Awet Abraha

The Replicative Fitness of Primary Human Immunodeficiency Virus Type 1 (HIV-1) Group M, HIV-1 Group O, and HIV-2 Isolates

ABSTRACT The main (M) group of human immunodeficiency virus type 1 (HIV-1) is responsible for the global AIDS epidemic while HIV-1 group O (outlier) and HIV type 2 are endemic only in west and central Africa. The failure of HIV-2 and especially HIV-1 group O to spread following the initial zoonotic jumps is not well understood. This study was designed to examine the relative replicative capacities between these human lentiviruses. A pairwise competition experiment was performed with peripheral blood mononuclear cells with eight HIV-2 isolates, 6 group O viruses, and 15 group M viruses of subtype A (2 viruses), B (5 viruses), C (4 viruses), D (2 viruses) and CRF01_AE (2 viruses). HIV-1 group M isolates of any subtype were typically 100-fold-more fit than group O or HIV-2 strains when competed in peripheral blood mononuclear cells from various humans. This order in replicative fitness was also observed when virus pairs were added to human dendritic cells and then cocultured with primary, quiescent T cells, which is the model for HIV-1 transmission. These results suggest that reduced replicative and transmission fitness may be contributing to the low prevalence and limited geographical spread of HIV-2 and group O HIV-1 in the human population.

Changes in Human Immunodeficiency Virus Type 1 Fitness and Genetic Diversity during Disease Progression

ABSTRACT This study examined the relationship between ex vivo human immunodeficiency virus type 1 (HIV-1) fitness and viral genetic diversity during the course of HIV-1 disease. Primary HIV-1 isolates from 10 patients at different time points were competed against control HIV-1 strains in peripheral blood mononuclear cell (PBMC) cultures to determine relative fitness values. Patient HIV-1 isolates sequentially gained fitness during disease at a significant rate that directly correlated with viral load and HIV-1 env C2V3 diversity. A loss in both fitness and viral diversity was observed upon the initiation of antiretroviral therapy. A possible relationship between genotype and phenotype (virus replication efficiency) is supported by the parallel increases in ex vivo fitness and viral diversity during disease, of which the correlation is largely based on specific V3 sequences. Syncytium-inducing, CXCR4-tropic HIV-1 isolates did have higher relative fitness values than non-syncytium-inducing, CCR5-tropic HIV-1 isolates, as determined by dual virus competitions in PBMC, but increases in fitness during disease were not solely powered by a gradual switch in coreceptor usage. These data provide in vivo evidence that increasing HIV-1 replication efficiency may be related to a concomitant increase in HIV-1 diversity, which in turn may be a determining factor in disease progression.

Comparing the ex vivo fitness of CCR5-tropic human immunodeficiency virus type 1 isolates of subtypes B and C.

ABSTRACT Continual human immunodeficiency virus type 1 (HIV-1) evolution and expansion within the human population have led to unequal distribution of HIV-1 group M subtypes. In particular, recent outgrowth of subtype C in southern Africa, India, and China has fueled speculation that subtype C isolates may be more fit in vivo. In this study, nine subtype B and six subtype C HIV-1 isolates were added to peripheral blood mononuclear cell cultures for a complete pairwise competition experiment. All subtype C HIV-1 isolates were less fit than subtype B isolates (P < 0.0001), but intrasubtype variations in HIV-1 fitness were not significant. Increased fitness of subtype B over subtype C was also observed in primary CD4+ T cells and macrophages from different human donors but not in skin-derived human Langerhans cells. Detailed analysis of the retroviral life cycle during several B and C virus competitions indicated that the efficiency of host cell entry may have a significant impact on relative fitness. Furthermore, phyletic analyses of fitness differences suggested that, for a recombined subtype B/C HIV-1 isolate, higher fitness mapped to the subtype B env gene rather than the subtype C gag and pol genes. These results suggest that subtype B and C HIV-1 may be transmitted with equal efficiency (Langerhans cell data) but that subtype C isolates may be less fit following initial infection (T-cell and macrophage data) and may lead to slower disease progression.

Variable fitness impact of HIV-1 escape mutations to cytotoxic T lymphocyte (CTL) response

Human lymphocyte antigen (HLA)-restricted CD8+ cytotoxic T lymphocytes (CTL) target and kill HIV-infected cells expressing cognate viral epitopes. This response selects for escape mutations within CTL epitopes that can diminish viral replication fitness. Here, we assess the fitness impact of escape mutations emerging in seven CTL epitopes in the gp120 Env and p24 Gag coding regions of an individual followed longitudinally from the time of acute HIV-1 infection, as well as some of these same epitopes recognized in other HIV-1-infected individuals. Nine dominant mutations appeared in five gp120 epitopes within the first year of infection, whereas all four mutations found in two p24 epitopes emerged after nearly two years of infection. These mutations were introduced individually into the autologous gene found in acute infection and then placed into a full-length, infectious viral genome. When competed against virus expressing the parental protein, fitness loss was observed with only one of the nine gp120 mutations, whereas four had no effect and three conferred a slight increase in fitness. In contrast, mutations conferring CTL escape in the p24 epitopes significantly decreased viral fitness. One particular escape mutation within a p24 epitope was associated with reduced peptide recognition and high viral fitness costs but was replaced by a fitness-neutral mutation. This mutation appeared to alter epitope processing concomitant with a reduced CTL response. In conclusion, CTL escape mutations in HIV-1 Gag p24 were associated with significant fitness costs, whereas most escape mutations in the Env gene were fitness neutral, suggesting a balance between immunologic escape and replicative fitness costs.

Relationships between Infectious Titer, Capsid Protein Levels, and Reverse Transcriptase Activities of Diverse Human Immunodeficiency Virus Type 1 Isolates

ABSTRACT Most studies on human immunodeficiency virus type 1 (HIV-1) replication kinetics or fitness must rely on a particular assay to initially standardize inocula from virus stocks. The most accurate measure of infectious HIV-1 titers involves a limiting dilution-infection assay and a calculation of the dose required for 50% infectivity of susceptible cells in tissue culture (TCID50). Surrogate assays are now commonly used to measure the amount of p24 capsid, the endogenous reverse transcriptase (RT) activity, or the amount of viral genomic RNA in virus particles. However, a direct comparison of these surrogate assays and actual infectious HIV-1 titers from TCID50 assays has not been performed with even the most conserved laboratory strains, let alone the highly divergent primary HIV-1 isolates of different subtypes. This study indicates that endogenous RT activity, not p24 content or viral RNA load, is the best surrogate measure of infectious HIV-1 titer in both cell-free supernatants and viruses purified on sucrose cushions. Sequence variation between HIV-1 subtypes did not appear to affect the function or activity of the RT enzyme in this endogenous assay but did affect the detection of p24 capsid by both enzyme immunoassays and Western blots. Clear groupings of non-syncytium-inducing (NSI), CCR5-tropic (R5), and SI/CXCR4-tropic (X4) HIV-1 isolates were observed when we compared the slopes derived from correlations of RT activity with infectious titers. Finally, the replication efficiency or fitness of both the NSI/R5 and SI/X4 HIV-1 isolates was not linked to the titers of the virus stocks.

Differences in the Fitness of Two Diverse Wild-Type Human Immunodeficiency Virus Type 1 Isolates Are Related to the Efficiency of Cell Binding and Entry

ABSTRACT The ability of one primary human immunodeficiency virus type 1 (HIV-1) isolate to outcompete another in primary CD4+ human lymphoid cells appears to be mediated by the efficiency of host cell entry. This study was designed to test the role of entry on fitness of wild-type HIV-1 isolates (e.g., replicative capacity) and to examine the mechanism(s) involved in differential entry efficiency. The gp120 coding regions of two diverse HIV-1 isolates (the more-fit subtype B strain, B5-91US056, and less-fit C strain, C5-97ZA003) were cloned into a neutral HIV-1 backbone by using a recently described yeast cloning technique. The fitness of the primary B5 HIV-1 isolates and its env gene cloned into the NL4-3 laboratory strain had similar fitness, and both were more fit than the C5 primary isolate and its env/NL4-3 chimeric counterpart. Increased fitness of the B5 over C5 virus was mediated by the gp120 coding region of the env gene. An increase in binding/fusion, as well as decreased sensitivity to entry inhibitors (PSC-RANTES and T-20), was observed in cell fusion assays mediated by B5 gp120 compared to C5 gp120. Competitive binding assays using a novel whole virus-cell system indicate that the primary or chimeric B5 had a higher avidity for CD4/CCR5 on host cells than the C5 counterpart. This increased avidity of an HIV-1 isolate for its cell receptors may be a significant factor influencing overall replicative capacity or fitness.

CCR5- and CXCR4-Tropic Subtype C Human Immunodeficiency Virus Type 1 Isolates Have a Lower Level of Pathogenic Fitness than Other Dominant Group M Subtypes: Implications for the Epidemic

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) subtype C is the dominant subtype globally, due largely to the incidence of subtype C infections in sub-Saharan Africa and east Asia. We compared the relative replicative fitness (ex vivo) of the major (M) group of HIV-1 subtypes A, B, C, D, and CRF01_AE and group O isolates. To estimate pathogenic fitness, pairwise competitions were performed between CCR5-tropic (R5) or CXCR4-tropic (X4) virus isolates in peripheral blood mononuclear cells (PBMC). A general fitness order was observed among 33 HIV-1 isolates; subtype B and D HIV-1 isolates were slightly more fit than the subtype A and dramatically more fit than the 12 subtype C isolates. All group M isolates were more fit (ex vivo) than the group O isolates. To estimate ex vivo transmission fitness, a subset of primary HIV-1 isolates were examined in primary human explants from penile, cervical, and rectal tissues. Only R5 isolates and no X4 HIV-1 isolates could replicate in these tissues, whereas the spread to PM1 cells was dependent on active replication and passive virus transfer. In tissue competition experiments, subtype C isolates could compete with and, in some cases, even win over subtype A and D isolates. However, when the migratory cells from infected tissues were mixed with a susceptible cell line, the subtype C isolates were outcompeted by other subtypes, as observed in experiments with PBMC. These findings suggest that subtype C HIV-1 isolates might have equal transmission fitness but reduced pathogenic fitness relative to other group M HIV-1 isolates.

PSC-RANTES Blocks R5 Human Immunodeficiency Virus Infection of Langerhans Cells Isolated from Individuals with a Variety of CCR5 Diplotypes

ABSTRACT Topical microbicides that effectively block interactions between CCR5+ immature Langerhans cells (LC) residing within genital epithelia and R5 human immunodeficiency virus (HIV) may decrease sexual transmission of HIV. Here, we investigated the ability of synthetic RANTES analogues (AOP-, NNY-, and PSC-RANTES) to block R5 HIV infection of human immature LC by using a skin explant model. In initial experiments using activated peripheral blood mononuclear cells, each analogue compound demonstrated marked antiviral activity against two R5 HIV isolates. Next, we found that 20-min preincubation of skin explants with each RANTES analogue blocked R5 HIV infection of LC in a dose-dependent manner (1 to 100 nM) and that PSC-RANTES was the most potent of these compounds. Similarly, preincubation of LC with each analogue was able to block LC-mediated infection of cocultured CD4+ T cells. Competition experiments between primary R5 and X4 HIV isolates showed blocking of R5 HIV by PSC-RANTES and no evidence of increased propagation of X4 HIV, data that are consistent with the specificity of PSC-RANTES for CCR5 and the CCR5+ CXCR4− phenotype of immature LC. Finally, when CCR5 genetic polymorphism data were integrated with results from the in vitro LC infection studies, PSC-RANTES was found to be equally effective in inhibiting R5 HIV in LC isolated from individuals with CCR5 diplotypes known to be associated with low, intermediate, and high cell surface levels of CCR5. In summary, PSC-RANTES is a potent inhibitor of R5 HIV infection in immature LC, suggesting that it may be useful as a topical microbicide to block sexual transmission of HIV.

High Prevalence of Antiretroviral Resistance in Treated Ugandans Infected with Non-subtype B Human Immunodeficiency Virus Type 1

This study examined the emergence and prevalence of drug-resistant mutations in reverse transcriptase and protease coding regions in human immunodeficiency virus type 1 (HIV-1)-infected Ugandans treated with antiretroviral drugs (ARV). Genotypic resistance testing was performed on 50 and 16 participants who were enrolled in a cross-sectional and longitudinal observational cohort, respectively. The majority of the 113 HIV-1 PR-RT sequences were classified as subtypes A and D. Drug resistance mutations were prevalent in 52% of ARV-experienced individuals, and 17 of 27 ARV-resistant isolates had three mutations or more in reverse transcriptase. Resistance mutations in protease were less prevalent but only 17 of the 50 patients were receiving a protease inhibitor upon sample collection. Mutations conferring drug resistance were also selected in 3 of 16 participants in the longitudinal cohort, i.e., less than 8 months after the initiation of ARV treatment. Rapid emergence of ARV resistance was associated with poor adherence to treatment regimens, which was related to treatment costs. ARV resistance did, however, appear at a slightly higher prevalence in HIV-1 subtype D (21 of 33) than subtype A (7 of 25) infected individuals. Overall, this observational study suggests that ARV-resistant HIV-1 isolates are emerging rapidly in ARV-treated individual in Uganda and possibly other developing countries.

In Vitro Intersubtype Recombinants of Human Immunodeficiency Virus Type 1: Comparison to Recent and Circulating In Vivo Recombinant Forms

ABSTRACT The increased prevalence of human immunodeficiency virus type 1 (HIV-1) intersubtype recombinants (ISRs) is shaping HIV-1 evolution throughout the world and will have an impact on both therapeutic and vaccine strategies. This study was designed to generate and compare in vitro ISRs to those isolated from HIV-infected individuals throughout the world. Human peripheral blood mononuclear cells were dually infected with seven pairs of HIV-1 isolates from different subtypes (i.e., A to F). Recombinant crossover sites were mapped to specific regions in the envelope (env) gene by using a cloning-hybridization technique and subtype-specific probes. In vitro intersubtype recombination was at least twofold more frequent in the V1-to-V3 region than in any other env fragment, i.e., C1 to V1, V3 to V5, or V5 to gp41. Sequence and recombination site analyses suggested the C2 env domain as a “hot region” for recombination and selection of replication-competent ISRs during the 15-day incubation. In addition to these regional preferences for env recombination, homopolymeric nucleotide tracts, i.e., sequences known to pause reverse transcriptase and promote template switching, were found in most in vitro crossover sites. ISRs, originating from recent dual infections and limited transmission events, partly retained this in vitro regional or sequence preference for recombination sites. However, a shift to crossover sites flanking the gp120-coding sequence was evident in the stable circulating recombinant forms of HIV-1. Based on these findings, HIV-1 recombinants generated from these dual infections may be used as a model for in vivo intersubtype recombination and for the design of various diagnostic assays and vaccine constructs.