CHARACTERIZATION of HIV and SARS-CoV-2 COINFECTION: ROLE of IL-10

Background: Interaction between HIV and SARS-CoV-2 infection has not yet been fully characterized. To this purpose, an in-vitro HIV/SARS-CoV-2 coinfection assay was set up. Furthermore, the results obtained in the in-vitro model were verified in a cohort of HIV/SARS-CoV-2 coinfected young individuals. Methods: We designed an in-vitro SARS-CoV-2/HIV coinfection. We challenged PBMCs derived from 10 healthy volunteers with 1 ng/1×106 cells of HIV-1BaL and subsequently co-cultured them with a human lung epithelial cell line (CaLu3) infected with SARS-CoV-2 at 0.015 MOI. At 96 hours post HIV-1 infection, both PBMCs and CaLu3 cells were harvested for mRNA expression and proteomic analysis. Furthermore, we enrolled 85 ART-treated HIV-vertically transmitted patients (mean age 22.4 years) followed at the Unit of Pediatric Infectious Diseases, Sacco Hospital in Milan, Italy. Real-time PCR was performed to detect SARS-CoV-2 and plasma samples were tested for anti-SARS-CoV-2-specific IgG (Euroimmun Kit). The subjects who contracted SARS-CoV-2 infection (H+/S+) were compared to the HIV-positive, SARS-CoV-2 negative ones (H+/S-) and to a cohort of SARS-CoV-2 positive, HIV-negative age-matched patients (H-/S+, mean age 22.8 years). We evaluated mRNA expression of factors involved in the anti-viral immune response on PBMCs upon stimulation with SARS-CoV-2 antigens (Quantigene Plex assay) and secreted cytokines/chemokines on plasma (Multiplex Cytokine Array). Results: We observed a significant reduction of SARS-CoV-2 replication on CaLu3 cells when exposed to HIV-pre-infected PBMCs in-vitro. IL-10 expression and production were significantly higher in the coinfected condition, in both CaLu3 cells and PBMCs. The upregulation of IL-10 was associated to higher expression levels of STAT3. In the HIV-vertically transmitted cohort, 4 out of 85 subjects contracted SARS-CoV-2 infection (H+/S+). All H+/S+ patients were asymptomatic. Similarly to the data obtained in-vitro, a significant increase in both expression and production of IL-10 emerged in comparison to H+/S-and H-/S+. Conclusion: In-vitro, a dampening in SARS-CoV-2 replication, along with a higher IL-10 mRNA expression and production, have been observed in the HIV/SARS-CoV-2 coinfected condition. Presumably, IL-10 exerted its activity through the STAT3 pathway. These results were confirmed in HIV/SARS-CoV-2 coinfected subjects in which an upregulation of IL-10 was observed. Our data might be useful defining HIV/SARS-CoV-2 coinfected young individuals pathogenesis.

THE EFFECTS of TYPE i & II IFNs DISCLOSE A ROLE for DOPAMINE in SARS-CoV-2 INFECTION

Background: Recent studies highlight the dynamic nature of virus-host interaction during SARS-CoV-2 infection, raising intriguing questions about the role and timing of interferon (IFN) responses. In fact, SARS-CoV-2 delays/antagonizes Type-I, and to a definitely lesser extent, Type II-IFNs. While paving the way for potential antiviral therapies based on immune activation, the molecular mechanisms linking different IFN pathways to SARS-CoV-2 susceptibility remain to be elucidated. The present study investigates the role of Type-I &-II IFNs in SARS-CoV-2 replication in human lung cells, with a focus on molecular pathways related with innate and adaptive immunity. Methods: Human lung carcinoma cells (CaLu3) were pretreated with IFN-α,-β or-γ (from 1 to 1000 U/mL), O.N. Cells were infected with SARS-CoV-2 (MOI 0.05) for 3h, and IFNs were added during infection. In another set of experiments, IFNs were added only p.i. Supernatants were harvested at 24 and 48h p.i. to assess viral replication by RT-qPCR, and to quantify the levels of cytokines/chemokines through Multiplex assay. At 48h post-infection, cells were collected and RNA was retrotranscribed to investigate a variety of transcriptional targets. Cell viability was assessed by MTT. Results are presented as the average of the relative expression units to the GAPDH gene, calculated by the 2-ΔΔCt equation. Statistical analyses were performed through the Student t-test. Results: Pretreatment with both Type-I &-II IFNs dramatically reduces SARS-CoV-2 replication in the absence of cell toxicity. Such an effect is maintained, though at a lower magnitude, when IFNs are added only p.i. The antireplicative effects of Type-I &-II IFNs are associated with both convergent and divergent mechanisms. Both Types decrease the expression and/or protein levels of most pro-inflammatory mediators while augmenting anti-inflammatory and anti-apoptotic factors. Surprisingly, IFN-γ shows the strongest effect in potentiating antiviral effectors besides boosting adaptive immunity pathways. Remarkably, a convergent effect of both IFN Types is observed upon the expression of genes associated with DA activity, including DA receptors (D1-D5) and the DA transporter (DAT), which are dramatically altered by SARS-CoV-2. Conclusion: Both Type-I &-II IFNs halt SARS-CoV-2 replication by acting through complementary mechanisms. Their effects also disclose a potential role for DA activity, and neuromodulators in general, in host immunity during SARS-CoV-2 infection in pulmonary cells.

SARS-CoV-2 mRNA vaccine BNT162b2 triggers a consistent cross-variant humoral and cellular response.

As the SARS-CoV-2 pandemic continues to rage worldwide, the emergence of numerous variants of concern (VOC) represents a challenge for the vaccinal protective efficacy and the reliability of commercially available high-throughput immunoassays. Our study demonstrates the administration of two doses of the BNT162b2 vaccine that elicited a robust SARS-CoV-2-specific immune response which was assessed up to 3 months after full vaccination in a cohort of 37 health care workers (HCWs). SARS-CoV-2-specific antibody response, evaluated by four commercially available chemiluminescence immunoassays (CLIA), was qualitatively consistent with the results provided by the gold-standard in vitro neutralization assay (NTA). However, we could not observe a correlation between the quantity of the antibody detected by CLIA assays and their neutralizing activity tested by NTA. Almost all subjects developed a SARS-CoV-2-specific T-cell response. Moreover, vaccinated HCWs developed a similar protective neutralizing antibodies response against the EU (B.1), Alpha (B.1.1.7), Gamma (P.1), and Eta (B.1.525) SARS-CoV-2 variants, while Beta (B.1.351) and Delta (B.1.617.2) strains displayed a consistent partial immune evasion. These results underline the importance of a solid vaccine-elicited immune response and a robust antibody titre. We believe that these relevant results should be taken into consideration in the definition of future vaccinal strategies.

Lung Allograft Dysfunction in a COVID-19 Transplanted Patient is Associated with a Peculiar Immunopathological Phenotype

Purpose Lung transplantation (LT) after severe SARS-CoV-2 infection is emerging as a life-saving medical procedure for selected patients who experience acute respiratory distress syndrome (ARDS). We present the first immunopathological evaluation of a lung allograft rejection in a patient who underwent LT because of irreversible ARDS related to COVID-19. Methods Two male patients with irreversible ARDS caused by COVID-19 underwent bilateral LT at our Institution. A surveillance transbronchial biopsy (TBB) was performed 2 months after LT in the first patient (Pt#1), while the second patient (Pt#2) died because of allograft rejection at day 62 post LT and explanted lungs were retrieved. CT imaging of the lungs was performed three days before death. Morphological examination was performed by H&E, whereas the immunophenotyping was performed by immunohistochemistry. Results Imaging and morphological examination of Pt#2 lungs indicated the presence of a graft dysfunction with features of a restrictive, widespread usual interstitial pneumonia-like syndrome (Fig. 1A, B). The immunophenotyping showed that B-lymphocytes (CD20-positive) were nearly absent, CD8-T-cells were not particularly expanded (mean positive cells within the lung stroma=13.8%;Fig. 1C), and the CD4/CD8 ratio was not decreased (Fig. 1D). The T-regs (Foxp3-positive) were 6% of the overall population (Fig. 1E). Analysis of the immune checkpoint molecules PD1, Tigit, CTLA4 and PDL1 showed that the expression of PD-L1 alone was highly increased in vases and in alveolar cells of rejected lungs, whereas it was nearly undetectable in the TBB from Pt#1 (Fig. 1F, G). Conclusion PDL1 expression in vases was previously documented as a sign of indirect ARDS. Together with our preliminary data, we can hypothesize that PDL1 may play a role in tissue effacement and graft failure, possibly indicating poor allograft prognosis.