SARS-CoV-2 awakens ancient retroviral genes and the expression of proinflammatory HERV-W envelope protein in COVID-19 patients.

Patients with COVID-19 may develop abnormal inflammatory response, followed in some cases by severe disease and long-lasting syndromes. We show here that in vitro exposure to SARS-CoV-2 activates the expression of the human endogenous retrovirus (HERV) HERV-W proinflammatory envelope protein (ENV) in peripheral blood mononuclear cells from a subset of healthy donors, in ACE2 receptor and infection-independent manner. Plasma and/or sera of 221 COVID-19 patients from different cohorts, infected with successive SARS-CoV-2 variants including the Omicron, had detectable HERV-W ENV, which correlated with ENV expression in T lymphocytes and peaked with the disease severity. HERV-W ENV was also found in postmortem tissues of lungs, heart, gastrointestinal tract, brain olfactory bulb, and nasal mucosa from COVID-19 patients. Altogether, these results demonstrate that SARS-CoV-2 could induce HERV-W envelope protein expression and suggest its involvement in the immunopathogenesis of certain COVID-19-associated syndromes and thereby its relevance in the development of personalized treatment of patients.

Development and Testing of a Low-Cost Inactivation Buffer That Allows for Direct SARS-CoV-2 Detection in Saliva.

Massive testing is a cornerstone in efforts to effectively track infections and stop COVID-19 transmission, including places with good vaccination coverage. However, SARS-CoV-2 testing by RT-qPCR requires specialized personnel, protection equipment, commercial kits, and dedicated facilities, which represent significant challenges for massive testing in resource-limited settings. It is therefore important to develop testing protocols that are inexpensive, fast, and sufficiently sensitive. Here, we optimized the composition of a buffer (PKTP), containing a protease, a detergent, and an RNase inhibitor, which is compatible with the RT-qPCR chemistry, allowing for direct SARS-CoV-2 detection from saliva without extracting RNA. PKTP is compatible with heat inactivation, reducing the biohazard risk of handling samples. We assessed the PKTP buffer performance in comparison to the RNA-extraction-based protocol of the US Centers for Disease Control and Prevention in saliva samples from 70 COVID-19 patients finding a good sensitivity (85.7% for the N1 and 87.1% for the N2 target) and correlations (R = 0.77, p < 0.001 for N1, and R = 0.78, p < 0.001 for N2). We also propose an auto-collection protocol for saliva samples and a multiplex reaction to minimize the PCR reaction number per patient and further reduce costs and processing time of several samples, while maintaining diagnostic standards in favor of massive testing.

Longitudinal variation in human immunodeficiency virus long terminal repeat methylation in individuals on suppressive antiretroviral therapy.

BACKGROUND: Persistence of latent, replication-competent provirus in CD4+ T cells of human immunodeficiency virus (HIV)-infected individuals on antiretroviral treatment (ART) is the main obstacle for virus eradication. Methylation of the proviral 5' long terminal repeat (LTR) promoter region has been proposed as a possible mechanism contributing to HIV latency; however, conflicting observations exist regarding its relevance. We assessed 5'-LTR methylation profiles in total CD4+ T cells from blood of 12 participants on short-term ART (30 months) followed up for 2 years, and a cross-sectional group of participants with long-term ART (6-15 years), using next generation sequencing. We then looked for associations between specific 5'-LTR methylation patterns and baseline and follow-up clinical characteristics. RESULTS: 5'-LTR methylation was observed in all participants and behaved dynamically. The number of 5'-LTR variants found per sample ranged from 1 to 13, with median sequencing depth of 16270× (IQR 4107×-46760×). An overall significant 5'-LTR methylation increase was observed at month 42 compared to month 30 (median CpG Methylation Index: 74.7% vs. 0%, p = 0.025). This methylation increase was evident in a subset of participants (methylation increase group), while the rest maintained fairly high and constant methylation (constant methylation group). Persons in the methylation increase group were younger, had higher CD4+ T cell gain, larger CD8% decrease, and larger CD4/CD8 ratio change after 48 months on ART (all p < 0.001). Using principal component analysis, the constant methylation and methylation increase groups showed low evidence of separation along time (factor 2: p = 0.04). Variance was largely explained (21%) by age, CD4+/CD8+ T cell change, and CD4+ T cell subpopulation proportions. Persons with long-term ART showed overall high methylation (median CpG Methylation Index: 78%; IQR 71-87%). No differences were observed in residual plasma viral load or proviral load comparing individuals on short-term (both at 30 or 42 months) and long-term ART. CONCLUSIONS: Our study shows evidence that HIV 5'-LTR methylation in total CD4+ T cells is dynamic along time and that it can follow different temporal patterns that are associated with a combination of baseline and follow-up clinical characteristics. These observations may account for differences observed between previous contrasting studies.

Identification and characterization of HIV-specific resident memory CD8+ T cells in human lymphoid tissue.

Current paradigms of CD8+ T cell-mediated protection in HIV infection center almost exclusively on studies of peripheral blood, which is thought to provide a window into immune activity at the predominant sites of viral replication in lymphoid tissues (LTs). Through extensive comparison of blood, thoracic duct lymph (TDL), and LTs in different species, we show that many LT memory CD8+ T cells bear phenotypic, transcriptional, and epigenetic signatures of resident memory T cells (TRMs). Unlike their circulating counterparts in blood or TDL, most of the total and follicular HIV-specific CD8+ T cells in LTs also resemble TRMs Moreover, high frequencies of HIV-specific CD8+ TRMs with skewed clonotypic profiles relative to matched blood samples are present in LTs of individuals who spontaneously control HIV replication in the absence of antiretroviral therapy (elite controllers). Single-cell RNA sequencing analysis confirmed that HIV-specific TRMs are enriched for effector-related immune genes and signatures compared with HIV-specific non-TRMs in elite controllers. Together, these data indicate that previous studies in blood have largely failed to capture the major component of HIV-specific CD8+ T cell responses resident within LTs.