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Biomedical subjects

Gerald H Learn

Publications and source records attributed to Gerald H Learn.

16 recordsLinked to original sources
PubMed2006

CD8+ T-cell responses to different HIV proteins have discordant associations with viral load.

Selection of T-cell vaccine antigens for chronic persistent viral infections has been largely empirical. To define the relationship, at the population level, between the specificity of the cellular immune response and viral control for a relevant human pathogen, we performed a comprehensive analysis of the 160 dominant CD8(+) T-cell responses in 578 untreated HIV-infected individuals from KwaZulu-Natal, South Africa. Of the HIV proteins targeted, only Gag-specific responses were associated with lowering viremia. Env-specific and Accessory/Regulatory protein-specific responses were associated with higher viremia. Increasing breadth of Gag-specific responses was associated with decreasing viremia and increasing Env breadth with increasing viremia. Association of the specific CD8(+) T-cell response with low viremia was independent of HLA type and unrelated to epitope sequence conservation. These population-based data, suggesting the existence of both effective immune responses and responses lacking demonstrable biological impact in chronic HIV infection, are of relevance to HIV vaccine design and evaluation.

Adult↗
PubMed2006

Large-scale amplification, cloning and sequencing of near full-length HIV-1 subtype C genomes.

Full-length HIV-1 genome sequencing provides important data needed to address several vaccine design, molecular epidemiologic and pathogenesis questions. A protocol is presented for obtaining near full-length genomes (NFLGs) from subjects infected with HIV-1 subtype C. This protocol was used to amplify NFLGs from 244 of 366 (67%) samples collected at two clinics in Durban, South Africa (SK and PS). Viral load was directly associated with frequency of successful NFLG amplification for both cohorts (PS; p = 0.005 and SK; p < 0.001). Seventeen of 38 initially NFLG-negative SK samples had variation within the PCR primer binding sites, however only 3 of these were successfully re-amplified using re-designed primers homologous to the target viruses. NFLGs were obtained from 7 of 24 PBMC samples processed from subjects whose plasma did not yield a NFLG. Stable plasmid clones were obtained from all 244 NFLG-positive PCR products, and both strands of each genome were sequenced, using a primary set of 46 primers. These methods thus allow the large-scale collection of HIV-1 NFLGs from populations infected primarily with subtype C. The methods are readily adaptable to other HIV-1 subtypes, and provide materials for viral functional analyses and population-based molecular epidemiology studies that include analysis of viral genome chimerization.

Base Sequence↗
PubMed2006

Conflicting selective forces affect T cell receptor contacts in an immunodominant human immunodeficiency virus epitope.

Cytotoxic T lymphocytes (CTLs) are critical for the control of human immunodeficiency virus, but containment of virus replication can be undermined by mutations in CTL epitopes that lead to virus escape. We analyzed the evolution in vivo of an immunodominant, HLA-A2-restricted CTL epitope and found two principal, diametrically opposed evolutionary pathways that exclusively affect T cell-receptor contact residues. One pathway was characterized by acquisition of CTL escape mutations and the other by selection for wild-type amino acids. The pattern of CTL responses to epitope variants shaped which variant(s) prevailed in the virus population. The pathways notably influenced the amount of plasma virus, as patients with efficient CTL selection had lower plasma viral loads than did patients without efficient selection. Thus, viral escape from CTL responses does not necessarily correlate with disease progression.

Adult↗
PubMed2006

Human immunodeficiency virus type 1 env evolves toward ancestral states upon transmission to a new host.

Selecting human immunodeficiency virus (HIV) sequences for inclusion within vaccines has been a difficult problem, as circulating HIV strains evolve relentlessly and become increasingly divergent over time. We report an assessment of this divergence from three perspectives: (i) across different hosts as a function of time of infection, (ii) between donors and recipients in known transmission pairs, and (iii) within individual hosts over time in relation to the initially replicating virus and to the deduced ancestral sequence of the intrahost viral population. Surprisingly, we consistently found less divergence between viruses from different individuals sampled in primary infection than in individuals sampled at more advanced stages of illness. Furthermore, longitudinal analysis of intrahost divergence revealed a 2- to 3-year period of evolution toward a common ancestral sequence at the start of infection, indicating that HIV recovers certain ancestral features when infecting a new host. These results have important implications for the study of HIV population genetics and rational vaccine design, including favoring the inclusion of viral gene sequences taken early in infection.

Evolution, Molecular↗
PubMed2005

Evidence that low-level viremias during effective highly active antiretroviral therapy result from two processes: expression of archival virus and replication of virus.

Episodes of low-level viremia (LLV), with plasma human immunodeficiency virus type 1 (HIV-1) RNA levels ranging from 50 to 400 copies (c)/ml, occur commonly during highly active antiretroviral therapy (HAART). LLV has been associated with virologic failure of HAART in some studies, while in others LLV did not appear to affect the clinical outcome. To understand the processes leading to LLV, genetic analyses were used to determine whether plasma virions emanated from archived or from newly evolved viral genomes. Episodes of LLV (plasma HIV-1 RNA, 50 to 379 [median, 77] c/ml) were detected in 21/37 (57%) HIV-1-infected children with median plasma HIV-1 RNA levels of <50 c/ml during 79 patient years of HAART. Viral sequences were derived by direct sequencing of PCR products from 21 plasma specimens diluted to end point. In phylogenetic analysis, LLV viral sequences grouped with virus from early in the course of infection in 8/11 subjects. Six specimens had multiple identical viral sequences, suggesting origin from clonally expanded infected cells. LLV plasma virus evolved over time, indicating viral replication, in 3/11 subjects. Two of these had frequent LLV, including the selection of drug-resistant mutants. In summary, plasma virus from episodes of LLV during effective HAART appeared to originate from two distinct processes, (i) clonal outgrowth from long-lived HIV-1-infected cells, presumably following activation and proliferation of these cells, and (ii) ongoing viral replication that included the selection of new drug-resistant mutants. These observations provide a plausible explanation for the divergent clinical outcomes previously associated with LLV.

Amino Acid Sequence↗
PubMed2004

Molecular clock-like evolution of human immunodeficiency virus type 1.

The molecular clock hypothesis states that the rate of nucleotide substitution per generation is constant across lineages. If generation times were equal across lineages, samples obtained at the same calendar time would have experienced the same number of generations since their common ancestor. However, if sequences are not derived from contemporaneous samples, differences in the number of generations may be misinterpreted as variation in substitution rates and hence may lead to false rejection of the molecular clock hypothesis. A recent study has called into doubt the validity of clock-like evolution for HIV-1, using molecular sequences derived from noncontemporaneous samples. However, after separating their within-individual data according to sampling time, we found that what appeared to be nonclock-like behavior could be attributed, in most cases, to noncontemporaneous sampling, with contributions also likely to derive from recombination. Natural selection alone did not appear to obscure the clock-like evolution of HIV-1.

Evolution, Molecular↗
PubMed2004

Persistence of low levels of simian immunodeficiency virus in macaques that were transiently viremic by conventional testing.

Transient SIV viremia after experimental SIV challenge has been documented. Whether SIV persists in these transiently viremic macaques remains unclear. In the present study, we applied a sensitive PCR and found persistent low levels of SIVmne infection (LLSI) (range: 0.1-5.3 SIV DNA copies/10(6) PBMC) in seven macaques that were transiently positive by conventional assays, which was 10(2)- to 10(6)-fold less than those of SIVmne infected monkeys with typical disease progression. SIV envelope V1 sequences remained homogeneous in these macaques for the 6-year study period, with a mean evolution rate of 0.005% per site per year, which was not different from zero (P = 0.612) and significantly lower than that (0.56-1.18%) in macaques with progressive infection of SIVmne. LLSI macaques have remained free from SIV-associated illness, and are still alive 10 years after virus inoculation. Understanding the mechanisms underlying this outcome may provide valuable insight into therapy and vaccine development.

Amino Acid Sequence↗
PubMed2004

Dual HIV-1 infection associated with rapid disease progression.

Infection with two strains of HIV-1 has implications for understanding HIV transmission and vaccine development; however, frequency and pathogenic consequences of dual infection are unknown. We assessed 64 patients for dual infection with heteroduplex mobility assay, viral sequencing, and phylogenetic methods. HIV disease outcomes were available in 34 patients. Five of these with AIDS endpoints had dual infection with HIV-1: four were cases of coinfection and one was superinfection. In all five, time from seroconversion to clinical AIDS or to CD4+ T-cell count less than 200 cells per microL was very rapid (<3.4 and <3.1 years, respectively). Our findings should prompt larger studies to assess the effect of dual infection at the population level.

Acquired Immunodeficiency Syndrome↗
PubMed2004

Immunogen sequence: the fourth tier of AIDS vaccine design.

While worldwide efforts to develop an effective HIV-1 vaccine are underway, the virus continues to spread, particularly in developing countries where the delivery of antiviral therapies presents formidable challenges. Vaccine research has largely focused on three general aspects: vectors, adjuvants, and immunization schedules. Our group favor the use of computational methods to design potential immunogens that capture the genetic and biological features of circulating viruses. These methods allow researchers to predict, in silico, the presence of potential glycosylation sites, humoral immune responses, and epitope coverage. This review shall compare three computational approaches for immunogen design: the consensus sequence, which has at each site the modal nucleotide or amino acid residue across a sequence alignment; the most recent common ancestor, the sequence estimated at the basal node of the clades seen in the HIV-1 phylogeny; and the center of tree method, which minimizes the evolutionary distance to all sequences in the data set.

AIDS Vaccines↗
PubMed2003

Predicting demographic group structures based on DNA sequence data.

The ability to infer relationships between groups of sequences, either by searching for their evolutionary history or by comparing their sequence similarity, can be a crucial step in hypothesis testing. Interpreting relationships of human immunodeficiency virus type 1 (HIV-1) sequences can be challenging because of their rapidly evolving genomes, but it may also lead to a better understanding of the underlying biology. Several studies have focused on the evolution of HIV-1, but there is little information to link sequence similarities and evolutionary histories of HIV-1 to the epidemiological information of the infected individual. Our goal was to correlate patterns of HIV-1 genetic diversity with epidemiological information, including risk and demographic factors. These correlations were then used to predict epidemiological information through analyzing short stretches of HIV-1 sequence. Using standard phylogenetic and phenetic techniques on 100 HIV-1 subtype B sequences, we were able to show some correlation between the viral sequences and the geographic area of infection and the risk of men who engage in sex with men. To help identify more subtle relationships between the viral sequences, the method of multidimensional scaling (MDS) was performed. That method identified statistically significant correlations between the viral sequences and the risk factors of men who engage in sex with men and individuals who engage in sex with injection drug users or use injection drugs themselves. Using tree construction, MDS, and newly developed likelihood assignment methods on the original 100 samples we sequenced, and also on a set of blinded samples, we were able to predict demographic/risk group membership at a rate statistically better than by chance alone. Such methods may make it possible to identify viral variants belonging to specific demographic groups by examining only a small portion of the HIV-1 genome. Such predictions of demographic epidemiology based on sequence information may become valuable in assigning different treatment regimens to infected individuals.

Adolescent↗
PubMed2003

Multiple viral genetic analyses detect low-level human immunodeficiency virus type 1 replication during effective highly active antiretroviral therapy.

To evaluate human immunodeficiency virus type 1 (HIV-1) replication and selection of drug-resistant viruses during seemingly effective highly active antiretroviral therapy (HAART), multiple HIV-1 env and pol sequences were analyzed and viral DNA levels were quantified from nucleoside analog-experienced children prior to and during a median of 5.1 (range, 1.8 to 6.4) years of HAART. Viral replication was detected at different rates, with apparently increasing sensitivity: 1 of 10 by phylogenetic analysis; 2 of 10 by viral evolution with increasing genetic distances from the most recent common ancestor (MRCA) of infection; 3 of 10 by selection of drug-resistant mutants; and 6 of 10 by maintenance of genetic distances from the MRCA. When four- or five-drug antiretroviral regimens were given to these children, persistent plasma viral rebound did not occur despite the accumulation of highly drug-resistant genotypes. Among the four children without genetic evidence of viral replication, a statistically significant decrease in the genetic distance to the MRCA was detected in three, indicating the persistence of a greater number of early compared to recent viruses, and their HIV-1 DNA decreased by > or =0.9 log(10), resulting in lower absolute DNA levels (P = 0.007). This study demonstrates the variable rates of viral replication when HAART has suppressed plasma HIV-1 RNA for years to a median of <50 copies/ml and that combinations of four or five antiretroviral drugs suppress viral replication even after short-term virologic failure of three-drug HAART and despite ongoing accumulation of drug-resistant mutants. Furthermore, the decrease of cellular HIV-1 DNA to low absolute levels in those without genetic evidence of viral replication suggests that monitoring viral DNA during HAART may gauge low-level replication.

Antiretroviral Therapy, Highly Active↗
PubMed2003

Persistence of extraordinarily low levels of genetically homogeneous human immunodeficiency virus type 1 in exposed seronegative individuals.

Some individuals remain inexplicably seronegative and lack evidence for human immunodeficiency virus type 1 (HIV-1) infection by conventional serologic or virologic testing despite repeated high-risk virus exposures. Here, we examined 10 exposed seronegative (ES) individuals exhibiting HIV-1-specific cytotoxicity for the presence of HIV-1. We discovered HIV-1 DNA in resting CD4(+) T cells (mean, 0.05 +/- 0.01 copies per million cells) at multiple visits spanning 69 to 130 weeks in two ES individuals at levels that were on average 10(4)- to 10(6)-fold lower than those of other HIV-1-infected populations reported. Sequences of HIV-1 envelope and gag genes remained markedly homogeneous, indicating little to undetectable virus replication. These results provide the evidence for HIV-1 infection in ES individuals below the detection limit of standard assays, suggesting that extraordinary control of infection can occur. The two HIV-infected ES individuals remained healthy and were not superinfected with other HIV-1 strains despite continued high-risk sexual exposures to multiple HIV-infected partners. Understanding the mechanisms that confer diminished replicative capacity of HIV-1 in these hosts is paramount to developing strategies for protection against and control of HIV-1 infection.

Amino Acid Sequence↗
PubMed2002

Curiously modern DNA for a "250 million-year-old" bacterium.

Studies of ancient DNA have attracted considerable attention in scientific journals and the popular press. Several of the more extreme claims for ancient DNA have been questioned on biochemical grounds (i.e., DNA surviving longer than expected) and evolutionary grounds (i.e., nucleotide substitution patterns not matching theoretical expectations for ancient DNA). A recent letter to Nature from Vreeland et al. (2000), however, tops all others with respect to age and condition of the specimen. These researchers extracted and cultured a bacterium from an inclusion body from what they claim is a 250 million-year (Myr)-old salt crystal. If substantiated, this observation could fundamentally alter views about bacterial physiology, ecology and evolution. Here we report on molecular evolutionary analyses of the 16S rDNA from this specimen. We find that 2-9-3 differs from a modern halophile, Salibacillus marismortui, by just 3 unambiguous bp in 16S rDNA, versus the approximately 59 bp that would be expected if these bacteria evolved at the same rate as other bacteria. We show, using a Poisson distribution, that unless it can be shown that S. marismortui evolves 5 to 10 times more slowly than other bacteria for which 16S rDNA substitution rates have been established, Vreeland et al.'s claim would be rejected at the 0.05 level. Also, a molecular clock test and a relative rates test fail to substantiate Vreeland et al.'s claim that strain 2-9-3 is a 250-Myr-old bacterium. The report of Vreeland et al. thus falls into a long series of suspect ancient DNA studies.

Bacteria↗
PubMed2002

Virus population homogenization following acute human immunodeficiency virus type 1 infection.

Understanding the properties of human immunodeficiency virus type 1 (HIV-1) variants capable of establishing infection is critical to the development of a vaccine against AIDS. Previous studies of men have shown that the HIV-1 env gene is homogeneous early in infection, leading to the suggestion that infection is established by a single transmitted variant. However, we report here that all of eight homosexual men evaluated beginning 3.7 to 9 weeks following onset of symptoms of acute infection harbored diverse virus populations in their blood, with median genetic distances averaging 1.08% in the env C2V5 region and 0.81% in the gag p17 gene. Within another 4.7 to 11 weeks, the variant lineage in env became more homogeneous, while gag sequences continued to diversify. Thus, the homogenization that has been reported to characterize acute infection is actually preceded by the replication of multiple virus variants. This early selective process focuses on viral properties within Env but not Gag p17. Hence, the viral homogeneity observed early in HIV-1 infection results from a selective process that occurs during the establishment of infection.

Acute Disease↗