ACE2 and TMPRSS2 are key players on SARS-CoV-2 entry into host cells. However, it is still unclear whether expression levels of these factors could reflect disease severity. Here, a case–control study was conducted with 213 SARS-CoV-2 positive individuals where cases were defined as COVID-19 patients with respiratory distress requiring oxygen support (N = 38) and controls were those with mild to moderate symptoms of the disease who did not need oxygen therapy along the entire clinical course (N = 175). ACE2 and TMPRSS2 mRNA levels were evaluated in nasopharyngeal swab samples by RT-qPCR and logistic regression analyzes were applied to estimate associations with respiratory outcomes. ACE2 and TMPRSS2 levels positively correlated with age, which was also strongly associated with respiratory distress. Increased nasopharyngeal ACE2 levels showed a protective effect against this outcome (adjOR = 0.30; 95% CI 0.09–0.91), while TMPRSS2/ACE2 ratio was associated with risk (adjOR = 4.28; 95% CI 1.36–13.48). On stepwise regression, TMPRSS2/ACE2 ratio outperformed ACE2 to model COVID-19 severity. When nasopharyngeal swabs were compared to bronchoalveolar lavages in an independent cohort of COVID-19 patients under mechanical ventilation, similar expression levels of these genes were observed. These data suggest nasopharyngeal TMPRSS2/ACE2 as a promising candidate for further prediction models on COVID-19.

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Background Despite antiretroviral treatment efficacy, it does not lead to the complete eradication of HIV infection. Consequently, reactivation of the virus from latently infected cell reservoirs is a major challenge toward cure efforts. Two strategies targeting viral latency are currently under investigation: the “shock and kill” and the “block and lock.” The “Block and Lock” methodology aims to control HIV-1 latency reactivation, promoting a functional cure. We utilized the CRISPR/dCas9-KRAB platform, which was initially developed to suppress cellular genes transcription, to block drug-induced HIV-1 reactivation in latently infected T cells and myeloid cells. Results We identified a set of five sgRNAs targeting the HIV-1 proviral genome (LTR1-LTR5), having the lowest nominated off-target activity, and transduced them into the latently infected lymphoid (J-Lat 10.6) and myeloid (U1) cell lines. One of the sgRNAs (LTR5), which binds specifically in the HIV-1 LTR NFκB binding site, was able to promote robust repression of HIV-1 reactivation in latently infected T cells stimulated with Phorbol 12-Myristate 13-Acetate (PMA) and Ingenol B (IngB), both potent protein kinase C (PKC) stimulators. Reactivation with HDAC inhibitors, such as SAHA and Panobinostat, showed the same strong inhibition of reactivation. Additionally, we observed a hundred times reduction of HIV-1 RNA expression levels in the latently infected myeloid cell line, U1 induced with IngB. Conclusion Taken together, our results show that the KRAB fused CRISPR/dCas9 system can robustly prevent the HIV-1 latency reactivation process, mediated by PMA or IngB and SAHA or Panobinostat, both in myeloid and lymphoid HIV-1 latently infected cells. In addition, we demonstrated that KRAB repressor protein is crucial to reactivation resistance phenotype, and we have identified some useful hotspots sequences in HIV-1 LTR for the design sgRNAs.

The use of combined antiretroviral therapy (cART) has resulted in a remarkable reduction in morbidity and mortality of people living with HIV worldwide. Nevertheless, interindividual variations in drug response often impose a challenge to cART effectiveness. Although personalized therapeutic regimens may help overcome incidence of adverse reactions and therapeutic failure attributed to host factors, pharmacogenetic studies are often restricted to a few populations. Latin American countries accounted for 2.1 million people living with HIV and 1.4 million undergoing cART in 2020-21. The present review describes the state of art of HIV pharmacogenetics in this region and highlights that such analyses remain to be given the required relevance. A broad analysis of pharmacogenetic markers in Latin America could not only provide a better understanding of genetic structure of these populations, but might also be crucial to develop more informative dosing algorithms, applicable to non-European populations.

Tweetable abstract Pharmacogenetic tests are a promising strategy to improve safety and effectiveness of HIV therapy. However, implementation can be challenging in populations with complex genetic structure.

Background: Despite antiretroviral treatment efficacy, it does not lead to the complete eradication of HIV infection. In addition, HIV-1 latency reactivation is a major challenge towards cure efforts. Two strategies to cure HIV-1 infection, the “shock and kill” is based on the eradication of the HIV-1 from the patient, and the “block and lock". The “Block and Lock” methodology aims to control HIV-1 latency reactivation, promoting a functional cure. The KRAB fused CRISPR/dCas9 (pdCas9KRAB) system was previously produced to control cell transcription. Based on this construct we developed a CRISPR RNAs (sgRNAs), to guide the pdCas9KRAB up to five different sites in HIV-1 provirus sites to block HIV-1 latency reactivation. This process was mediated by phorbol esters and HDAC inhibitors.Results: We found five sites in the HIV-1 provirus genome (LTR1-LTR5) that minimize CRISPR off-targets and transduced them in the lymphoid and myeloid HIV-1 latency models. One of the five sgRNAs (LTR5) which binds specifically in the HIV-1 LTR NFκB binding site was able to promote a robust repression of reactivation pattern in a HIV-1 latency lymphoid model stimulated with Phorbol 12-Myristate 13-Acetate (PMA) and Ingenol B (IngB), both potent protein kinase C (PKC) stimulators. Reactivation with HDAC inhibitors, such as SAHA and Panobinostat, showed the same strong inhibition of reactivation. Additionally, we observed a reduction of 100 times in HIV-1 RNA molecules, when reactivated IngB in myeloid HIV-1 latently infected U1 cells.Conclusion: Taken together, our results show that the KRAB fused CRISPR/dCas9 system can robustly prevent the HIV-1 latency reactivation process, mediated by PMA or IngB and SAHA or Panobinostat, both in myeloid and lymphoid HIV-1 latency. In addition, we demonstrated that KRAB repressor protein is crucial to reactivation resistance phenotype, and we also have shown some useful hotspots sequences in HIV-1 LTR to design sgRNAs.