Characterization update of HIV-1 M subtypes diversity and proposal for subtypes A and D sub-subtypes reclassification.

BACKGROUND: The large and constantly evolving HIV-1 pandemic has led to an increasingly complex diversity. Because of some taxonomic difficulties among the most diverse HIV-1 subtypes, and taking advantage of the large amount of sequence data generated in the recent years, we investigated novel lineage patterns among the main HIV-1 subtypes. RESULTS: All HIV full-length genomes available in public databases were analysed (n = 2017). Maximum likelihood phylogenies and pairwise genetic distance were obtained. Clustering patterns and mean distributions of genetic distances were compared within and across the current groups, subtypes and sub-subtypes of HIV-1 to detect and analyse any divergent lineages within previously defined HIV lineages. The level of genetic similarity observed between most HIV clades was deeply consistent with the current classification. However, both subtypes A and D showed evidence of further intra-subtype diversification not fully described by the nomenclature system at the time and could be divided into several distinct sub-subtypes. CONCLUSIONS: With this work, we propose an updated nomenclature of sub-types A and D better reflecting their current genetic diversity and evolutionary patterns. Allowing a more accurate nomenclature and classification system is a necessary step for easier subtyping of HIV strains and a better detection or follow-up of viral epidemiology shifts

HIV-2 diversity displays two clades within group A with distinct geographical distribution and evolution

Genetic diversity of HIV-2 groups A and B has not yet been fully described, especially in a few Western Africa countries such as Ivory-Coast or Mali. We collected 444 pol, 152 vif, 129 env, and 74 LTR sequences from patients of the French ANRS CO5 HIV-2 cohort completed by 221 pol, 18 vif, 377 env, and 63 LTR unique sequences from public databases. We performed phylogenetic reconstructions and revealed two distinct lineages within HIV-2 group A, herein called A1 and A2, presenting non-negligible genetic distances and distinct geographic distributions as A1 is related to coastal Western African countries and A2 to inland Western countries. Estimated early diversification times for groups A and B in human populations were 1940 [95% higher probability densitiy: 1935-53] and 1961 [1952-70]. A1 experienced an early diversification in 1942 [1937-58] with two distinct early epidemics in Guinea-Bissau or Senegal, raising the possibility of group A emergence in those countries from an initial introduction from Ivory-Coast to Senegal, two former French colonies. Changes in effective population sizes over time revealed that A1 exponentially grew concomitantly to Guinea-Bissau independence war, but both A2 and B lineages experienced a latter growth, starting during the 80s economic crisis. This large HIV-2 genetic analysis provides the existence of two distinct subtypes within group A and new data about HIV-2 early spreading patterns and recent epidemiologic evolution for which data are scarce outside Guinea-Bissau

Tetravalent SARS-CoV-2 S1 Subunit Protein Vaccination Elicits Robust Humoral and Cellular Immune Responses in SIV-Infected Rhesus Macaque Controllers

The study provides important insights into the immunogenicity and efficacy of a tetravalent protein subunit vaccine candidate against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The vaccine induced both humoral and cellular immune responses in nonhuman primates with controlled SIVagm infection and was able to generate Omicron variant-specific antibodies without specifically vaccinating with Omicron. These findings suggest that the tetravalent composition of the vaccine candidate could provide broad protection against multiple SARS-CoV-2 variants while minimizing the risk of immune escape and the emergence of new variants. Additionally, the use of rhesus macaques with controlled SIVsab infection may better represent vaccine immunogenicity in humans with chronic viral diseases, highlighting the importance of preclinical animal models in vaccine development. Overall, the study provides valuable information for the development and implementation of coronavirus disease 2019 vaccines, particularly for achieving global vaccine equity and addressing emerging variants

Vaccine breakthrough hypoxemic COVID-19 pneumonia in patients with auto-Abs neutralizing type I IFNs

Life-threatening `breakthrough' cases of critical COVID-19 are attributed to poor or waning antibody response to the SARS- CoV-2 vaccine in individuals already at risk. Pre-existing autoantibodies (auto-Abs) neutralizing type I IFNs underlie at least 15% of critical COVID-19 pneumonia cases in unvaccinated individuals; however, their contribution to hypoxemic breakthrough cases in vaccinated people remains unknown. Here, we studied a cohort of 48 individuals ( age 20-86 years) who received 2 doses of an mRNA vaccine and developed a breakthrough infection with hypoxemic COVID-19 pneumonia 2 weeks to 4 months later. Antibody levels to the vaccine, neutralization of the virus, and auto- Abs to type I IFNs were measured in the plasma. Forty-two individuals had no known deficiency of B cell immunity and a normal antibody response to the vaccine. Among them, ten (24%) had auto-Abs neutralizing type I IFNs (aged 43-86 years). Eight of these ten patients had auto-Abs neutralizing both IFN-a2 and IFN-., while two neutralized IFN-omega only. No patient neutralized IFN-ss. Seven neutralized 10 ng/mL of type I IFNs, and three 100 pg/mL only. Seven patients neutralized SARS-CoV-2 D614G and the Delta variant (B.1.617.2) efficiently, while one patient neutralized Delta slightly less efficiently. Two of the three patients neutralizing only 100 pg/mL of type I IFNs neutralized both D61G and Delta less efficiently. Despite two mRNA vaccine inoculations and the presence of circulating antibodies capable of neutralizing SARS-CoV-2, auto-Abs neutralizing type I IFNs may underlie a significant proportion of hypoxemic COVID-19 pneumonia cases, highlighting the importance of this particularly vulnerable population

COVID-19 symptoms at hospital admission vary with age and sex: results from the ISARIC prospective multinational observational study

Background: The ISARIC prospective multinational observational study is the largest cohort of hospitalized patients with COVID-19. We present relationships of age, sex, and nationality to presenting symptoms. Methods: International, prospective observational study of 60 109 hospitalized symptomatic patients with laboratory-confirmed COVID-19 recruited from 43 countries between 30 January and 3 August 2020. Logistic regression was performed to evaluate relationships of age and sex to published COVID-19 case definitions and the most commonly reported symptoms. Results: ‘Typical’ symptoms of fever (69%), cough (68%) and shortness of breath (66%) were the most commonly reported. 92% of patients experienced at least one of these. Prevalence of typical symptoms was greatest in 30- to 60-year-olds (respectively 80, 79, 69%; at least one 95%). They were reported less frequently in children (≤ 18 years: 69, 48, 23; 85%), older adults (≥ 70 years: 61, 62, 65; 90%), and women (66, 66, 64; 90%; vs. men 71, 70, 67; 93%, each P < 0.001). The most common atypical presentations under 60 years of age were nausea and vomiting and abdominal pain, and over 60 years was confusion. Regression models showed significant differences in symptoms with sex, age and country. Interpretation: This international collaboration has allowed us to report reliable symptom data from the largest cohort of patients admitted to hospital with COVID-19. Adults over 60 and children admitted to hospital with COVID-19 are less likely to present with typical symptoms. Nausea and vomiting are common atypical presentations under 30 years. Confusion is a frequent atypical presentation of COVID-19 in adults over 60 years. Women are less likely to experience typical symptoms than men

Impact of genetic variability within Long Terminal Repeat region and integrase gene on HIV-2 replication

Le VIH-2 est souvent considéré comme un modèle d’infection rétrovirale atténuée, du fait de sa faible réplication virale, des faibles taux de transmission et de la progression plus lente vers le stade SIDA des patients infectés. Les mécanismes causaux de cette moindre physiopathologie sont encore mal connus. Dans ce travail, nous nous sommes intéressés à la diversité du VIH-2, notamment dans deux régions génomiques : l’intégrase et la région Long Terminal Repeat (LTR). Nous avons tout d’abord amélioré la technique classique d’isolement de souches virales afin de disposer de davantage de souches de VIH-2, en purifiant les échantillons de patients avec des billes anti-CD44. Ensuite, nous avons utilisé ces souches pour étudier la sensibilité aux inhibiteurs de transfert de brin de l’intégrase, ou INSTI. Au cours de ce travail, nous avons mis en évidence un nouveau mécanisme de résistance aux INSTI chez le VIH-2, l’insertion de 5 acides aminés après le codon 231 du gène de l’intégrase. Cette insertion est responsable d’une résistance importante au raltegravir (RAL), à l’elvitegravir (EVG), au cabotegravir (CAB), ainsi qu’une résistance modérée au dolutegravir (DTG). La sensibilité au bictegravir (BIC) était conservée pour certains isolats porteurs de cette insertion. Une insertion de deux acides aminés a aussi été observée transitoirement chez un patient. Nous avons confirmé ces observations phénotypiques en construisant des virus porteurs de ces insertions par mutagénèse dirigée. L’insertion, qu’elle soit de 2 ou de 5 acides aminés, n’était pas responsable d’une perte de capacité réplicative, à l’exception du mutant avec l’insertion GIRGK.En revanche, la structure prédite de l’intégrase est fortement modifiée, avec notamment la perte d’un des deux feuillets bêta composant le tonneau bêta du domaine C-terminal.La sensibilité phénotypique des mutants a été déterminée, pour les insertions de 5 acides aminés, les résultats étaient similaires à ceux obtenus avec les isolats cliniques. Nous avons aussi pu déterminer la sensibilité du mutant avec l’insertion GK, qui était sensible au BIC et au DTG, mais déjà pleinement résistant à RAL et au CAB. Dans la dernière partie de notre travail, nous avons étudié la variabilité dans la région LTR des provirus de 66 patients naïfs d’antirétroviraux, inclus dans la cohorte ANRS CO5 VIH-2. La variabilité génétique était plus importante dans les séquences du groupe B, du fait notamment de nombreuses insertions et délétions dans la sous-région «régulatrice» comprenant l’ensemble des sites de fixation de facteurs de transcription cellulaire. Ces virus du groupe B présentaient une délétion de quelques nucléotides dans la région contenant les sites de fixation PuB1 et pets, causant une perte de ce dernier site de fixation. De plus, 4 provirus du groupe B présentaient une délétion du premier site de fixation du facteur de transcription ubiquitaire Sp1. Cette variabilité entraînait des conséquences sur l’activité transcriptionnelle de ces LTR. Les LTR du groupe A et du groupe B présentaient la même activité transcriptionnelle basale mais, dans les cellules Jurkat, après activation cellulaire, l’activité transcriptionnelle des LTR du groupe B et d’un LTR de groupe A dans lequel la zone contenant la délétion de pets a été insérée était 10 fois plus faible que celle du LTR de groupe A. La délétion du premier site de fixation de Sp1 diminuait l’activité transcriptionnelle basale dans les cellules Jurkat et la réponse à la transactivation par la protéine virale Tat dans les cellules HEK293T.HIV-2 is oftenconsidered as an attenuated model of HIV-1 infection. Indeed, HIV-2 is characterized by its lower viral replication, reduced transmission rates and a slower progression towards AIDS of infected-patients. Mechanisms implied in HIV-2 reduced pathogenicity are not entirely understood. In our work, we have focused on HIV-2 diversity, especially in two genomic regions: the integrase and the Long Terminal Repeat (LTR) region.We have improved the culture method, by purifying clinical samples using anti-CD44 paramagnetic beads. This allowed us to increase the number of viral strains in our collection. Then, we used those strains to study phenotypic susceptibility to integrase strand transfer inhibitors (INSTI). During this study, we identified a new molecular mechanism of resistance to INSTI, a 5 amino-acids insertion after codon 231 of HIV-2 integrase. This insertion was responsible for a high level of resistance to raltegravir (RAL), elvitegravir (EVG), cabotegravir (CAB), and a slight increase in the resistance todolutegravir (DTG). Certain isolates harboring this insertion remained susceptible to bictegravir (BIC).A 2 amino-acids insertion was also transiently observed in one patient. We have confirmed those phenotypic data by constructing HIV-2 mutants by site-directed mutagenesis. Whether the insertion was composed of 2 or 5 amino-acids, replicative capacitywas similar to the wild-type virus, except for the mutant with a GIRGK insertion. However, the predicted structure of mutated HIV-2 integrases was modified, notably in the C-terminal domain, due to the loss of one of the two beta sheets composing the beta barrel. Phenotypic susceptibility of mutants was determined and results for HIV-2 mutants witha 5 amino-acids insertion were similar to those obtained with clinical isolates.We also determined the susceptibility of the mutant with a GK insertion. It was susceptible to BIC and DTG, but already resistant to RAL and CAB. In the last part of our work, we have characterized genetic variability within LTR region in 66 antiretroviral-naïve patients, included in the French ANRS CO5 HIV-2 cohort.Genetic variability was higher among group B sequences, due to a high number of insertions and deletions in the «regulatory» sub-region that encompasses all transcription factor binding sites. All group B viruses presented a short deletion in the region encompassing PuB1 andpets binding sites, causing the loss of the pets binding site.Furthermore, 4 group B viruses had also a deletion of the first binding site of Sp1 transcription factor. This variability impacted LTR transcriptional activity. Group A and B LTRs presented asimilar basal transcriptional activity but, in Jurkat cells, after cellular activation, transcriptional activities of group B LTR and a group A LTR in which the deletion of pets binding site has been introduced were 10-fold lower than the transcriptional activity of an intact group A LTR. The deletion of the first Sp1 binding site reduced the basal transcriptional activity in Jurkat cells and the response to transactivation by the viral protein Tat in HEK293T

Impact de la variabilité génétique dans la région Long Terminal Repeat et le gène de l'intégrase sur la réplication du VIH-2

HIV-2 is oftenconsidered as an attenuated model of HIV-1 infection. Indeed, HIV-2 is characterized by its lower viral replication, reduced transmission rates and a slower progression towards AIDS of infected-patients. Mechanisms implied in HIV-2 reduced pathogenicity are not entirely understood. In our work, we have focused on HIV-2 diversity, especially in two genomic regions: the integrase and the Long Terminal Repeat (LTR) region.We have improved the culture method, by purifying clinical samples using anti-CD44 paramagnetic beads. This allowed us to increase the number of viral strains in our collection. Then, we used those strains to study phenotypic susceptibility to integrase strand transfer inhibitors (INSTI). During this study, we identified a new molecular mechanism of resistance to INSTI, a 5 amino-acids insertion after codon 231 of HIV-2 integrase. This insertion was responsible for a high level of resistance to raltegravir (RAL), elvitegravir (EVG), cabotegravir (CAB), and a slight increase in the resistance todolutegravir (DTG). Certain isolates harboring this insertion remained susceptible to bictegravir (BIC).A 2 amino-acids insertion was also transiently observed in one patient. We have confirmed those phenotypic data by constructing HIV-2 mutants by site-directed mutagenesis. Whether the insertion was composed of 2 or 5 amino-acids, replicative capacitywas similar to the wild-type virus, except for the mutant with a GIRGK insertion. However, the predicted structure of mutated HIV-2 integrases was modified, notably in the C-terminal domain, due to the loss of one of the two beta sheets composing the beta barrel. Phenotypic susceptibility of mutants was determined and results for HIV-2 mutants witha 5 amino-acids insertion were similar to those obtained with clinical isolates.We also determined the susceptibility of the mutant with a GK insertion. It was susceptible to BIC and DTG, but already resistant to RAL and CAB. In the last part of our work, we have characterized genetic variability within LTR region in 66 antiretroviral-naïve patients, included in the French ANRS CO5 HIV-2 cohort.Genetic variability was higher among group B sequences, due to a high number of insertions and deletions in the «regulatory» sub-region that encompasses all transcription factor binding sites. All group B viruses presented a short deletion in the region encompassing PuB1 andpets binding sites, causing the loss of the pets binding site.Furthermore, 4 group B viruses had also a deletion of the first binding site of Sp1 transcription factor. This variability impacted LTR transcriptional activity. Group A and B LTRs presented asimilar basal transcriptional activity but, in Jurkat cells, after cellular activation, transcriptional activities of group B LTR and a group A LTR in which the deletion of pets binding site has been introduced were 10-fold lower than the transcriptional activity of an intact group A LTR. The deletion of the first Sp1 binding site reduced the basal transcriptional activity in Jurkat cells and the response to transactivation by the viral protein Tat in HEK293T.Le VIH-2 est souvent considéré comme un modèle d’infection rétrovirale atténuée, du fait de sa faible réplication virale, des faibles taux de transmission et de la progression plus lente vers le stade SIDA des patients infectés. Les mécanismes causaux de cette moindre physiopathologie sont encore mal connus. Dans ce travail, nous nous sommes intéressés à la diversité du VIH-2, notamment dans deux régions génomiques : l’intégrase et la région Long Terminal Repeat (LTR). Nous avons tout d’abord amélioré la technique classique d’isolement de souches virales afin de disposer de davantage de souches de VIH-2, en purifiant les échantillons de patients avec des billes anti-CD44. Ensuite, nous avons utilisé ces souches pour étudier la sensibilité aux inhibiteurs de transfert de brin de l’intégrase, ou INSTI. Au cours de ce travail, nous avons mis en évidence un nouveau mécanisme de résistance aux INSTI chez le VIH-2, l’insertion de 5 acides aminés après le codon 231 du gène de l’intégrase. Cette insertion est responsable d’une résistance importante au raltegravir (RAL), à l’elvitegravir (EVG), au cabotegravir (CAB), ainsi qu’une résistance modérée au dolutegravir (DTG). La sensibilité au bictegravir (BIC) était conservée pour certains isolats porteurs de cette insertion. Une insertion de deux acides aminés a aussi été observée transitoirement chez un patient. Nous avons confirmé ces observations phénotypiques en construisant des virus porteurs de ces insertions par mutagénèse dirigée. L’insertion, qu’elle soit de 2 ou de 5 acides aminés, n’était pas responsable d’une perte de capacité réplicative, à l’exception du mutant avec l’insertion GIRGK.En revanche, la structure prédite de l’intégrase est fortement modifiée, avec notamment la perte d’un des deux feuillets bêta composant le tonneau bêta du domaine C-terminal.La sensibilité phénotypique des mutants a été déterminée, pour les insertions de 5 acides aminés, les résultats étaient similaires à ceux obtenus avec les isolats cliniques. Nous avons aussi pu déterminer la sensibilité du mutant avec l’insertion GK, qui était sensible au BIC et au DTG, mais déjà pleinement résistant à RAL et au CAB. Dans la dernière partie de notre travail, nous avons étudié la variabilité dans la région LTR des provirus de 66 patients naïfs d’antirétroviraux, inclus dans la cohorte ANRS CO5 VIH-2. La variabilité génétique était plus importante dans les séquences du groupe B, du fait notamment de nombreuses insertions et délétions dans la sous-région «régulatrice» comprenant l’ensemble des sites de fixation de facteurs de transcription cellulaire. Ces virus du groupe B présentaient une délétion de quelques nucléotides dans la région contenant les sites de fixation PuB1 et pets, causant une perte de ce dernier site de fixation. De plus, 4 provirus du groupe B présentaient une délétion du premier site de fixation du facteur de transcription ubiquitaire Sp1. Cette variabilité entraînait des conséquences sur l’activité transcriptionnelle de ces LTR. Les LTR du groupe A et du groupe B présentaient la même activité transcriptionnelle basale mais, dans les cellules Jurkat, après activation cellulaire, l’activité transcriptionnelle des LTR du groupe B et d’un LTR de groupe A dans lequel la zone contenant la délétion de pets a été insérée était 10 fois plus faible que celle du LTR de groupe A. La délétion du premier site de fixation de Sp1 diminuait l’activité transcriptionnelle basale dans les cellules Jurkat et la réponse à la transactivation par la protéine virale Tat dans les cellules HEK293T

Future of Antiretroviral Drugs and Evolution of HIV-1 Drug Resistance

Highly active antiretroviral (ARV) therapy has been used for many years, but the use in low- and middle-income countries of antiretroviral drugs with low genetic barrier to resistance, combined with limited availability of viral load testing, has led to higher rates of acquired drug resistance, sustaining the rate of transmitted drug resistance. Here, we describe the evolution of ARV drugs with the ongoing development of injectable long-acting forms and the requirements regarding all new ARV drugs (i.e., no transmitted drug resistance, no cross-resistance and high genetic barrier to resistance). Then, we report the evolution of both transmitted and acquired resistance regarding new ARV drugs. The WHO has set very ambitious but motivating goals for HIV testing, treatment and viral suppression, aiming to achieve rates of 95% for all three by 2025. Reaching these goals requires a wide implementation and use of close virological monitoring in LMICs

New insights are game-changers in HIV-2 disease management

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