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.     

Phylodynamic Profile of HIV-1 Subtype B,CRF01_AE and the Recently Emerging CRF51_01B among Men Who Have Sex with Men (MSM) in Singapore

HIV-1 subtype B and CRF01_AE are the predominant infecting subtypes among men who have sex with men (MSM) in Singapore. The genetic history, population dynamics and pattern of transmission networks of these genotypes remain largely unknown. We delineated the phylodynamic profiles of HIV-1 subtype B, CRF01_AE and the recently characterized CRF51_01B strains circulating among the MSM population in Singapore. A total of 105 (49.5%) newly-diagnosed treatment-naïve MSM were recruited between February 2008 and August 2009. Phylogenetic reconstructions of the protease gene (HXB2: 2239 – 2629), gp120 (HXB2: 6942 – 7577) and gp41 (HXB2: 7803 – 8276) of the env gene uncovered five monophyletic transmission networks (two each within subtype B and CRF01_AE and one within CRF51_01B lineages) of different sizes (involving 3 – 23 MSM subjects, supported by posterior probability measure of 1.0). Bayesian coalescent analysis estimated that the emergence and dissemination of multiple sub-epidemic networks occurred between 1995 and 2005, driven largely by subtype B and later followed by CRF01_AE. Exponential increase in effective population size for both subtype B and CRF01_AE occurred between 2002 to 2007 and 2005 to 2007, respectively. Genealogical estimates suggested that the novel CRF51_01B lineages were probably generated through series of recombination events involving CRF01_AE and multiple subtype B ancestors. Our study provides the first insight on the phylodynamic profiles of HIV-1 subtype B, CRF01_AE and CRF51_01B viral strains circulating among MSM in Singapore.     

Higher frequency of premature stop codon mutations at vpu gene of human immunodeficiency virus type 1 CRF01_AE compared with those of other subtypes

Our previous study demonstrated the anti-apoptosis function of the human immunodeficiency virus type 1 (HIV-1) vpu gene product in normal CD4+ T lymphocytes. In this study, using sequences obtained from the HIV sequence database, we compared vpu sequences from 184 preparations of various subtypes of HIV-1 from diverse geographical regions. Our analysis revealed that CRF01_AE isolates had premature stop codon mutations at the vpu gene at a much higher rate (36%) than other subtypes (0-9%). The premature stop codon mutations in vpu existed mostly at two amino acid residues: the methionine initiation codon and the boundary between the transmembrane (TM) and cytoplasmic domains. The mutations at the latter site were more often detected in CRF01_AE. The higher mutation rates at vpu in CRF01_AE were confirmed by sequence comparison of polymerase chain reaction products newly obtained directly from the DNA extracted from peripheral blood mononuclear cells (PBMCs), but not from the RNA from the plasma, in CRF01_AE- and subtype B-infected individuals. This finding may indicate the possibility that the more abundant population of HIV-1 CRF01_AE is able to induce apoptosis in CD4+ T lymphocytes than the populations of other subtypes.     

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.     

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.     

The Effect of HLA Polymorphisms on the Recognition of Gag Epitopes in HIV-1 CRF01_AE Infection

Introduction

The design of a globally effective vaccine rests on the identification of epitopes capable of eliciting effective cytotoxic T lymphocyte (CTL) responses across multiple HIV clades in different populations. This study aims to discern the effect of HLA polymorphisms and the cross-clade reactivity or clade-specificity of epitopes in Thailand where HIV-1 CRF01_AE is circulating.

Materials and Methods

14 peptides based on consensus HIV-1 CRF01_AE amino acid sequences were designed for use in IFN-γ ELISpot assays and ⁵¹Cr release assays among 66 HIV-1 CRF01_AE-infected Thai patients. For ELISpot responders carrying HLA alleles currently unknown to restrict CRF01_AE epitopes, in silico epitope-HLA prediction was performed.

Results

29/66 (43.9%) patients recognized at least one peptide. In total 79 responses were seen against all 14 peptides. 28/79 (35.4%) of the responses were in patients with HLA alleles previously reported to restrict CRF01_AE epitopes, 24/79 (30.4%) responses were in individuals with HLA alleles previously reported to restrict epitopes of HIV clades other than CRF01_AE, and the remaining 27/79 (34.2%) responses were not associated with HLA alleles previously known to restrict HIV epitopes. In silico epitope prediction detected 19 novel, epitope-HLA combinations, and 11/19 (57.9%) were associated with HLA-C alleles. We further confirmed a novel HLA restriction of a previously identified HIV-1 Gag epitope [p24_122–130: PPIPVGDIY (PY9)] by HLA-B*40:01 with a standard ⁵¹Cr release assay.

Discussion

CTL recognition sites in HIV-1 Gag were similar among different clades but the HLA restriction differed in Thai patients. This disparity in HLA restriction along different populations illustrated the importance of clade- and population-specific HLA analysis prior to CTL vaccine design.     

Molecular evolution of HIV-1 CRF01_AE Env in Thai patients

Background

The envelope glycoproteins (Env), gp120 and gp41, are the most variable proteins of human immunodeficiency virus type 1 (HIV-1), and are the major targets of humoral immune responses against HIV-1. A circulating recombinant form of HIV-1, CRF01_AE, is prevalent throughout Southeast Asia; however, only limited information regarding the immunological characteristics of CRF01_AE Env is currently available. In this study, we attempted to examine the evolutionary pattern of CRF01_AE Env under the selection pressure of host immune responses.

Methodology/Principal Findings

Peripheral blood samples were collected periodically over 3 years from 15 HIV-1-infected individuals residing in northern Thailand, and amplified env genes from the samples were subjected to computational analysis. The V5 region of gp120 showed highest variability in several samples over 3 years, whereas the V1/V2 and/or V4 regions of gp120 also showed high variability in many samples. In addition, the N-terminal part of the C3 region of gp120 showed highest amino acid diversity among the conserved regions of gp120. Chronological changes in the numbers of amino acid residues in gp120 variable regions and potential N-linked glycosylation (PNLG) sites are involved in increasing the variability of Env gp120. Furthermore, the C3 region contained several amino acid residues potentially under positive selection, and APOBEC3 family protein-mediated G to A mutations were frequently detected in such residues.

Conclusions/Significance

Several factors, including amino acid substitutions particularly in gp120 C3 and V5 regions as well as changes in the number of PNLG sites and in the length of gp120 variable regions, were revealed to be involved in the molecular evolution of CRF01_AE Env. In addition, a similar tendency was observed between CRF01_AE and subtype C Env with regard to the amino acid variation of gp120 V3 and C3 regions. These results may provide important information for understanding the immunological characteristics of CRF01_AE Env.     

Increased rate of CD4+ T-cell decline and faster time to antiretroviral therapy in HIV-1 subtype CRF01_AE infected seroconverters in Singapore

Background

It remains controversial as to whether HIV-1 subtypes influence disease progression. Singapore offers a unique opportunity to address this issue due to the presence of co-circulating subtypes. We compared subtype CRF01_AE and non-CRF01_AE infected patients, with regards to estimated annual rate of CD4+ T-cell loss and time from estimated data of seroconversion (EDS) to antiretroviral therapy (ART).

Methods

We recruited ART-naive patients with known dates of seroconversion between October 2002 and December 2007 at the Singapore Communicable Disease Centre, the national reference treatment centre. Multilevel mixed-effects models were used to analyse the rate of CD4+ T-cell decline. Time from EDS to ART was analyzed with the Kaplan-Meier survival method and compared with Cox proportional hazards models.

Results

54 patients with previously assigned HIV-1 subtypes (24 CRF01_AE, 17 B, 8 B'', 1 CRF33_01B, 3 CRF34_01B and 1 G) were observed for 89 patient-years. Subtype CRF01_AE and non-CRF01_AE infected patients did not differ in age, gender, risk factor, rate of symptomatic seroconversion, baseline CD4+ T-cell count, log₁₀ viral load or haemoglobin concentration. The estimated annual rate of CD4+ T-cell loss was 58 cells/mm³/year (95% CI: 7 to 109; P = 0.027) greater in subtype CRF01_AE infected patients compared to non-CRF01_AE patients, after adjusting for age, baseline CD4+ T-cell count and baseline log₁₀ viral load. The median time from EDS to ART was 1.8 years faster comparing CRF01_AE to non-CRF01_AE infected patient with a 2.5 times (95% CI: 1.2-5.0; P = 0.013) higher hazard for ART initiation, after controlling for age, baseline CD4+ T-cell count and baseline log₁₀ viral load.

Conclusions

Infecting subtype significantly impacted the rate of CD4+ T-cell loss and time to treatment in this cohort. Studies to understand the biological basis for this difference could further our understanding of HIV pathogenesis.     

Susceptibility of HIV-1 subtypes B', CRF07_BC and CRF01_AE that are predominantly circulating in China to HIV-1 entry inhibitors

Background

The B′, CRF07_BC and CRF01_AE are the predominant HIV-1 subtypes in China. It is essential to determine their baseline susceptibility to HIV entry inhibitors before these drugs are used in China.

Methodology/Principal Findings

The baseline susceptibility of 14 representative HIV-1 isolates (5 CRF07_BC, 4 CRF01_AE, and 5 B′), most of which were R5 viruses, obtained from drug-naïve patients to HIV entry inhibitors, including two fusion inhibitors (enfuvirtide and C34), two CCR5 antagonists (maraviroc and TAK779) and one CXCR4 antagonist (AMD3100), were determined by virus inhibition assay. The sequences of their env genes were amplified and analyzed. These isolates possessed similar susceptibility to C34, but they exhibited different sensitivity to enfuvirtide, maraviroc or TAK779. CRF07_BC isolates, which carried polymorphisms of A578T and V583I in the N-terminal heptad repeat and E630Q, E662A, K665S, A667K and S668N in the C-terminal heptad repeat of gp41, were about 5-fold less sensitive than B′ and CRF01_AE isolates to enfuvirtide. Subtype B′ isolates with a unique polymorphism site of F317W in V3 loop, were about 4- to 5-fold more sensitive than CRF07_BC and CRF01_AE isolates to maraviroc and TAK779. AMD3100 at the concentration as high as 5 µM exhibited no significant inhibitory activity against any of the isolates tested.

Conclusion

Our results suggest that there are significant differences in baseline susceptibility to HIV entry inhibitors among the predominant HIV-1 subtypes in China and the differences may partly result from the naturally occurring polymorphisms in these subtypes. This study provides useful information for rational design of optimal therapeutic regimens for HIV-1-infected patients in China.