Phd in ADVANCES IN INFECTIOUS DISEASES, MICROBIOLOGY, LEGAL
MEDICINE AND PUBLIC HEALTH SCIENCES

Thesis title: Evaluation of innate immunity and antibody production induced by SARS-CoV-2 vaccination in people living with HIV (PLWH)

Anti-SARS-CoV-2 mRNA vaccines elicit a strong adaptive immune response against the viral spike (S) glycoprotein, even in PLWH, but their potential in modulating innate immunity remains largely unexplored. In HIV-infected individuals, it is important to collect data on the immunogenicity of vaccines, as the impairment of cell-mediated immunity and B lymphocytes that occurs during the course of HIV infection may reduce the magnitude of the response and the duration of vaccine protection. Given the pre-existing high levels of IFNs that characterize PLWH and that these cytokines are activated by vaccination, administration of the BNT162b2 mRNA vaccine could further increase these levels, posing a health risk to these individuals. While chronic IFN activation contributes to HIV immunopathogenesis, a rapid and early interferon response is critical in counteracting the virus during SARS-CoV-2 infection. Indeed, anti-IFN autoantibodies were found in some life-threatening patients with SARS-CoV-2 infection characterized by severe pneumonia and were associated with reduced expression of ISGs and increased biomarkers of disease severity. Given the role of IFN-I in stimulating both B and T lymphocyte responses, it was hypothesized that an increase in IFN-I levels induced by SARS-CoV-2 vaccination could enhance anti-S antibodies production. Therefore, to further our understanding of the immunogenicity of these vaccines, the aim of my PhD project was to investigate whether vaccination in PLWH activates a strong anti-S antibody response and induces changes in the IFN-mediated innate immune response. Specifically, 82 PLWH on stable and effective cART were enrolled in this study to assess the impact of SARS-CoV-2 vaccination on both antibody production and IFN-I molecular signature. Blood samples were taken at four different time points related to the vaccination protocol: T0 (before the first dose), T1 (three weeks after the first dose, at the second vaccine dose administration), T2 (approximately one month after the second vaccine dose) and T3 (approximately one year after T2). All patients received a third dose of vaccine between T2 and T3. To avoid confounding in the analysis of IFN-I response, all patients were first screened for the presence of autoantibodies that can bind the active site of IFN and neutralize its biological activity. Anti-S IgG antibody levels were measured longitudinally at all time points using a second-generation chemiluminescence immunoassay and compared to a HIV-negative control group. The results were further validated by an in vitro antibody neutralization assay using the S-VSV pseudovirus on Vero E6 cells. In parallel, gene expression levels of IFN-α2, IFN-β and IFN-ω were assessed in PBMCs. The results showed that four patients had anti-IFN antibodies with broad specificity against IFN-α2b (with titers 22757, 11378, 7964 and 249 TRU/ml, respectively) at T3. Three of them also developed anti-IFN-α2a antibodies (with titers 498, 498 and 249 TRU/ml, respectively) at T3. Quantitative measurement of anti-S IgG antibodies in patients without anti-IFN autoantibodies showed a progressive increase in the titer of these antibodies over time (T0 vs T1 and T1 vs T2: p < 0.0001; T2 vs T3: p = 0.0002). The in vitro biological assay based on the Vero E6/S-VSV system for the detection of anti-S nAbs confirmed the longitudinal increase in antibody titer against SARS-CoV-2 S protein in PLWH, showing positive correlation with the results of the immunoassay (T1: ρ = 0,6461, p < 0.0001; T2: ρ = 0,7776, p < 0.0001; T3: ρ = 0,6138, p < 0.0001). Comparing PLWH with a HIV-negative control group, a similar trend in the longitudinal production of anti-S antibodies was observed. Furthermore, a negative correlation was observed between the number of days elapsed between each timepoint and the anti-S IgG titer over time (ρ = - 0.0082, p = 0.003), while other factors such as age, sex, CD4+ T cell count at nadir and T0, time of exposure to cART, expression of IFN-α2, IFN-β, and IFN-ω genes at T0 did not show any correlation with antibody production. In addition, IFNs-I gene analysis performed in the study population showed that BNT162b2 mRNA vaccine administration did not induce an increase in the expression of IFN-α2, IFN-β and IFN-ω in PLWH. As for the anti-S antibody analysis of patients with anti-IFN neutralizing antibodies, they had a similar anti-S antibody production to those who were negative to autoantibodies and showed a reduction in IFN-induced genes expression. However, one of these four patients failed to develop adequate levels of anti-S antibodies at T1 and T2, while only at T3 his antibody levels were comparable to those of the other patients. This individual had a nadir CD4+ T cell blood count of 90 cells/µL, and, despite being treated with an effective cART for 5 years, his CD4+ T cell blood count was < 200 cells/µL over the study period. By contrast, the other three patients had CD4+ T cell counts above 600 cells/µL at all time points. In conclusion, this study shows that vaccination against SARS-CoV-2 is effective even in PLWH with good CD4+ T lymphocyte counts, who exhibit anti-S antibody production similar to HIV-negative individuals. Although IFNs appear to play a key role in generating a proper immune response during vaccination, it was observed that BNT162b2 mRNA vaccine does not appear to increase them excessively in PLWH. Thus, SARS-CoV-2 vaccination seems to be successful in inducing an appropriate anti-S antibody response in this cohort of individuals, without causing overexpression of innate immunity components that are generally elevated in PLWH and could be detrimental. Therefore, vaccination appears to be both effective and immunologically safe in the PLWH population.