Socio-demographic and drug use factors associated with HIV-1 recombinants and dual infections in Northern Thai drug users: associations of risk with genetic complexity.

BACKGROUND: Dual infection with diverse HIV strains can foster the emergence of recombinants. The resulting increase in viral genetic diversity is a major challenge for vaccine development HIV treatment. In this study we aim to investigate the socio demographic factors associated with an increasing level of genetic diversity among HIV strains in a population of drug-users in Northern Thailand. METHODS: From 1999 through 2000, 2231 volunteers were enrolled in the Opiate-Users Research in Chiang Mai, Thailand. HIV subtype analysis was conducted among those HIV-1 seropositive (n=347) using a multi-region hybridization assay. Social and demographic variables were assessed using a structured questionnaire. RESULTS: Overall, 336/347 (96.8%) of the samples could be typed. 81.8% were CRF01_AE, 3.9% were subtype B, 9.2% were recombinants (mostly between CRF01_AE and B) and 5.1% were dual infections. Dual infections were more frequent among those with a lower education level (AOR: 5.2; 95% CI 1.4-20.3), those who have initiated injecting in the last 3 years (AOR: 3.9; 95% CI 1.1-14.6), and those reporting frequent needle sharing in the last 3 months (AOR: 7.0; 95% CI 1.5-34.1). Both recombinant strains and dual infection were more frequent among those reporting frequent needle sharing in the last 3 months (AOR: 5.3; 95% CI 1.6-17.1). CONCLUSION: To limit the expanding complexity of HIV-1 strains, early intervention should be aimed at reduction in needle sharing, especially among new intravenous drug users.

High prevalence of HIV infection among rural tea plantation residents in Kericho, Kenya.

Human immunodeficiency virus type 1 (HIV-1) epidemiology among residents of a rural agricultural plantation in Kericho, Kenya was studied. HIV-1 prevalence was 14.3%, and was higher among women (19.1%) than men (11.3%). Risk factors associated with HIV-1 for men were age (>or=25 years), marital history (one or more marriages), age difference from current spouse (>or=5 years), Luo ethnicity, sexually transmitted infection (STI) symptoms in the past 6 months, circumcision (protective), and sexual activity (>or=7 years). Among women, risk factors associated with HIV-1 were age (25-29 years, >or=35 years), marital history (one or more marriages), age difference from current spouse (>or=10 years), Luo ethnicity, STI symptoms in the past 6 months, and a STI history in the past 5 years. Most participants (96%) expressed a willingness to participate in a future HIV vaccine study. These findings will facilitate targeted intervention and prevention measures for HIV-1 infection in Kericho.

Evaluation of aldrithiol-2-inactivated preparations of HIV type 1 subtypes A, B, and D as reagents to monitor T cell responses.

The development of HIV vaccines is an urgent priority and there is need to generate reagents representing multiple subtypes that can be used to screen HIV-1-specific responses. We used Aldrithiol-2 (AT-2), a mild oxidizing reagent, to eliminate the infectivity of HIV while maintaining its structure and ability to be processed for presentation to T cells. Inactivated subtype A, B, and D viruses were evaluated for their ability to stimulate T cell responses in PBMC samples from 18 U.S. subjects infected with HIV-1 subtype B and 32 Ugandan subjects infected with subtypes A and D or recombinants AC and AD. Five HIV-1-negative samples were also analyzed. T cell responses to AT-2-inactivated viral isolates were monitored by interferon-gamma (IFN-gamma) intracellular cytokine secretion (ICS) analysis; matched microvesicle preparations served as negative controls. Among the 18 subtype B infected subjects, 39% had CD3(+) CD4 (+) IFN-gamma responses and 67% had CD3(+) CD8(+) IFN-gamma responses. Of the 32 Ugandan subjects, 34% demonstrated CD3(+) CD4(+) IFN-gamma responses and 78% demonstrated CD3(+) CD8(+) IFN-gamma responses. Both subtype-specific and cross-reactive responses were observed. Responses to the AT-2 viruses tended to be lower in magnitude than those detected by a set of overlapping gag peptides. Robust lymphoproliferative responses to AT-2 viruses were seen in a subset of subjects. In conclusion, AT-2-inactivated HIV-1 virions stimulated both CD4 and CD8 HIV-1-specific responses and may provide an additional reagent for screening HIV-1-specific responses in HIV seropositives and vaccinees.

HLA class I diversity among rural rainforest inhabitants in Cameroon: identification of A*2612-B*4407 haplotype.

The population distribution of alleles of the classical HLA class I loci in Cameroon has not been well studied but is of particular interest given the AIDS and malarial epidemics afflicting this population. We investigated the genetic diversity of HLA-A, HLA-B and HLA-C alleles in remote populations of Cameroon. Subjects from seven small, isolated, indigenous populations (N = 274) in the rainforest of southern Cameroon were typed for HLA-A, HLA-B and HLA-C alleles using a polymerase chain reaction/sequence-specific oligonucleotide probe assay and sequence analysis. Multiple alleles of the HLA-A (N = 28), HLA-B (N = 41) and HLA-C (N = 21) loci were identified, of which A*2301[allele frequency (AF) = 12.8%], B*5802 (AF = 10.9%) and Cw*0401 (AF = 16.6%) were the most frequent individual alleles and A*02 (AF = 19.0%), B*58 (AF = 15.9%) and Cw*07 (AF = 22.4%) the most common serologically defined groups of alleles. Twenty-six (28.9%) alleles with a frequency of less than 1% (AF < 1%), 39 (43%) with a frequency of 2.0-15.0% (AF = 2.0-15.0%), three globally uncommon alleles [A*2612 (AF = 2.0%), B*4016 (AF = 0.7%) and B*4407 (AF = 1.4%)], and the A*2612-Cw*0701/06/18-B*4407 haplotype (haplotype frequency = 1.3%) were also identified. Heterozygosity values of 0.89, 0.92 and 0.89 were determined for HLA-A, HLA-B and HLA-C, respectively. The extensive allelic and haplotypic diversity observed in this population may have resulted from varied natural selective pressures on the population, as well as intermingling of peoples from multiple origins. Thus, from an anthropologic perspective, these data highlight the challenges in T-cell-based vaccine development, the identification of allogeneic transplant donors and the understanding of infectious disease patterns in different populations.

In-depth analysis of a heterosexually acquired human immunodeficiency virus type 1 superinfection: evolution, temporal fluctuation, and intercompartment dynamics from the seronegative window period through 30 months postinfection.

Human immunodeficiency virus type 1 (HIV-1) superinfection refers to the acquisition of another strain by an already infected individual. Here we report a comprehensive genetic analysis of an HIV-1 superinfection acquired heterosexually. The infected individual was in a high-risk cohort in Tanzania, was exposed to multiple subtypes, and was systematically evaluated every 3 months with a fluorescent multi-region genotyping assay. The subject was identified in the window period and was first infected with a complex ACD recombinant strain, became superinfected 6 to 9 months later with an AC recombinant, and was monitored for >2.5 years. The plasma viral load exceeded 400,000 copies/ml during the first 9 months of infection but resolved to the set point of 67,000 copies/ml by 3 months after superinfection; the CD4 cell count was 377 cells/mul at 30 months. Viral diversity was evaluated with techniques designed to fully sample the quasi-species, permitting direct observation of the evolution, temporal fluctuation, and intercompartment dynamics of the initial and superinfecting strains and recombinants derived from them. Within 3 months of superinfection, seven different molecular forms were detected in gag and six were detected in env. The proportions of forms fluctuated widely over time in plasma and peripheral blood mononuclear cells, illustrating how challenging the detection of dually infected individuals can be. Strain-specific nested PCR confirmed that the superinfecting strain was not present until the 9 month follow-up. This study further defines the parameters and dynamics of superinfection and will foster appropriate studies and approaches to gain a more complete understanding of risk factors for superinfection and its impact on clinical progression, epidemiology, and vaccine design.

In-depth, longitudinal analysis of viral quasispecies from an individual triply infected with late-stage human immunodeficiency virus type 1, using a multiple PCR primer approach.

Co-infections with more than one human immunodeficiency virus type 1 (HIV-1) subtype appear to be the source of new recombinant strains and may be commonplace in high-risk cohorts exposed to multiple subtypes. Many potential dual infections have been identified during the HIV Superinfection Study in Mbeya, Tanzania, where 600 female bar workers who are highly exposed to subtypes A, C, and D have been evaluated every 3 months for over 3 years by use of the MHAacd HIV-1 genotyping assay. Here we describe an in-depth, longitudinal analysis of the viral quasispecies in a woman who was triply infected with HIV-1 and who developed AIDS and passed away 15 months after enrollment. The MHA results obtained at 0, 3, 6, 9, and 12 months revealed dual-probe reactivities and shifts in subtype over time, indicating a potential dual infection and prompting further investigation. The multiple infection was confirmed by PCR amplification of three genome regions by a multiple primer approach, followed by molecular cloning and sequencing. A highly complex viral quasispecies was found, including several recombinant forms, with vpu/gp120 being the most diverse region. A significant fluctuation in molecular forms over time was observed, showing that the serial sample format is highly desirable, if not essential, for the identification of multiple infections. In a separate experiment, we confirmed that the detection of co-infections is more efficient with the use of multiple amplification primers to overcome the primer bias that results from the enormous diversity in the HIV-1 genome.

Documentation of subtype C HIV Type 1 strains in Argentina, Paraguay, and Uruguay.

HIV subtypes B, F, and BF recombinants have been previously reported in South America. This report describes the presence of HIV-1 subtype C infection in the countries of Argentina, Uruguay, and Paraguay dating back to at least 1999. Surveillance for uncommon non-B/non-F subtype viruses circulating in South America has been conducted in samples obtained from nine countries. Peripheral blood mononuclear cells (PBMC), dried filter paper (FP), and fresh blood (FB) samples were collected from HIV-positive patients from Ecuador, Colombia, Venezuela, Peru, Chile, Bolivia, Argentina, Uruguay, and Paraguay. From a total of 2962 HIV seropositive samples examined during a 9-year period (1995-2003), only 11 (0.4%) were found to be infected with non-B/non-F HIV variants. Eight of these 11 strains were determined to be subtype C by heteroduplex mobility assay (HMA). Five of these 8 strains were further characterized by sequencing and phylogenetic analysis of the protease (Pro) and reverse transcriptase (RT) region of the genome and two were sequenced full length. One of the strains was found to be a unique BC recombinant. The spread of a third subtype of HIV, subtype C, should raise the question of its potential future role in the HIV epidemic in this region.

HLA class I serotypes and cytotoxic T-lymphocyte responses among human immunodeficiency virus-1-uninfected Thai volunteers immunized with ALVAC-HIV in combination with monomeric gp120 or oligomeric gp160 protein boosting.

Antigen-induced cellular immunogenicity may vary between populations due to differences in human leukocyte antigen (HLA) diversity and, hence, may play a critical role in the protection afforded by vaccines. In the setting of two, phase I/II human immunodeficiency virus-1 vaccine trials of a recombinant canarypox prime, and boosting with either recombinant monomeric gp120 or oligomeric gp160, we assessed the association between specific human leukocyte antigen (HLA) class I serotypes and the presence of cytotoxic T-lymphocyte response measured by 51Cr-release assay. HLA class I serotypes A11, A24, A33, B46, and B75 were the most common, present in 10% or more of 245 individuals studied. Forty of 187 (21.4%) Thai adults who received either ALVAC-HIV with gp120 or oligomeric gp160 or ALVAC alone had a precursor cytolytic CD8 T-cell response (pCTL). HLA-B44 was positively and significantly associated with a pCTL response (odds ratio 7.6, 95% CI: 2.7-21.2), whereas B46 was negatively associated but not robust when adjusted for multiple comparisons. Responses to Env proteins accounted for the majority (nine of 11) of pCTL activity among those persons with B44. This HLA class I serotype occurred in 9.4% of participants overall (including the placebo group), less commonly than what is reported from populations of European ancestry. These results strengthen the importance of assessing HLA class I distributions in conjunction with studies of vaccines designed to elicit cellular immunity in different populations.

Circulating recombinant form CRF02_AG in South America.

With the objective of monitoring the distribution of HIV-1 subtypes and circulating recombinant forms (CRFs)in South America, population-based surveillance studies were performed in seven countries. Peripheral blood mononuclear cell, filter paper, fresh blood, and cocultivation samples were collected from HIV-positive patients from Colombia, Ecuador, Peru, Bolivia, Chile, Argentina, and Uruguay, during a 7-year period(1995-2001). DNA was prepared and HIV envelope subtypes were determined by heteroduplex mobility as-say and DNA sequencing from 1289 HIV-positive samples. While subtypes B and F were the most commonly observed subtypes, two CRF02_AG strains were detected, in Ecuador. This is the first report of the existence of this CRF in South America.

Precise mapping of recombination breakpoints suggests a common parent of two BC recombinant HIV type 1 strains circulating in China.

Two different BC recombinant HIV-1 strains have arisen and begun to circulate among intravenous drug users in China. The recombinants are mostly subtype C with a few small subtype B segments. Additional full-genome sequences of the two recombinants, termed CRF07_BC and CRFO&_BC, are now available for analysis. Four CRF07_BC strains, including c54, 97CNU01, 98CN009, and a new strain CNGL-179, described here, and four CRF08_BC strains, including 97CNGX-6, 97CNGX-7, 97CNGX-9, and 98CN006, were compared for their recombination breakpoints by bootscanning and software for fine mapping of recombinants. The four CRF07_BC strains shared an identical recombination structure and the four CRF08_BC strains shared an identical, but different, recombination structure. The two CRFs share five precise subtype B/C boundaries, although although other segments differ between them, suggesting that they shared a common ancestor, itself a BC recombinant that separately "back-crossed" onto different subtype C strains. Both CRFs are broadly distributed from north to south in western China and have maintained low interpatient diversity.

Diverse BF recombinants have spread widely since the introduction of HIV-1 into South America.

OBJECTIVE: To describe the genetic diversity of HIV-1 in South America by full genome sequencing and analysis. METHODS: Purified peripheral blood mononuclear cell DNA from HIV-infected individuals in Argentina, Uruguay and Bolivia was used to amplify full HIV-1 genomes. These were sequenced using the ABI 3100 automated sequencer and phylogenetically analysed. RESULTS: Twenty-one HIV-1 strains from three South American countries, 17 of which were pre-screened by envelope heteroduplex mobility assay (HMA), were studied. Ten out of 10 HMA subtype F and four out of seven HMA subtype B strains were actually BF recombinants upon full genome analysis. Two BF recombinants from Argentina and two from Uruguay had the same structure, representing a new circulating recombinant form termed CRF12_BF(ARMA159). Twelve other BF recombinants had structures related to CRF12 but with additional segments of subtype B; each was unique. BF recombinants were temporally and geographically widespread, found as early as 1986-1987 in vertically infected Argentinian children and in Argentina, Uruguay, and Bolivia.

Cytotoxic T lymphocyte responses to vaccinia virus antigens but not HIV-1 subtype E envelope protein seen in HIV-1 seronegative Thais.

The HIV-1 prime boost phase I/II vaccine trial using a recombinant canarypox vector, vCP1521, containing subtype E env (gp120), and subtype B env (gp41), gag and protease has started in Thailand. We have demonstrated that although 4 from 15 human immunodeficiency virus type 1 (HIV-1) seronegative Individuals showed cytotoxic T lymphocyte (CTL) responses to vaccinia virus antigens, none of them showed specific CTL responses to subtype E Env after in vitro stimulation. This preliminary study suggests that specific CTL responses to subtype E envelope detected in HIV-1 seronegative Individuals after vaccination should be considered as specific responses to the immunization.

Human immunodeficiency virus type 1 DNA sequences genetically damaged by hypermutation are often abundant in patient peripheral blood mononuclear cells and may be generated during near-simultaneous infection and activation of CD4(+) T cells.

G-to-A hypermutation has been sporadically observed in human immunodeficiency virus type 1 (HIV-1) proviral sequences from patient peripheral blood mononuclear cells (PBMC) and virus cultures but has not been systematically evaluated. PCR primers matched to normal and hypermutated sequences were used in conjunction with an agarose gel electrophoresis system incorporating an AT-binding dye to visualize, separate, clone, and sequence hypermutated and normal sequences in the 297-bp HIV-1 protease gene amplified from patient PBMC. Among 53 patients, including individuals infected with subtypes A through D and at different clinical stages, at least 43% of patients harbored abundant hypermutated, along with normal, protease genes. In 70 hypermutated sequences, saturation of G residues in the GA or GG dinucleotide context ranged from 20 to 94%. Levels of other mutants were not elevated, and G-to-A replacement was entirely restricted to GA or GG, and not GC or GT, dinucleotides. Sixty-nine of 70 hypermutated and 3 of 149 normal sequences had in-frame stop codons. To investigate the conditions under which hypermutation occurs in cell cultures, purified CD4(+) T cells from normal donors were infected with cloned NL4-3 virus stocks at various times before and after phytohemagglutinin (PHA) activation. Hypermutation was pronounced when HIV-1 infection occurred simultaneously with, or a few hours after, PHA activation, but after 12 h or more after PHA activation, most HIV-1 sequences were normal. Hypermutated sequences generated in culture corresponded exactly in all parameters to those obtained from patient PBMC. Near-simultaneous activation and infection of CD4(+) T cells may represent a window of susceptibility where the informational content of HIV-1 sequences is lost due to hypermutation.

Selective increases in HIV-specific neutralizing antibody and partial reconstitution of cellular immune responses during prolonged, successful drug therapy of HIV infection.

Because the immune response to HIV depends on viral gene expression, we examined the HIV-specific immune responses in persons whose viral load after highly active antiretroviral therapy (HAART) was <400 on at least 3 occasions over a 12-month interval. Eleven patients were identified. While there was little change in mean HIV-binding antibody (Ab) titers in this group, two persons mounted increases in HIV envelope-specific binding antibody. Neutralizing antibody (NAb) titers against a panel of HIV-1 primary isolates (BZ167, US1, and CM237) increased post-HAART (80% neutralization titer against US1, p = 0.06; against CM237, p = 0.04). The two persons with large increases in binding antibody also had increases in primary isolate NAb. Roughly half of HAART recipients had significant increases in neutralizing antibody to the primary isolates US1 and CM237. Compared with CD4-matched, non-HAART controls, there were significant increases in NAb against the subtype B primary isolate US1 (p < 0.0009); no increases were seen against more easily neutralized primary isolate BZ167. There were no differences after HAART in antibody-directed cellular cytotoxicity (ADCC). HAART resulted in a partial restoration of lymphoproliferative responses to recall antigens (tetanus and diphtheria). New responses developed to HIV Gag p24. No patient responded to HIV Env gp160 or gp120 either before or after HAART. The data underscore the lack of functional reconstitution of HIV-specific, CD4-mediated responses despite durable suppression of viral replication. In the setting of stable anti-HIV Ab levels, the development of increased NAb in certain individuals suggests that control of the virus by HAART may assist in immune control of HIV.

High proportion of unrelated HIV-1 intersubtype recombinants in the Mbeya region of southwest Tanzania.

BACKGROUND: In Mbeya, a rural region of southwest Tanzania, HIV-1 subtypes A, C and D have been co-circulating since the early 1990s. OBJECTIVE: To define to what extent the co-existence of subtypes has led to recombinant HIV-1 strains and whether there is evidence for epidemic spread of any circulating recombinant form. METHODS: Nine HIV-1-seropositive young adults from Mbeya Town with no evident high-risk behaviour contributed peripheral blood mononuclear cells for this study. Nine virtually full-length-genome-sequences were amplified from this DNA and phylogenetically analysed. RESULTS: Out of the nine samples, two were subtype A (22%), two were subtype C (22%) and five were recombinants (56%): four A/C recombinants and one C/D recombinant. None of the recombinants were related to each other; all of them had different mosaic structures. Most of the genome in the recombinants was subtype C. CONCLUSION: A high proportion of unrelated intersubtype recombinants, none of them apparently spreading in the population, may be present in southwest Tanzania.

HIV-1 seroconversion in United States Army active duty personnel, 1985-1999.

OBJECTIVE: To monitor HIV-1 infection trends among United States Army personnel, a predominantly young population group, tested between 1985 and 1999 for HIV-1 infection. DESIGN: Demographic correlates of HIV-1 infection were assessed in the cohort via epidemiologic analysis. METHODS: Annual seroconversion incidence rates were calculated per 1000 person-years (PY) of follow-up. Poisson regression was used to assess demographic correlates of HIV-1 seroconversion risk. RESULTS: There were 1275 seroconverters among 2 004 903 active duty Army personnel accounting for 7 700 231 PY of follow-up. The HIV-1 incidence rate (IR) was 0.17/1000 PY [95% confidence interval (CI), 0.16-0.17]. The highest IR was observed in the first year of testing (IR, 0.43/1000 PY; 95% CI, 0.33-0.52). The IR for male and female soldiers was 0.18/1000 PY and 0.08/1000 PY, respectively. HIV-1 incidence declined with age. Significant risk of HIV-1 seroconversion was associated with age [> 30 years old relative risk (RR), 1.51], race (Black RR, 4.61; Hispanic RR, 2.76), gender (male RR, 3.12), marital status (unmarried RR, 2.01) and rank (enlisted RR, 2.50). CONCLUSIONS: HIV-1 seroconversions in the US Army have been low and stable since the early 1990s. Continued HIV-1 incidence surveillance in the US Army provides information on the status of the epidemic in the Army, as well as important corroborative data on HIV-1 infections throughout the US.

The AG recombinant IbNG and novel strains of group M HIV-1 are common in Cameroon.

The genetic diversity of group M HIV-1 is highest in west central Africa. Blood samples from four locations in Cameroon were collected to determine the molecular epidemiology of HIV-1. The C2-V5 region of envelope was sequenced from 39 of the 40 samples collected, and 7 samples were sequenced across the genome. All strains belonged to group M of HIV-1. The circulating recombinant form CRF02 AG (IbNG) was the most common strain (22/39, 56%). Two of these were confirmed by full genome analysis. Four samples (4/39, 10%) clustered with the sub-subtype F2 and one of these was confirmed by full genome sequencing. Recombinant forms, each different but containing subtype A, accounted for the next most common form (7/39, 18%). Among these recombinants, those combining subtypes A and G were the most common (4/7, 57%). Also found were 3 subtype A, 2 subtype G, and 1 subtype B strain. Many recombination break points were shared between IbNG and the other AG recombinants, though none of these other AG recombinants included IbNG as a parent. This suggests that there was an ancestral AG recombinant that gave rise to CRF02 AG (IbNG), the successful circulating recombinant form, and to others that were less successful and are now rare.

Clinical prognosis of patients with early-stage human immunodeficiency virus (HIV) disease: contribution of HIV-1 RNA and T lymphocyte subset quantitation.

Systems for the staging of individuals with human immunodeficiency virus type 1 (HIV-1) infection were developed 15 years ago. Subsequently, assays for quantitating HIV-1 RNA and immunophenotyping of lymphocyte subsets have been developed and validated. The utility of these assays for improved staging in early disease was evaluated in 256 HIV-infected adults (52% minority) with CD4 counts > or = 400 cells/microL followed in U.S. military medical centers before the highly active anti-retroviral therapy era. HIV viral load (RNA) was quantitated; the frequencies of select CD4+ immunophenotypes were determined in 112 subjects. The results were analyzed in relation to three outcome measures: death, first acquired immunodeficiency syndrome-defining opportunistic infection, and CD4 count < or = 200 cells/microL. Serum RNA level and CD4 count were each found to be predictive of all three outcomes. In addition, increases in the T-cell subsets CD28-CD4+ and CD29+CD26-CD4+ were found to be independently predictive of more rapid progression. The classification of early-stage HIV patients is improved by the quantitation of both viral RNA and T-lymphocyte subsets.