Trimeric HIV-1-Env Structures Define Glycan Shields from Clades A, B, and G.

The HIV-1-envelope (Env) trimer is covered by a glycan shield of ∼90 N-linked oligosaccharides, which comprises roughly half its mass and is a key component of HIV evasion from humoral immunity. To understand how antibodies can overcome the barriers imposed by the glycan shield, we crystallized fully glycosylated Env trimers from clades A, B, and G, visualizing the shield at 3.4-3.7 Å resolution. These structures reveal the HIV-1-glycan shield to comprise a network of interlocking oligosaccharides, substantially ordered by glycan crowding, that encase the protein component of Env and enable HIV-1 to avoid most antibody-mediated neutralization. The revealed features delineate a taxonomy of N-linked glycan-glycan interactions. Crowded and dispersed glycans are differently ordered, conserved, processed, and recognized by antibody. The structures, along with glycan-array binding and molecular dynamics, reveal a diversity in oligosaccharide affinity and a requirement for accommodating glycans among known broadly neutralizing antibodies that target the glycan-shielded trimer.

Broad and Potent Neutralizing Antibodies Recognize the Silent Face of the HIV Envelope.

Broadly neutralizing antibodies (bNAbs) against HIV-1 envelope (Env) inform vaccine design and are potential therapeutic agents. We identified SF12 and related bNAbs with up to 62% neutralization breadth from an HIV-infected donor. SF12 recognized a glycan-dominated epitope on Env's silent face and was potent against clade AE viruses, which are poorly covered by V3-glycan bNAbs. A 3.3Å cryo-EM structure of a SF12-Env trimer complex showed additional contacts to Env protein residues by SF12 compared with VRC-PG05, the only other known donor-derived silentface antibody, explaining SF12's increased neutralization breadth, potency, and resistance to Env mutation routes. Asymmetric binding of SF12 was associated with distinct N-glycan conformations across Env protomers, demonstrating intra-Env glycan heterogeneity. Administrating SF12 to HIV-1-infected humanized mice suppressed viremia and selected for viruses lacking the N448gp120 glycan. Effective bNAbs can therefore be raised against HIV-1 Env's silent face, suggesting their potential for HIV-1 prevention, therapy, and vaccine development.

Glycine Substitution at Helix-to-Coil Transitions Facilitates the Structural Determination of a Stabilized Subtype C HIV Envelope Glycoprotein.

Advances in HIV-1 envelope glycoprotein (Env) design generate native-like trimers and high-resolution clade A, B, and G structures and elicit neutralizing antibodies. However, a high-resolution clade C structure is critical, as this subtype accounts for the majority of HIV infections worldwide, but well-ordered clade C Env trimers are more challenging to produce due to their instability. Based on targeted glycine substitutions in the Env fusion machinery, we defined a general approach that disfavors helical transitions leading to post-fusion conformations, thereby favoring the pre-fusion state. We generated a stabilized, soluble clade C Env (16055 NFL) and determined its crystal structure at 3.9 Å. Its overall conformation is similar to SOSIP.664 and native Env trimers but includes a covalent linker between gp120 and gp41, an engineered 201-433 disulfide bond, and density corresponding to 22 N-glycans. Env-structure-guided design strategies resulted in multiple homogeneous cross-clade immunogens with the potential to advance HIV vaccine development.

Particulate Array of Well-Ordered HIV Clade C Env Trimers Elicits Neutralizing Antibodies that Display a Unique V2 Cap Approach.

The development of soluble envelope glycoprotein (Env) mimetics displaying ordered trimeric symmetry has ushered in a new era in HIV-1 vaccination. The recently reported native, flexibly linked (NFL) design allows the generation of native-like trimers from clinical isolates at high yields and homogeneity. As the majority of infections world-wide are of the clade C subtype, we examined responses in non-human primates to well-ordered subtype C 16055 trimers administered in soluble or high-density liposomal formats. We detected superior germinal center formation and enhanced autologous neutralizing antibodies against the neutralization-resistant (tier 2) 16055 virus following inoculation of liposome-arrayed trimers. Epitope mapping of the neutralizing monoclonal antibodies (mAbs) indicated major contacts with the V2 apex, and 3D electron microscopy reconstructions of Fab-trimer complexes revealed a horizontal binding angle to the Env spike. These vaccine-elicited mAbs target the V2 cap, demonstrating a means to accomplish tier 2 virus neutralization by penetrating the dense N-glycan shield.

Prevalence and Phenotypic Susceptibility to Doravirine of the HIV-1 Reverse Transcriptase V106I Polymorphism in B and Non-B Subtypes.

BACKGROUND: Limited data are available regarding the susceptibility of the reverse transcriptase V106 polymorphism to doravirine. METHODS: Doravirine susceptibility was measured in site-directed mutants (SDMs) containing V106I, V106A, V106M, and Y188L mutations in subtype B (NL4-3, HXB2) and CRF02_AG background and in recombinant viruses with RT harboring V106I alone derived from 50 people with HIV. RESULTS: HIV-1 B subtype was detected in 1523 of 2705 cases. Prevalence of V106I was 3.2% in B and 2.5% in non-B subtypes, and was higher in subtype F (8.1%) and D (14.3%). Fold-changes (FC) in susceptibility for SDMs were below doravirine biological cutoff (3.0) for V106I, but not for V106A, V106M, and Y188L. Clinically derived viruses tested included 22 B (median FC, 1.2; interquartile range [IQR], 0.9-1.6) and 28 non-B subtypes (median FC, 1.8; IQR, 0.9-3.0). Nine (18%) viruses showed FC values equal or higher than the doravirine biological FC cutoff. CONCLUSIONS: The prevalence of the HIV-1 RT V106I polymorphism in MeditRes HIV consortium remains low, but significantly more prevalent in subtypes D and F. V106I minimally decreased the susceptibility to doravirine in SDMs and most clinical isolates. Reduced susceptibility seems to occur at increased frequency in subtype F1; however, the clinical impact remains to be investigated. CLINICAL TRIALS REGISTRATION: NCT04894357.

Sensitivity and specificity of the new Bio-Rad HIV screening test, Access HIV combo V2.

Diagnosing of human immunodeficiency virus (HIV) types 1 and 2 requires a screening with a highly sensitive and specific enzyme immunoassay and a low detection limit for the HIV-1 p24 antigen to minimize the diagnostic window. The objective of the study was to determine the sensitivity, specificity, and p24 limit of detection of the Access HIV combo V2 assay. Retrospective part of sensitivity: 452 HIV-1 positive samples from 403 chronic (9 different HIV-1 group M subtypes, 22 different HIV-1 group M CRFs, and 3 HIV-1 group O), 49 primary HIV-1 infections, 103 HIV-2 positive samples assessed at Pitié-Salpêtrière Hospital, 600 untyped HIV-1, 10 subtype-D, and 159 untyped HIV-2 samples assessed in Bio-Rad Laboratories. Prospective part of clinical specificity: all consecutive samples in two blood donor facilities and Pitié-Salpêtrière (6,570 patients) tested with Access HIV combo V2 and respectively Prism HIV O Plus (Abbott) or Architect HIV Ag/Ab Combo (Abbott) for Ag/Ab screening, and Procleix Ultrio (Gen Probe) for HIV RNA screening. Limit of detection for p24 antigen was assessed on recombinant virus-like particles (10 HIV-1 group M subtypes/CRFs, HIV-1 group O). Sensitivity [95% confidence interval (CI)] of Access HIV combo V2 was 100% (99.63-100) for HIV-1 chronic infection, 100% (98.55-100) for HIV-2 chronic infection, and 100% (93.00-100) for HIV-1 primary infection. Specificity (95% CI) was 99.98 (99.91-100). Limit of detection for p24 antigen was around 0.43 IU/mL [interquartile range (0.38-0.56)], and consistent across the 11 analyzed subtypes/CRFs. Hence, with both high sensitivity and specificity, Access HIV combo V2 is a suitable screening assay for HIV-1/2 infection. IMPORTANCE: Bio-Rad is one of the leading human immunodeficiency virus (HIV) screening test manufacturers. This laboratory released in 2021 their new version of the Access combo HIV test. However, to date, there have been no studies regarding its performance, especially its limit of detection of the diverse p24 antigen. We present the sensitivity (chronic and primary HIV-1 infection and HIV-2 chronic infection), specificity (blood donors and hospitalized patients), and raw data for the p24/seroconversion panels the manufacturer gave to the European agencies.

Performance of the Applied Biosystems HIV-1 Genotyping Kit with Integrase.

HIV genotyping is used to assess HIV susceptibility to antiretroviral drugs. The Applied Biosystems HIV-1 Genotyping Kit with Integrase (AB kit, Thermo Fisher Scientific) detects resistance-associated mutations (RAMs) in HIV protease (PR), reverse transcriptase (RT), and integrase (IN). We compared results from the AB kit with results obtained previously with the ViroSeq HIV-1 Genotyping System. DNA amplicons from the AB kit were also analyzed using next-generation sequencing (NGS). HIV RNA was extracted using the MagNA Pure 24 instrument (Roche Diagnostics; 96 plasma samples, HIV subtype B, viral load range: 530-737,741 copies/mL). FASTA files were generated from AB kit data using Exatype (Hyrax Biosciences). DNA amplicons from the AB kit were also analyzed by NGS using the Nextera XT kit (Illumina). Drug resistance was predicted using the Stanford HIV Drug Resistance Database. The mean genetic distance for sequences from ViroSeq and the AB kit was 0.02% for PR/RT and 0.04% for IN; 103 major RAMs were detected by both methods. Four additional major RAMs were detected by the AB kit only. These four major RAMs were also detected by NGS (detected in 18.1%-38.2% of NGS reads). NGS detected 27 major RAMs that were not detected with either of the Sanger sequencing-based kits. All major RAMs detected with ViroSeq were detected with the AB kit; additional RAMs were detected with the AB kit only. DNA amplicons from the AB kit can be used for NGS for more sensitive detection of RAMs.

Relative resistance of patient-derived envelope sequences to SERINC5-mediated restriction of HIV-1 infectivity.

IMPORTANCE: Pathogenesis of HIV-1 is enhanced through several viral-encoded proteins that counteract a range of host restriction molecules. HIV-1 Nef counteracts the cell membrane protein SERINC5 by downregulating it from the cell surface, thereby enhancing virion infectivity. Some subtype B reference Envelope sequences have shown the ability to bypass SERINC5 infectivity restriction independent of Nef. However, it is not clear if and to what extent circulating HIV-1 strains can exhibit resistance to SERINC5 restriction. Using a panel of Envelope sequences isolated from 50 Tanzanians infected with non-B HIV-1 subtypes, we show that the lentiviral reporters pseudotyped with patient-derived Envelopes have reduced sensitivity to SERINC5 and that this sensitivity differed among viral subtypes. Moreover, we found that SERINC5 sensitivity within patient-derived Envelopes can be modulated by separate regions, highlighting the complexity of viral/host interactions.

V3 tip determinants of susceptibility to inhibition by CD4-mimetic compounds in natural clade A human immunodeficiency virus (HIV-1) envelope glycoproteins.

IMPORTANCE: CD4-mimetic compounds (CD4mcs) are small-molecule inhibitors of human immunodeficiency virus (HIV-1) entry into host cells. CD4mcs target a pocket on the viral envelope glycoprotein (Env) spike that is used for binding to the receptor, CD4, and is highly conserved among HIV-1 strains. Nonetheless, naturally occurring HIV-1 strains exhibit a wide range of sensitivities to CD4mcs. Our study identifies changes distant from the binding pocket that can influence the susceptibility of natural HIV-1 strains to the antiviral effects of multiple CD4mcs. We relate the antiviral potency of the CD4mc against this panel of HIV-1 variants to the ability of the CD4mc to activate entry-related changes in Env conformation prematurely. These findings will guide efforts to improve the potency and breadth of CD4mcs against natural HIV-1 variants.

The origin and spread of HIV-1 CRF59_01B epidemic in China: A molecular network and phylogeographic analysis.

Human immunodeficiency virus type 1 CRF59_01B, identified in China in 2013, has been detected nationwide, exhibiting notably high prevalence in Guangzhou and its vicinity. This study aimed to unravel its origin and migration. A data set was established, incorporating all available CRF59_01B pol gene sequences and their metadata from Guangzhou and the public database. Bayesian phylogeographic analysis demonstrated that CRF59_01B originated in Shenzhen, the neighboring city of Guangzhou, around 1998 with posterior probability of 0.937. Molecular network analysis detected 1131 transmission links and showed a remarkably high clustering rate (78.9%). Substantial inter-city transmissions (26.5%, 300/1131) were observed between Shenzhen and Guangzhou while inter-region transmissions linked Guangzhou with South (46) and Southwest (64) China. The centre of Guangzhou was the hub of CRF59_01B transmission, including the inflow from Shenzhen (3.57 events/year) and outflow to the outskirts of Guangzhou (>2 events/year). The large-scale analysis revealed significant migration from Shenzhen to Guangzhou (5.08 events/year) and North China (0.59 events/year), and spread from Guangzhou to Central (0.47 events/year), East (0.42 events/year), South (0.76 events/year), Southwest China (0.76 events/year) and Shenzhen (1.89 events/year). Shenzhen and Guangzhou served as the origin and the hub of CRF59_01B circulation, emphasizing inter-city cooperation and data sharing to confine its nationwide diffusion.

Differences in neutralizing antibody sensitivities and envelope characteristics indicate distinct antigenic properties of Nigerian HIV-1 subtype G and CRF02_AG.

The magnitude of the HIV-1 epidemic in Nigeria is second only to the subtype C epidemic in South Africa, yet the subtypes prevalent in Nigeria require further characterization. A panel of 50 subtype G and 18 CRF02_AG Nigerian HIV-1 pseudoviruses (PSV) was developed and envelope coreceptor usage, neutralization sensitivity and cross-clade reactivity were characterized. These PSV were neutralized by some antibodies targeting major neutralizing determinants, but potentially important differences were observed in specific sensitivities (eg. to sCD4, MPER and V2/V3 monoclonal antibodies), as well as in properties such as variable loop lengths, number of potential N-linked glycans and charge, demonstrating distinct antigenic characteristics of CRF02_AG and subtype G. There was preferential neutralization of the matched CRF/subtype when PSV from subtype G or CRF02_AG were tested using pooled plasma. These novel Nigerian PSV will be useful to study HIV-1 CRF- or subtype-specific humoral immune responses for subtype G and CRF02_AG.

Impact of subtype C-specific amino acid variants on HIV-1 Tat-TAR interaction: insights from molecular modelling and dynamics.

BACKGROUND: HIV-1 produces Tat, a crucial protein for transcription, viral replication, and CNS neurotoxicity. Tat interacts with TAR, enhancing HIV reverse transcription. Subtype C Tat variants (C31S, R57S, Q63E) are associated with reduced transactivation and neurovirulence compared to subtype B. However, their precise impact on Tat-TAR binding is unclear. This study investigates how these substitutions affect Tat-TAR interaction. METHODS: We utilized molecular modelling techniques, including MODELLER, to produce precise three-dimensional structures of HIV-1 Tat protein variants. We utilized Tat subtype B as the reference or wild type, and generated Tat variants to mirror those amino acid variants found in Tat subtype C. Subtype C-specific amino acid substitutions were selected based on their role in the neuropathogenesis of HIV-1. Subsequently, we conducted molecular docking of each Tat protein variant to TAR using HDOCK, followed by molecular dynamic simulations. RESULTS: Molecular docking results indicated that Tat subtype B (TatWt) showed the highest affinity for the TAR element (-262.07), followed by TatC31S (-261.61), TatQ63E (-256.43), TatC31S/R57S/Q63E (-238.92), and TatR57S (-222.24). However, binding free energy analysis showed higher affinities for single variants TatQ63E (-349.2 ± 10.4 kcal/mol) and TatR57S (-290.0 ± 9.6 kcal/mol) compared to TatWt (-247.9 ± 27.7 kcal/mol), while TatC31S and TatC31S/R57SQ/63E showed lower values. Interactions over the protein trajectory were also higher for TatQ63E and TatR57S compared to TatWt, TatC31S, and TatC31S/R57SQ/63E, suggesting that modifying amino acids within the Arginine/Glutamine-rich region notably affects TAR interaction. Single amino acid mutations TatR57S and TatQ63E had a significant impact, while TatC31S had minimal effect. Introducing single amino acid variants from TatWt to a more representative Tat subtype C (TatC31S/R57SQ/63E) resulted in lower predicted binding affinity, consistent with previous findings. CONCLUSIONS: These identified amino acid positions likely contribute significantly to Tat-TAR interaction and the differential pathogenesis and neuropathogenesis observed between subtype B and subtype C. Additional experimental investigations should prioritize exploring the influence of these amino acid signatures on TAR binding to gain a comprehensive understanding of their impact on viral transactivation, potentially identifying them as therapeutic targets.

Molecular transmission network analysis of newly diagnosed HIV-1 infections in Nanjing from 2019 to 2021.

OBJECTIVE: The objective of this study was to conduct a comprehensive analysis of the molecular transmission networks and transmitted drug resistance (TDR) patterns among individuals newly diagnosed with HIV-1 in Nanjing. METHODS: Plasma samples were collected from newly diagnosed HIV patients in Nanjing between 2019 and 2021. The HIV pol gene was amplified, and the resulting sequences were utilized for determining TDR, identifying viral subtypes, and constructing molecular transmission network. Logistic regression analyses were employed to investigate the epidemiological characteristics associated with molecular transmission clusters. RESULTS: A total of 1161 HIV pol sequences were successfully extracted from newly diagnosed individuals, each accompanied by reliable epidemiologic information. The analysis revealed the presence of multiple HIV-1 subtypes, with CRF 07_BC (40.57%) and CRF01_AE (38.42%) being the most prevalent. Additionally, six other subtypes and unique recombinant forms (URFs) were identified. The prevalence of TDR among the newly diagnosed cases was 7.84% during the study period. Employing a genetic distance threshold of 1.50%, the construction of the molecular transmission network resulted in the identification of 137 clusters, encompassing 613 nodes, which accounted for approximately 52.80% of the cases. Multivariate analysis indicated that individuals within these clusters were more likely to be aged ≥ 60, unemployed, baseline CD4 cell count ≥ 200 cells/mm3, and infected with the CRF119_0107 (P < 0.05). Furthermore, the analysis of larger clusters revealed that individuals aged ≥ 60, peasants, those without TDR, and individuals infected with the CRF119_0107 were more likely to be part of these clusters. CONCLUSIONS: This study revealed the high risk of local HIV transmission and high TDR prevalence in Nanjing, especially the rapid spread of CRF119_0107. It is crucial to implement targeted interventions for the molecular transmission clusters identified in this study to effectively control the HIV epidemic.

Tracing HIV-1 strains that imprint broadly neutralizing antibody responses.

Understanding the determinants of broadly neutralizing antibody (bNAb) evolution is crucial for the development of bNAb-based HIV vaccines1. Despite emerging information on cofactors that promote bNAb evolution in natural HIV-1 infections, in which the induction of bNAbs is genuinely rare2, information on the impact of the infecting virus strain on determining the breadth and specificity of the antibody responses to HIV-1 is lacking. Here we analyse the influence of viral antigens in shaping antibody responses in humans. We call the ability of a virus strain to induce similar antibody responses across different hosts its antibody-imprinting capacity, which from an evolutionary biology perspective corresponds to the viral heritability of the antibody responses. Analysis of 53 measured parameters of HIV-1-binding and neutralizing antibody responses in a cohort of 303 HIV-1 transmission pairs (individuals who harboured highly related HIV-1 strains and were putative direct transmission partners or members of an HIV-1 transmission chain) revealed that the effect of the infecting virus on the outcome of the bNAb response is moderate in magnitude but highly significant. We introduce the concept of bNAb-imprinting viruses and provide evidence for the existence of such viruses in a systematic screening of our cohort. The bNAb-imprinting capacity can be substantial, as indicated by a transmission pair with highly similar HIV-1 antibody responses and strong bNAb activity. Identification of viruses that have bNAb-imprinting capacities and their characterization may thus provide the potential to develop lead immunogens.

HIV Coinfection Provides Insights for the Design of Vaccine Cocktails to Elicit Broadly Neutralizing Antibodies.

Induction of broadly neutralizing antibodies (bNAbs) to HIV and other diverse pathogens will likely require the use of multiple immunogens. An understanding of the dynamics of antibody development to multiple diverse but related antigens would facilitate the rational design of immunization strategies. Here, we characterize, in detail, the development of neutralizing antibodies in three individuals coinfected with several divergent HIV variants. Two of these coinfected individuals developed additive or cross-neutralizing antibody responses. However, interference was observed in the third case, with neutralizing antibody responses to one viral variant arising to the near exclusion of neutralizing responses to the other. Longitudinal characterization of the diversity in the Envelope glycoprotein trimer (Env) structure showed that in the individual who developed the broadest neutralizing antibodies, circulating viruses shared a conserved epitope on the trimer apex that was targeted by cross-neutralizing antibodies. In contrast, in the other two individuals, diversity was distributed across Env. Taken together, these data highlight that multiple related immunogens can result in immune interference. However, they also suggest that immunogen cocktails presenting shared, conserved neutralizing epitopes in a variable background may focus broadly neutralizing antibody responses to these targets. IMPORTANCE Despite being the focus of extensive research, we still do not know how to reproducibly elicit cross-neutralizing antibodies against variable pathogens by vaccination. Here, we characterize the antibody responses in people coinfected with more than one HIV variant, providing insights into how the use of antigen "cocktails" might affect the breadth of the elicited neutralizing antibody response and how the relatedness of the antigens may shape this.

Phylodynamics of HIV in the Mexico City Metropolitan Region.

Evolutionary analyses of viral sequences can provide insights into transmission dynamics, which in turn can optimize prevention interventions. Here, we characterized the dynamics of HIV transmission within the Mexico City metropolitan area. HIV pol sequences from persons recently diagnosed at the largest HIV clinic in Mexico City (between 2016 and 2021) were annotated with demographic/geographic metadata. A multistep phylogenetic approach was applied to identify putative transmission clades. A data set of publicly available sequences was used to assess international introductions. Clades were analyzed with a discrete phylogeographic model to evaluate the timing and intensity of HIV introductions and transmission dynamics among municipalities in the region. A total of 6,802 sequences across 96 municipalities (5,192 from Mexico City and 1,610 from the neighboring State of Mexico) were included (93.6% cisgender men, 5.0% cisgender women, and 1.3% transgender women); 3,971 of these sequences formed 1,206 clusters, involving 78 municipalities, including 89 clusters of ≥10 sequences. Discrete phylogeographic analysis revealed (i) 1,032 viral introductions into the region, over one-half of which were from the United States, and (ii) 354 migration events between municipalities with high support (adjusted Bayes factor of ≥3). The most frequent viral migrations occurred between northern municipalities within Mexico City, i.e., Cuauhtémoc to Iztapalapa (5.2% of events), Iztapalapa to Gustavo A. Madero (5.4%), and Gustavo A. Madero to Cuauhtémoc (6.5%). Our analysis illustrates the complexity of HIV transmission within the Mexico City metropolitan area but also identifies a spatially active transmission area involving a few municipalities in the north of the city, where targeted interventions could have a more pronounced effect on the entire regional epidemic. IMPORTANCE Phylogeographic investigation of the Mexico City HIV epidemic illustrates the complexity of HIV transmission in the region. An active transmission area involving a few municipalities in the north of the city, with transmission links throughout the region, is identified and could be a location where targeted interventions could have a more pronounced effect on the entire regional epidemic, compared with those dispersed in other manners.

Selection of HIV Envelope Strains for Standardized Assessments of Vaccine-Elicited Antibody-Dependent Cellular Cytotoxicity-Mediating Antibodies.

Antibody-dependent cellular cytotoxicity (ADCC) has been correlated with reduced risk of human immunodeficiency virus type 1 (HIV-1) infection in several preclinical vaccine trials and in the RV144 clinical trial, indicating that this is a relevant antibody function to study. Given the diversity of HIV-1, the breadth of vaccine-induced antibody responses is a critical parameter to understand if a universal vaccine is to be realized. Moreover, the breadth of ADCC responses can be influenced by different vaccine strategies and regimens, including adjuvants. Therefore, to accurately evaluate ADCC and to compare vaccine regimens, it is important to understand the range of HIV Envelope (Env) susceptibility to these responses. These evaluations have been limited because of the complexity of the assay and the lack of a comprehensive panel of viruses for the assessment of these humoral responses. Here, we used 29 HIV-1 infectious molecular clones (IMCs) representing different Envelope subtypes and circulating recombinant forms to characterize susceptibility to ADCC from antibodies in plasma from infected individuals, including 13 viremic individuals, 10 controllers, and six with broadly neutralizing antibody responses. We found in our panel that ADCC susceptibility of the IMCs in our panel did not cluster by subtype, infectivity, level of CD4 downregulation, level of shedding, or neutralization sensitivity. Using partitioning around medoids (PAM) clustering to distinguish smaller groups of IMCs with similar ADCC susceptibility, we identified nested panels of four to eight IMCs that broadly represent the ADCC susceptibility of the entire 29-IMC panel. These panels, together with reagents developed to specifically accommodate circulating viruses at the geographical sites of vaccine trials, will provide a powerful tool to harmonize ADCC data generated across different studies and to detect common themes of ADCC responses elicited by various vaccines. IMPORTANCE Antibody-dependent cellular cytotoxicity (ADCC) responses were found to correlate with reduced risk of infection in the RV144 trial of the only human HIV-1 vaccine to show any efficacy to date. However, reagents to understand the breadth and magnitude of these responses across preclinical and clinical vaccine trials remain underdeveloped. In this study, we characterize HIV-1 infectious molecular clones encoding 29 distinct Envelope strains (Env-IMCs) to understand factors that impact virus susceptibility to ADCC and use statistical methods to identify smaller nested panels of four to eight Env-IMCs that accurately represent the full set. These reagents can be used as standardized reagents across studies to fully understand how ADCC may affect efficacy of future vaccine studies and how studies differ in the breadth of responses developed.

Viral and Cellular Factors Leading to the Loss of CD4 Homeostasis in HIV-1 Viremic Nonprogressors.

Human immunodeficiency virus type 1 (HIV-1) viremic nonprogressors (VNPs) represent a very rare HIV-1 extreme phenotype. VNPs are characterized by persistent high plasma viremia and maintenance of CD4+ T-cell counts in the absence of treatment. However, the causes of nonpathogenic HIV-1 infection in VNPs remain elusive. Here, we identified for the first time two VNPs who experienced the loss of CD4+ homeostasis (LoH) after more than 13 years. We characterized in deep detail viral and host factors associated with the LoH and compared with standard VNPs and healthy controls. The viral factors determined included HIV-1 coreceptor usage and replicative capacity. Changes in CD4+ and CD8+ T-cell activation, maturational phenotype, and expression of CCR5 and CXCR6 in CD4+ T-cells were also evaluated as host-related factors. Consistently, we determined a switch in HIV-1 coreceptor use to CXCR4 concomitant with an increase in replicative capacity at the LoH for the two VNPs. Moreover, we delineated an increase in the frequency of HLA-DR+CD38+ CD4+ and CD8+ T cells and traced the augment of naive T-cells upon polyclonal activation with LoH. Remarkably, very low and stable levels of CCR5 and CXCR6 expression in CD4+ T-cells were measured over time. Overall, our results demonstrated HIV-1 evolution toward highly pathogenic CXCR4 strains in the context of very limited and stable expression of CCR5 and CXCR6 in CD4+ T cells as potential drivers of LoH in VNPs. These data bring novel insights into the correlates of nonpathogenic HIV-1 infection. IMPORTANCE The mechanism behind nonpathogenic human immunodeficiency virus type 1 (HIV-1) infection remains poorly understood, mainly because of the very low frequency of viremic nonprogressors (VNPs). Here, we report two cases of VNPs who experienced the loss of CD4+ T-cell homeostasis (LoH) after more than 13 years of HIV-1 infection. The deep characterization of viral and host factors supports the contribution of viral and host factors to the LoH in VNPs. Thus, HIV-1 evolution toward highly replicative CXCR4 strains together with changes in T-cell activation and maturational phenotypes were found. Moreover, we measured very low and stable levels of CCR5 and CXCR6 in CD4+ T-cells over time. These findings support viral evolution toward X4 strains limited by coreceptor expression to control HIV-1 pathogenesis and demonstrate the potential of host-dependent factors, yet to be fully elucidated in VNPs, to control HIV-1 pathogenesis.

Cuban history of CRF19 recombinant subtype of HIV-1.

CRF19 is a recombinant form of HIV-1 subtypes D, A1 and G, which was first sampled in Cuba in 1999, but was already present there in 1980s. CRF19 was reported almost uniquely in Cuba, where it accounts for ∼25% of new HIV-positive patients and causes rapid progression to AIDS (∼3 years). We analyzed a large data set comprising ∼350 pol and env sequences sampled in Cuba over the last 15 years and ∼350 from Los Alamos database. This data set contained both CRF19 (∼315), and A1, D and G sequences. We performed and combined analyses for the three A1, G and D regions, using fast maximum likelihood approaches, including: (1) phylogeny reconstruction, (2) spatio-temporal analysis of the virus spread, and ancestral character reconstruction for (3) transmission mode and (4) drug resistance mutations (DRMs). We verified these results with a Bayesian approach. This allowed us to acquire new insights on the CRF19 origin and transmission patterns. We showed that CRF19 recombined between 1966 and 1977, most likely in Cuban community stationed in Congo region. We further investigated CRF19 spread on the Cuban province level, and discovered that the epidemic started in 1970s, most probably in Villa Clara, that it was at first carried by heterosexual transmissions, and then quickly spread in the 1980s within the "men having sex with men" (MSM) community, with multiple transmissions back to heterosexuals. The analysis of the transmission patterns of common DRMs found very few resistance transmission clusters. Our results show a very early introduction of CRF19 in Cuba, which could explain its local epidemiological success. Ignited by a major founder event, the epidemic then followed a similar pattern as other subtypes and CRFs in Cuba. The reason for the short time to AIDS remains to be understood and requires specific surveillance, in Cuba and elsewhere.

HIV-1 at 25.

In 1981, the acquired immunodeficiency syndrome (AIDS) appeared insidiously and mystified doctors and scientists alike. No one could have predicted then that it would become, arguably, the worst plague in human history. Today, 33 million persons are living with infection by the human immunodeficiency virus type 1 (HIV-1), the causative agent of AIDS, while another 25 million have already died of this disease.