Transmitted Drug Resistance and HIV Diversity Among Adolescents Newly Diagnosed With HIV in Spain.

BACKGROUND: Virologic characterization of newly HIV-diagnosed adolescents could help to improve their specific needs. The objective was to describe the transmitted drug resistance mutations (TDR) and its transmission by clusters in this population in Spain. METHODS: TDR to retrotranscriptase and protease inhibitors included in the WHO TDR list 2009 implemented in the Calibrated Population Resistance tool v8.0 (Stanford) were studied in HIV pol sequences from all HIV-diagnosed adolescents (12-19-year-old) enrolled during 2004-2019 period in the Spanish pediatric and adult (CoRISpe-CoRIS) cohorts. The found TDR were compared with the provided by the Stanford algorithm v9.0 2021. HIV-1 variants and transmission clusters were also studied. RESULTS: Among 410 HIV-1 adolescents diagnosed, 141 (34.4%) had available ART-naive sequences. They were mostly male (81.6%), Spanish (55.3%) and with behavioral risk (92.2%), mainly male-to-male sexual contact (63.1%). TDR prevalence was significantly higher by Stanford versus WHO list (18.4% vs. 7.1%; P = 0.004). The most prevalent TDR by the WHO list was K103N (3.6%) and by Stanford E138A (6.6%), both at retrotranscriptase. E138A, related to rilpivirine/etravirine resistance, was absent in the WHO list. One in 4 adolescents carried HIV-1 non-B variants. We described 5 transmission clusters, and 2 carried TDR mutations. CONCLUSIONS: Our data suggest a high TDR prevalence in adolescents with a new HIV diagnosis in Spain, similar to adults, 2 active TDR transmission clusters, and the need for the WHO TDR list update. These findings could have implications for the options of the recently available rilpivirine-related long-acting treatment and in first-line regimen election.

Potent Induction of Envelope-Specific Antibody Responses by Virus-Like Particle Immunogens Based on HIV-1 Envelopes from Patients with Early Broadly Neutralizing Responses.

Longitudinal studies in HIV-1-infected individuals have indicated that 2 to 3 years of infection are required to develop broadly neutralizing antibodies. However, we have previously identified individuals with broadly neutralizing activity (bNA) in early HIV-1 infection, indicating that a vaccine may be capable of bNA induction after short periods of antigen exposure. Here, we describe 5 HIV-1 envelope sequences from individuals who have developed bNA within the first 100 days of infection (early neutralizers) and selected two of them to design immunogens based on HIV-1-Gag virus-like particles (VLPs). These VLPs were homogeneous and incorporated the corresponding envelopes (7 to 9 µg of gp120 in 1010 VLPs). Both envelopes (Envs) bound to well-characterized broadly neutralizing antibodies (bNAbs), including trimer-specific antibodies (PGT145, VRC01, and 35022). For immunogenicity testing, we immunized rabbits with the Env-VLPs or with the corresponding stabilized soluble envelope trimers. A short immunization protocol (105 days) was used to recapitulate the early nAb induction observed after HIV-1 infection in these two individuals. All VLP and trimeric envelope immunogens induced a comparably strong anti-gp120 response despite having immunized rabbits with 30 times less gp120 in the case of the Env-VLPs. In addition, animals immunized with VLP-formulated Envs induced antibodies that cross-recognized the corresponding soluble stabilized trimer and vice versa, even though no neutralizing activity was observed. Nevertheless, our data may provide a new platform of immunogens, based on HIV-1 envelopes from patients with early broadly neutralizing responses, with the potential to generate protective immune responses using vaccination protocols similar to those used in classical preventive vaccines. IMPORTANCE It is generally accepted that an effective HIV-1 vaccine should be able to induce broad-spectrum neutralizing antibodies. Since most of these antibodies require long periods of somatic maturation in vivo, several groups are developing immunogens, based on the HIV envelope protein, that require complex and lengthy immunization protocols that would be difficult to implement in the general population. Here, we show that rabbits immunized with new envelopes (VLP formulated) from two individuals who demonstrated broadly neutralizing activity very early after infection induced specific HIV-1 antibodies after a short immunization protocol. This evidence provides the basis for generating protective immune responses with classic vaccination protocols with vaccine prototypes based on HIV envelope sequences from individuals who have developed early broadly neutralizing responses.

Prevalence of M184V and K65R in proviral DNA from PBMCs in HIV-infected youths with lamivudine/emtricitabine exposure.

OBJECTIVES: We analysed the prevalence of M184V/I and/or K65R/E/N mutations archived in proviral DNA (pDNA) in youths with perinatal HIV, virological control and who previously carried these resistance mutations in historic plasma samples. METHODS: We included vertically HIV-infected youths/young adults aged ≥10 years in the Madrid Cohort of HIV-1 Infected Children and Adolescents, exposed to lamivudine and/or emtricitabine, with M184V/I and/or K65R/E/N in historic plasma samples, on antiretroviral therapy (ART), virologically suppressed (HIV-1 RNA <50 copies/mL), and with available PBMCs in the Spanish HIV BioBank. Genomic DNA was extracted from PBMCs and HIV-1 RT gene was amplified and sequenced for resistance testing by Stanford HIV Resistance tool. RESULTS: Among the 225 patients under follow-up in the study cohort, 13 (5.8%) met selection criteria, and RT sequences were recovered in 12 (92.3%) of them. All but one were Spaniards, carrying subtype B, with a median age at PBMCs sampling of 21.3 years (IQR: 15.6-23.1) with 4 years (IQR 2.1-6.5) of suppressed viral load (VL). Nine (75%) youths did not present M184V/I in pDNA after at least 1 year of viral suppression. In December 2019, the remaining three subjects carrying M184V/I in pDNA maintained suppressed viraemia, and two still used emtricitabine in ART. CONCLUSIONS: The prevalence of resistance mutations to lamivudine and emtricitabine in pDNA in a cohort of youths perinatally infected with HIV who remain with undetectable VL, previously lamivudine and/or emtricitabine experienced, was infrequent. Our results indicate that ART including lamivudine or emtricitabine may also be safe and successful in youths with perinatal HIV with previous experience of and resistances to these drugs detected in plasma.

Early versus deferred anti-SARS-CoV-2 convalescent plasma in patients admitted for COVID-19: A randomized phase II clinical trial.

BACKGROUND: Convalescent plasma (CP), despite limited evidence on its efficacy, is being widely used as a compassionate therapy for hospitalized patients with COVID-19. We aimed to evaluate the efficacy and safety of early CP therapy in COVID-19 progression. METHODS AND FINDINGS: The study was an open-label, single-center randomized clinical trial performed in an academic medical center in Santiago, Chile, from May 10, 2020, to July 18, 2020, with final follow-up until August 17, 2020. The trial included patients hospitalized within the first 7 days of COVID-19 symptom onset, presenting risk factors for illness progression and not on mechanical ventilation. The intervention consisted of immediate CP (early plasma group) versus no CP unless developing prespecified criteria of deterioration (deferred plasma group). Additional standard treatment was allowed in both arms. The primary outcome was a composite of mechanical ventilation, hospitalization for >14 days, or death. The key secondary outcomes included time to respiratory failure, days of mechanical ventilation, hospital length of stay, mortality at 30 days, and SARS-CoV-2 real-time PCR clearance rate. Of 58 randomized patients (mean age, 65.8 years; 50% male), 57 (98.3%) completed the trial. A total of 13 (43.3%) participants from the deferred group received plasma based on clinical aggravation. We failed to find benefit in the primary outcome (32.1% versus 33.3%, odds ratio [OR] 0.95, 95% CI 0.32-2.84, p > 0.999) in the early versus deferred CP group. The in-hospital mortality rate was 17.9% versus 6.7% (OR 3.04, 95% CI 0.54-17.17 p = 0.246), mechanical ventilation 17.9% versus 6.7% (OR 3.04, 95% CI 0.54-17.17, p = 0.246), and prolonged hospitalization 21.4% versus 30.0% (OR 0.64, 95% CI, 0.19-2.10, p = 0.554) in the early versus deferred CP group, respectively. The viral clearance rate on day 3 (26% versus 8%, p = 0.204) and day 7 (38% versus 19%, p = 0.374) did not differ between groups. Two patients experienced serious adverse events within 6 hours after plasma transfusion. The main limitation of this study is the lack of statistical power to detect a smaller but clinically relevant therapeutic effect of CP, as well as not having confirmed neutralizing antibodies in donor before plasma infusion. CONCLUSIONS: In the present study, we failed to find evidence of benefit in mortality, length of hospitalization, or mechanical ventilation requirement by immediate addition of CP therapy in the early stages of COVID-19 compared to its use only in case of patient deterioration. TRIAL REGISTRATION: NCT04375098.

Insights into neutralizing antibody responses in individuals exposed to SARS-CoV-2 in Chile.

Chile has one of the worst numbers worldwide in terms of SARS-CoV-2 positive cases and COVID-19-related deaths per million inhabitants; thus, characterization of neutralizing antibody (NAb) responses in the general population is critical to understanding of immunity at the local level. Given our inability to perform massive classical neutralization assays due to the scarce availability of BSL-3 facilities in the country, we developed and fully characterized an HIV-based SARS-CoV-2 pseudotype, which was used in a 96-well plate format to investigate NAb responses in samples from individuals exposed to SARS-CoV-2 or treated with convalescent plasma. We also identified samples with decreased or enhanced neutralization activity against the D614G spike variant compared with the wild type, indicating the relevance of this variant in host immunity. The data presented here represent the first insights into NAb responses in individuals from Chile, serving as a guide for future studies in the country.

Accuracy of a RT-qPCR SARS-CoV-2 detection assay without prior RNA extraction.

The current COVID-19 pandemic constitutes a threat to the population worldwide with over 21 million infected people. There is an urgent need for the development of rapid and massive detection tools as well as the identification and isolation of infected individuals. we sought to evaluate different RT-qPCR kits and protocols to evaluate the best approach to be used omitting an RNA extraction step. We have investigated the sensitivity and performance of different commercially available RT-qPCR kits in detecting SARS-CoV-2 using 80 extracted RNA and NSS from COVID-19 diagnosed patients. We evaluated the ability of each kit to detect viral RNA from both kit-extracted or directly from a pre-boiled NSS observing that direct RNA detection is possible when Ct values are lower than 30 with the three kits tested. Since SARS-CoV-2 testing in most locations occurs once COVID-19 symptoms are evident and, therefore, viral loads are expected to be high, our protocol will be useful in supporting SARS-CoV-2 diagnosis, especially in America where COVID-19 cases have exploded in the recent weeks as well as in low- and middle-income countries, which would not have massive access to kit-based diagnosis. The information provided in this work paves the way for the development of more efficient SARS-CoV-2 detection approaches avoiding an RNA extraction step.

Virological outcome among HIV infected patients transferred from pediatric care to adult units in Madrid, Spain (1997-2017).

The aim of this transversal study was to describe the virological and immunological features of HIV-infected youths transferred from pediatric to adult care units since 1997 vs. the non-transferred patients from the Madrid Cohort of HIV-infected children and adolescents in Spain. We included 106 non-transferred and 184 transferred patients under clinical follow-up in 17 public hospitals in Madrid by the end of December 2017. Virological and immunological outcomes were compared in transferred vs. non-transferred patients. ART drug resistance mutations and HIV-variants were analyzed in all subjects with available resistance pol genotypes and/or genotypic resistance profiles. Among the study cohort, 133 (72.3%) of 184 transferred and 75 (70.7%) of 106 non-transferred patients had available resistance genotypes. Most (88.9%) of transferred had ART experience at sampling. A third (33.3%) had had a triple-class experience. Acquired drug resistance (ADR) prevalence was significantly higher in pretreated transferred than non-transferred patients (71.8% vs. 44%; p = 0.0009), mainly to NRTI (72.8% vs. 31.1%; p < 0.0001) and PI (29.1% vs. 12%; p = 0.0262). HIV-1 non-B variants were less frequent in transferred vs. non-transferred (6.9% vs. 32%; p < 0.0001). In conclusion, the frequent resistant genotypes found in transferred youths justifies the reinforcement of HIV resistance monitoring after the transition to avoid future therapeutic failures.

Development of an Isavirus minigenome system to study the function of the pocket RNA-binding domain of the viral nucleoprotein (NP) in salmon cells.

The Isavirus is an orthomyxovirus with a genome composed of eight segments of negative single-strand RNA (-ssRNA). It has been proposed that the eight genomic segments of the Isavirus are organized as a ribonucleoprotein (RNP) complex called a minigenome, which contains all the viral RNA segments, a viral heterotrimeric polymerase and multiple copies of the viral nucleoprotein (NP). Here, we develop an Isavirus minigenome system and show the importance of the formation of active RNPs and the role of viral NP R189, R194, R302 and K325 residues in the NP RNA-binding domain in the context of RNPs. The results indicate it is possible to generate a minigenome in salmon cells, a composite ISAV RNPs with EGFP-based chimeric vRNA with heterotrimeric polymerase (PB1, PB2, PA) and NP protein using CMV-based auxiliary plasmids. It was also shown that NP R189, R194, R302 and K325 residues are important to generate viral mRNA from the constituted RNPs and a detectable reporter protein. This work is the first salmon cell-based minigenome assay for the Isavirus, which was evaluated by a bioinformatic and functional study of the NP protein in viral RNPs, which showed that correct NP-vRNA interaction is key to the functioning of RNPs.

Guiding the humoral response against HIV-1 toward a MPER adjacent region by immunization with a VLP-formulated antibody-selected envelope variant.

Preventive HIV-1 vaccine strategies rely on the elicitation of broadly neutralizing antibody (bNAb) responses, but their induction in vivo by vaccination remains challenging. Considering that the ability of an epitope to elicit effective humoral immunity depends on its exposure on the virion, we have used a reverse genetics approach to select variants from an HIV-1 AC10_29 randomly mutated envelope library that showed increased affinity for a selected bNAb (4E10 bNAb targeting the HIV-1 MPER region). Isolated envelope sequences were analyzed by deep-sequencing showing a small number of dominant changes, including the loss of four potential N-linked glycosylation sites and disruption of the V1/V2 loop. Accordingly, the dominant variant (LR1-C1), showed not only increased affinity for MPER bNAbs 4E10 and 2F5, but also higher affinity for an additional antibody targeting the V3 loop (447-52D) that could be a consequence of an open conformation tier 1-like Env. Furthermore, the amino acids specific for the selected variant are associated with an increased sensitivity for 4E10 and 2F5 antibodies. In vivo studies showed that sera from mice immunized with LR1-C1 viruses possessed an improved neutralizing activity compared to the wild-type AC10_29 env. While Virus Like Particles (VLPs) carrying this envelope were unable to induce detectable neutralizing activity in immunized rabbits, one animal showed antibody response to the 4E10-proximal region. Our data establish a novel approach that has the potential to yield HIV envelope immunogen sequences that direct antibody responses to specific envelope regions.