Genetic characteristics, coreceptor usage potential and evolution of Nigerian HIV-1 subtype G and CRF02_AG isolates

HIV-1 CRF02_AG and subtype G (HIV-1G) account for most HIV infections in Nigeria, but their evolutionary trends have not been well documented. To better elucidate the dynamics of the epidemic in Nigeria we characterised the gag and env genes of North-Central Nigerian HIV-1 isolates from pregnant women. Of 28 samples sequenced in both genes, the predominant clades were CRF02_AG (39%) and HIV-1G (32%). Higher predicted proportion of CXCR4-tropic (X4) HIV-1G isolates was noted compared to CRF02_AG (p = 0.007, Fisher''s exact test). Phylogenetic and Bayesian analysis conducted on our sequences and all the dated available Nigerian sequences on the Los Alamos data base showed that CRF02_AG and HIV-1G entered into Nigeria through multiple entries, with presence of HIV-1G dating back to early 1980s. This study underlines the genetic complexity of the HIV-1 epidemic in Nigeria, possible subtype-specific differences in co-receptor usage, and the evolutionary trends of the predominant HIV-1 strains in Nigeria, which may have implications for the design of biomedical interventions and better understanding of the epidemic.     

Recombination confounds the early evolutionary history of human immunodeficiency virus type 1: subtype G is a circulating recombinant form

Human immunodeficiency virus type 1 (HIV-1) is classified in nine subtypes (A to D, F, G, H, J, and K), a number of subsubtypes, and several circulating recombinant forms (CRFs). Due to the high level of genetic diversity within HIV-1 and to its worldwide distribution, this classification system is widely used in fields as diverse as vaccine development, evolution, epidemiology, viral fitness, and drug resistance. Here, we demonstrate how the high recombination rates of HIV-1 may confound the study of its evolutionary history and classification. Our data show that subtype G, currently classified as a pure subtype, has in fact a recombinant history, having evolved following recombination between subtypes A and J and a putative subtype G parent. In addition, we find no evidence for recombination within one of the lineages currently classified as a CRF, CRF02_AG. Our analysis indicates that CRF02_AG was the parent of the recombinant subtype G, rather than the two having the opposite evolutionary relationship, as is currently proposed. Our results imply that the current classification of HIV-1 subtypes and CRFs is an artifact of sampling history, rather than reflecting the evolutionary history of the virus. We suggest a reanalysis of all pure subtypes and CRFs in order to better understand how high rates of recombination have influenced HIV-1 evolutionary history.     

HIV-1 genetic diversity in Western Brittany,France

We describe human immunodeficiency virus type 1 (HIV-1) diversity in Western Brittany, France, and trace the dissemination of HIV-1 non-B subtype infection. The strategy for HIV-1 subtyping used involved subtype specific enzyme immunoassays, heteroduplex mobility assays and phylogenetic analysis of the sequences of env encoding the V3 loop region. Samples were obtained from 567 patients: 465 (82%) were of subtype B and 66 (11.6%) were not (20 were subtype A, 11 subtype C, four subtype D, seven subtype F, five subtype G and 19 others with circulating recombinant forms: 4CRF01_AE, 11CRF02_AG, 1H, 3CRF11_cpx). These findings are consistent with other studies of French populations. There is an epidemiological correlation between subtype B and homosexual or heterosexual contamination in France and between non-B subtype and heterosexual contamination in Africa.     

Molecular Epidemiology of HIV-1 in Jilin Province,Northeastern China: Emergence of a New CRF07_BC Transmission Cluster and Intersubtype Recombinants

Objective

To investigate the HIV-1 molecular epidemiology among newly diagnosed HIV-1 infected persons living in the Jilin province of northeastern China.

Methods

Plasma samples from 189 newly diagnosed HIV-1 infected patients were collected between June 2010 and August 2011 from all nine cities of Jilin province. HIV-1 nucleotide sequences of gag P17–P24 and env C2–C4 gene regions were amplified using a multiplex RT-PCR method and sequenced. Phylogenetic and recombination analyses were used to determine the HIV-1 genotypes.

Results

Based on all sequences generated, the subtype/CFR distribution was as follows: CRF01_AE (58.1%), CRF07_BC (13.2%), subtype B’ (13.2%), recombinant viruses (8.1%), subtype B (3.7%), CRF02_AG (2.9%), subtype C (0.7%). In addition to finding CRF01_AE strains from previously reported transmission clusters 1, 4 and 5, a new transmission cluster was described within the CRF07_BC radiation. Among 11 different recombinants identified, 10 contained portions of gene regions from the CRF01_AE lineage. CRF02_AG was found to form a transmission cluster of 4 in local Jilin residents.

Conclusions

Our study presents a molecular epidemiologic investigation describing the complex structure of HIV-1 strains co-circulating in Jilin province. The results highlight the critical importance of continuous monitoring of HIV-infections, along with detailed socio-demographic data, in order to design appropriate prevention measures to limit the spread of new HIV infections.     

Distinct human immunodeficiency virus type 1 subtype A virus circulating in West Africa: sub-subtype A3

Phylogenetic analyses demonstrate significant diversity in worldwide circulating strains of human immunodeficiency virus type 1 (HIV-1). Detailed studies have revealed a complex pattern of intersubtype recombinations, as well as evidence of sub-subtypes circulating in various populations. In this study, we characterized an HIV-1 strain that had previously been identified as a distinct subcluster within the subtype A radiation based on partial sequence data. These viruses were of particular interest given that we recently found that their prevalence was significantly higher in dually infected individuals compared to women who were singly infected with HIV-1. Five viruses isolated from commercial sex workers in Dakar, Senegal, were full-length PCR amplified and sequenced. Phylogenetic analyses indicated that, whereas three of these viruses were closely related and clustered overall within the HIV-1 subtype A radiation, they were distinct from previously characterized sub-subtype A1 and A2 viruses. The clustering pattern was maintained in the individual gag, pol, and env regions of the genome. Distance calculations between these viruses, which we termed A3, and other reference sub-subtype A1 and A2 viruses fell in the range of distances between previously characterized sub-subtype groups. In addition, we found evidence of two A3-containing recombinants in our cohort. These recombinants are mosaics composed of sequence from both sub-subtype A3 and CRF02_AG, the major circulating recombinant form in this West African population. Based on phylogenetic analyses, we propose that the group of viruses found in the Dakar sex worker cohort, previously referred to as HIV-1 A subcluster 2, be referred to as HIV-1 sub-subtype A3.     

B cell depletion in HIV-1 subtype A infected Ugandan adults: relationship to CD4 T cell count, viral load and humoral immune responses

To better understand the nature of B cell dysfunctions in subjects infected with HIV-1 subtype A, a rural cohort of 50 treatment-naïve Ugandan patients chronically infected with HIV-1 subtype A was studied, and the relationship between B cell depletion and HIV disease was assessed. B cell absolute counts were found to be significantly lower in HIV-1+ patients, when compared to community matched negative controls (p<0.0001). HIV-1-infected patients displayed variable functional and binding antibody titers that showed no correlation with viral load or CD4+ T cell count. However, B cell absolute counts were found to correlate inversely with neutralizing antibody (NAb) titers against subtype A (p = 0.05) and subtype CRF02_AG (p = 0.02) viruses. A positive correlation was observed between subtype A gp120 binding antibody titers and NAb breadth (p = 0.02) and mean titer against the 10 viruses (p = 0.0002). In addition, HIV-1 subtype A sera showed preferential neutralization of the 5 subtype A or CRF02_AG pseudoviruses, as compared with 5 pseudoviruses from subtypes B, C or D (p<0.001). These data demonstrate that in patients with chronic HIV-1 subtype A infection, significant B cell depletion can be observed, the degree of which does not appear to be associated with a decrease in functional antibodies. These findings also highlight the potential importance of subtype in the specificity of cross-clade neutralization in HIV-1 infection.     

The origin and evolutionary history of HIV-1 subtype C in Senegal

Background

The classification of HIV-1 strains in subtypes and Circulating Recombinant Forms (CRFs) has helped in tracking the course of the HIV pandemic. In Senegal, which is located at the tip of West Africa, CRF02_AG predominates in the general population and Female Sex Workers (FSWs). In contrast, 40% of Men having Sex with Men (MSM) in Senegal are infected with subtype C. In this study we analyzed the geographical origins and introduction dates of HIV-1 C in Senegal in order to better understand the evolutionary history of this subtype, which predominates today in the MSM population

Methodology/Principal Findings

We used a combination of phylogenetic analyses and a Bayesian coalescent-based approach, to study the phylogenetic relationships in pol of 56 subtype C isolates from Senegal with 3,025 subtype C strains that were sampled worldwide. Our analysis shows a significantly well supported cluster which contains all subtype C strains that circulate among MSM in Senegal. The MSM cluster and other strains from Senegal are widely dispersed among the different subclusters of African HIV-1 C strains, suggesting multiple introductions of subtype C in Senegal from many different southern and east African countries. More detailed analyses show that HIV-1 C strains from MSM are more closely related to those from southern Africa. The estimated date of the MRCA of subtype C in the MSM population in Senegal is estimated to be in the early 80''s.

Conclusions/Significance

Our evolutionary reconstructions suggest that multiple subtype C viruses with a common ancestor originating in the early 1970s entered Senegal. There was only one efficient spread in the MSM population, which most likely resulted from a single introduction, underlining the importance of high-risk behavior in spread of viruses.     

Broad antibody mediated cross-neutralization and preclinical immunogenicity of new codon-optimized HIV-1 clade CRF02_AG and G primary isolates

Creation of an effective vaccine for HIV has been an elusive goal of the scientific community for almost 30 years. Neutralizing antibodies are assumed to be pivotal to the success of a prophylactic vaccine but previous attempts to make an immunogen capable of generating neutralizing antibodies to primary "street strain" isolates have resulted in responses of very limited breadth and potency. The objective of the study was to determine the breadth and strength of neutralizing antibodies against autologous and heterologous primary isolates in a cohort of HIV-1 infected Nigerians and to characterize envelopes from subjects with particularly broad or strong immune responses for possible use as vaccine candidates in regions predominated by HIV-1 CRF02_AG and G subtypes. Envelope vectors from a panel of primary Nigerian isolates were constructed and tested with plasma/sera from the same cohort using the PhenoSense HIV neutralizing antibody assay (Monogram Biosciences Inc, USA) to assess the breadth and potency of neutralizing antibodies. The immediate goal of this study was realized by the recognition of three broadly cross-neutralizing sera: (NG2-clade CRF02_AG, NG3-clade CRF02_AG and NG9- clade G). Based on these findings, envelope gp140 sequences from NG2 and NG9, complemented with a gag sequence (Clade G) and consensus tat (CRF02_AG and G) antigens have been codon-optimized, synthesized, cloned and evaluated in BALB/c mice. The intramuscular administration of these plasmid DNA constructs, followed by two booster DNA immunizations, induced substantial specific humoral response against all constructs and strong cellular responses against the gag and tat constructs. These preclinical findings provide a framework for the design of candidate vaccine for use in regions where the HIV-1 epidemic is driven by clades CRF02_AG and G.     

Profile of the HIV Epidemic in Cape Verde: Molecular Epidemiology and Drug Resistance Mutations among HIV-1 and HIV-2 Infected Patients from Distinct Islands of the Archipelago

HIV-1 and HIV-2 have been detected in Cape Verde since 1987, but little is known regarding the genetic diversity of these viruses in this archipelago, located near the West African coast. In this study, we characterized the molecular epidemiology of HIV-1 and HIV-2 and described the occurrence of drug resistance mutations (DRM) among antiretroviral therapy naïve (ARTn) patients and patients under treatment (ARTexp) from different Cape Verde islands. Blood samples, socio-demographic and clinical-laboratory data were obtained from 221 HIV-positive individuals during 2010–2011. Phylogenetic and bootscan analyses of the pol region (1300 bp) were performed for viral subtyping. HIV-1 and HIV-2 DRM were evaluated for ARTn and ARTexp patients using the Stanford HIV Database and HIV-GRADE e.V. Algorithm Homepage, respectively. Among the 221 patients (169 [76.5%] HIV-1, 43 [19.5%] HIV-2 and 9 [4.1%] HIV-1/HIV-2 co-infections), 67% were female. The median ages were 34 (IQR = 1–75) and 47 (IQR = 12–84) for HIV-1 and HIV-2, respectively. HIV-1 infections were due to subtypes G (36.6%), CRF02_AG (30.6%), F1 (9.7%), URFs (10.4%), B (5.2%), CRF05_DF (3.0%), C (2.2%), CRF06_cpx (0.7%), CRF25_cpx (0.7%) and CRF49_cpx (0.7%), whereas all HIV-2 infections belonged to group A. Transmitted DRM (TDRM) was observed in 3.4% (2/58) of ARTn HIV-1-infected patients (1.7% NRTI, 1.7% NNRTI), but not among those with HIV-2. Among ARTexp patients, DRM was observed in 47.8% (33/69) of HIV-1 (37.7% NRTI, 37.7% NNRTI, 7.4% PI, 33.3% for two classes) and 17.6% (3/17) of HIV-2-infections (17.6% NRTI, 11.8% PI, 11.8% both). This study indicates that Cape Verde has a complex and unique HIV-1 molecular epidemiological scenario dominated by HIV-1 subtypes G, CRF02_AG and F1 and HIV-2 subtype A. The occurrence of TDRM and the relatively high level of DRM among treated patients are of concern. Continuous monitoring of patients on ART, including genotyping, are public policies to be implemented.     

Refined identification of neutralization-resistant HIV-1 CRF02_AG viruses

We studied neutralization of CRF02_AG HIV-1-infected plasma samples. In contrast to previous reports, these samples neutralized CRF02_AG viruses better than other viruses. This included six of eight CRF02_AG viruses previously designated resistant (tier 2/3 or 3). Only viruses 253-11 and 278-50 remained highly resistant, but they were sensitive to membrane-proximal external region (MPER)-specific monoclonal antibodies, suggesting neutralization targets for even these viruses. We propose using high-neutralizing-within-subtype samples for evaluation of neutralization resistance of viruses.     

Molecular Evolution of the HIV-1 Thai Epidemic between the Time of RV144 Immunogen Selection to the Execution of the Vaccine Efficacy Trial

The RV144 HIV-1 vaccine trial (Thailand, 2003 to 2009), using immunogens genetically matched to the regional epidemic, demonstrated the first evidence of efficacy for an HIV-1 vaccine. Here we studied the molecular evolution of the HIV-1 epidemic from the time of immunogen selection to the execution of the efficacy trial. We studied HIV-1 genetic diversity among 390 volunteers who were deferred from enrollment in RV144 due to preexisting HIV-1 infection using a multiregion hybridization assay, full-genome sequencing, and phylogenetic analyses. The subtype distribution was 91.7% CRF01_AE, 3.5% subtype B, 4.3% B/CRF01_AE recombinants, and 0.5% dual infections. CRF01_AE strains were 31% more diverse than the ones from the 1990s Thai epidemic. Sixty-nine percent of subtype B strains clustered with the cosmopolitan Western B strains. Ninety-three percent of B/CRF01_AE recombinants were unique; recombination breakpoint analysis showed that these strains were highly embedded within the larger network that integrates recombinants from East/Southeast Asia. Compared to Thai sequences from the early 1990s, the distance to the RV144 immunogens increased 52% to 68% for CRF01_AE Env immunogens and 12% to 29% for subtype B immunogens. Forty-three percent to 48% of CRF01_AE sequences differed from the sequence of the vaccine insert in Env variable region 2 positions 169 and 181, which were implicated in vaccine sieve effects in RV144. In conclusion, compared to the molecular picture at the early stages of vaccine development, our results show an overall increase in the genetic complexity of viruses in the Thai epidemic and in the distance to vaccine immunogens, which should be considered at the time of the analysis of the trial results.     

Detailed Molecular Epidemiologic Characterization of HIV-1 Infection in Bulgaria Reveals Broad Diversity and Evolving Phylodynamics

Limited information is available to describe the molecular epidemiology of HIV-1 in Bulgaria. To better understand the genetic diversity and the epidemiologic dynamics of HIV-1 we analyzed 125 new polymerase (pol) sequences from Bulgarians diagnosed through 2009 and 77 pol sequences available from our previous study from persons infected prior to 2007. Epidemiologic and demographic information was obtained from each participant and phylogenetic analysis was used to infer HIV-1 evolutionary histories. 120 (59.5%) persons were infected with one of five different HIV-1 subtypes (A1, B, C, F1 and H) and 63 (31.2%) persons were infected with one of six different circulating recombinant forms (CRFs; 01_AE, 02_AG, 04_cpx, 05_DF, 14_BG, and 36_cpx). We also for the first time identified infection with two different clusters of unique A-like and F-like sub-subtype variants in 12 persons (5.9%) and seven unique recombinant forms (3.5%), including a novel J/C recombinant. While subtype B was the major genotype identified and was more prevalent in MSM and increased between 2000–2005, most non-B subtypes were present in persons ≥45 years old. CRF01_AE was the most common non-B subtype and was higher in women and IDUs relative to other risk groups combined. Our results show that HIV-1 infection in Bulgaria reflects the shifting distribution of genotypes coincident with the changing epidemiology of the HIV-1 epidemic among different risk groups. Our data support increased public health interventions targeting IDUs and MSM. Furthermore, the substantial and increasing HIV-1 genetic heterogeneity, combined with fluctuating infection dynamics, highlights the importance of sustained and expanded surveillance to prevent and control HIV-1 infection in Bulgaria.     

The N276 Glycosylation Site Is Required for HIV-1 Neutralization by the CD4 Binding Site Specific HJ16 Monoclonal Antibody

Immunogen design for HIV-1 vaccines could be based on epitope identification of naturally occurring neutralizing antibodies in infected patients. A tier 2 neutralizing monoclonal antibody (mAb), HJ16 recognizes a new epitope in the CD4 binding site (CD4bs) region that only partially overlaps with the b12 epitope. We aimed to identify the critical binding site by resistance induction in a sensitive primary CRF02_AG strain. In four independent dose-escalation studies, the N276D mutation was consistently the only alteration found and it was confirmed to be responsible for resistance to HJ16 by site-directed mutagenesis in envelopes (envs) of the homologous CRF02_AG, as well as of a subtype A and a subtype C primary isolate. This mutation removes an N-linked glycosylation site. The effect of N276D was very selective, as it failed to confer resistance to a range of other entry inhibitors. Remarkably, sensitivity to the CD4bs VRC01 and VRC03 mAbs was increased in the N276D mutated viruses. These data indicate that binding of the CD4bs specific HJ16 mAb critically depends on the interaction with the N276-glycan, thus indicating that HJ16 is the first glycan dependent CD4bs-specific mAb.     

Origin and Population Dynamics of a Novel HIV-1 Subtype G Clade Circulating in Cape Verde and Portugal

The human immunodeficiency virus type 1 (HIV-1) subtype G is the most prevalent and second most prevalent HIV-1 clade in Cape Verde and Portugal, respectively; but there is no information about the origin and spatiotemporal dispersal pattern of this HIV-1 clade circulating in those countries. To this end, we used Maximum Likelihood and Bayesian coalescent-based methods to analyze a collection of 578 HIV-1 subtype G pol sequences sampled throughout Portugal, Cape Verde and 11 other countries from West and Central Africa over a period of 22 years (1992 to 2013). Our analyses indicate that most subtype G sequences from Cape Verde (80%) and Portugal (95%) branched together in a distinct monophyletic cluster (here called G_CV-PT). The G_CV-PT clade probably emerged after a single migration of the virus out of Central Africa into Cape Verde between the late 1970s and the middle 1980s, followed by a rapid dissemination to Portugal a couple of years later. Reconstruction of the demographic history of the G_CV-PT clade circulating in Cape Verde and Portugal indicates that this viral clade displayed an initial phase of exponential growth during the 1980s and 1990s, followed by a decline in growth rate since the early 2000s. Our data also indicate that during the exponential growth phase the G_CV-PT clade recombined with a preexisting subtype B viral strain circulating in Portugal, originating the CRF14_BG clade that was later disseminated to Spain and Cape Verde. Historical and recent human population movements between Angola, Cape Verde and Portugal probably played a key role in the origin and dispersal of the G_CV-PT and CRF14_BG clades.     

Autochthonous horizontal transmission of a CRF02_AG strain revealed by a human immunodeficiency virus type 1 diversity survey in a small city in inner state of Rio de Janeiro, Southeast Brazil

As part of an ongoing study on the features of AIDS spread towards small cities and rural areas, we present a molecular survey of human immunodeficiency virus type 1 (HIV-1) polymerase sequences recovered between 2004 and 2006 from 71 patients receiving care in the city of Saquarema, inner state of Rio de Janeiro. Phylogenetic reconstructions found the two prevalent lineages in the state (subtypes B [59 strains, 83.1%], F1 [6 strains; 8.4%], and BF1 recombinants [four strains; 5.6%]), as well as two (2.8%) CRF02_AG strains, which seems to be an emerging lineage in the capital. These CRF02_AG sequences were recovered from a married heterosexual couple who never traveled abroad, thus providing the first molecular evidence of autochthonous horizontal transmission of this lineage of major global importance. Also, three phylogenetic clusters of strains recovered from a total of 18.3% of the cohort were uncovered. Their close genetic relatedness suggests they were recovered from patients who probably took part in the same chain of viral spread. In conjunction with our previous surveys from inner Rio de Janeiro, these results suggest that although small cities harbor unique molecular features of HIV-1 infection, they also clearly reflect and may rapidly absorb the diversity recorded in large urban centers.     

A Comprehensive Mapping of HIV-1 Genotypes in Various Risk Groups and Regions across China Based on a Nationwide Molecular Epidemiologic Survey

Background

China is experiencing a dynamic HIV/AIDS epidemic. While serology based surveillance systems have reported the spread of HIV/AIDS, detailed tracking of its transmission in populations and regions is not possible without mapping it at the molecular level. We therefore conducted a nationwide molecular epidemiology survey across the country.

Methods

HIV-1 genotypes were determined from 1,408 HIV-positive persons newly diagnosed in 2006. The prevalence of each genotype was estimated by weighting the genotype’s prevalence from each province- and risk-specific subpopulation with the number of reported cases in the corresponding subgroups in that year.

Results

CRF07_BC (35.5%), CRF01_AE (27.6%), CRF08_BC (20.1%), and subtype B'' (9.6%) were the four main HIV-1 strains in China. CRF07_BC and CRF08_BC were the primary drivers of infection among injecting drug users in northeastern and southeastern China, respectively, and subtype B'' remained dominant among former plasma donors in central China. In contrast, all four strains occurred in significant proportions among heterosexuals nationwide, pointing to an expansion of the HIV-1 epidemic from high-risk populations into the general population. CRF01_AE also replaced subtype B as the principal driver of infection among men-who-have-sex-with-men.

Conclusions

Our study provides the first comprehensive baseline data on the diversity and characteristics of HIV/AIDS epidemic in China, reflecting unique region- and risk group-specific transmission dynamics. The results provide information critical for designing effective prevention measures against HIV transmission.