Investigating the impact of respiratory viral superinfections on HIV-1 pathogenesis: insights from in vitro models of HIV-1/H1N1 and HIV-1/RSV superinfections

Background: Respiratory viruses are frequent causes of acute respiratory illness in PLWH, particularly in HIV-endemic areas, and may influence HIV pathogenesis through poorly understood mechanisms. Given the limited information on respiratory viral infections in PLWH, in vitro models can help uncover the molecular dynamics of HIV and viral superinfections. Methods: Herein, we developed in vitro models of HIV-1/ H1N1 (H+H+) and HIV-1/RSV (H+R+) superinfections. Three days post HIV-infection (1 ng/1×106 cells of HIV-1BaL ) PBMCs from 10 healthy volunteers were co-cultured, in a 24 transwell plate, with a human lung epithelial cell line (CaLu3), infected with H1N1 or RSV (0.015 MOI). HIV-1, H1N1 or RSV replication was monitored by p24 ELISA and Real-Time PCR. Five days post HIV-1 infection, both PBMCs and CaLu3 cells were harvested for mRNA expression of antiviral/inflammatory factors by Real−time PCR. T lymphocyte subpopulations, along with Perforin-and IFNγ-producing CD8+ lymphocytes were investigated by flow cytometry. Results: In the H+H+ superinfection, replication rate of H1N1 was significantly hindered in the presence of HIV-1 compared to H1N1-single infection (p=0.027). In Calu3 cells, the H+H+ condition exhibited a decline in the expression of several anti-viral factors, mainly RIG-I levels compared to H1N1-single infected cells (p=0.041). Furthermore, a unique immunophenotype was identified in H+H+ PBMCs, characterized by higher percentages of Th2 lymphocytes (CD4+GATA3+, p=0.022) and lower frequencies of Th1 cells (CD4+Tbet+, p=0.05), compared to HIV-1-infected PBMCs. Conversely, in the H+R+ superinfection, HIV-1 replication rate exhibited a marked increase in the presence of RSV in comparison to HIV-1-single infection (p=0.048). HIV-1-infected PBMCs exposed to RSV exhibited significant increased expression levels of IFITM1 (p=0.05) and IFITM3 (p<0.001), and higher expression levels of CCR5 compared to the other conditions. Increased frequencies in Th17 (CD4+RORγT+, p=0.05) and in IFNγ-producing CD8+ (p=0.023), along with a diminished expression of HLA-DRII on CD4+ lymphocytes (p=0.03), were observed compared to HIV-1 single-infected PBMCs. Conclusions: These findings reveal virus-specific interactions: HIV-1 dampens H1N1 replication while RSV enhances HIV-1 replication, each shaping distinct immune responses. This underscores the importance of evaluating respiratory viral superinfections in PLWH and highlights in vitro models as valuable tools for understanding HIV-viral interactions and their impact on pathogenesis.