non-B Human Immunodeficiency Virus Type 1 Research Articles

Reduced and highly diverse peripheral HIV-1 reservoir in virally suppressed patients infected with non-B HIV-1 strains in Uganda

BackgroundOur understanding of the peripheral human immunodeficiency virus type 1 (HIV-1) reservoir is strongly biased towards subtype B HIV-1 strains, with only limited information available from patients infected with non-B HIV-1 subtypes, which are the predominant viruses seen in low- and middle-income countries (LMIC) in Africa and Asia.ResultsIn this study, blood samples were obtained from well-suppressed ART-experienced HIV-1 patients monitored in Uganda (n = 62) or the U.S. (n = 50), with plasma HIV-1 loads < 50 copies/ml and CD4+ T-cell counts > 300 cells/ml. The peripheral HIV-1 reservoir, i.e., cell-associated HIV-1 RNA and proviral DNA, was characterized using our novel deep sequencing-based EDITS assay. Ugandan patients were slightly younger (median age 43 vs 49 years) and had slightly lower CD4+ counts (508 vs 772 cells/ml) than U.S. individuals. All Ugandan patients were infected with non-B HIV-1 subtypes (31% A1, 64% D, or 5% C), while all U.S. individuals were infected with subtype B viruses. Unexpectedly, we observed a significantly larger peripheral inducible HIV-1 reservoir in U.S. patients compared to Ugandan individuals (48 vs. 11 cell equivalents/million cells, p < 0.0001). This divergence in reservoir size was verified measuring proviral DNA (206 vs. 88 cell equivalents/million cells, p < 0.0001). However, the peripheral HIV-1 reservoir was more diverse in Ugandan than in U.S. individuals (8.6 vs. 4.7 p-distance, p < 0.0001).ConclusionsThe smaller, but more diverse, peripheral HIV-1 reservoir in Ugandan patients might be associated with viral (e.g., non-B subtype with higher cytopathicity) and/or host (e.g., higher incidence of co-infections or co-morbidities leading to less clonal expansion) factors. This highlights the need to understand reservoir dynamics in diverse populations as part of ongoing efforts to find a functional cure for HIV-1 infection in LMICs.

Increasing prevalence and local transmission of non-B HIV-1 subtypes in the French Antilles and French Guiana between 1995 and 2018.

The Caribbean and South American French Overseas Territories (CSAFOT) are the regions most heavily affected by the Human Immunodeficiency Virus type 1 (HIV-1) epidemic in France. Although dominated by HIV-1 subtype B, the detection of non-B subtypes and the great proportion of HIV-positive persons born abroad demonstrated the potential for local spread of non-B subtype strains in CSAFOT. To reconstruct the epidemiologic dynamics of major non-B subtype clusters spreading in CSAFOT, we conducted phylogenetic and evolutionary analyses of 2,523 HIV-1 pol sequences collected from patients living in Martinique, Guadeloupe, and French Guiana from 1995 to 2018. A large variety of HIV-1 non-B subtype strains (eight subtypes, twelve CRFs, and multiple URFs) have been introduced in CSAFOT and their prevalence significantly increases over time in Martinique and Guadeloupe. We identified twelve major transmission networks of non-B subtypes (CRF02_AG and subtypes A3, C, D, and F1) that probably arose in Guadeloupe, Martinique, French Guiana, and mainland France between the late 1970s and the middle 2000s. Phylogeographic analyses support frequent non-B subtype viral transmissions within CSAFOT as well as transatlantic transmission between CSAFOT and mainland France. Domestic transmission networks of non-B subtype variants in CSAFOT comprise both men having sex with men and heterosexual individuals from different age groups. Different HIV-1 non-B subtype variants were sequentially introduced in CSAFOT between the late 1970s and the middle 2000s and are currently spreading through domestic, regional, and/or transatlantic networks of individuals from different age and risk groups.

Large-Scale Analysis of the Prevalence and Geographic Distribution of HIV-1 Non-B Variants in the United States

The genetic diversity of human immunodeficiency virus type 1 (HIV-1) has significant implications for diagnosis, vaccine development, and clinical management of patients. Although HIV-1 subtype B is predominant in the United States, factors such as global travel, immigration, and military deployment have the potential to increase the proportion of non-subtype B infections. Limited data are available on the prevalence and distribution of non-B HIV-1 strains in the United States. We sought to retrospectively examine the prevalence, geographic distribution, diversity, and temporal trends of HIV-1 non-B infections in samples obtained by ARUP Laboratories, a national reference laboratory, from all regions of the United States. HIV-1 pol sequences from 24,386 specimens collected from 46 states between 2004 and September 2011 for drug resistance genotyping were analyzed using the REGA HIV-1 Subtyping Tool, version 2.0. Sequences refractory to subtype determination or reported as non-subtype B by this tool were analyzed by PHYLIP version 3.5 and Simplot version 3.5.1. Non-subtype B strains accounted for 3.27% (798/24,386) of specimens. The 798 non-B specimens were received from 37 states and included 5 subtypes, 23 different circulating recombinant forms (CRFs), and 39 unique recombinant forms (URFs). The non-subtype B prevalence varied from 0% in 2004 (0/54) to 4.12% in 2011 (201/4,884). This large-scale analysis reveals that the diversity of HIV-1 in the United States is high, with multiple subtypes, CRFs, and URFs circulating. Moreover, the geographic distribution of non-B variants is widespread. Data from HIV-1 drug resistance testing have the potential to significantly enhance the surveillance of HIV-1 variants in the United States.

P04-11. Prevalence of neutralizing antibody responses in chronic clades A and D human immunodeficiency virus type 1 (HIV-1) infections

Background Characterizing neutralizing antibody (Nab) responses in non-B HIV-1 subtypes is essential, because the data may illustrate key genetic and antibody properties that could impact the design and testing of Nab vaccine candidates in countries where these subtypes circulate. The principle objective of this study is to document the magnitude, breadth and prevalence of neutralizing antibodies in a cohort of non-B HIV chronically infected individuals in rural Uganda.

Evaluation of an in-house genotyping resistance test for HIV-1 drug resistance interpretation and genotyping

Introduction The human immunodeficiency virus type 1 (HIV-1) genotyping resistance test (GRT) has been considered essential for HIV-1 drug resistance monitoring. However, it is not commonly used in some developing countries in Asia and Africa due to its high running cost. Objective This study aims to evaluate a new low-cost in-house GRT for both subtype B and non-B HIV-1. Study design The in-house GRT sequenced the entire protease and 410 codons of reverse transcriptase (RT) in the pol gene. Its performance on drug resistance interpretation was evaluated against the FDA-approved ViroSeq™ HIV-1 Genotyping System. Particularly, a panel of 235 plasma samples from 205 HIV-1-infected patients in Hong Kong was investigated. The HIV-1 drug resistance-related mutations detected by the two systems were compared. The HIV-1 subtypes were analyzed through the REGA HIV-1 Genotyping Tool and env phylogenetic analysis. Results Among the 235 samples, 229 (97.4%) were successfully amplified by both in-house and ViroSeq™ systems. All PCR-negative samples harbored viral RNA at <400 copies/mL. The in-house and ViroSeq™ system showed identical drug resistance-related mutation patterns in 216 out of 229 samples (94.3%). The REGA pol genotyping results showed 93.9% (215/229) concordance with the env phylogenetic results including HIV-1 subtype A1, B, C, D, G, CRF01_AE, CRF02_AG, CRF06_cpx, CRF07_BC, CRF08_BC, CRF15_01B and other recombinant strains. The cost of running the in-house GRT is only 25% of that for the commercial system, thus making it suitable for the developing countries in Asia and Africa. Conclusions Overall, our in-house GRT provided comparable results to those of the commercial ViroSeq™ genotyping system on diversified HIV-1 subtypes at a more affordable price which make it suitable for HIV-1 monitoring in developing countries.

Use of sequence data generated in the Bayer Tru Gene genotyping assay to recognize and characterize non-subtype-b human immunodeficiency virus type 1 strains.

Human immunodeficiency virus type 1 (HIV-1) protease (PR) and reverse transcriptase (RT) gene sequences obtained during antiretroviral resistance testing with a commercial genotyping assay (Tru Gene; Bayer Corp.) were analyzed to assess the utility of these data for detecting and characterizing non-subtype-B HIV-1 strains. A total of 125 viral sequences obtained from patients believed to have acquired their HIV-1 infection in Africa were analyzed, of which 121 were determined to belong to non-B subtypes. Utilizing Tru Gene sequence data alone, 92 (76%) of these viruses could be subtyped by conventional phylogenetic analysis. The addition of supplemental RT sequence data enabled a further 28 (23.1%) viruses to be classified, while one (0.9%) sample could not be classified conclusively. Two internet-accessible databases that generate HIV-1 subtypes from PR and RT sequences (HIV-SEQ and Geno 2 Pheno) were also evaluated, and both achieved 88% concordance (106/120) with phylogenetic analysis. Non-subtype-B and B-subtype HIV-1 sequences could be readily discriminated by tallying silent polymorphisms listed on the Tru Gene research report. The mean number of silent polymorphisms in the non-B HIV-1 sequences identified in this study was 58.3 (95% confidence interval [CI], 41.1 to 75.5), compared with 20.7 (95% CI, 9.9 to 31.5) for the four subtype B viruses in the study cohort and 118 case-matched B-subtype controls. Sequence data generated in the Tru Gene HIV-1 genotyping assay could, therefore, provide a ready means of tracking the prevalence and identity of non-B subtypes in HIV-1-infected populations undergoing routine antiretroviral resistance testing.

Broad Cross-Clade T-Cell Responses to Gag in Individuals Infected with Human Immunodeficiency Virus Type 1 Non-B Clades (A to G): Importance of HLA Anchor Residue Conservation

We aimed to identify cross-clade human immunodeficiency virus type 1 (HIV-1) specific T-cell responses among 10 HLA-typed individuals who were infected with non-B HIV-1 strains (A, AG, C, D, G, or F) and to correlate these responses with genetic variation in documented T-cell epitopes. T-cell reactivity was tested against peptide pools spanning clade B Gag, Pol, Nef, Rev, and Tat consensus, with Gag and Nef providing the highest responses. Nine individuals who responded to clade B Gag demonstrated cross-reactive T-cell responses against clade A and C Gag pools, while six of seven responders to Nef-B reacted to clade A and C Nef pools. An inverse correlation between the height of the T-cell responses and the sequence divergence of the HLA class I-restricted epitopes was identified when we compared autologous Gag and Nef sequences with the reactive consensus pools. This could be explained for the Gag sequences through observed variations in the HLA anchor residues. Through mapping of 30 amino acid cross-clade-reactive regions using Gag-B pools, we were able to link 58% (14/24) of the T-cell responses to regions containing previously described HLA class I-restricted epitopes. Forty-two percent (10/24) of the responses were directed to regions containing new epitopes, for which predicted HLA class I motifs could be recognized in 70% (7/10) of individuals. We demonstrate here that cross-clade T-cell responses are frequently induced in individuals infected with distinct HIV-1 clades, suggesting that interclade variation outside of HLA anchor residues may have less impact on vaccine-induced T-cell reactivity than previously thought.

Characterization of human immunodeficiency virus type-1 from HIV-1 seropositive cases with undetectable viremia.

Human immunodeficiency virus type 1 (HIV-1) viral load has become a standard of care among HIV-1-infected patients; however, a small number of patients have undetectable viral load even though they have never been treated. By using RT-PCR and DNA-PCR, and followed by sequencing and phylogenetic analyses, a detailed molecular characterization was carried out from five HIV-1-seropositive patients who had undetectable viral load by commercially available ultrasensitive viral load assays. Of the four patients whose plasmas were available, viral RNAs were detected in three of them by using an in-house RT-PCR in at least one of the three regions (integrase, protease or envgp41). The fourth patient had positive RT-PCR signals in these regions only when RNA isolated from the supernatant of cocultivated patient PBLs with PHA-stimulated HIV-1 negative donor PBLs was used. Further analysis of DNA extracted from the PBMCs revealed that four of the five patients had detectable proviral sequences in at least two of the three regions. The fifth patient had only positive PCR results in all three regions when DNA isolated from PHA-stimulated patient's PBLs was used. Phylogenetic analysis of protease and envgp41 regions revealed that three patients were infected with subtype B viruses while the remaining two patients were infected with subtype C and CRF02_AG viruses. These subtypes coincided with geographic origin and known molecular epidemiology of HIV-1 infection. These data provide evidence that both subtype B and non-B HIV-1 infection can result in undetectable viral load in HIV-1-infected patients and that efforts should continue to further characterize these viruses.

Human immunodeficiency virus type 1 primary isolate neutralization resistance is associated with the syncytium-inducing phenotype and lower CD4 cell counts in subtype CRF01_AE-infected patients.

A number of human immunodeficiency virus type 1 (HIV-1) non-B-subtype products have been developed for present or future vaccine trials; in Thailand, several studies using subtype B and/or CRF01_AE vaccines have been conducted. To better characterize the biologic properties of these subtypes, 70 HIV-1 subtype B and E isolates were phenotyped as syncytium-inducing (SI) or non-syncytium-inducing (NSI) isolates and assessed for sensitivity to neutralizing antibody (NAb). A significantly higher number of NSI subtype E viruses were neutralization sensitive than SI subtype E viruses (P = 0.009), while no association between viral phenotype and sensitivity to NAb was observed for subtype B (P = 0.856), suggesting a difference in the neutralization patterns of subtypes B and E. Strikingly, concurrent CD4 T-cell numbers were significantly lower for subtype E-infected patients whose isolates were more resistant to NAb, both for the overall study group (P < 0.001) as well as for the 22 patients with NSI isolates (P = 0.013). Characterization of the evolution of biologic properties of both B and non-B HIV-1 subtypes will provide a clearer understanding of the repertoire of antibodies that must be elicited for a vaccine to be effective against all phenotypes and subtypes.

Underestimation of HIV-1 plasma viral burden in patients who acquire infection abroad: the experience in a community hospital clinic.

In a suburban teaching hospital clinic, we diagnosed human immunodeficiency virus type 1 (HIV-1) infection in 10 patients who acquired the infection in Africa or Asia. All the patients had low viral plasma HIV RNA levels. This finding likely represents underestimation of true plasma viral burden in patients infected with non-B serotypes of HIV-1. Clinicians caring for immigrants with HIV infection need to be aware of the limitations of current commercial assays for the quantification of HIV-1 plasma viral burden, and better assays are needed for non-B HIV-1 serotypes.

Erroneously low or undetectable plasma human immunodeficiency virus type 1 (HIV-1) ribonucleic acid load, determined by polymerase chain reaction, in West African and American patients with non-B subtype HIV-1 infection.

The polymerase chain reaction (PCR) assay for plasma human immunodeficiency virus type 1 (HIV-1) ribonucleic acid (RNA) inadequately quantitates virus load for some non-B HIV-1 subtypes because of genetic diversity in the gag region targeted by the PCR primers. Unexpectedly low or undetectable plasma HIV-1 RNA findings by PCR were a clue to non-B HIV-1 infections in patients in whom plasma HIV-1 RNA was found to be substantially higher when determined by a branched-chain deoxyribonucleic acid assay.

The molecular epidemiology of HIV type 1 of men in Mexico.

Genotypic characteristics of human immunodeficiency virus type 1 (HIV-1) in Mexico were investigated in a multicenter study that involved centers in five geographic regions of the country. Study samples (n = 65) collected from male patients in 1998-1999 were sequenced within the C2-V5 region of the gp120 env gene. Phylogenetic analysis revealed that subtype B predominates in Mexico. The level of interpatient nucleotide diversity (mean value of 8.9%) was congruent with multiple introductions of the virus and the "aging" epidemic in Mexico. One-third of samples (30.8% of cases) showed polymorphism within the crown of the V3 loop demonstrating non-GPGR motifs. Two new motifs in the V3 loop crown - HPGG and GPEG - were observed. The evolution of the AIDS epidemic in Mexico should be closely monitored since non-B HIV-1 subtypes might be introduced. The nucleotide sequences were deposited in the GenBank under accession numbers AF200855-AF200869, AF200871-AF200892, and AF200894-AF200921.

Detection of human immunodeficiency virus type 1 (HIV-1) DNA and RNA sequences in HIV-1 antibody-positive blood donors in Uganda by the Roche AMPLICOR assay

The ability of commercially available PCR-based assays to accurately detect or quantitate human immunodeficiency virus type 1 (HIV-1) DNA or RNA in individuals predominantly infected with HIV-1 subtypes A and D is not known. Therefore, peripheral leukocytes from 43 individuals in Kampala, Uganda, positive for HIV by the Western blot (immunoblot) assay were tested by using the Roche AMPLICOR HIV-1 assay for the detection of DNA gag sequences. Plasma from these same individuals was tested by using the Roche HIV-1 AMPLICOR MONITOR HIV-1 assay for the quantitation of HIV-1 RNA gag sequences. In addition, peripheral leukocytes were tested for HIV-1 DNA by using a lower annealing temperature or a different primer pair for the HIV-1 pol region. The proportions of individuals with detectable HIV-1 DNA and RNA gag sequences by the Roche assays were 74 and 90%, respectively. The proportions positive for HIV-1 DNA sequences by using a 50 degrees C annealing temperature or the pol primer pair were 71 and 98%, respectively. In summary, the standard Roche assay did not detect HIV-1 DNA sequences in a significant number of HIV-1-infected individuals in Uganda. However, use of a pol primer pair increased the sensitivity of the assay to 98%. The sensitivity of the Roche AMPLICOR MONITOR assay for the detection and quantitation of HIV-1 RNA sequences was significantly higher than that of the DNA-based assay, but the efficiency of the assay, and hence, the accuracy of the values obtained with RNA, is not known. Modifications to existing assays are needed to enhance the sensitivities and accuracies of these commercially available assays for use in developing countries where non-B HIV-1 subtypes predominate.