0.05, **p< 0.01, ***p< 0.001. ‘ns’ not significant.

Figure 2

Comparison of the activation and exhaustion profile of HCoV-specific CD4⁺ and CD8⁺ T cells in COVID-19 convalescent HIV-infected and HIV-uninfected Individuals and healthy controls. Blood samples collected between 1- and 28-days post infection during the second (beta) and third (delta) waves were used to detect activated and exhausted T cells in HIV-uninfected (HIV-, n = 46), PLWH (HIV+, n = 20) and healthy controls (n = 11). (A) Representative flow cytometry plots for the identification of activated (HLA-DR⁺CD38⁺) CD4⁺ and CD8⁺ T cells. (B) Summary plots of the frequency of activated CD4⁺ and CD8⁺ T cells based on the expression of HLA-DR, CD38. Correlation of T cell activation of SARS-CoV-2 and HCoV-specific (C) CD4⁺ and (D) CD8⁺ T cells in HIV-infected and HIV-uninfected individuals. (E) Representative flow cytometry plots and (F) summary data for the identification of exhausted (PD-1⁺) CD4⁺ and CD8⁺ T cells. Significance was determined by two-tailed Mann-Whitney test and these two-tailed nonparametric Spearman test was used for correlation analysis, p< 0.05 was considered statistically significant. *p< 0.05, **p< 0.01, ***p< 0.001, ****p< 0.0001. ‘ns’ not significant.

Figure 3

Comparison of plasma cytokine and chemokine levels in cells in convalescent HIV-infected and HIV-uninfected individuals and healthy controls. Serum samples collected between 1- and 28-days post infection were used to measure cytokine and chemokine levels by the Bio-Plex assay. (A) Heatmap showing normalized cytokine and chemokine levels (in percentages) in convalescent HIV-infected (HIV+/S+, n = 5) and HIV-uninfected (HIV-/S+, n = 8) individuals and healthy controls (HIV-/S-, n = 8). Summary plots of (B) IL-1b, (C) IL-1ra, (D) IL-2, (E) IL-4, (F) IL-5, (G) IL-9, (H) IL-10, (I) IL-12(p70), (J) IL-13, (K) IL-17, (L) FGF basic, (M) G-CSF, (N) GM-CSF, (O) IFN-g, (P) MIP-1a and (Q) RANTES. Significance was determined by two-tailed Mann-Whitney test, p< 0.05 was considered statistically significant. *p< 0.05, **p< 0.01, ***p< 0.001. ‘ns’ not significant.

Figure 4

Comparison of IgG concentrations in convalescent HIV-infected and HIV-uninfected individuals. Serum samples collected between 1- and 28-days post infection were used to measure spike-specific responses by ELISA. (A) Comparison of anti-RBD IgG antibody OD values in convalescent HIV-infected (red bars, n = 10) and HIV-uninfected (green bars, n = 24) individuals. (B) Aggregate data of anti-RBD antibodies in HIV infected and HIV-uninfected individuals. (C) Correlation of anti-RBD antibodies and SARS-CoV-2 specific IFNγ secreting CD4⁺ and CD8⁺ T cells. (D) Correlation of anti-RBD antibodies and SARS-CoV-2 specific TNF-α secreting CD4⁺ and CD8⁺ T cells. The dotted line denotes OD values ≤ 0.7 that represent a negative ELISA test. ELISA tests are positive if the average OD value is > 0.7. Significance was determined by two-tailed Mann-Whitney test and the two-tailed nonparametric Spearman test was used for correlation analysis, p< 0.05 was considered statistically significant.

Figure 5

Comparison of anti-SARS-CoV-2 IgG antibodies and ACE2 blocking potential in healthy controls and COVID-19 convalescent HIV-infected and HIV-uninfected individuals. Serum samples collected between 1- and 22-days post infection were used to measure anti-SARS-CoV-2 IgG antibodies and ACE2 blocking in HIV-infected (HIV+, n = 6), HIV-uninfected (HIV-, n = 6) and healthy controls (n = 6) by the MSD V=Plex assays. (A) Summary plots of SARS-CoV-2-specific IgG antibody concentrations in the three groups. (B) Summary plots showing ACE2 blocking of SARS-CoV-2-specific antigens in the three groups.