Biomedical subjects
Publications and source records attributed to B T Korber.
SUMMARY: This program compares sequence sets to a reference sequence, tallies G --> A hypermutations, and presents the results in various tables and graphs, which include dinucleotide context, summaries of all observed nucleotide changes, and stop codons introduced by hypermutation. AVAILABILITY: www.hiv.lanl.gov/HYPERMUT/hypermut.html
BACKGROUND: In patients infected with human immunodeficiency virus type 1 (HIV-1), combination antiretroviral therapy can result in sustained suppression of plasma levels of the virus. However, replication-competent virus can still be recovered from latently infected resting memory CD4 lymphocytes; this finding raises serious doubts about whether antiviral treatment can eradicate HIV-1. METHODS: We looked for evidence of residual HIV-1 replication in eight patients who began treatment soon after infection and in whom plasma levels of HIV-1 RNA were undetectable after two to three years of antiretroviral therapy. We examined whether there had been changes over time in HIV-1 proviral sequences in peripheral-blood mononuclear cells, which would indicate residual viral replication. We also performed in situ hybridization studies on tissues from one patient to identify cells actively expressing HIV-1 RNA. We estimated the rate of decrease of latent, replication-competent HIV-1 in resting CD4 lymphocytes on the basis of the decrease in the numbers of proviral sequences identified during primary infection and direct sequential measurements of the size of the latent reservoir. RESULTS: Six of the eight patients had no significant variations in proviral sequences during treatment. However, in two patients there was sequence evolution but no evidence of drug-resistant viral genotypes. In one patient, extensive in situ studies provided additional evidence of persistent viral replication in lymphoid tissues. Using two independent approaches, we estimated that the half-life of the latent, replication-competent virus in resting CD4 lymphocytes was approximately six months. CONCLUSIONS: These findings suggest that combination antiretroviral regimens suppress HIV-1 replication in some but not all patients. Given the half-life of latently infected CD4 lymphocytes of about six months, it may require many years of effective antiretroviral treatment to eliminate this reservoir of HIV-1.
Significant diversity exists in amino acid sequences encoding HIV-1 protease in individuals naive for protease inhibitors, which could influence the rate of evolution of resistance. High-level resistance to indinavir requires multiple substitutions among at least 11 amino acid sites, and no single substitution was observed in all of 29 resistant isolates obtained from patients on long-term indinavir monotherapy. We have analyzed the evolution of PR in these sequences. The divergence from the baseline amino acid sequence by week 24 was 4%, increasing more than 7% by week 60. The mean difference between sequences from different patients at baseline was 6% (3-9%), rising to 10% after 40 weeks (3-16%), although at all time points nonsynonymous substitutions were less frequent than synonymous nucleotide changes. Analysis of associations between variants at different amino acid sites using a mutual information statistic revealed four pairs of sites to be significantly associated. In three cases these associations included residue 82. Clusters of baseline and week 24 amino acid sequences identified by maximum parsimony did not correlate significantly with the IC95 to indinavir, although a weak correlation of baseline clusters with phenotype at the week 24 time point was suggested.
In a prospective cohort study, clinical and biologic factors that contribute to maternal-child transmission of human immunodeficiency virus type 1 (HIV-1) were studied. HIV-infected pregnant women and their infants were evaluated prospectively according to a standardized protocol. Of 204 evaluable women, 81% received zidovudine during their pregnancy. The infection rate among the 209 evaluable infants was 9.1%. By univariate analysis, histologic chorioamnionitis, prolonged rupture of membranes, and a history of genital warts were significantly associated with transmission. Additional factors associated with transmission that approached significance included a higher maternal virus load at delivery and the presence of cocaine in the urine. In a logistic regression model, histologic chorioamnionitis was the only independent predictor of transmission. Despite a significantly higher transmission rate at one site, no unique viral genotype was found at any site. Thus, chorioamnionitis was found to be the major risk factor for transmission among women receiving zidovudine.
There are natural mutations in the coding and noncoding regions of the human immunodeficiency virus type 1 (HIV-1) CC-chemokine coreceptor 5 (CCR5) and in the related CCR2 protein (the CCR2-64I mutation). Individuals homozygous for the CCR5-Delta32 allele, which prevents CCR5 expression, strongly resist HIV-1 infection. Several genetic polymorphisms have been identified within the CCR5 5' regulatory region, some of which influence the rate of disease progression in adult AIDS study cohorts. We genotyped 1,442 infants (1,235 uninfected and 207 HIV-1 infected) for five CCR5 and CCR2 polymorphisms: CCR5-59353-T/C, CCR5-59356-C/T CCR5-59402-A/G, CCR5-Delta32, and CCR2-64I. The clinical significance of each genotype was assessed by measuring whether it influenced the rate of perinatal HIV-1 transmission among 667 AZT-untreated mother-infant pairs (554 uninfected and 113 HIV-1 infected). We found that the mutant CCR5-59356-T allele is relatively common in African-Americans (20.6% allele frequency among 552 infants) and rare in Caucasians and Hispanics (3.4 and 5.6% of 174 and 458 infants, respectively; P < 0.001). There were 38 infants homozygous for CCR5-59356-T, of whom 35 were African-Americans. Among the African-American infants in the AZT-untreated group, there was a highly significant increase in HIV-1 transmission to infants with two mutant CCR5-59356-T alleles (47.6% of 21), compared to those with no or one mutant allele (13.4 to 14.1% of 187 and 71, respectively; P < 0.001). The increased relative risk was 5.9 (95% confidence interval, 2.3 to 15.3; P < 0.001). The frequency of the CCR5-59356-T mutation varies between population groups in the United States, a low frequency occurring in Caucasians and a higher frequency occurring in African-Americans. Homozygosity for CCR5-59356-T is strongly associated with an increased rate of perinatal HIV-1 transmission.
Host immunologic factors, including human immunodeficiency virus (HIV)-specific cytotoxic T lymphocytes (CTL), are thought to contribute to the control of HIV type 1 (HIV-1) replication and thus delay disease progression in infected individuals. Host immunologic factors are also likely to influence perinatal transmission of HIV-1 from infected mother to infant. In this study, the potential role of CTL in modulating HIV-1 transmission from mother to infant was examined in 11 HIV-1-infected mothers, 3 of whom transmitted virus to their offspring. Frequencies of HIV-1-specific human leukocyte antigen class I-restricted CTL responses and viral epitope amino acid sequence variation were determined in the mothers and their infected infants. Maternal HIV-1-specific CTL clones were derived from each of the HIV-1-infected pregnant women. Amino acid substitutions within the targeted CTL epitopes were more frequently identified in transmitting mothers than in nontransmitting mothers, and immune escape from CTL recognition was detected in all three transmitting mothers but in only one of eight nontransmitting mothers. The majority of viral sequences obtained from the HIV-1-infected infant blood samples were susceptible to maternal CTL. These findings demonstrate that epitope amino acid sequence variation and escape from CTL recognition occur more frequently in mothers that transmit HIV-1 to their infants than in those who do not. However, the transmitted virus can be a CTL susceptible form, suggesting inadequate in vivo immune control.
There is considerable genetic diversity among viruses of different subtypes (designated A to J) in the major group of human immunodeficiency virus type 1 (HIV-1), the form of HIV that is dominant in the global epidemic. If available, HIV-1 sequences pre-dating the recognition of AIDS could be crucial in defining the time of origin and the subsequent evolution of these viruses in humans. The oldest known case of HIV-1 infection was reported to be that of a sailor from Manchester who died of an AIDS-like illness in 1959; however, the authenticity of this case has not been confirmed. Genetic analysis of sequences from clinical materials obtained from 1971 to 1976 from members of a Norwegian family infected earlier than 1971 showed that they carried viruses of the HIV-1 outlier group, a variant form that is mainly restricted to West Africa. Here we report the amplification and characterization of viral sequences from a 1959 African plasma sample that was previously found to be HIV-1 seropositive. Multiple phylogenetic analyses not only authenticate this case as the oldest known HIV-1 infection, but also place its viral sequence near the ancestral node of subtypes B and D in the major group, indicating that these HIV-1 subtypes, and perhaps all major-group viruses, may have evolved from a single introduction into the African population not long before 1959.
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The ARIEL Project for the Prevention of HIV Transmission from Mother to Infant was established to evaluate virologic and immunologic parameters during vertical transmission. To determine the strength and breadth of the cytotoxic T lymphocyte (CTL) response and its correlation with human immunodeficiency virus (HIV) transmission, a cross-sectional study was done of 31 HIV-infected pregnant women, of whom 15 transmitted and 16 did not transmit HIV to their infants. The precursor frequencies of CTL specific for HIV-1 gag, pol, nef, and env from 5 different isolates of the clade B of HIV-1 were determined by limiting dilution analysis. Results showed that variable levels of HIV-specific CTL response were present in HIV-infected pregnant women during and after pregnancy. In addition, CTL precursor frequencies specific for pol and nef were higher during pregnancy in nontransmitters than in transmitters. Thus, CTL responding to different HIV antigens may not be contributing equally to the prevention of vertical transmission.
Among 2099 uninfected subjects in phase I and II trials of candidate AIDS vaccines, 23 were diagnosed with intercurrent human immunodeficiency virus type 1 (HIV-1) infection. High-risk sexual exposures accounted for 17 infections, and intravenous drug use accounted for 6. Four subjects received placebo, 13 received a complete immunization schedule (> or = 3 injections), and 6 were partially immunized (< or = 2 injections). There was no significant difference between vaccine recipients and control groups in incidence of HIV-1 infection, virus load, CD4 lymphocyte count, or V3 loop amino acid sequence. In summary, 19 vaccinated subjects acquired HIV-1 infection during phase I and II trials, indicating that immunization with the products described is < 100% effective in preventing or rapidly clearing infection. Laboratory analysis suggested that vaccine-induced immune responses did not significantly affect the genotypic or phenotypic characteristics of transmitted virus or the early clinical course of HIV-1 infection.
We have studied 18 participants in phase I/II clinical trials of recombinant gp120 (rgp120) subunit vaccines (MN and SF-2) who became infected with human immunodeficiency virus type 1 (HIV-1) during the course of the trials. Of the 18 individuals, 2 had received a placebo vaccine, 9 had been immunized with MN rgp120, and seven had been immunized with SF-2 rgp120. Thirteen of the 18 infected vaccinees had received three or four immunizations prior to becoming infected. Of these, two were placebo recipients, six had received MN rgp120, and five had received SF-2 rgp120. Only 1 of the 11 rgp120 recipients who had multiple immunizations failed to develop a strong immunoglobulin G antibody response to the immunogen. However, the antibody response to rgp120 was transient, typically having a half-life of 40 to 60 days. No significant neutralizing activity against the infecting strain was detected in any of the infected individuals at any time prior to infection. Antibody titers in subjects infected despite vaccination and in noninfected subjects were not significantly different. Envelope-specific cytotoxic T-lymphocyte responses measured after infection were infrequent and weak in the nine vaccinees who were tested. HIV-1 was isolated successfully from all 18 individuals. Sixteen of these strains had a non-syncytium-inducing (NSI) phenotype, while two had a syncytium-inducing (SI) phenotype. NSI strains used the CCR5 coreceptor to enter CD4+ cells, while an SI strain from one of the vaccinees also used CXCR4. Viruses isolated from the blood of rgp120 vaccinees were indistinguishable from viruses isolated from control individuals in terms of their inherent sensitivity to neutralization by specific monoclonal antibodies and their replication rates in vitro. Furthermore, genetic sequencing of the env genes of strains infecting the vaccinees did not reveal any features that clearly distinguished these viruses from contemporary clade B viruses circulating in the United States. Thus, despite rigorous genetic analyses, using various breakdowns of the data sets, we could find no evidence that rgp120 vaccination exerted selection pressure on the infecting HIV-1 strains. The viral burdens in the infected rgp120 vaccine recipients were also determined, and they were found to be not significantly different from those in cohorts of placebo-vaccinated and nonvaccinated individuals. In summary, we conclude that vaccination with rgp120 has had,to date, no obvious beneficial or adverse effects on the individuals we have studied.
Most HIV-1 infections of children result from mother-to-infant transmission, which may occur perinatally or postnatally, as a consequence of breast feeding. In this study, the influence of maternal viral load on transmission of infection to infants from non-breast-feeding mothers was examined using samples of plasma and peripheral blood mononuclear cells (PBMCs) collected at several time points during pregnancy and the 6-month period after delivery. These samples were analyzed by several quantitative methods, including virus cultures of PBMCs and polymerase chain reaction (PCR) assays for HIV-1 RNA in plasma and DNA in PBMCs. The risk of transmission increased slightly with a higher viral load, but transmission and nontransmission occurred over the entire range of values for each assay. No threshold value of virus load was identified which discriminated between transmitters and nontransmitters. We also noted a significant rise in viral load and a decline in CD4+ lymphocytes in the six months after delivery. These findings suggest that a high maternal viral load is insufficient to fully explain vertical transmission of HIV-1. Additional studies are needed to examine the post-partum increase in viremia.
The rate of development of disease varies considerably among human immunodeficiency virus type 1 (HIV-1)-infected children. The reasons for these observed differences are not clearly understood but most probably depend on the dynamic interplay between the HIV-1 quasispecies virus population and the immune constraints imposed by the host. To study the relationship between disease progression and genetic diversity, we analyzed the evolution of viral sequences within six perinatally infected children by examining proviral sequences spanning the C2 through V5 regions of the viral envelope gene by PCR of blood samples obtained at sequential visits. PCR product DNAs from four sample time points per child were cloned, and 10 to 13 clones from each sample were sequenced. Greater genetic distances relative to the time of infection were found for children with low virion-associated RNA burdens and slow progression to disease relative to those found for children with high virion-associated RNA burdens and rapid progression to disease. The greater branch lengths observed in the phylogenetic reconstructions correlated with a higher accumulation rate of nonsynonymous base substitutions per potential nonsynonymous site, consistent with positive selection for change rather than a difference in replication kinetics. Viral sequences from children with slow progression to disease also showed a tendency to form clusters that associated with different sampling times. These progressive shifts in the viral population were not found in viral sequences from children with rapid progression to disease. Therefore, despite the HIV-1 quasispecies being a diverse, rapidly evolving, and competing population of genetic variants, different rates of genetic evolution could be found under different selective constraints. These data suggest that the evolutionary dynamics exhibited by the HIV-1 quasispecies virus populations are compatible with a Darwinian system evolving under the constraints of natural selection.
The rate of progression to disease varies considerably among individuals infected with human immunodeficiency virus-type 1 (HIV-1). Analyses of semiannual blood samples obtained from six infected men showed that a rapid rate of CD4 T cell loss was associated with relative evolutionary stasis of the HIV-1 quasispecies virus population. More moderate rates of CD4 T cell loss correlated with genetic evolution within three of four subjects. Consistent with selection by the immune constraints of these subjects, amino acid changes were apparent within the appropriate epitopes of human leukocyte antigen class I-restricted cytotoxic T lymphocytes. Thus, the evolutionary dynamics exhibited by the HIV-1 quasispecies virus populations under natural selection are compatible with adaptive evolution.
Human immunodeficiency virus type 1 (HIV-1) sequences are accumulating in the literature at a rapid pace. For this ever-expanding resource to be maximally useful, it is critical that researchers strive to maintain a high level of quality assurance, both in experimental design and conduct and in analyses. Here we present detailed analyses of problematic sets of HIV-1 sequences in the database that include sequence anomalies suggestive of mislabeling or sample contamination problems. These data are examined in the context of currently available HIV-1 sequence information to provide an example of how to identify potentially flawed data. Indicators of potential problems with sequences are (i) sequences that are nearly identical that are supposed to be derived from unlinked individuals and that are markedly distinct from other sequences from the putative source or (ii) sequences that are nearly identical to those of laboratory strains. We provide an outline of methods that researchers can use to perform preliminary laboratory and computational analyses that could help identify problematic data and thus help ensure the integrity of sequence databases.
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