Characteristics of HIV type 1 (HIV-1) glycoprotein 120 env sequences in mother-infant pairs infected with HIV-1 subtype CRF01_AE

We analyzed the characteristics of the envelope genes of human immunodeficiency virus type 1 in 17 mother-infant pairs infected with variants of the CRF01_AE clade. A total of 353 sequences covering almost the entire glycoprotein (gp) 120 region were available for analysis. We found that, even if the virus population in the mother was complex, only viruses of a restricted subset were transmitted to her infant, independently of whether transmission occurred in utero or during the intrapartum period. We did not find that shorter gp120 regions or fewer potential N-glycosylation sites (PNGS) were characteristic of viruses transmitted from mother to infant. However, our data suggest that a limited number of PNGS that seem to be conserved in all variants in infants but are not uniformly present in variants in mothers may confer an advantage for transmission of the virus, thereby highlighting the potentially important role of the "glycan shield." This finding was particularly significant for the PNGS at positions N301 and N384.     

Heterosexual transmission of novel CRF01_AE and subtype B recombinant forms of HIV type 1 in northern Thailand

The increased proportion of CRFO1_AE/subtype B recombinant infections among injecting drug users raised a concern that such recombinant forms may also spread in a heterosexual population in Thailand. Using the BootScan method, we reanalyzed pol gene sequences among 114 heterosexually infected individuals in northern Thailand, who were tested for a drug-resistance genotype between July 2000 and July 2001. Two individuals were suspected of carrying a recombinant HIV-1. Thus we analyzed a nearly full-length HIV genome in the two individuals and their spouses. An identical recombinant form of CRF01_AE and subtype B was found in one couple, indicating that this recombinant virus was heterosexually transmitted. Interestingly, this recombinant form had multiple breakpoints in the core protein of Gag and both infected individuals had a high CD4+ cell count without antiretroviral therapy. CRFO1/subtype B recombinant forms exist in a heterosexual population in northern Thailand. Some recombinant virus may be associated with a slow rate of HIV disease progression.     

Heterosexually acquired CRF01_AE/B recombinant HIV type 1 found in Thailand

CRF01_AE and subtype B are circulating in Thailand and the strains have become intermixed in some high-risk groups, establishing the possibility of intersubtype recombination. The first such recombinant, mostly B with gp120 from CRF01_AE, was recently identified. Here we report a heterosexually acquired recombinant of different structure, with most of the genome from CRF01_AE but almost the entire envelope from subtype B. Surveillance by V3 serotype and genotype in multiple regions, followed by full-genome sequencing, was used to identify this strain. Pending vaccine trials in Thailand require knowledge of the presence of such strains in the population, and these recombinants provide valuable reagents for the laboratory evaluation of cross-subtype immunity. Studies are underway to determine whether either recombinant is circulating widely.     

Isolation and characterization of replication-competent molecular DNA clones of HIV type 1 CRF01_AE with different coreceptor usages

We have isolated replication-competent molecular clones of HIV-1 circulating recombinant form CRF01_AE with different coreceptor usages. After lambda phage cloning of unintegrated circular proviral DNAs derived from a CRF01_AE strain (HIV-1NH1), isolated in Japan, the infectious molecular clone, designated p93JP-NH1, was reconstituted. 93JP-NH1 showed an X4 and R5 phenotype in NP2 cell-based coreceptor utilization assays and exerted robust replication in human T cell lines, including MT2, M8166, and PM1 cells, whereas it propagated modestly in peripheral blood mononuclear cells. The CRF01_AE molecular clone with R5 phenotype (p93JP-NH2env) was then constructed by replacing the env gene of p93JP-NH1 with that of a nearly isogenic CRF01_AE R5 strain isolated from an epidemiologically linked case. The phylogeny and recombination break-point analysis confirmed that these clones shared an A/E recombinant structure similar to that of the prototype CRF01_AE strain, CM240. These replication-competent CRF01_AE molecular clones with different coreceptor usages would be useful tools for the study of CRF01_AE, one of the most prevalent strains in Asia.     

Coreceptor utilization of HIV type 1 subtype E viral isolates from Thai men with HIV type 1-infected and uninfected wives.

HIV-1 coreceptors CCR5 and CXCR4 play an important role in viral entry and pathogenesis. To better understand the role of viral tropism in HIV-1 transmission, we examined the coreceptor utilization of viral isolates obtained from men enrolled in a study of heterosexual transmission in northern Thailand. Viral isolates were obtained from HIV-1-positive males who had either HIV-1-infected spouses (RM; n = 5) or HIV-1-uninfected spouses (HM; n = 10). Viral isolates from 1 of the 5 RM males and 2 of the 10 HM males were CCR5 tropic, whereas isolates from 3 RM males and 6 of the HM male isolates were CXCR4 tropic. Of the nine X4-tropic isolates, seven also used at least one of the following coreceptors: CCR8, CCR1, CCR2b, or CX3CR1, and none employed CCR5 as an additional coreceptor. More importantly, three isolates, RM-15, HM-13, and HM-16 (one from a transmitter and two from nontransmitter), did not infect GHOST4.cl.34 cells expressing any of the known coreceptors. Further analysis using MAGI-plaque assays, which allow visualization of infected cells, revealed that RM-15 had low numbers of infected cells in MAGI-R5 and MAGI-X4 cultures, whereas HM-13 and HM-16 had high levels of plaques in MAGI-X4 cultures. Replication kinetics using activated lymphocytes revealed that these three isolates replicated in CCR5(+/+) as well as CCR5(-/-) peripheral blood mononuclear cells, suggesting that these isolates did not have an absolute requirement of CCR5 for viral entry. All three isolates were sensitive to the X4-antagonistic compounds T-22 and AMD3100. Analysis of the C2V3 region did not reveal any significant structural differences between any of the Thai subtype E isolates. Thus, there was no association between the pattern of coreceptor usage and transmissibility among these subtype E HIV-1 isolates.     

Conserved neutralizing epitopes of HIV type 1 CRF01_AE against primary isolates in long-term nonprogressors

Linear conserved B cell epitopes in envelope glycoprotein of long-term nonprogressors (LTNPs) HIV-1 CRF01_AE were determined. The envelope sequences of HIV-1 subtype E from Thailand were aligned to define consensus sequences. Then the peptides corresponding to these predicted regions were synthesized as peptides represent C1, C2, C3, C5, V2, V3, and gp41 regions. After that, the neutralizing B cell epitopes were determined by neutralized competitive assay with pool sera of typical progressor and LTNP HIV-1 CRF01_AE patients against HIV-1 CRF01_AE 24 primary isolates (PI) and laboratory strains (TCLA). We found that the strength and breadth of neutralization were greater for sera from LTNPs compared with sera from typical progressors. Peptides C1E and C2E could inhibit primary isolates but not the TCLA strain in LTNP sera. The new B cell epitopes, which were located in the C1 and C2 regions of CRF01_AE against primary HIV-1 isolates, were identified in HIV-1 CRF01_AE LTNPs. This may be important in HIV-1 vaccine development and trial.     

Differential prevalence of HIV type 1 subtype B and CRF01_AE among different sexual transmission groups in Tokyo, Japan, as revealed by subtype-specific PCR

We determined the subtype of HIV-1 in 89 infected individuals attending three reference hospitals located in the Tokyo metropolitan area of Japan. Subtyping was performed with subtype-specific polymerase chain re-action (PCR) distinguishing subtype A, B, C, and CRF01_AE and/or phylogenetic analysis of HIV-1 env C2V3C3 sequences. Subtype-specific PCR provided unequivocal results in 97% of samples. Sixty-five subjects were infected with subtype B, 16 with CRFO1_AE, 4 with subtype A, 1 with CRF02_AG, and 3 with subtype C. Among 31 Japanese individuals infected through heterosexual contact, 13 were infected with subtype Band 12 with CRFO1_AE. All of the 41 Japanese men infected through homosexual contact harbored subtype B. These results indicate that subtype B is exclusively predominant in a homosexually transmitted group, whereas subtype B and CRFO1_AE are evenly predominant in a heterosexually transmitted group.     

HIV type 1 integrase polymorphisms in treatment-naive and treatment-experienced HIV type 1-infected patients in Thailand where HIV type 1 subtype A/E predominates

Integrase inhibitor (INI) is a novel antiretroviral drug recommended for both treatment-naive and treatment-experienced HIV-1-infected patients. Limited data are available on INI resistance in Thailand, where HIV-1 subtype A/E predominates. We aimed to investigate INI resistance-associated mutations (RAMs) among treatment-naive patients and patients who experienced treatment failure with NNRTI-based or PI-based antiretroviral therapy (ART) in Thailand. One hundred and eight plasma samples of 58 treatment-naive and 50 treatment-experienced HIV-1-infected individuals were collected. The HIV-1 integrase coding region was sequenced. Polymorphisms were compared between subtype A/E and B circulating in Thailand and between treatment-naive and treatment-experienced groups. Resulting amino acids were interpreted for drug resistance according to Stanford algorithms. Ninety-seven samples were HIV-1 subtype A/E, 10 were subtype B, and one was subtype C. Age, gender, and CD4 cell counts were similar between treatment-naive and treatment-experienced groups, while the treatment-failure group showed a statistically significant longer awareness time of HIV-1 infection and lower viral load than the treatment-naive group. Major INI-RAM was not found in this study, but some minor INI-RAMs, such asV54I, L68I, L74M, T97A, and S230N, were found. Comparing INI-RAMs between subtype A/E and B, the prevalence of V54I and V72I was higher in subtype B than subtype E, while V201I was found in all sequences of subtype A/E. In subtype A/E, integrase polymorphisms were not different between treatment-naive and treatment-experienced groups. However, the number of amino acid substitutions was significantly higher in the treatment-experienced group (p=0.009). One NNRTI-based ART-treated patient was found to have potential low-level INI-RAMs. INI-RAMs are rare in both treatment-naive and treatment-experienced patients in Thailand. This suggested that INI should be active in patients who are naive to INI in Thailand.     

HIV type 1-specific inter- and intrasubtype cellular immune responses in HIV type 1-infected Ugandans

Investigations concerning the extent and nature of subtype-specific and intersubtype immune responses in HIV-1-infected persons are necessary for the development of appropriate candidate vaccines. In the cross-sectional study described here, 26 HIV-1-positive Ugandan patients were tested for their ability to mount HIV antigen-specific cellular immune responses. Subjects were infected with either HIV-1 subtypes A, C, or D. Recombinant vaccinia virus (rVV)-based and peptide-based enzyme-linked immunospot (Elispot) assays were used to evaluate HIV-1-specific gamma-interferon (IFN-gamma) cellular responses. rVV expressing gag, pol, or env proteins derived from HIV-1 subtypes A, B, and D were evaluated for their ability to induce whole HIV-1-protein-specific IFN-gamma responses in 14 patients. A panel of previously identified HLA class I-restricted peptides based on representative sequences from HIV-1 subtypes A, B, C, and D and restricted through HLA-A2, -A29, -B42, -B53, and -B57 alleles were used to evaluate the presence of HIV-1-peptide-specific T cells in 19 patients. Using rVV, 27 of a possible 38 subtype-specific responses (71%) and 56 of a possible 110 intersubtype responses (51%) were observed. When appropriate peptides were used 18 of 39 (46.2%) subtype-specific and 13 of 39 (33.3%) intersubtype responses were observed. Peptide responses were higher quantitatively than those seen when rVV were used. In 7 patients, both rVV and specific peptides were evaluated; in 3 of 7 individuals, global responses were seen despite a lack of measurable HLA-restricted peptide-specific responses demonstrating the need to evaluate a broader range of HIV-specific immune responses.     

Molecular epidemiology of HIV type 1 in Singapore and identification of novel CRF01_AE/B recombinant forms

To investigate HIV-1 molecular epidemiology in Singapore, we sequenced portions of three regions of the HIV-1 genome (protease HXB2: 2163 to 2620, gp120 HXB2: 6904 to 7628, and gp41 HXB2: 7817 to 8264) from 212 plasma samples collected between February 2008 and August 2009. From these samples, 109 (51.4%) generated interpretable data in all regions. Sixty-one (56.0%) were identified as CRF01_AE, 26 (23.9%) as subtype B and 14 (12.8%) as possible novel recombinant forms. The main novel recombinant pattern, detected in 13 sequences, had subtype B in protease and gp41 and CRF01_AE in gp120. There was intermixing of subtypes within transmission risk groups. However, 85% of subjects infected with the novel recombinant forms self-identified as men who have sex with men or bisexuals compared with only 41% of individuals infected with CRF01_AE and 62% infected with subtype B (p = 0.001).     

Binding antibody to neutralizing epitope gp41 in HIV-1 subtype CRF 01_AE infection related to stage of disease

The responsiveness of gp41 antibody against epitope ELDKWA in HIV-1 infected subjects is of importance in neutralizing viral infectivity and for being related to disease progression. In this study, antibody titers to this neutralizing epitope from HIV-1 infected subjects at asymptomatic and AIDS stages in Thailand were investigated by peptide ELISA. The results showed that the frequency of antibody production against this neutralizing epitope was low (15-35%). Moreover, antibody titers to this epitope in sera from AIDS patients were significantly lower than those in sera from asymptomatic subjects which were collected in the same year (p=0.001). Comparison between the past (1992-1994) and present (2002) sera from asymptomatic infected individuals revealed that the earlier panel contained lower antibody titers than the later panel did (p = 0.05). In addition, random sera for HIV-1 infected subjects who were infected by diverse genetic subtypes, (A through G) including CRF 01_AE, had low titers of antibody to this region as well. It is assumed that antibody production to this epitope is low and related to the stage of HIV-1 infection.     

Emergence of a New HIV Type 1 CRF01_AE Variant in Guangxi, Southern China

Abstract The distribution of HIV-1 subtypes and genetic characterization of CRF01_AE in Guangxi, southern China were identified. The distribution of HIV-1 genotypes based on gag, pol, and partial env sequences (n=349) was as follows: CRF01_AE (66.5%), CRF08_BC (19.2%), CRF07_BC (7.2%), URF (4.6%), subtype B (1.7%), and subtype B' (0.9%). CRF01_AE predominated in all geographic regions and risk populations and there were multiple introductions of CRF01_AE strains in Guangxi. We found a peculiar CRF01_AE monophyletic lineage distinct from other CRF01_AE viruses, and we designated it "CRF01_AE-v" for convenience. CRF01_AE-v circulating in both heterosexuals and injecting drug users (IDUs) had accounted for 39.7% of CRF01_AE. It showed a selective advantage in the Guangxi population and formed its own characteristic compared with all the CRF01_AE references. Our results suggested that CRF01_AE-v was a new variant of CRF01_AE and it might lead to a new epidemic in Guangxi.     

Full-length sequences of two CRF01_ae (subtype e) HIV type 1 isolates from 1995 samples of patients with sexually transmitted diseases in Thailand.

We isolated two CRF01_AE human immunodeficiency virus type 1 (95TNIH022 and 95TNIH047) from the 1995 blood samples derived from asymptomatic carriers in Ubonratchatani province of northeastern Thailand. Both isolates can replicate in peripheral blood mononuclear cells, but not in several T cell lines examined. The full-length sequences recovered from proviruses in infected cells by long-range polymerase chain reaction were determined. Phylogenetic analyses of these sequences at individual genes showed them to be closely related to those of reported CRF01_AE HIV-1, such as 1990 isolate CM240 and 1993 isolate 93TH253. Two isolates in this study also showed a similar pattern of CRF01_AE mosaicism and a similar structure at the long terminal repeat, i.e., a copy number of NF-kappaB binding sites, sequence at the TATA box, and the putative secondary structure of stem-loop in the transactivation response region. Our results showed that 1995 Thai E isolates could contribute to our understanding of the epidemiology, pathogenesis, and diagnostics of HIV-1 CRF01_AE and further to vaccine development.     

Tat-specific binding IgG and disease progression in HIV type 1-infected Ugandans

There are data to suggest that both the humoral and cellular immune responses directed against Tat are beneficial in delaying HIV disease progression. We examined the association between the occurrence of Tat-specific binding antibodies (Abs) and different parameters of HIV-1 disease progression. We generated eight Tat proteins, derived from HIV-1 subtypes A, B, C, and D, and circulating recombinant form CRF01_AE. These proteins were used to screen for Tat-specific binding Abs by an ELISA. Using five Tat proteins, we investigated whether the occurrence of Tat-specific Abs within 2 years after seroconversion for the majority, affected disease progression over time among 126 participants using survival analysis and rate of CD4 decline. Of these, 52 participants with a sample at 1.5 and 4.5 years after seroconversion were further examined to study the effect of Tat-specific Ab loss or maintenance on disease progression. Finally, using all the eight Tat proteins, we also investigated whether specific Abs to these Tat proteins among 48 participants, grouped as rapid progressors (RP, n = 26) and long-term survivors (LTS, n = 22) according to their CD4 decline over time, affected disease progression. Survival analysis did not reveal any evidence of protection from progression by Tat-specific Abs. Comparison of rate of CD4 declines between individuals with and without Abs to any Tat protein showed only a small and borderline significant advantage of having Tat-specific Abs (p = 0.043). There was no correlation between either loss or maintenance of Tat-specific Abs and disease progression. Comparison of LTS with RP showed no evidence that Tat-specific Abs slows participants' disease progression. This study showed no evidence of a protective effect of having Tat-specific Abs among these Ugandan subjects.     

Determination of HIV type 1 CRF01_AE gag p17 and env-V3 consensus sequences for HIV/AIDS vaccine design

A molecular epidemiological study of the gag p17 and env-V3 regions on HIV-infected drug users and blood donors was carried out in northern Thailand from 1998 through 2002 to determine the predominant subtype and consensus sequence (CS) for circulating HIV-1 strains. CRF01_AE was concluded to be a predominant strain and the nucleotide CSs in gag p17 and env-V3 showed only 1.26% and no difference from CS in the Los Alamos database, respectively. Our env-V3 CS was identical to the previously published CSs, suggesting that the CS was very conserved from 1990 through 2002 in Thailand. Gag p17 and env-V3 nucleotide sequences of seroconvertors in our subjects were quite similar to the CS and conserved for at least 9 and 6 years postinfection, respectively. These results suggest that the CS approach to the HIV-1 antigen design could overcome HIV diversity and help us develop an effective HIV/AIDS vaccine.     

Maintaining low HIV type 1 env genetic diversity among injection drug users infected with a B/C recombinant and CRF01_AE HIV type 1 in southern China

HIV-1 outbreaks in Guangxi Province, southern China were initiated from two separate border cities in 1996 and 1997. Drug users in Pingxiang City, which borders Vietnam, were infected with CRF01_AE HIV-1, and drug users in Baise City, which borders Yunan Province, were infected with a novel B/C recombinant HIV-1. Since 1997, HIV-1 has been rapidly spreading in Guangxi, including its capital city Nanning. Survey data indicated that HIV-1 prevalence among IDUs in new outbreak regions increased 8 to 42% within 1 year. The B/C recombinants obtained from five separate regions in Guangxi, which span a 4-year time frame, were remarkable for their low intersubject env V3 diversity, less than 0.2%. Similarly, the CRF01_AE from IDUs over a 3-year time frame had low intersubject env V3 diversity of less than 1.6%. Different patterns of sequence variations in the V3 and V4 regions were observed for the B/C recombinant and the CRF01_AE HIV1. The rapid spreading of homogeneous HIV-1 strains in Guangxi may have important implications for HIV transmission as well as vaccine development and evaluation.     

No differences in cellular immune responses between asymptomatic HIV type 1- and type 2-infected Gambian patients

Fewer people infected with human immunodeficiency virus (HIV) type 2 progress to acquired immunodeficiency syndrome, compared with those infected with HIV-1. To understand the immune mechanisms leading to slow progression in HIV-2 infection, cell-mediated immune responses were compared between the 2 infections in asymptomatic subjects with a CD4 cell count > or =20%. Interferon- gamma release from T lymphocytes and the cytotoxicity of CD8+ T lymphocytes were measured by ELISPOT and 51Cr release assays. The level of responses and the proportion of responders were similar in the 2 infections, despite a 20-fold difference in their geometric mean plasma virus loads. The proliferation of CD4+ T helper cells, which was evaluated by thymidine incorporation, was not different between the 2 infections. Contrary to widely held views, our results suggest that nonprogression in HIV-2 infection may not be due to more vigorous immune responses.     

Construction and characterization of an infectious molecular clone derived from the CRF01_AE primary isolate of HIV type 1

An infectious molecular clone (named p95TNIH022) was constructed using long-range polymerase chain reaction products derived from a clinical isolate (95TNIH022) of HIV-1 CRF01_AE obtained from an asymptomatic Thai carrier in 1995. The virus in the supernatant from p95TNIH022-transfected 293T cells showed infectivity in peripheral blood mononuclear cells (PBMCs) as well as in MAGIC5 cells, which express CD4 and CCR5, but not in the original MAGI cells, indicating that p95TNIH022 is an infectious molecular clone with CCR5 tropism. Interestingly, p95TNIH022-derived virus induced profound cell killing in infected PBMCs, as in cells infected with the parental isolate.