Characterization of humoral and SARS-CoV-2 specific T cell responses in people living with HIV

Abstract

There is an urgent need to understand the nature of immune responses against SARS-CoV-2, to inform risk-mitigation strategies for people living with HIV (PLWH). Here we show that the majority of PLWH with ART suppressed HIV viral load, mount a detectable adaptive immune response to SARS-CoV-2. Humoral and SARS-CoV-2-specific T cell responses are comparable between HIV-positive and negative subjects and persist 5-7 months following predominately mild COVID-19 disease. T cell responses against Spike, Membrane and Nucleoprotein are the most prominent, with SARS-CoV-2-specific CD4 T cells outnumbering CD8 T cells. We further show that the overall magnitude of SARS-CoV-2-specific T cell responses relates to the size of the naive CD4 T cell pool and the CD4:CD8 ratio in PLWH. These findings suggest that inadequate immune reconstitution on ART, could hinder immune responses to SARS-CoV-2 with implications for the individual management and vaccine effectiveness in PLWH.

Fig. 1. Antibody response in HIV-positive and -negative donors recovered from COVID-19 disease.

a Seropositivity screen of plasma samples for antibodies against the external Spike antigen, using a recombinant Spike S1₁₋₅₃₀ subunit protein (S1), and against the full-length internal Nucleoprotein (N) antigen to confirm prior infection in HIV-negative and -positive donors. A sample absorbance >4-fold above the average background of the assay was regarded as positive. Black dots denote laboratory-confirmed cases and gray dots suspected/household contacts. b Comparison of S1 IgG and N IgG antibody titers in HIV-negative (n = 29) and c HIV-positive donors (n = 30). Red dots: hospitalized cases; black dots: mild (non-hospitalized cases); blue dots: asymptomatic cases. Plots show geometric mean. d Correlation between S1 IgG and N IgG titers in HIV-negative and -positive donors. e Neutralization titers in HIV-negative (n = 29) and -positive (n = 30) donors. Dotted lines indicate detection limit, minimum ID50 and potent levels >1000. f Proportion of HIV-negative (n = 29) and -positive (n = 30) donors with neutralizing antibodies within the given ranges. g Correlation between S1 IgG titers and neutralization titers in HIV-negative and -positive donors. The non-parametric Spearman test was used for correlation analysis. Two-tailed Mann–Whitney U test was used for group comparisons. *p < 0.05, **p < 0.01.

Fig. 2. Similar SARS-CoV-2-specific T-cell responses by IFN-γ-ELISpot in HIV-positive and -negative donors.

a Genome organization of SARS-CoV-2. b Dominance of the IFN-γ-ELISpot responses. Heatmap depicting the magnitude of the IFN-γ-ELISpot responses to the different SARS-CoV-2 peptide pools in HIV-negative and HIV-positive individuals (n = 30 in each group). c Magnitude of the IFN-γ-ELISpot responses. IFN-γ SFU/10⁶ PBMCs are shown for SARS-CoV-2 Spike (S), Membrane (M) and Nucleocapsid (N) between HIV negative (green) and HIV positive (red) (n = 30 per group). Plots show geometric mean. d Magnitude of the IFN-γ-ELISpot responses for Total SARS-CoV-2 responses (S, M, N, ORF3a, ORF6, ORF7, ORF8 and Env), FEC and HIV Gag between HIV negative (green) and HIV positive (red) (n = 30 per group). Plots show geometric mean. e Hierarchy of the IFNγ-ELISpot responses. IFN-γ SFU/10⁶ PBMCs responses in order of magnitude within each group with the contribution of the responses to a specific pool shown by color legend. f Diversity of the IFN-γ-ELISpot responses. Number of pools each of the donors (n = 30 per group) has shown positive responses in the IFN-γ-ELISpot assay. The total of SARS-CoV-2 pools tested was 8. Plots show mean with error bars indicating SD. g Proportion of T-cell response magnitude in the HIV-negative and HIV-positive individuals. h Correlation between CD4:CD8 ratio in HIV-infected individuals with their total SARS-CoV-2 responses, depicting disease severity per each donor (red dots: hospitalized cases; Black dots: non-hospitalized cases; blue dots: asymptomatic cases). The non-parametric Spearman test was used for correlation analysis. For multiple groups significance was assessed by two-way ANOVA with multiple comparisons. Two-tailed *p < 0.05, **p < 0.01, ****p < 0.0001.

Fig. 3. Interrelations between T-cell and antibody responses in HIV-positive and -negative donors.

a Correlation of total SARS-CoV-2 responses with S1 IgG titers in HIV negative and b HIV positive. c Correlation of total SARS-CoV-2 responses with N IgG titers in HIV-negative and d HIV-positive subjects. Red dots: hospitalized cases; black dots: non-hospitalized cases. e Correlation of neutralization titers with Spike-specific SARS-CoV-2 responses in HIV-negative and f HIV-positive donors. g Correlation of neutralization titers with total SARS-CoV-2 responses in HIV-negative and h HIV-positive donors. i Hierarchy of the T-cell responses ordered by the neutralizing capacity by their antibody titers in HIV-negative and j HIV-positive donors. The non-parametric Spearman test was used for correlation analysis. Two-tailed *p < 0.05.

Fig. 4. Composition of SARS-CoV-2-specific T cells in convalescent HIV-negative and HIV-positive individuals.

Intracellular cytokine staining (ICS) was performed to detect cytokine-producing T cells to the indicated peptide pools in HIV-negative (HIV−, n = 12) and HIV-positive individuals (HIV+, n = 11). a Representative flow cytometric plots for the identification of antigen-specific CD4 T cells based on double expression (CD154⁺IFN-γ⁺, CD154⁺IL-2⁺, and CD154⁺TNF-α⁺) following 6-h stimulation with media alone (control) or overlapping SARS-CoV-2 peptides against Spike pool 1 and 2 (Spike), Nucleoprotein (N), and Membrane protein (M) directly ex vivo. b Frequency of aggregated CD4 T-cell responses (CD154⁺IFN-γ⁺, CD154⁺IL-2⁺, and CD154⁺TNF-α⁺) against Spike, M/N or combined (Spike and M/N) peptide pools (HIV−, n = 12; HIV+, n = 11). Error bars represent SEM. c Pie charts representing the relative proportions of Spike, M/N, or total (combined Spike and M/N) CD4 T-cell responses for one (gray), two (green) or three (dark blue) cytokines, and pie arcs denoting IFN-γ, TNF-α and IL-2. d Representative flow cytometric plots for the identification of antigen-specific CD8 T cells based on the expression of (IFN-γ⁺, TNF-α⁺, and IL-2⁺) against the specified peptide pools or media alone (control). e Proportion of aggregated CD8 T-cell responses against Spike, M/N or combined (Spike and M/N) responses in HIV− (n = 12) and HIV+ (n = 11) donors. Error bars represent SEM. f Pie charts representing the relative proportions of Spike, M/N and combined CD8 T-cell responses for one (gray), two (green) or three (dark blue) cytokines, and pie arcs showing IFN-γ, TNF-α and IL-2. g Comparison of the frequencies of summed SARS-CoV-2-specific CD4 and CD8 T-cell responses against Spike in n = 12 HIV− donors (p = 0.0004) and n = 11 HIV+ (p = 0.001) and M/N proteins (HIV−, p = 0.042; HIV+, p = 0.0009). Error bars represent SEM. h Correlation between the frequency of total SARS-CoV-2-specific CD4 T cells and overall T-cell responses detected by IFN-γ ELISpot responses or i ID50 neutralization titer (log10) in HIV-negative (n = 12) and HIV-positive (n = 11) individuals. The non-parametric Spearman test was used for correlation analysis (two-tailed); p values for individual correlation analysis within groups, HIV− (green) or HIV+ (red) or combined correlation analysis (black) are presented. Significance determined by two-tailed Mann−Whitney U test or Wilcoxon matched-pairs signed rank test, *p < 0.05, **p < 0.01, ***p < 0.001. SPICE was used for polyfunctional analysis.

Fig. 5. Phenotypic characterization of SARS-CoV-2-specific CD4 and CD8 T cells from convalescent HIV-negative and HIV-positive subjects.

a Representative flow plots and b pie charts representing the proportion of antigen-specific CD4 T cell with a CD45RA⁻/CCR7⁺ central memory (CM), CD45RA⁺/CCR7⁺ naïve, CD45RA⁺/CCR7⁻ terminally differentiated effector memory (TEMRA) and CD45RA⁻/CCR7⁻ effector memory (EM) phenotype from HIV-negative (HIV−, n = 12) and HIV-positive individuals (HIV+, n = 11) against SARS-CoV-2 Spike, M, N, CMV pp65 and HIV gag. c Representative flow plots and d pie charts representing the proportion of CD45RA⁻/CCR7⁺ central memory (CM), CD45RA⁺/CCR7⁺ naïve, CD45RA⁺/CCR7⁻ terminally differentiated effector memory (TEMRA) and CD45RA⁻/CCR7⁻ effector memory (EM) antigen-specific CD8 T-cell subsets against SARS-CoV-2 Spike, M, N, CMV pp65 and HIV gag. e Representative flow plots from an HIV-negative donor (HIV−) and an HIV-positive donor (HIV+) showing expression of CD154 and IFN-γ production from PD1+ and PD1− SARS-CoV-2-specific CD4 T cells and paired analysis of responses in HIV-negative (HIV−, n = 12, p = 0.0005) and HIV-positive (HIV+, n = 11, p = 0.001) individuals. f Correlation between frequency of PD-1⁺CD154⁺IFN-γ⁺ SARS-CoV-2-specific CD4 T cells and DPSO in both groups. Significance determined by Wilcoxon matched-pairs signed rank test, *p < 0.05, ***p < 0.001. The non-parametric Spearman test was used for correlation analysis; two-tailed p values for individual correlation analysis within groups, HIV−, HIV+, or combined correlation analysis (black) are presented.

Fig. 6. Immune profile relationships between convalescent HIV-positive and -negative individuals.

a viSNE analysis of CD3 T cells in HIV-negative (top panel) and HIV-positive donors (lower panel). Each point on the high-dimensional mapping represents an individual cell, and color intensity represents the expression of selected markers. b Frequency of CD4 and CD8 T cells out of total lymphocytes in SARS-CoV-2 convalescent HIV-negative (HIV−, n = 26) and HIV-positive individuals (HIV+, n = 19) via traditional gating (p < 0.0001). Plots show the geometric mean. c Summary data of the proportion of CD45RA⁻/CCR7⁺ central memory (CM), CD45RA⁺/CCR7⁺ naïve, CD45RA⁺/CCR7⁻ terminally differentiated effector memory (TEMRA) and CD45RA⁻/CCR7⁻ effector memory (EM) CD4 and CD8 T-cell subsets in the study groups (HIV−, n = 26; HIV+, n = 19). Higher frequencies of naïve CD4 and CD8 T cells and lower frequencies of CD8 TEMRA in the HIV− compared to the HIV+ group are depicted (p = 0.0195, p = 0.0006 and p = 0.0036 respectively). Plots show the geometric mean. d Correlation between CD4:CD8 ratio and frequency of naïve CD4 T cells in HIV-positive individuals. e Correlation between frequency of naïve CD4 T cells and total SARS-CoV-2 T-cell responses, detected via ELISpot, in HIV-positive individuals. f Correlation between frequency of naïve CD4 T cells and g naïve CD8 T cells and age in HIV-negative individuals. h Correlation between frequency of naïve CD4 T cells and i naïve CD8 T cells age in HIV-positive donors. Significance determined by two-tailed Mann−Whitney U test, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. The non-parametric Spearman test was used for correlation analysis (two-tailed).