[show abstract][hide abstract] ABSTRACT: A successful HIV vaccine will likely induce both humoral and cell-mediated immunity, however, the enormous diversity of HIV has hampered the development of a vaccine that effectively elicits both arms of the adaptive immune response. To tackle the problem of viral diversity, T cell-based vaccine approaches have focused on two main strategies (i) increasing the breadth of vaccine-induced responses or (ii) increasing vaccine-induced responses targeting only conserved regions of the virus. The relative extent to which set-point viremia is impacted by epitope-conservation of CD8(+) T cell responses elicited during early HIV-infection is unknown but has important implications for vaccine design. To address this question, we comprehensively mapped HIV-1 CD8(+) T cell epitope-specificities in 23 ART-naïve individuals during early infection and computed their conservation score (CS) by three different methods (prevalence, entropy and conseq) on clade-B and group-M sequence alignments. The majority of CD8(+) T cell responses were directed against variable epitopes (p<0.01). Interestingly, increasing breadth of CD8(+) T cell responses specifically recognizing conserved epitopes was associated with lower set-point viremia (r = - 0.65, p = 0.009). Moreover, subjects possessing CD8(+) T cells recognizing at least one conserved epitope had 1.4 log10 lower set-point viremia compared to those recognizing only variable epitopes (p = 0.021). The association between viral control and the breadth of conserved CD8(+) T cell responses may be influenced by the method of CS definition and sequences used to determine conservation levels. Strikingly, targeting variable versus conserved epitopes was independent of HLA type (p = 0.215). The associations with viral control were independent of functional avidity of CD8(+) T cell responses elicited during early infection. Taken together, these data suggest that the next-generation of T-cell based HIV-1 vaccines should focus on strategies that can elicit CD8(+) T cell responses to multiple conserved epitopes of HIV-1.
PLoS ONE 01/2013; 8(5):e64405. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: HIV-1 transmission and viral evolution in the first year of infection were studied in 11 individuals representing four transmitter-recipient pairs and three independent seroconverters. Nine of these individuals were enrolled during acute infection; all were men who have sex with men (MSM) infected with HIV-1 subtype B. A total of 475 nearly full-length HIV-1 genome sequences were generated, representing on average 10 genomes per specimen at 2 to 12 visits over the first year of infection. Single founding variants with nearly homogeneous viral populations were detected in eight of the nine individuals who were enrolled during acute HIV-1 infection. Restriction to a single founder variant was not due to a lack of diversity in the transmitter as homogeneous populations were found in recipients from transmitters with chronic infection. Mutational patterns indicative of rapid viral population growth dominated during the first 5 weeks of infection and included a slight contraction of viral genetic diversity over the first 20 to 40 days. Subsequently, selection dominated, most markedly in env and nef. Mutants were detected in the first week and became consensus as early as day 21 after the onset of symptoms of primary HIV infection. We found multiple indications of cytotoxic T lymphocyte (CTL) escape mutations while reversions appeared limited. Putative escape mutations were often rapidly replaced with mutually exclusive mutations nearby, indicating the existence of a maturational escape process, possibly in adaptation to viral fitness constraints or to immune responses against new variants. We showed that establishment of HIV-1 infection is likely due to a biological mechanism that restricts transmission rather than to early adaptive evolution during acute infection. Furthermore, the diversity of HIV strains coupled with complex and individual-specific patterns of CTL escape did not reveal shared sequence characteristics of acute infection that could be harnessed for vaccine design.
Journal of Virology 05/2011; 85(15):7523-34. · 5.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: Improved understanding of the dynamics of host immune responses and viral evolution is critical for effective HIV-1 vaccine design. We comprehensively analyzed Cytotoxic T-lymphocyte (CTL)-viral epitope dynamics in an antiretroviral therapy-naïve subject over the first four years of HIV-1 infection. We found that CTL responses developed sequentially and required constant antigenic stimulation for maintenance. CTL responses exerting strong selective pressure emerged early and led to rapid escape, proliferated rapidly and were predominant during acute/early infection. Although CTL responses to a few persistent epitopes developed over the first two months of infection, they proliferated slowly. As CTL epitopes were replaced by mutational variants, the corresponding responses immediately declined, most rapidly in the cases of strongly selected epitopes. CTL recognition of epitope variants, via cross-reactivity and de novo responses, was common throughout the period of study. Our data demonstrate that HIV-specific CTL responses, especially in the critical acute/early stage, were focused on regions that are prone to escape. Failure of CTL responses to strongly target functional or structurally critical regions of the virus, as well as the sequential cascade of CTL responses, followed closely by viral escape and decline of the corresponding responses, likely contribute to a lack of sustainable viral suppression. Focusing early and rapidly proliferating CTL on persistent epitopes may be essential for durable viral control in HIV-1 infection.
PLoS ONE 01/2011; 6(1):e15639. · 3.73 Impact Factor
[show abstract][hide abstract] ABSTRACT: DIVEIN is a web interface that performs automated phylogenetic and other analyses of nucleotide and amino acid sequences. Starting with a set of aligned sequences, DIVEIN estimates evolutionary parameters and phylogenetic trees while allowing the user to choose from a variety of evolutionary models; it then reconstructs the consensus (CON), most recent common ancestor (MRCA), and center of tree (COT) sequences. DIVEIN also provides tools for further analyses, including condensing sequence alignments to show only informative sites or private mutations; computing phylogenetic or pairwise divergence from any user-specified sequence (CON, MRCA, COT, or existing sequence from the alignment); computing and outputting all genetic distances in column format; calculating summary statistics of diversity and divergence from pairwise distances; and graphically representing the inferred tree and plots of divergence, diversity, and distance distribution histograms. DIVEIN is available at http://indra.mullins.microbiol.washington.edu/DIVEIN.
[show abstract][hide abstract] ABSTRACT: HLA alleles B57/58, B27, and B35 have the strongest genetic associations with HIV-1 disease progression. The mechanisms of these relationships may be host control of HIV-1 infection via CD8(+) T-cell responses. We examined these immune responses in subjects from the Seattle Primary Infection Cohort with these alleles. CD8(+) T-cell responses to conserved HIV epitopes within B57/58 alleles (TW10 and KF11) and B27 alleles (KK10 and FY10) delayed declines in CD4(+) T-cell counts (4 to 8 times longer), while responses to variable epitopes presented by B35 alleles (DL9 and IL9) resulted in more rapid progression. The plasma viral load was higher in B57/58(+) and B27(+) subjects lacking the conserved B57/58- and B27-restricted responses. The presence of certain B57/58-, B27-, and B35-restricted HIV-specific CD8(+) T-cell responses after primary HIV-1 infection better defined disease progression than the HLA genotype alone, suggesting that it is the HIV-specific CD8(+) T cells and not the presence of a particular HLA allele that determine disease progression. Further, the most effective host CD8(+) T-cell responses to HIV-1 were prevalent within an HLA allele, represented a high total allele fraction of the host CD8(+) T-cell response, and targeted conserved regions of HIV-1. These data suggest that vaccine immunogens should contain only conserved regions of HIV-1.
Journal of Virology 02/2010; 84(9):4461-8. · 5.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: The epitope specificities and antiviral activities of class I HLA-restricted CD8(+) T cells, especially those induced during human immunodeficiency virus type 1 (HIV-1) primary infection, are important considerations in designing HIV-1 vaccines. Conserved epitopes may be more commonly and persistently recognized than variable epitopes, as they may be more likely to be present in infecting viruses. However, some studies have shown preferential or similar targeting of variable versus conserved epitopes during primary infection.
We analyzed cytotoxic T-lymphocyte (CTL) responses toward predefined conserved and variable epitopes in 45 subjects during primary (n = 34) and/or chronic infection (n = 16).
Conserved and variable CTL epitopes were recognized with similar probabilities, whereas conserved epitopes generally elicited subdominant responses during both primary and chronic infection. During primary infection, CTL responses against Gag versus responses against Env and variable epitopes tended to be associated with lower and higher viral loads, respectively. During chronic infection, Env-specific responses tended to be associated with lower CD4(+) cell counts.
Subdominant CTL recognition of conserved HIV-1 epitopes commonly occurs from the primary through chronic stages of HIV-1 infection. These findings underscore the challenge in designing T cell-based vaccines that can induce immunodominant CTL responses to conserved HIV-1 regions.
The Journal of Infectious Diseases 10/2009; 200(12):1825-33. · 5.85 Impact Factor
[show abstract][hide abstract] ABSTRACT: Understanding the mechanisms underlying potential altered susceptibility to human immunodeficiency virus type 1 (HIV-1) infection in highly exposed seronegative (ES) individuals and the later clinical consequences of breakthrough infection can provide insight into strategies to control HIV-1 with an effective vaccine. From our Seattle ES cohort, we identified one individual (LSC63) who seroconverted after over 2 years of repeated unprotected sexual contact with his HIV-1-infected partner (P63) and other sexual partners of unknown HIV-1 serostatus. The HIV-1 variants infecting LSC63 were genetically unrelated to those sequenced from P63. This may not be surprising, since viral load measurements in P63 were repeatedly below 50 copies/ml, making him an unlikely transmitter. However, broad HIV-1-specific cytotoxic T-lymphocyte (CTL) responses were detected in LSC63 before seroconversion. Compared to those detected after seroconversion, these responses were of lower magnitude and half of them targeted different regions of the viral proteome. Strong HLA-B27-restricted CTLs, which have been associated with disease control, were detected in LSC63 after but not before seroconversion. Furthermore, for the majority of the protein-coding regions of the HIV-1 variants in LSC63 (except gp41, nef, and the 3' half of pol), the genetic distances between the infecting viruses and the viruses to which he was exposed through P63 (termed the exposed virus) were comparable to the distances between random subtype B HIV-1 sequences and the exposed viruses. These results suggest that broad preinfection immune responses were not able to prevent the acquisition of HIV-1 infection in LSC63, even though the infecting viruses were not particularly distant from the viruses that may have elicited these responses.
Journal of Virology 09/2009; 83(20):10821-9. · 5.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: 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.
[show abstract][hide abstract] ABSTRACT: The envelope gene (env) of human immunodeficiency virus type 1 (HIV-1) undergoes rapid divergence from the transmitted sequence and increasing diversification during the prolonged course of chronic infection in humans. In about half of infected individuals or more, env evolution leads to expansion of the use of entry coreceptor from CCR5 alone to CCR5 and CXCR4. The stochastic nature of this coreceptor switch is not well explained by host selective forces that should be relatively constant between infected individuals. Moreover, differences in the incidence of coreceptor switching among different HIV-1 subtypes suggest that properties of the evolving virus population drive the switch. We evaluated the functional properties of sequential env clones from a patient with evidence of coreceptor switching at 5.67 years of infection. We found an abrupt decline in the ability of viruses to use CCR5 for entry at this time, manifested by a 1- to 2-log increase in susceptibility to CCR5 inhibitors and a reduced ability to infect cell lines with low CCR5 expression. There was an abnormally rapid 5.4% divergence in env sequences from 4.10 to 5.76 years of infection, with the V3 and V4/V5 regions showing the greatest divergence and evidence of positive selection. These observations suggest that a decline in the fitness of R5 virus populations may be one driving force that permits the emergence of R5X4 variants.
Journal of Virology 10/2008; 82(23):11758-66. · 5.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: Whether there is selection for specific viral Env variants upon HIV-1 transmission is controversial. We examined the V1V2 and V1V4 regions of Env in 10 new and 8 previously described transmission pairs infected with HIV-1 subtype B, including a total of 9 pairs in which the infecting partner had developed substantial viral diversity prior to transmission. We found that during transmission of HIV-1 subtype B, as well as for other subtypes reported in the past, viral populations in recipients undergo substantial genetic bottlenecks, as well as weak evidence for a propensity to replicate viruses with shorter variable loops and fewer potential N-linked glycosylation sites.
[show abstract][hide abstract] ABSTRACT: Cytolytic T lymphocytes (CTL) play a major role in controlling human immunodeficiency virus type 1 (HIV-1) infection. To evade immune pressure, HIV-1 is selected at targeted CTL epitopes, which may consequentially alter viral replication fitness. In our longitudinal investigations of the interplay between T-cell immunity and viral evolution following acute HIV-1 infection, we observed in a treatment-naïve patient the emergence of highly avid, gamma interferon-secreting, CD8(+) CTL recognizing an HLA-Cw*0102-restricted epitope, NSPTRREL (NL8). This epitope lies in the p6(Pol) protein, located in the transframe region of the Gag-Pol polyprotein. Over the course of infection, an unusual viral escape mutation arose within the p6(Pol) epitope through insertion of a 3-amino-acid repeat, NSPT(SPT)RREL, with a concomitant insertion in the p6(Gag) late domain, PTAPP(APP). Interestingly, this p6(Pol) insertion mutation is often selected in viruses with the emergence of antiretroviral drug resistance, while the p6(Gag) late-domain PTAPP motif binds Tsg101 to permit viral budding. These results are the first to demonstrate viral evasion of immune pressure by amino acid insertions. Moreover, this escape mutation represents a novel mechanism whereby HIV-1 can alter its sequence within both the Gag and Pol proteins with potential functional consequences for viral replication and budding.
Journal of Virology 02/2008; 82(1):495-502. · 5.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: In HIV-1 evolution, a 100-100,000 fold discrepancy between census size and effective population size (Ne) has been noted. Although it is well known that selection can reduce Ne, high in vivo mutation and recombination rates complicate attempts to quantify the effects of selection on HIV-1 effective size.
We use the inbreeding coefficient and the variance in allele frequency at a linked neutral locus to estimate the reduction in Ne due to selection in the presence of mutation and recombination. With biologically realistic mutation rates, the reduction in Ne due to selection is determined by the strength of selection, i.e., the stronger the selection, the greater the reduction. However, the dependence of Ne on selection can break down if recombination rates are very high (e.g., r > or = 0.1). With biologically likely recombination rates, our model suggests that recurrent selective sweeps similar to those observed in vivo can reduce within-host HIV-1 effective population sizes by a factor of 300 or more.
Although other factors, such as unequal viral reproduction rates and limited migration between tissue compartments contribute to reductions in Ne, our model suggests that recurrent selection plays a significant role in reducing HIV-1 effective population sizes in vivo.
[show abstract][hide abstract] ABSTRACT: CD8(+) cytotoxic T lymphocytes (CTL) are strong mediators of human immunodeficiency virus type 1 (HIV-1) control, yet HIV-1 frequently mutates to escape CTL recognition. In an analysis of sequences in the Los Alamos HIV-1 database, we show that emerging CTL escape mutations were more often present at lower frequencies than the amino acid(s) that they replaced. Furthermore, epitopes that underwent escape contained amino acid sites of high variability, whereas epitopes persisting at high frequencies lacked highly variable sites. We therefore infer that escape mutations are likely to be associated with weak functional constraints on the viral protein. This was supported by an extensive analysis of one subject for whom all escape mutations within defined CTL epitopes were studied and by an analysis of all reported escape mutations of defined CTL epitopes in the HIV Immunology Database. In one of these defined epitopes, escape mutations involving the substitution of amino acids with lower database frequencies occurred, and the epitope soon reverted back to the sensitive form. We further show that this escape mutation substantially diminished viral fitness in in vitro competition assays. Coincident with the reversion in vivo, we observed the fixation of a mutation 3 amino acids C terminal to the epitope, coincident with the ablation of the corresponding CTL response. The C-terminal mutation did not restore replication fitness reduced by the escape mutation in the epitope and by itself had little effect on replication fitness. Therefore, this C-terminal mutation presumably impaired the processing and presentation of the epitope. Finally, for one persistent epitope, CTL cross-reactivity to a mutant form may have suppressed the mutant to undetected levels, whereas for two other persistent epitopes, each of two mutants showed poor cross-reactivity and appeared in the subject at later time points. Thus, a viral dynamic exists between the advantage of immune escape, peptide cross-reactivity, and the disadvantage of lost replication fitness, with the balance playing an important role in determining whether a CTL epitope will persist or decline during infection.
Journal of Virology 12/2007; 81(22):12179-88. · 5.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: Typically during human immunodeficiency virus type 1 (HIV-1) infection, a nearly homogeneous viral population first emerges and then diversifies over time due to selective forces that are poorly understood. To identify these forces, we conducted an intensive longitudinal study of viral genetic changes and T-cell immunity in one subject at < or =17 time points during his first 3 years of infection, and in his infecting partner near the time of transmission. Autologous peptides covering amino acid sites inferred to be under positive selection were powerful for identifying HIV-1-specific cytotoxic-T-lymphocyte (CTL) epitopes. Positive selection and mutations resulting in escape from CTLs occurred across the viral proteome. We detected 25 CTL epitopes, including 14 previously unreported. Seven new epitopes mapped to the viral Env protein, emphasizing Env as a major target of CTLs. One-third of the selected sites were associated with epitopic mutational escapes from CTLs. Most of these resulted from replacement with amino acids found at low database frequency. Another one-third represented acquisition of amino acids found at high database frequency, suggesting potential reversions of CTL epitopic sites recognized by the immune system of the transmitting partner and mutation toward improved viral fitness in the absence of immune targeting within the recipient. A majority of the remaining selected sites occurred in the envelope protein and may have been subjected to humoral immune selection. Hence, a majority of the amino acids undergoing selection in this subject appeared to result from fitness-balanced CTL selection, confirming CTLs as a dominant selective force in HIV-1 infection.
Journal of Virology 11/2006; 80(19):9519-29. · 5.08 Impact Factor
[show abstract][hide abstract] ABSTRACT: HIV-1 is one of the fastest evolving entities known. Given that census population sizes of HIV-1 within individuals are much greater than the inverse mutation rate, every possible single point mutation in the viral genome occurs each generation. This enormous capability to generate genetic variation allows for escape from immune surveillance and antiviral therapy. However, compared to this potential, populations of HIV-1 within individuals exhibit little genetic variation. This discrepancy between the known mutation rate of HIV-1 and the average level of genetic variation in the env gene observed in vivo is reflected in comparisons of the actual numbers of productively infected cells, estimated as 10(7), and the effective population size, estimated as 10(3). Using approximate Bayesian computation, we evaluated several hypotheses based on a variety of selective and demographic processes to explain the low effective population size of HIV-1. Of the models we examined, the metapopulation model, in which HIV-1 evolves within an individual as a large collection of small subpopulations subject to frequent migration, extinction, and recolonization, was most consistent with the observed levels of genetic variation and the average frequencies of those variants. The metapopulation model links previous studies of viral dynamics and population genetics.
[show abstract][hide abstract] ABSTRACT: Eliciting maximal immune responses to highly divergent viruses is a challenge and a focus in AIDS vaccine development. Another challenge is to identify the immune correlates of protective immunity. Recent AIDS vaccine design approaches attempt to use reconstructed centralized viral sequences that minimize genetic differences to circulating viruses. Using these approaches, we derive and analyze consensus (CON), ancestral (ANC), and center-of-tree (COT) sequences to represent intra-individual HIV-1 env variants encoding a range of diversities and phylogenetic structures. Each reconstructed sequence significantly minimized genetic distances to extant sequences throughout the first 5 years of infection of an individual. Interestingly, ANC sequences diverged and were not significantly better than extant sequences in minimizing genetic distances at later stages of infection and disease, likely due to the development of a substantially asymmetric phylogeny. COT or CON sequences derived from autologous virus samplings may be useful for increasing the sensitivity of assessments of immune reactivity against HIV.
[show abstract][hide abstract] ABSTRACT: We characterized the variation in the reconstructed ancestor of 118 HIV-1 envelope gene sequences arising from the methods used for (a) estimating and (b) rooting the phylogenetic tree, and (c) reconstructing the ancestor on that tree, from (d) the sequence format, and from (e) the number of input sequences. The method of rooting the tree was responsible for most of the sequence variation both among the reconstructed ancestral sequences and between the ancestral and observed sequences. Variation in predicted 3-D structural properties of the ancestors mirrored their sequence variation. The observed sequence consensus and ancestral sequences from center-rooted trees were most similar in all predicted attributes. Only for the predicted number of N-glycosylation sites was there a difference between MP and ML methods of reconstruction. Taxon sampling effects were observed only for outgroup-rooted trees, not center-rooted, reflecting the occurrence of several divergent basal sequences. Thus, for sequences exhibiting a radial phylogenetic tree, as does HIV-1, most of the variation in the estimated ancestor arises from the method of rooting the phylogenetic tree. Those investigating the ancestors of genes exhibiting such a radial tree should pay particular attention to alternate rooting methods in order to obtain a representative sample of ancestors.
[show abstract][hide abstract] ABSTRACT: 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.