PWH with conserved CD4+ T cell counts mount robust antibody responses against SARS-CoV-2 without clinical worsening of COVID-19
We aimed to study the impact of HIV infection over the immune response against SARS-CoV-2 in COVID-19 convalescent individuals. The median age of PWH was 44 years (IQR: 34.7-53.5) and 22 out of 29 individuals (75.8%) were male. Median CD4+ T cell count was 513 cells/µL (IQR: 351-873). Otherwise, the median age of HIV-negative donors was 41 years (IQR: 35-51.5) and 11/29 (37.9%) were male. All individuals met the confirmed case definition of COVID-19 (positive for SARS-CoV-2 by RT-PCR). In both groups 23 out of 29 individuals showed a mild to moderate COVID-19 presentation, whereas the remaining individuals presented severe forms of the disease (defined by the presence of related complications such as pneumonia, hypoxemia or need of oxygen (2)). The mean time from symptoms onset to sampling was 69 days (IQR: 31-93) for PWH and 41 days (IQR: 18.5-55) for HIVneg. Regarding the available clinical data, comorbidities could be documented in 3 PWH (1 case of arterial hypertension -AHT- + diabetes mellitus + hypothyroidism, 1 case of AHT and 1 case of diabetes mellitus) and in 5 HIVneg donors (1 case of AHT+ hypothyroidism, 1 case of AHT + asthma, 1 case of AHT + pituitary adenoma, 1 case of thrombocytopenic purpura and 1 case of obesity). In addition, both groups displayed similar symptom patterns with no statistical differences (Chi square test with Yates correction, significance level: 0.05; not significant for any symptom analyzed, Figure 1A). We could observe that 65.5% of PWH and 79.3% of HIVneg individuals showed detectable SARS-CoV-2-specific antibodies (Figure 1B), with similar amounts of total IgG, IgG titers and anti-SARS-CoV-2 neutralizing antibodies between groups (Figure 1C, D and E). Finally, a thorough analysis was performed among PWH, revealing that neutralization capacity correlated with IgG titers (r:0.70, p<0.001) and NOD values (r:0.65, p<0.001). Additionally, IgG titers were associated with NOD values (r:0.87, p:0.0001) and CD8+ TL counts (r:0.82, p<0.001) (Figure 1F), as was also observed by us in HIVneg donors ((2) and Figure 7).
PWH display a distinctive immune profile of peripheral leucocytes’ populations
We next aimed to investigate the impact of HIV infection over the cell populations involved in SARS-CoV-2 immune response. Therefore, the phenotype of T, B, NK cells and monocytes was investigated by flow cytometry in peripheral blood samples from the donors involved in the study. Gating strategy and population definitions developed during this study is depicted in Supplementary Figure 1. Diminished percentages of circulating antibody secreting cells (ASC) were observed among PWH compared to HIVneg, with similar B cell proportions between groups (Figure 2A and B). Regarding the phenotype of CD4+ T cell populations, Th1 lymphocytes were expanded in PWH compared to HIVneg, whereas both groups presented similar TH17 and Th1* (CD3+/CD4+/CCR6+/CXCR3+) populations (Figure 2C). Although PWH displayed augmented percentages of Tfh CD4+ cells, the proportion of CXCR3-expressing Tfh subset, a population involved in antibody responses against viral infections and vaccination (12), were similar between groups (Figure 2D and E). Nevertheless, when examining the phenotype of Tfh cells by UMAP high dimensional analysis, we observed differences between PWH and HIVneg in the reduction 2D representations (Figure 2F), mainly due to the upregulation of CD27 and CXCR3 expression in several Tfh clusters (Supplementary Figure 2). Importantly, we observed a negative correlation between IgG titers and Tfh proportions in the PWH group, therefore highlighting the importance of this population on the immune response against SARS-CoV-2 (Figure 2G).
The frequencies of Tregs were similar for both groups (Supp. Figure 3A). As regards the exhaustion/activation profile of CD4+ T lymphocytes, we observed augmented PD-1+ frequency but not of CD57 nor of PD-1/CD57 co-expression proportions within the PWH group, therefore showing an increased activation state, without enhanced exhaustion/senescence among these individuals (Figure 3A) (13). In addition, a slight but significant increase of CD4+ TCM cells was detected, as depicted in Supplementary Figure 3B. Concerning the phenotype of CD8+ T cells during the convalescent phase of COVID-19, a significant increment in the proportion of HLA-DR+ CD8+ T lymphocytes in PWH compared to HIV-negative individuals was detected (Figure 3B). No differences were observed neither in the CD38+ proportion nor the exhaustion/senescent profile or CXCR5+ distributions of CD8+ T cells between groups, although regarding the memory/effector profile, a significant lesser proportion of CD8+ TTM cells was detected (Supp. Figure 3C and D, and data not shown).
Continuing our analysis, similar proportions of NK cells were observed in both groups as determined by the expression of CD56 (Figure 4A). Augmented proportions of CD25, CD95, NKP46, HLA-DR and HLA-DR/CD38 co-expressing cells among the PWH group were detected, therefore indicating an enhanced activation/exhaustion of peripheral NK cells in these individuals (Figure 4B to E, respectively). Finally, increased percentages of CD14++CD16- classical peripheral monocytes in the PWH group were observed compared to HIVneg individuals, whereas there weren’t any differences regarding the proportion of total CD14+, CD14+CD16bright non-classical or intermediate CD14++CD16dim monocytes between groups (Supplementary Figure 4), therefore showing minor differences on monocyte’s distribution or abundance (14).
Plasma cytokines / chemokines are differentially modulated in convalescent COVID-19 PWH
Continuing our task on characterizing the immune profile during the COVID-19 convalescence phase, plasma concentrations of several cytokines and chemokines were determined by flow cytometry using a multiplex assay. A marked decrease of IL-8/CCL8 and increased levels of IP-10/CXCL10 were observed in PWH individuals compared to HIV-negative COVID-19 convalescents (Figure 5), whereas any differences were found on MCP-1/CCL2, MIG/CXCL9 or RANTES/CCL5 plasma concentrations between groups (data not shown). Moreover, statistically diminished levels of IFN-γ, TNF-α, IL-17A, IL-6 and IL-10 plasma concentrations were noticed in PWH compared to HIVneg COVID-19 convalescent individuals, as shown in Figure 5. Notably, IL-8/CCL8 couldn’t be detected in 65.2% of the PWH tested, whereas in the HIV negative group was undetectable in 22.9% of individuals (p = 0,0005, Chi square test with Yates correction).
Cellular immune responses against SARS-CoV-2 are diminished in PWH
Advancing on our studies, we aimed to determine T cell responses against SARS-CoV-2 antigens and peptide pools, as it was described elsewhere (9, 15). Hence, PBMCs from each donor were stimulated in the presence of proteins or peptide pools encompassing both Spike and Nucleocapside proteins and afterwards the IFN-γ-producing cells were determined by ELISpot. Our data demonstrated an overall diminished response against SARS-CoV-2 antigens, specifically against Spike, RBD and Nucleocapside whole proteins in the PWH group, with no differences in T cell responses against Spike or Nucleocapside peptide pools (Figure 6A and B). These data show that although PWH presented lower T cell responses against SARS-CoV-2 compared to HIV negative donors, these cells were able to collaborate to mount robust humoral immune responses against the pathogen.
Analysis of associations of immune cell subsets, serological determinations, cytokine/chemokines assessments and Ag-specific cellular immune responses in PWH and HIV negative participants
Finally, to examine differences in the immune landscape between PWH and HIV-negative participant groups, associations between clinical, serological, cytokine/chemokine plasma levels, numerous immune cell populations and SARS-CoV-2-specific cellular immune response parameters were performed by Spearman correlation analysis. Results can be evinced in Figure 7 for statistical significances and Supplementary Figure 5 for Spearman R values. All in all, we could observe distinctive patterns of associations when comparing both cohorts. Several correlations were statistically significant in PWH but not in HIVneg: i) On comparing serological parameters and Tfh frequency, a positive correlation between ASC and Tfh proportions and negative correlations between nAb titers and ASC/Tfh percentages were found; ii) Among CD4+ T cell activation/exhaustion/memory-effector markers, positive correlations between PD1 and PD1/CD57 with Treg proportions or between exhaustion markers and CD4+ TCM, CD4+ TTM, CD4+ TEM, and CD4+ TEMRA, Th1, Th1* and Th17 percentages were discovered, as well as negative correlations between Treg and CD4+ TNaive or Th2 cells; iii) Numerous cytokines/chemokines levels correlated with Ag-specific cellular immune responses, finding negative associations between CCL5 concentration and Spike protein responses, IL-6 and Spike peptide pool–specific IFN-γ producing cells, or TNFα/ IL-10 levels and RBD or Nucleocapsid protein responses; iv) Ag-specific immune cell responses correlated with the number of symptoms, B lymphocytes, several CD8+ TL and NK activated/exhausted populations, and lastly, v) different cytokines/chemokines levels were associated to ASC/Tfh frequencies (Figure 7 and Supp. Figure 5). We couldn’t observe any significant correlation between the magnitude of the cellular immune responses against viral antigens and anti-SARS-CoV-2 IgG titers neither in PWH nor HIVneg donors. These differences reveal the overall impact of HIV infection over the memory response against SARS-CoV-2 after COVID-19 infection.