Francine E. McCutchan

Emergence of unique primate T-lymphotropic viruses among central African bushmeat hunters.

The human T-lymphotropic viruses (HTLVs) types 1 and 2 originated independently and are related to distinct lineages of simian T-lymphotropic viruses (STLV-1 and STLV-2, respectively). These facts, along with the finding that HTLV-1 diversity appears to have resulted from multiple cross-species transmissions of STLV-1, suggest that contact between humans and infected nonhuman primates (NHPs) may result in HTLV emergence. We investigated the diversity of HTLV among central Africans reporting contact with NHP blood and body fluids through hunting, butchering, and keeping primate pets. We show that this population is infected with a wide variety of HTLVs, including two previously unknown retroviruses: HTLV-4 is a member of a phylogenetic lineage that is distinct from all known HTLVs and STLVs; HTLV-3 falls within the phylogenetic diversity of STLV-3, a group not previously seen in humans. We also document human infection with multiple STLV-1-like viruses. These results demonstrate greater HTLV diversity than previously recognized and suggest that NHP exposure contributes to HTLV emergence. Our discovery of unique and divergent HTLVs has implications for HTLV diagnosis, blood screening, and potential disease development in infected persons. The findings also indicate that cross-species transmission is not the rate-limiting step in pandemic retrovirus emergence and suggest that it may be possible to predict and prevent disease emergence by surveillance of populations exposed to animal reservoirs and interventions to decrease risk factors, such as primate hunting.

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

ABSTRACT 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.

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

ObjectiveTo describe the genetic diversity of HIV-1 in South America by full genome sequencing and analysis. MethodsPurified 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. ResultsTwenty-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_BFARMA159. 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.

CD8 T-Cell Recognition of Multiple Epitopes within Specific Gag Regions Is Associated with Maintenance of a Low Steady-State Viremia in Human Immunodeficiency Virus Type 1-Seropositive Patients

ABSTRACT The importance of HLA class I-restricted CD8 T-cell responses in the control of human immunodeficiency virus (HIV) infection is generally accepted. While several studies have shown an association of certain HLA class I alleles with slower disease progression, it is not fully established whether this effect is mediated by HIV-specific CD8 T-cell responses restricted by these alleles. In order to study the influence of the HLA class I alleles on the HIV-specific CD8 T-cell response and on viral control, we have assessed HIV-specific epitope recognition, plasma viral load, and expression of HLA class I alleles in a cohort of HIV-seropositive bar workers. Possession of the HLA class I alleles B5801, B8101, and B0702 was associated with a low median viral load and simultaneously with a broader median recognition of Gag epitopes compared to all other HLA alleles (twofold increase) (P = 0.0035). We further found an inverse linear relationship between the number of Gag epitopes recognized and the plasma viral load (R = −0.36; P = 0.0016). Particularly, recognition of multiple epitopes within two regions of Gag (amino acids [aa] 1 to 75 and aa 248 to 500) was associated with the maintenance of a low steady-state viremia, even years after acute infection.

Detection of diverse HIV-1 genetic subtypes in the USA

Of the nine genetic subtypes of HIV-1 that exist world wide, subtype B predominates in North America and Europe. Thus, most knowledge about HIV-1 and most vaccine development efforts are based on subtype B viruses. We document here the detection of HIV-1 subtypes A, D, and E in five US servicemen who acquired these non-subtype-B infections during overseas deployments. The dispersal of diverse HIV-1 subtypes into regions of the world with previously restricted genetic diversity may have important implications for the epidemiology of the epidemic and for the design and implementation of vaccine trials.

Forty-one near full-length HIV-1 sequences from Kenya reveal an epidemic of subtype A and A-containing recombinants.

ObjectiveTo further define the genetic diversity of HIV-1 in Kenya using approaches that clearly distinguish subtypes from inter-subtype recombinants. DesignNear full genome sequencing and analysis were used, including sensitive new tools for detection and mapping of recombinants. MethodsPurified peripheral blood mononuclear cell DNA from 41 HIV-1 positive blood donations collected from six hospitals across southern Kenya was used to amplify near full-length genomes by nested PCR. These were sequenced on an ABI 3100 automated sequencer and analyzed phylogenetically. ResultsAmong 41 near full-length genomes, 25 were non-recombinant (61%) and 16 were recombinant (39%). Of the 25 pure subtypes, 23 were subtype A, one was subtype C and one was subtype D. Most recombinants consisted of subtype A and either subtype C or subtype D; a few contained A2, a recently identified sub-subtype. Two A2/D recombinants had identical breakpoints and may represent a circulating recombinant form. A third A2/D recombinant had the same structure as a previously described Korean isolate, and these may constitute a second A2-containing circulating recombinant form. ConclusionsIn Kenya, 93% of HIV-1 genomes were subtype A or A-containing recombinant strains. Almost 40% of all strains were recombinant. Vaccine candidates tested in Kenya should be based on subtype A strains, but the methods used for evaluation of breakthrough infections during future vaccine trials should be capable of identifying non-A subtypes, the A2 sub-subtype, and recombinants.

Biologic and Genetic Characterization of a Panel of 60 Human Immunodeficiency Virus Type 1 Isolates, Representing Clades A, B, C, D, CRF01_AE, and CRF02_AG, for the Development and Assessment of Candidate Vaccines

ABSTRACT A critical priority for human immunodeficiency virus type 1 (HIV-1) vaccine development is standardization of reagents and assays for evaluation of immune responses elicited by candidate vaccines. To provide a panel of viral reagents from multiple vaccine trial sites, 60 international HIV-1 isolates were expanded in peripheral blood mononuclear cells and characterized both genetically and biologically. Ten isolates each from clades A, B, C, and D and 10 isolates each from CRF01_AE and CRF02_AG were prepared from individuals whose HIV-1 infection was evaluated by complete genome sequencing. The main criterion for selection was that the candidate isolate was pure clade or pure circulating recombinant. After expansion in culture, the complete envelope (gp160) of each isolate was verified by sequencing. The 50% tissue culture infectious dose and p24 antigen concentration for each viral stock were determined; no correlation between these two biologic parameters was found. Syncytium formation in MT-2 cells and CCR5 or CXCR4 coreceptor usage were determined for all isolates. Isolates were also screened for neutralization by soluble CD4, a cocktail of monoclonal antibodies, and a pool of HIV-1-positive patient sera. The panel consists of 49 nonsyncytium-inducing isolates that use CCR5 as a major coreceptor and 11 syncytium-inducing isolates that use only CXCR4 or both coreceptors. Neutralization profiles suggest that the panel contains both neutralization-sensitive and -resistant isolates. This collection of HIV-1 isolates represents the six major globally prevalent strains, is exceptionally large and well characterized, and provides an important resource for standardization of immunogenicity assessment in HIV-1 vaccine trials.

Detection of HIV-1 subtypes, recombinants, and dual infections in east Africa by a multi-region hybridization assay

Objective: To enable more rapid and efficient genotyping of HIV-1 in East Africa, where subtypes A, C, and D and their recombinants are co-circulating. Design: Full-genome sequencing of HIV-1 provides complete discrimination of subtypes and recombinant forms but is costly and low-throughput compared to other genotyping approaches. Here we describe the development and evaluation of a Multi-region Hybridization Assay (MHA) for the efficient determination of HIV-1 subtypes A, C, D, recombinants, and dual infections. Methods: Five genome regions containing clustered mutations distinguishing subtypes A, C, and D were identified and used to design subtype-specific probes. DNA from primary peripheral blood mononuclear cells was used as template for real-time PCR using the fluorescent, subtype-specific probes. Results: A panel of 45 clinical samples from Uganda, Kenya, and Tanzania, previously characterized by full-genome sequencing and including 26 pure subtypes and 19 recombinant strains, was evaluated by MHA. The MHA provided 90% sensitivity and 98% specificity for the three subtypes, efficiently discriminated subtypes from recombinant forms, and detected several dual infections. Conclusions: Accurate and efficient genotyping of HIV-1 strains in vaccine trial populations in East Africa, ascertainment of dual infections, and elucidation of the genesis of recombinant forms in individuals can be facilitated by the application of MHA.

Drug Resistance Patterns, Genetic Subtypes, Clinical Features, and Risk Factors in Military Personnel with HIV-1 Seroconversion

Genetic variability is a central feature of HIV-1. The high frequency of mutations during HIV-1 replication leads to the development of viral quasi-species in vivo and contributes to genetic heterogeneity among HIV-1 isolates (1, 2). There is a growing appreciation that HIV-1 genetic diversity, including the existence of distinct genetic subtypes and the evolution of drug-resistant genotypes, can greatly affect the diagnosis and treatment of HIV-1 infection (1, 2). On the basis of DNA sequence analysis, HIV-1 has been classified into genetic subtypes, with subtypes A through I making up the major HIV-1 group (group M). The more genetically diverse groups, O and N, have also recently been described (1). For reasons that are not clear, HIV-1 subtypes are variably dispersed throughout the world. Some regions, such as central Africa and eastern Europe, have multiple circulating subtypes, whereas the distribution in other regions is more restricted (1, 3). Differences among HIV-1 subtypes can affect the sensitivity of some diagnostic assays of antibody (4) and plasma HIV-1 RNA (5, 6). Although non-subtype B infection has been reported in the United States, the prevalence in the U.S. population is unknown (7, 8). Widespread use of antiretroviral drugs has led to transmission of drug-resistant HIV-1, but the prevalence of resistant mutations in treatment-naive persons has not been thoroughly studied. Because all U.S. Navy and U.S. Marine Corps personnel are screened for HIV-1 infection at 1- to 3-year intervals, HIV-1 infection is often detected early and the seroconversion period can be estimated (9). This permits an investigation of the epidemiologic correlates and risk behaviors associated with the acquisition of non-subtype B and drug-resistant infections. Thus, we determined the prevalence of non-subtype B infection and genotypes associated with antiretroviral drug resistance in a well-characterized cohort of military personnel with recently acquired HIV-1 infection. Methods Study Design United States Navy or Marine Corps personnel with HIV-1 seroconversion who are assigned to military bases west of the Mississippi River in the United States or in the Pacific region overseas are referred to the Navy Medical Center San Diego for initial and follow-up HIV-1 evaluations. Between February 1997 and February 1998, 99 of 141 personnel referred were eligible on the basis of documented seroconversion within the past 3 years. Ninety-five of 99 patients enrolled and signed a consent form approved by the institutional review board of the Navy Medical Center San Diego. Thirty-two patients were being referred for their first HIV evaluation, and 63 were enrolled during a follow-up visit. Blood was obtained to determine genetic subtype and the presence of antiretroviral drug resistance. All patients completed a self-administered risk factor questionnaire labeled with a unique code number. Surveys were sealed in an envelope and placed in a locked drop box that was emptied weekly by off-site data entry personnel. Clinical research staff extracted clinical and laboratory information from the medical record and sent this information, identified by code number, to the data entry site. Laboratory Analysis Seroconversion was documented by a previous negative result on whole-virus HIV-1 enzyme immunoassay followed by a positive result on enzyme immunoassay and a confirmatory positive result on Western blot assay. Lymphocyte subset analysis was performed by flow cytometry, and plasma HIV-1 RNA (available after July 1996) was measured by quantitative reverse transcription polymerase chain reaction assay (Amplicor HIV-1 Monitor assay, Roche Molecular Systems, Branchburg, New Jersey). Genetic subtyping of HIV-1 was performed by using a two-step algorithm. Sera were screened by using a competitive binding enzyme immunoassay with peptides derived from the third variable loop of the HIV-1 envelope glycoprotein. For samples that were serologically reactive to non-subtype B peptides, DNA was extracted from corresponding peripheral blood mononuclear cells, and sequence analysis was performed over a 640-base pair segment of the HIV-1 envelope gene (10). Testing for viral drug resistance was successfully performed in 31 of the 32 therapy-naive patients. Plasma-derived viral RNA was reverse transcribed into complementary DNA, amplified by polymerase chain reaction, and directly sequenced by using an automated ABI Sequencer (Applied Biosystems, Foster City, California). Consensus DNA sequences for the protease and reverse transcriptase genes from each participant were examined for mutations associated with HIV-1 antiretroviral resistance (11). Statistical Analysis Descriptive analysis of demographics, risk behaviors, and laboratory results were performed. Duration of HIV infection was calculated starting from the time of onset, which we estimated as the midpoint between the last negative and first positive result on HIV enzyme immunoassay. Bivariate analyses using odds ratios and 95% CIs determined by the Fisher exact method were done to compare the treatment-naive patients with drug-resistant infection and those with drug-sensitive infection. Statistical analyses were done by using Epi-Info, version 6.02 (Centers for Disease Control and Prevention, Atlanta, Georgia). Results Patients Characteristics and risk exposures of the cohort are summarized in Table 1. Duration of infection and laboratory results are presented for the entire cohort on initial evaluation and for the treatment-naive cohort at the time of enrollment and antiretroviral testing. Risk behaviors known to be associated with acquisition of HIV-1 were reported during the period of seroconversion in all but two patients. Table 1. Characteristics and Risk Exposures of Military Personnel with HIV-1 Seroconversion Laboratory Analysis The CD4 cell counts, plasma HIV RNA levels, and results of syphilis and hepatitis serologic testing in study patients are summarized in Table 1. Combined serologic and genetic analysis revealed that 7 of 95 patients were infected with HIV-1 genetic subtype E (7.4%); the remaining patients were infected with subtype B. Eight of 31 treatment-naive patients (26% [95% CI, 12% to 46%]) had one or more primary mutations that have been associated with phenotypic drug resistance (11) (Table 2). All reverse transcriptase mutations were associated with resistance to zidovudine, lamivudine, or nevirapine-delavirdine. Of the 4 patients with reverse transcriptase mutations, 2 (patients 1 and 2) had mutations that could confer resistance to both nucleoside and non-nucleoside reverse transcriptase inhibitors. Table 2. Drug-Resistant Mutations in Eight Treatment-Naive Patients Characteristics of Patients with Subtype E Infection and Drug-Resistant Mutations Infection with HIV-1 subtype E was documented in 6 men and 1 woman; all 6 men reported sexual contact during short deployments in Thailand. The female patient reported having sex with a man on active duty in the United States. Compared with subtype B-infected patients, subtype E-infected patients were more likely to be heterosexual (100% and 38%), to have had overseas exposure (86% and 27%), and to report sex with commercial sex workers (86% and 15%). Comparisons between patients with wild-type (n=23) and treatment-resistant (n=8) genotypes are listed in Table 1. Discussion We found a high prevalence of non-B genetic subtypes and antiretroviral drug-resistant mutations among treatment-naive military personnel with recently acquired HIV-1 infection. Seven of 95 patients (7.4%) were infected with HIV-1 subtype E. Eight of 31 treatment-naive patients (26%) had primary drug-resistant mutations; of these 8 patients, 3 had mutations in the reverse transcriptase gene only, 4 had mutations in the protease gene only, and 1 had mutations in both the reverse transcriptase and protease genes. Although our study is limited to a single clinical referral center and the sample is small, the results are generally similar to those of other studies that focused on selected drug-resistant mutations. Among several recent studies, 6% to 13% of treatment-naive patients had mutations for zidovudine resistance (12-14). Although data on the prevalence of mutations in the protease gene have not yet been published, transmission of HIV-1 that is resistant to multiple reverse transcriptase and protease inhibitors has been reported (15). Our data suggest that patients infected with drug-resistant virus were more likely to have acquired HIV in the United States and to report sexual contact with a person who is known to be infected with HIV; they were also less likely to be heterosexual. Prospective studies of the treatment responses of drug-naive patients with resistant genotypes have not been performed, but some data suggest that baseline nucleoside analogue mutations can diminish the potency and duration of viral suppression by commonly used nucleoside analogue combinations (16). In addition, several reports suggest an inherent decrease in susceptibility to reverse transcriptase or protease inhibitors in some HIV-1 genetic subtypes (17). Thus, both the transmission of acquired mutations and the genetic subtype of HIV-1 may have important implications for treatment of HIV-1 disease. Although we (7) and others (8) have previously described the introduction of non-subtype B infections into the United States, the current study is the first use of a large cohort of recently infected persons to describe the prevalence and associated risk factors for non-subtype B infection. Six of the seven patients infected with HIV-1 subtype E reported sexual contact during short deployments to Thailand; in contrast, most cases of subtype B infection were acquired in the United States. The acquisition of sexually transmitted diseases and HIV during overseas travel is not unique to the military; 5% to 20% of travelers report having sex with a new partner while abroad (18).

Characterization of subtype A Hiv-1 from Africa by full genome sequencing

OBJECTIVE To improve our understanding of the genetic complexity of HIV-1 subtype A by increasing the number of subtype A isolates that have been sequenced in their entirety. METHODS Nine HIV-1-seropositive patients from Africa living in Sweden contributed peripheral blood mononuclear cells (PBMC) for this study. Sequencing of the C2-V3 region of env had shown them to be subtype A. DNA from virus cultures was used for the amplification of virtually full-length proviral sequences, and the resulting fragment was sequenced. RESULTS Six of the nine viral isolates were subtype A throughout the genome, or non-recombinant, and all of these were from east Africa. One virus from the Ivory Coast had the AG(IbNG) genetic form, a recombinant form common in west Africa. Two of the isolates were novel recombinants: one was an A/C recombinant and the other was A/D. Analysis of gag reveals three subclusters within the A subtype: one containing the AG(IbNG) subtype viruses, one containing the AE(CM240) viruses and one containing the non-recombinant A viruses. These genetic clusters have different geographical distributions in Africa. CONCLUSION The prevailing view of HIV-1 subtype A forming a uniform band across the center of sub-Saharan Africa needs revision. In all probability, the most common subtype in west Africa and west central Africa is the AG recombinant, AG(IbNG), whereas in east central Africa it is the non-recombinant subtype A.