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Natural killer (NK) cells and a subset of T cells express families of receptors that are capable of detecting major histocompatibility complex (MHC) class I expression on the surface of cells. Molecules of the killer cell immunoglobulin-like receptor (KIR) family bind directly to MHC class I, while those of the CD94/NKG2 family recognize MHC class I signal sequences bound to HLA-E. Both the KIR and CD94/NKG2 families are composed of activating and inhibitory molecules that serve to regulate the function of NK cells as a result of their MHC class I recognition. Here we review the recently described KIR and CD94/NKG2 family members in the rhesus monkey.

Numerous complete human immunodeficiency virus type 1 (HIV-1) genomes have been characterized for contemporary viruses, but few isolates obtained early in the HIV-1 epidemic have been studied. In this article, we describe the molecular characterization of an HIV-1 isolate (83CD003) that was obtained from an AIDS patient in Kinshasa, Democratic Republic of Congo (DRC) in 1983. The complete 83CD003 genome was sequenced in its entirety and found to encode uninterrupted open reading frames for all viral genes. Phylogenetic analysis revealed 83CD003 was a member of the major (M) group of HIV -1, but did not group with any of the known subtypes. Rather, it formed an independent lineage in all regions of its genome that was roughly equidistant from representatives of all other subtypes. Similarly, 83CD003 also did not cluster with any of several unclassified group M sequences that have been reported more recently to circulate in the DRC, suggesting that it may represent an early group M lineage thai is either rare or has gone extinct. The molecular clone of 83CD003 yielded an infectious virus after transfection into mammalian cells and its biological properties can be further studied.

In earlier work, human immunodeficiency virus type 1 (HIV–1) sequences were analysed to estimate the timing of the ancestral sequence of the main group of HIV–1, the virus that is responsible for the acquired immune deficiency syndrome pandemic, yielding a best estimate of 1931 (95% confidence interval of 1915–1941). That work will be briefly reviewed, outlining how phylogenetic tools were extended to incorporate improved evolutionary models, how the molecular clock model was adapted to incorporate variable periods of latency, and how the approach was validated by correctly estimating the timing of two historically documented dates. The advantages, limitations, and assumptions of the approach will be summarized, with particular consideration of the implications of branch length uncertainty and recombination. We have recently undertaken new phylogenetic analysis of an extremely diverse set of human immunodeficiency virus envelope sequences from the Democratic Republic of the Congo (the DRC, formerly Zaire). This analysis both corroborates and extends the conclusions of our original study. Coalescent methods were used to infer the demographic history of the HIV–1 epidemic in the DRC, and the results suggest an increase in the exponential growth rate of the infected population through time.

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.

A recent report has provided information that helps to determine the age of a common viral ancestor of the AIDS epidemic. Korber et al . examine the strengths as well as limitations of molecular clock analysis and the implications of the findings for planning vaccines and forecasting the future of the epidemic.

Despite detailed analysis of the HIV-1-specific cytotoxic T lymphocyte response by various groups, its relation to viral load and viral sequence variation remains controversial. We analyzed HLA-A*0201 restricted cytotoxic T lymphocyte responses in 17 HIV-1-infected individuals with viral loads ranging from < 400 to 221,000 HIV RNA molecules per milliliter of plasma. In 13 out of 17 infected subjects, CTL responses against the SLYNTVATL epitope (p17 Gag; aa 77-85) were detectable, whereas two other HLA-A*0201 restricted epitopes (ILKEPVHGV, IV9; and VIYQYMDDL, VL9) were only recognized by six and five individuals out of 17 individuals tested, respectively. Naturally occurring variants of the SL9 epitope were tested for binding to HLA-A*0201 and for recognition by specific T cell clones generated from five individuals. Although these variants were widely recognized, they differed by up to 10,000-fold in terms of variant peptide concentrations required for lysis of target cells. A comparison of viral sequences derived from 10 HLA-A*0201-positive individuals to sequences obtained from 11 HLA-A*0201-negative individuals demonstrated only weak evidence for immune selective pressure and thus question the in vivo efficacy of immunodominant CTL responses present during chronic HIV-1 infection.

Detection of human immunodeficiency virus–type 1 (HIV-1) on only one or a few occasions in infants born to infected mothers has been interpreted to indicate that infection may be transient rather than persistent. Forty-two cases of suspected transient HIV-1 viremia among 1562 perinatally exposed seroreverting infants and one mother were reanalyzed. HIV-1envsequences were not found in specimens from 20; in specimens from 6, somatic genetic analysis revealed that specimens were mistakenly attributed to an infant; and in specimens from 17, phylogenetic analysis failed to demonstrate the expected linkage between the infant's and the mother's virus. These findings argue that transient HIV-1 infection, if it exists, will only rarely be satisfactorily documented.

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.

Despite recent advances in antiviral therapy for human immunodeficiency virus (HIV) infection, successful global intervention will require an effective vaccine. Expanding evidence suggests that cytotoxic T-lymphocyte (CTL) responses will be an important component of such a vaccine. The varying geographic distribution of HIV type 1 (HIV-1) clades, with the relative absence of clade B HIV-1 outside the developed world, is considered a major obstacle to the development of a single efficacious vaccine. An understanding of cross-reactive CTL responses between different HIV-1 clades is crucial in the design of a vaccine which will be broadly immunogenic. In this study, we examined the ability of HIV-1 Gag-, reverse transcriptase-, and Env-specific CTL clones isolated from individuals infected in the United States to recognize non-B clade viral sequences and found that all were cross-reactive with the majority of non-B clade viral sequences tested. We next studied HIV-1-specific CTL responses in African individuals infected with clade A, C, or G virus and evaluated cross-recognition of clade B virus. Of 14 persons evaluated, all demonstrated cross-reactivity with the U.S. clade B viral constructs. We conclude that significant CTL cross-reactivity exists between clade B and non-B epitopes, suggesting that CTL cross-recognition among HIV-1 clades is more widespread than anticipated and that a vaccine based on a single clade may be broadly applicable.

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.

Background Primer design for highly variable DNA sequences is difficult, and experimental success requires attention to many interacting constraints. The advent of next-generation sequencing methods allows the investigation of rare variants otherwise hidden deep in large populations, but requires attention to population diversity and primer localization in relatively conserved regions, in addition to recognized constraints typically considered in primer design. Results Design constraints include degenerate sites to maximize population coverage, matching of melting temperatures, optimizing de novo sequence length, finding optimal bio-barcodes to allow efficient downstream analyses, and minimizing risk of dimerization. To facilitate primer design addressing these and other constraints, we created a novel computer program (PrimerDesign) that automates this complex procedure. We show its powers and limitations and give examples of successful designs for the analysis of HIV-1 populations. Conclusions PrimerDesign is useful for researchers who want to design DNA primers and probes for analyzing highly variable DNA populations. It can be used to design primers for PCR, RT-PCR, Sanger sequencing, next-generation sequencing, and other experimental protocols targeting highly variable DNA samples.

The RV144 clinical trial of a prime/boost immunizing regimen using recombinant canary pox (ALVAC-HIV) and two gp120 proteins (AIDSVAX B and E) was previously shown to have a 31.2% efficacy rate. Plasma specimens from vaccine and placebo recipients were used in an extensive set of assays to identify correlates of HIV-1 infection risk. Of six primary variables that were studied, only one displayed a significant inverse correlation with risk of infection: the antibody (Ab) response to a fusion protein containing the V1 and V2 regions of gp120 (gp70-V1V2). This finding prompted a thorough examination of the results generated with the complete panel of 13 assays measuring various V2 Abs in the stored plasma used in the initial pilot studies and those used in the subsequent case-control study. The studies revealed that the ALVAC-HIV/AIDSVAX vaccine induced V2-specific Abs that cross-react with multiple HIV-1 subgroups and recognize both conformational and linear epitopes. The conformational epitope was present on gp70-V1V2, while the predominant linear V2 epitope mapped to residues 165–178, immediately N-terminal to the putative α4β7 binding motif in the mid-loop region of V2. Odds ratios (ORs) were calculated to compare the risk of infection with data from 12 V2 assays, and in 11 of these, the ORs were ≤1, reaching statistical significance for two of the variables: Ab responses to gp70-V1V2 and to overlapping V2 linear peptides. It remains to be determined whether anti-V2 Ab responses were directly responsible for the reduced infection rate in RV144 and whether anti-V2 Abs will prove to be important with other candidate HIV vaccines that show efficacy, however, the results support continued dissection of Ab responses to the V2 region which may illuminate mechanisms of protection from HIV-1 infection and may facilitate the development of an effective HIV-1 vaccine.

The HIV-1 envelope (Env) spike, which consists of a compact, heterodimeric trimer of the glycoproteins gp120 and gp41, is the target of neutralizing antibodies. However, the high mutation rate of HIV-1 and plasticity of Env facilitates viral evasion from neutralizing antibodies through various mechanisms. Mutations that are distant from the antibody binding site can lead to escape, probably by changing the conformation or dynamics of Env; however, these changes are difficult to identify and define mechanistically. Here we describe a network analysis-based approach to identify potential allosteric immune evasion mechanisms using three known HIV-1 Env gp120 protein structures from two different clades, B and C. First, correlation and principal component analyses of molecular dynamics (MD) simulations identified a high degree of long-distance coupled motions that exist between functionally distant regions within the intrinsic dynamics of the gp120 core, supporting the presence of long-distance communication in the protein. Then, by integrating MD simulations with network theory, we identified the optimal and suboptimal communication pathways and modules within the gp120 core. The results unveil both strain-dependent and -independent characteristics of the communication pathways in gp120. We show that within the context of three structurally homologous gp120 cores, the optimal pathway for communication is sequence sensitive, i.e. a suboptimal pathway in one strain becomes the optimal pathway in another strain. Yet the identification of conserved elements within these communication pathways, termed inter-modular hotspots, could present a new opportunity for immunogen design, as this could be an additional mechanism that HIV-1 uses to shield vulnerable antibody targets in Env that induce neutralizing antibody breadth., Author Summary The Env glycoproteins, gp120 and gp41, are the viral targets of HIV neutralizing antibodies. Accordingly, vaccine studies have focused on eliciting broadly neutralizing antibodies against epitopes in these proteins. Sequence diversity and the conformational flexibility of Env have made vaccine design efforts difficult. It is well documented that mutations distant from defined epitopes can lead to escape from neutralizing antibodies. In such cases, allostery within the Env protein could play a dominant role. In this study, we characterized the dynamical network in gp120 in terms of how spatially distant regions communicate with each other. We introduced an approach based on coupling computer simulations to compare gp120 core structures of three different virus strains from two clades, clade B and C. Our study finds that the long-distance collective motions in the protein are functionally relevant and are conserved across diverse strains of gp120, the communication pathways associated with these motions are sensitive to its sequence. Importantly, we find that gp120 exhibits communication modules (communities) with key residues (hotspots) serving as conduits for communication between different communities, a possible strategy to exploit in future vaccine design efforts.

Despite improved hepatitis C virus (HCV) treatments, vaccines remain an effective and economic option for curtailing the epidemic. Mosaic protein HCV genotype 1 vaccine candidates designed to address HCV diversity were immunogenic in mice. They elicited stronger T-cell responses to NS3-NS4a and E1-E2 proteins than did natural strains, as assessed with vaccine-matched peptides.

HIV-1 accumulates mutations in and around reactive epitopes to escape recognition and killing by CD8+ T cells. Measurements of HIV-1 time to escape should therefore provide information on which parameters are most important for T cell-mediated in vivo control of HIV-1. Primary HIV-1-specific T cell responses were fully mapped in 17 individuals, and the time to virus escape, which ranged from days to years, was measured for each epitope. While higher magnitude of an individual T cell response was associated with more rapid escape, the most significant T cell measure was its relative immunodominance measured in acute infection. This identified subject-level or "vertical" immunodominance as the primary determinant of in vivo CD8+ T cell pressure in HIV-1 infection. Conversely, escape was slowed significantly by lower population variability, or entropy, of the epitope targeted. Immunodominance and epitope entropy combined to explain half of all the variability in time to escape. These data explain how CD8+ T cells can exert significant and sustained HIV-1 pressure even when escape is very slow and that within an individual, the impacts of other T cell factors on HIV-1 escape should be considered in the context of immunodominance.

Simian immunodeficiency virus (SIV) infection of rhesus macaques causes immune depletion and disease closely resembling human AIDS and is well recognized as the most relevant animal model for the human disease. Experimental investigations of viral pathogenesis and vaccine protection primarily involve a limited set of related viruses originating in sooty mangabeys (SIVsmm). The diversity of human immunodeficiency virus type 1 (HIV-1) has evolved in humans in about a century; in contrast, SIV isolates used in the macaque model evolved in sooty mangabeys over millennia. To investigate the possible consequences of such different evolutionary histories for selection pressures and observed diversity in SIVsmm and HIV-1, we isolated, sequenced, and analyzed 20 independent isolates of SIVsmm, including representatives of 7 distinct clades of viruses isolated from natural infection. We found SIVsmm diversity to be lower overall than HIV-1 M group diversity. Reduced positive selection (i.e., less diversifying evolution) was evident in extended regions of SIVsmm proteins, most notably in Gag p27 and Env gp120. In addition, the relative diversities of proteins in the two lineages were distinct: SIVsmm Env and Gag were much less diverse than their HIV-1 counterparts. This may be explained by lower SIV-directed immune activity in mangabeys relative to HIV-1-directed immunity in humans. These findings add an additional layer of complexity to the interpretation and, potentially, to the predictive utility of the SIV/macaque model, and they highlight the unique features of human and simian lentiviral evolution that inform studies of pathogenesis and strategies for AIDS vaccine design.

The Thai Phase III clinical trial (RV144) showed modest efficacy in preventing HIV-1 acquisition. Plasma collected from HIV-1-uninfected trial participants completing all injections with ALVAC-HIV (vCP1521) prime and AIDSVAX B/E boost were tested for antibody responses against HIV-1 gp120 envelope (Env). Peptide microarray analysis from six HIV-1 subtypes and group M consensus showed that vaccination induced antibody responses to the second variable (V2) loop of gp120 of multiple subtypes. We further evaluated V2 responses by ELISA and surface plasmon resonance using cyclic (Cyc) and linear V2 loop peptides. Thirty-one of 32 vaccine recipients tested (97%) had antibody responses against Cyc V2 at 2 weeks postimmunization with a reciprocal geometric mean titer (GMT) of 1100 (range: 200–3200). The frequency of detecting plasma V2 antibodies declined to 19% at 28 weeks post-last injection (GMT: 110, range: 100–200). Antibody responses targeted the mid-region of the V2 loop that contains conserved epitopes and has the amino acid sequence KQKVHALFYKLDIVPI (HXB2 Numbering sequence 169–184). Valine at position 172 was critical for antibody binding. The frequency of V3 responses at 2 weeks postimmunization was modest (18/32, 56%) with a GMT of 185 (range: 100–800). In contrast, naturally infected HIV-1 individuals had a lower frequency of antibody responses to V2 (10/20, 50%; p=0.003) and a higher frequency of responses to V3 (19/20, 95%), with GMTs of 400 (range: 100–3200) and 3570 (range: 200–12,800), respectively. RV144 vaccination induced antibodies that targeted a region of the V2 loop that contains conserved epitopes. Early HIV-1 transmission events involve V2 loop interactions, raising the possibility that anti-V2 antibodies in RV144 may have contributed to viral inhibition.

We report the rational design and in vivo testing of mosaic proteins for a polyvalent pan-filoviral vaccine using a computational strategy designed for the Human Immunodeficiency Virus type 1 (HIV-1) but also appropriate for Hepatitis C virus (HCV) and potentially other diverse viruses. Mosaics are sets of artificial recombinant proteins that are based on natural proteins. The recombinants are computationally selected using a genetic algorithm to optimize the coverage of potential cytotoxic T lymphocyte (CTL) epitopes. Because evolutionary history differs markedly between HIV-1 and filoviruses, we devised an adapted computational technique that is effective for sparsely sampled taxa; our first significant result is that the mosaic technique is effective in creating high-quality mosaic filovirus proteins. The resulting coverage of potential epitopes across filovirus species is superior to coverage by any natural variants, including current vaccine strains with demonstrated cross-reactivity. The mosaic cocktails are also robust: mosaics substantially outperformed natural strains when computationally tested against poorly sampled species and more variable genes. Furthermore, in a computational comparison of cross-reactive potential a design constructed prior to the Bundibugyo outbreak performed nearly as well against all species as an updated design that included Bundibugyo. These points suggest that the mosaic designs would be more resilient than natural-variant vaccines against future Ebola outbreaks dominated by novel viral variants. We demonstrate in vivo immunogenicity and protection against a heterologous challenge in a mouse model. This design work delineates the likely requirements and limitations on broadly-protective filoviral CTL vaccines.

Previously we proposed two simplified models of early HIV-1 evolution. Both showed that under a model of neutral evolution and exponential growth, the mean Hamming distance (HD) between genetic sequences grows linearly with time. In this paper we describe a more realistic continuous-time, age-dependent mathematical model of infection and viral replication, and show through simulations that even in this more complex description, the mean Hamming distance grows linearly with time. This remains unchanged when we introduce recombination, though the confidence intervals of the mean HD obtained ignoring recombination are overly conservative.

One of the challenges of genetic data analysis is to combine information from sources that are distributed around the world and accessible through a wide array of different methods and interfaces. The HIV database and its footsteps, the hepatitis C virus (HCV) and hemorrhagic fever virus (HFV) databases, have made it their mission to make different data types easily available to their users. This involves a large amount of behind-the-scenes processing, including quality control and analysis of the sequences and their annotation. Gene and protein sequences are distilled from the sequences that are stored in GenBank; to this end, both submitter annotation and script-generated sequences are used. Alignments of both nucleotide and amino acid sequences are generated, manually curated, distilled into an alignment model, and regenerated in an iterative cycle that results in ever better new alignments. Annotation of epidemiological and clinical information is parsed, checked, and added to the database. User interfaces are updated, and new interfaces are added based upon user requests. Vital for its success, the database staff are heavy users of the system, which enables them to fix bugs and find opportunities for improvement. In this chapter we describe some of the infrastructure that keeps these heavily used analysis platforms alive and vital after nearly 25 years of use. The database/analysis platforms described in this chapter can be accessed at http://hiv.lanl.gov http://hcv.lanl.gov http://hfv.lanl.gov.

Background One of the major challenges in the development of an effective HIV-1 vaccine is the extraordinary genetic diversity of the virus. Immunizations of nonhuman primates using consensus and mosaic immunogens have been shown to elicit cross-reactive CD8+T lymphocyte responses that increase the depth of epitope recognition. However, one of the limitations of vaccine-induced epitope-specific CD8+T lymphocytes includes lack of protection against diverse strains and emergent forms of HIV-1 due to altered T cell receptor (TCR) affinity for variant peptide:MHC class I complexes. Methods In this study, we immunized a cohort of fifteen MamuA*01+ rhesus monkeys with either a 3-valent mosaic Env, group M consensus Env, or single clade B Env vaccine and compared the ability of the CD8+T lymphocyte populations elicited by each immunogen to recognize variants of an HIV-1 envelope epitope sequence p41A (YI9). We identified vaccine-induced CD8+T lymphocytes populations using tetramers constructed with 9 variants of p41A epitope. We assessed the ability of those variant peptides to activate CTL by measuring cytokine production and CD107a expression. We evaluated proliferation using carboxyfluorescein succinimidyl ester. Finally, we investigated the functional avidity of these CD8+T lymphocytes for the variant peptide:Mamu-A*01 complexes using surface plasmon resonance technology. Results Our data show that Env immunizations can generate cross-reactive CD8+T lymphocytes that recognize 2 of 9 (22%) of the variants of p41A epitope, with higher responses induced by the consensus and the 3-valent mosaic immunogens (variant from clade C and variant from clade A/E) compared to the single clade Env immunogen. Tetramer-binding data also show that CD8+ T lymphocytes from monkeys immunized with mosaic immunogen have a trend of higher binding to majority of the variant peptides tested.

Background A global HIV-1 vaccine will need to induce broadly reactive immune responses against conserved HIV-1 regions. It is currently unclear how best to elicit these responses by vaccination. We therefore compared the immunogenicity of a bivalent full-length HIV-1 Gag/Pol/Env mosaic vaccine, a trivalent full-length HIV-1 Gag/Pol/Env mosaic vaccine, and a bivalent mosaic vaccine containing only conserved HIV-1 Gag/Pol/Env epitopes in rhesus monkeys.

A global HIV-1 vaccine will likely need to induce immune responses against conserved HIV-1 regions to contend with the profound genetic diversity of HIV-1. Here we evaluated the capacity of immunogens consisting of only highly conserved HIV-1 sequences that are aimed at focusing cellular immune responses on these potentially critical regions. We assessed in rhesus monkeys the breadth and magnitude of T lymphocyte responses elicited by adenovirus vectors expressing either full-length HIV-1 Gag/Pol/Env immunogens or concatenated immunogens consisting of only highly conserved HIV-1 sequences. Surprisingly, we found that the full-length immunogens induced comparable breadth ( P = 1.0) and greater magnitude ( P = 0.01) of CD8 + T lymphocyte responses against conserved HIV-1 regions compared with the conserved-region-only immunogens. Moreover, the full-length immunogens induced a 5-fold increased total breadth of HIV-1-specific T lymphocyte responses compared with the conserved-region-only immunogens ( P = 0.007). These results suggest that full-length HIV-1 immunogens elicit a substantially increased magnitude and breadth of cellular immune responses compared with conserved-region-only HIV-1 immunogens, including greater magnitude and comparable breadth of responses against conserved sequences.

Hepatitis C virus (HCV) is present in the host with multiple variants generated by its error prone RNA-dependent RNA polymerase. Little is known about the initial viral diversification and the viral life cycle processes that influence diversity. We studied the diversification of HCV during acute infection in 17 plasma donors, with frequent sampling early in infection. To analyze these data, we developed a new stochastic model of the HCV life cycle. We found that the accumulation of mutations is surprisingly slow: at 30 days, the viral population on average is still 46% identical to its transmitted viral genome. Fitting the model to the sequence data, we estimate the median in vivo viral mutation rate is 2.5×10−5 mutations per nucleotide per genome replication (range 1.6–6.2×10−5), about 5-fold lower than previous estimates. To confirm these results we analyzed the frequency of stop codons (N = 10) among all possible non-sense mutation targets (M = 898,335), and found a mutation rate of 2.8–3.2×10−5, consistent with the estimate from the dynamical model. The slow accumulation of mutations is consistent with slow turnover of infected cells and replication complexes within infected cells. This slow turnover is also inferred from the viral load kinetics. Our estimated mutation rate, which is similar to that of other RNA viruses (e.g., HIV and influenza), is also compatible with the accumulation of substitutions seen in HCV at the population level. Our model identifies the relevant processes (long-lived cells and slow turnover of replication complexes) and parameters involved in determining the rate of HCV diversification., Author Summary Hepatitis C virus (HCV) is a RNA virus that infects over 170 million people across the world. It leads to a chronic infection in the majority of people who are infected (>70%). Most people only discover that they are infected long after initial infection. Thus, it is difficult to study the very early events in infection. Here we study 17 individuals during the earliest possible stages of infection, from before the virus is detectable in the plasma to around 35 days post-infection. We focus on understanding the viral kinetics and the diversification of HCV during this acute phase of infection. During chronic infection HCV is present in the host as a swarm of multiple variants generated by its error prone copying. We studied the early diversification of HCV during acute infection using a new mathematical model of HCV replication. We found that after a phase of fast increase in viral load, accompanied by viral diversification, there is a stabilization of viral load and diversity levels. Using our model, we were able to estimate for the first time the HCV mutation rate during acute infection. We estimated the median in vivo viral mutation rate is 2.5×10−5 mutations per nucleotide per genome replication (range 1.6–6.2×10−5), about 5-fold lower than previous estimates. We also used a different approach, based on results of classical genetics, to calculate HCV's mutation rate and obtained consistent results (2.8–3.2×10−5).

A precise molecular identification of transmitted hepatitis C virus (HCV) genomes could illuminate key aspects of transmission biology, immunopathogenesis and natural history. We used single genome sequencing of 2,922 half or quarter genomes from plasma viral RNA to identify transmitted/founder (T/F) viruses in 17 subjects with acute community-acquired HCV infection. Sequences from 13 of 17 acute subjects, but none of 14 chronic controls, exhibited one or more discrete low diversity viral lineages. Sequences within each lineage generally revealed a star-like phylogeny of mutations that coalesced to unambiguous T/F viral genomes. Numbers of transmitted viruses leading to productive clinical infection were estimated to range from 1 to 37 or more (median = 4). Four acutely infected subjects showed a distinctly different pattern of virus diversity that deviated from a star-like phylogeny. In these cases, empirical analysis and mathematical modeling suggested high multiplicity virus transmission from individuals who themselves were acutely infected or had experienced a virus population bottleneck due to antiviral drug therapy. These results provide new quantitative and qualitative insights into HCV transmission, revealing for the first time virus-host interactions that successful vaccines or treatment interventions will need to overcome. Our findings further suggest a novel experimental strategy for identifying full-length T/F genomes for proteome-wide analyses of HCV biology and adaptation to antiviral drug or immune pressures., Author Summary Hepatitis C virus infects as many as 170 million people worldwide. Globally, there are seven major genotypes of HCV that differ by approximately 30% in nucleotide sequence. Importantly, the natural history of HCV infection is variable, ranging from spontaneous resolution to persistent viremia and chronic disease. Factors responsible for this variability in clinical outcome are unknown but likely involve a combination of viral and host determinants. To this end, a precise molecular identification of transmitted HCV genomes could illuminate key aspects of transmission biology, immunopathogenesis and natural history. We used single genome sequencing of plasma viral RNA to identify transmitted viral genomes and their progeny in 17 subjects with acute infection. Numbers of transmitted viruses leading to productive clinical infection ranged from 1 to 37 or more (median = 4). Surprisingly, we found evidence of high multiplicity acute-to-acute HCV transmission in 3 of 17 subjects, which suggests that clinical transmission of HCV, like that of HIV-1, may be enhanced in early infection when virus titers are highest and neutralizing antibodies are absent. These results provide novel insight into HCV transmission and early virus diversification key to our understanding of virus natural history and response to drug selection and immune pressure.

The World Health Organization Global Programme on AIDS (WHO/GPA) is conducting a large-scale collaborative study of human immunodeficiency virus type 1 (HIV-1) variation, based in four potential va...

HIV-1 isolates were obtained from four countries within the framework of the WHO Network for HIV Isolation and Characterization. The use of standard HIV isolation procedures allowed us to compare the biological properties of 126 HIV-1 isolates spanning five genetic subtypes. In primary isolation cultures, viruses from Uganda and Brazil appeared early and replicated without delay, whereas the replication of Thai viruses was delayed by several weeks. Regardless of genetic subtype or country of origin, blood samples collected more than 2 years after seroconversion yielded virus that replicated efficiently in the primary isolation cultures. None of the isolates obtained from Thailand or Rwanda replicated in cell lines, whereas 5 of the 13 Brazilian isolates and 7 of the 11 Ugandan isolates replicated and induced syncytia in MT-2 cells. As expected for virus isolates obtained early in HIV-1 infection (within 2 years of seroconversion), all viruses from Brazil, Rwanda, and Thailand showed a slow/low replicative pattern. For the Ugandan samples, the time from seroconversion was known precisely for a few of the samples and only in one case was less than 2 years. This may explain why the five viruses that were able to replicate in all cell lines, and thus classified as rapid/high, were of Ugandan origin. Viruses able to induce syncytia in MT-2 cells, also induced syncytia in PBMC. However, 8 slow/low viruses (out of 27) gave discordant results, inducing syncytia in PBMC but not in MT-2 cells. Furthermore, using syncytium induction as a marker, changes in virus populations during early in vitro passage in PBMC could be observed

Highly variable international human immunodeficiency virus type 1 envelope sequences can be assigned to six major clades, or phylogenetically defined subtypes, designated A through F. These subtypes are approximately equidistant in terms of evolutionary distance measured by nucleotide sequences. This radiation from a common ancestral sequence may have been in step with the spread of the pandemic. In this study, V3 loop protein sequence relationships within these major clades are analyzed to determine how the different lineages might be evolving with respect to this biologically important domain. The V3 loop has been shown to influence viral phenotype and to elicit both humoral and cellular immune responses. To identify patterns in V3 loop amino acid evolution, we cluster the sequences by a phenetic principle which evaluates protein similarities on the basis of amino acid identities and similarities irrespective of evolutionary relationships. When phenetic clustering patterns are superimposed upon phylogenetic subtype classifications, two interesting mutational trends are revealed. First, a set of identical, or highly similar, V3 loop protein sequences can be identified within two otherwise dissimilar genetic subtypes, A and C. Second, the D subtype sequences are found to possess the most radically divergent set of V3 loop sequences. These and other patterns characteristic of the V3 loop reflect the acquisition of specific biological properties during the apparently recent evolution of the human immunodeficiency virus type 1 lineages.

Background The efficacy of the CTL component of a future HIV-1 vaccine will depend on the induction of responses with the most potent antiviral activity and broad HLA class I restriction. However, current HIV vaccine designs are largely based on viral sequence alignments only, not incorporating experimental data on T cell function and specificity. Methods Here, 950 untreated HIV-1 clade B or -C infected individuals were tested for responses to sets of 410 overlapping peptides (OLP) spanning the entire HIV-1 proteome. For each OLP, a "protective ratio" (PR) was calculated as the ratio of median viral loads (VL) between OLP non-responders and responders. Results For both clades, there was a negative relationship between the PR and the entropy of the OLP sequence. There was also a significant additive effect of multiple responses to beneficial OLP. Responses to beneficial OLP were of significantly higher functional avidity than responses to non-beneficial OLP. They also had superior in-vitro antiviral activities and, importantly, were at least as predictive of individuals' viral loads than their HLA class I genotypes. Conclusions The data thus identify immunogen sequence candidates for HIV and provide an approach for T cell immunogen design applicable to other viral infections.

The predominant mode of HIV-1 infection is heterosexual transmission, where a genetic bottleneck is imposed on the virus quasispecies. To probe whether limited genetic diversity in the genital tract (GT) of the transmitting partner drives this bottleneck, viral envelope sequences from the blood and genital fluids of eight transmission pairs from Rwanda and Zambia were analyzed. The chronically infected transmitting partner's virus population was heterogeneous with distinct genital subpopulations, and the virus populations within the GT of two of four women sampled longitudinally exhibited evidence of stability over time intervals on the order of weeks to months. Surprisingly, the transmitted founder variant was not derived from the predominant GT subpopulations. Rather, in each case, the transmitting variant was phylogenetically distinct from the sampled locally replicating population. Although the exact distribution of the virus population present in the GT at the time of transmission cannot be unambiguously defined in these human studies, it is unlikely, based on these data, that the transmission bottleneck is driven in every case by limited viral diversity in the donor GT or that HIV transmission is solely a stochastic event.

The emergence of whole genome sequencing (WGS) technologies as primary research tools has allowed for the detection of genetic diversity in Mycobacterium tuberculosis (Mtb) with unprecedented resolution. WGS has been used to address a broad range of topics, including the dynamics of evolution, transmission and treatment. Here, we have analyzed 55 publically available genomes to reconstruct the phylogeny of Mtb, and we have addressed complications that arise during the analysis of publically available WGS data. Additionally, we have reviewed the application of WGS to the study of Mtb and discuss those areas still to be addressed, moving from global (phylogeography), to local (transmission chains and circulating strain diversity), to the single patient (clonal heterogeneity) and to the bacterium itself (evolutionary studies). Finally, we discuss the current WGS approaches, their strengths and limitations.

Here we have identified HIV-1 B clade Envelope (Env) amino acid signatures from early in infection that may be favored at transmission, as well as patterns of recurrent mutation in chronic infection that may reflect common pathways of immune evasion. To accomplish this, we compared thousands of sequences derived by single genome amplification from several hundred individuals that were sampled either early in infection or were chronically infected. Samples were divided at the outset into hypothesis-forming and validation sets, and we used phylogenetically corrected statistical strategies to identify signatures, systematically scanning all of Env. Signatures included single amino acids, glycosylation motifs, and multi-site patterns based on functional or structural groupings of amino acids. We identified signatures near the CCR5 co-receptor-binding region, near the CD4 binding site, and in the signal peptide and cytoplasmic domain, which may influence Env expression and processing. Two signatures patterns associated with transmission were particularly interesting. The first was the most statistically robust signature, located in position 12 in the signal peptide. The second was the loss of an N-linked glycosylation site at positions 413–415; the presence of this site has been recently found to be associated with escape from potent and broad neutralizing antibodies, consistent with enabling a common pathway for immune escape during chronic infection. Its recurrent loss in early infection suggests it may impact fitness at the time of transmission or during early viral expansion. The signature patterns we identified implicate Env expression levels in selection at viral transmission or in early expansion, and suggest that immune evasion patterns that recur in many individuals during chronic infection when antibodies are present can be selected against when the infection is being established prior to the adaptive immune response., Author Summary A single virus most often establishes HIV-1 infection. As a consequence, virus sampled early in infection is usually very homogeneous. A few months into the infection, the virus begins to accumulate mutations as it evolves to evade HIV-specific immune responses mounted by the infected host. During chronic infection, the viral population diversifies, reflecting the history of mutations that arose within that infected individual. We hypothesized that particular amino acids might confer a selective advantage during transmission or early infection, and others might recur during chronic infection because they provide common and effective strategies of immune escape. We compared a large number of viral sequences from several hundred infected people sampled soon after transmission or during chronic infection to identify such infection-status “signature” patterns. A particularly robust signature was identified in the signal peptide of Envelope, a region that regulates its expression. Other signatures were found in regions of Envelope that interact with its cellular receptors, or are implicated in immune escape.

Epitopes that drive the initial autologous neutralizing antibody response in HIV-1-infected individuals could provide insights for vaccine design. Although highly strain specific, these epitopes are immunogenic, vulnerable to antibody attack on infectious virus, and could be involved in the ontogeny of broadly neutralizing antibody responses. To delineate such epitopes, we used site-directed mutagenesis, autologous plasma samples, and autologous monoclonal antibodies to map the amino acid changes that led to escape from the initial autologous neutralizing antibody response in two HIV-1 subtype B-infected individuals. Additional mapping of the epitopes was accomplished by using alanine scanning mutagenesis. Escape in the two individuals occurred by different pathways, but the responses in both cases appeared to be directed against the same region of gp120. In total, three amino acid positions were identified that were independently associated with autologous neutralization. Positions 295 and 332 are located immediately before and after the N- and C-terminal cysteines of the V3 loop, respectively, the latter of which affected an N-linked glycan that was critical to the neutralization epitope. Position 415 affected an N-linked glycan at position 413 in the C terminus of V4 that might mask epitopes near the base of V3. All three sites lie in close proximity on a four-stranded antiparallel sheet on the outer domain of gp120. We conclude that a region just below the base of the V3 loop, near the coreceptor binding domain of gp120, can be a target for autologous neutralization.

The sequence diversity of HIV-1 presents a challenge for the development of an effective HIV-1 vaccine, because such a vaccine must confer protection against diverse forms of the virus. The present studies were initiated to explore how vaccine-induced clonal populations of CD8+ T lymphocytes of rhesus monkeys recognize variants of an HIV-1 envelope epitope sequence. Evaluating a subset of variants of a selected epitope peptide that retain their binding to the MHC class I molecule of rhesus monkeys that presents this epitope peptide, we show that vaccine-elicited CD8+ T lymphocytes comparably recognize the wild-type and a number of variant epitope peptides as determined by tetramer binding assays. In fact, the same clonal populations of CD8+ T lymphocytes recognize the wild-type and variant epitope peptides. However, functional assays show that many of these variant epitope peptides stimulate suboptimal cytokine production by the vaccine-elicited CD8+ T lymphocytes. These findings suggest that vaccine-induced CD8+ T lymphocyte populations may recognize diverse forms of a viral epitope, but may not function optimally to confer protection against viruses expressing many of those variant sequences.

We characterized the evolution of simian immunodeficiency virus (SIV) in the male genital tract by examining blood- and semen-associated virus from experimentally and sham vaccinated rhesus monkeys during primary infection. At the time of peak virus replication, SIV sequences were intermixed between the blood and semen supporting a scenario of high-level virus “spillover” into the male genital tract. However, at the time of virus set point, compartmentalization was apparent in 4 of 7 evaluated monkeys, likely as a consequence of restricted virus gene flow between anatomic compartments after the resolution of primary viremia. These findings suggest that SIV replication in the male genital tract evolves to compartmentalization after peak viremia resolves., Author Summary Methods to reduce the transmission of HIV-1 are hindered by a lack of information regarding early viral dynamics and evolution in the male genital tract. In the present study, we show that SIV in the blood and genital tract are homogeneous during early infection, indicating facile virus gene flow between these compartments. Importantly, the coincidence of the resolution of primary viremia with the decreased virus levels in genital secretions suggest that the dramatic fall in virus replication during early infection underlies the development of viral compartmentalization. Our demonstration of early virus compartmentalization in the male genital tract has important implications for the understanding of early events leading to infection of the male genital tract and the nature of the transmitted virus during primary retrovirus infection.

In the present study, we analyzed the functional profile of CD8+ T-cell responses directed against autologous transmitted/founder HIV-1 isolates during acute and early infection, and examined whether multifunctionality is required for selection of virus escape mutations. Seven anti-retroviral therapy-naïve subjects were studied in detail between 1 and 87 weeks following onset of symptoms of acute HIV-1 infection. Synthetic peptides representing the autologous transmitted/founder HIV-1 sequences were used in multiparameter flow cytometry assays to determine the functionality of HIV-1-specific CD8+ T memory cells. In all seven patients, the earliest T cell responses were predominantly oligofunctional, although the relative contribution of multifunctional cell responses increased significantly with time from infection. Interestingly, only the magnitude of the total and not of the poly-functional T-cell responses was significantly associated with the selection of escape mutants. However, the high contribution of MIP-1β-producing CD8+ T-cells to the total response suggests that mechanisms not limited to cytotoxicity could be exerting immune pressure during acute infection. Lastly, we show that epitope entropy, reflecting the capacity of the epitope to tolerate mutational change and defined as the diversity of epitope sequences at the population level, was also correlated with rate of emergence of escape mutants., Author Summary An important role for the polyfunctional T-cell fraction of anti-HIV CD8 responses during chronic HIV infection has previously been suggested. This study characterized the role of polyfunctional T-cells directed against the transmitted/founder virus in the selection of viral escape mutants during acute HIV-1 infection within a unique cohort of individuals recruited within 3 weeks from the onset of symptoms at the time when the virus load was still declining. For the first time, the sequences of the transmitted/founder virus isolated from each patient were used. Interestingly, polyfunctionality was not found to be a pre-requisite for selection of escape mutations. A novel significant correlation is found between the order of appearance of escape mutations in different epitope sequences and both the magnitude of the CD8+ T-cell responses and the degree of entropy of the individual epitopes. A high proportion of the T-cells participating in the total response produced MIP-1β, suggesting that mechanisms not limited to the killing of infected cells might play a relevant role in early infection. This highlights the importance of measuring the quality of the CD8+ lymphocyte response and the sequence of the transmitted virus isolates to better understand the mechanisms of control of HIV replication during acute infection.

T cell directed HIV vaccines are based upon the induction of CD8+ T cell memory responses that would be effective in inhibiting infection and subsequent replication of an infecting HIV-1 strain, a process that requires a match or near-match between the epitope induced by vaccination and the infecting viral strain. We compared the frequency and specificity of the CTL epitope responses elicited by the replication-defective Ad5 gag/pol/nef vaccine used in the Step trial with the likelihood of encountering those epitopes among recently sequenced Clade B isolates of HIV-1. Among vaccinees with detectable 15-mer peptide pool ELISpot responses, there was a median of four (one Gag, one Nef and two Pol) CD8 epitopes per vaccinee detected by 9-mer peptide ELISpot assay. Importantly, frequency analysis of the mapped epitopes indicated that there was a significant skewing of the T cell response; variable epitopes were detected more frequently than would be expected from an unbiased sampling of the vaccine sequences. Correspondingly, the most highly conserved epitopes in Gag, Pol, and Nef (defined by presence in >80% of sequences currently in the Los Alamos database www.hiv.lanl.gov) were detected at a lower frequency than unbiased sampling, similar to the frequency reported for responses to natural infection, suggesting potential epitope masking of these responses. This may be a generic mechanism used by the virus in both contexts to escape effective T cell immune surveillance. The disappointing results of the Step trial raise the bar for future HIV vaccine candidates. This report highlights the bias towards less-conserved epitopes present in the same vaccine used in the Step trial. Development of vaccine strategies that can elicit a greater breadth of responses, and towards conserved regions of the genome in particular, are critical requirements for effective T-cell based vaccines against HIV-1. Trial Registration ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00849680","term_id":"NCT00849680"}}NCT00849680, A Study of Safety, Tolerability, and Immunogenicity of the MRKAd5 Gag/Pol/Nef Vaccine in Healthy Adults.

The authors would like to add the following to the funding information: "The study was supported by funding from the NIHR Oxford Biomedical Research Centre programme."

Background The occurrence of a genetic bottleneck in HIV sexual or mother-to-infant transmission has been well documented. This results in a majority of new infections being homogeneous, i.e., initiated by a single genetic strain. Early after infection, prior to the onset of the host immune response, the viral population grows exponentially. In this simple setting, an approach for estimating evolutionary and demographic parameters based on comparison of diversity measures is a feasible alternative to the existing Bayesian methods (e.g., BEAST), which are instead based on the simulation of genealogies. Results We have devised a web tool that analyzes genetic diversity in acutely infected HIV-1 patients by comparing it to a model of neutral growth. More specifically, we consider a homogeneous infection (i.e., initiated by a unique genetic strain) prior to the onset of host-induced selection, where we can assume a random accumulation of mutations. Previously, we have shown that such a model successfully describes about 80% of sexual HIV-1 transmissions provided the samples are drawn early enough in the infection. Violation of the model is an indicator of either heterogeneous infections or the initiation of selection. Conclusions When the underlying assumptions of our model (homogeneous infection prior to selection and fast exponential growth) are met, we are under a very particular scenario for which we can use a forward approach (instead of backwards in time as provided by coalescent methods). This allows for more computationally efficient methods to derive the time since the most recent common ancestor. Furthermore, the tool performs statistical tests on the Hamming distance frequency distribution, and outputs summary statistics (mean of the best fitting Poisson distribution, goodness of fit p-value, etc). The tool runs within minutes and can readily accommodate the tens of thousands of sequences generated through new ultradeep pyrosequencing technologies. The tool is available on the LANL website.

Elucidating virus-host interactions responsible for HIV-1 transmission is important for advancing HIV-1 prevention strategies. To this end, single genome amplification (SGA) and sequencing of HIV-1 within the context of a model of random virus evolution has made possible for the first time an unambiguous identification of transmitted/founder viruses and a precise estimation of their numbers. Here, we applied this approach to HIV-1 env analyses in a cohort of acutely infected men who have sex with men (MSM) and found that a high proportion (10 of 28; 36%) had been productively infected by more than one virus. In subjects with multivariant transmission, the minimum number of transmitted viruses ranged from 2 to 10 with viral recombination leading to rapid and extensive genetic shuffling among virus lineages. A combined analysis of these results, together with recently published findings based on identical SGA methods in largely heterosexual (HSX) cohorts, revealed a significantly higher frequency of multivariant transmission in MSM than in HSX [19 of 50 subjects (38%) versus 34 of 175 subjects (19%); Fisher's exact p = 0.008]. To further evaluate the SGA strategy for identifying transmitted/founder viruses, we analyzed 239 overlapping 5′ and 3′ half genome or env-only sequences from plasma viral RNA (vRNA) and blood mononuclear cell DNA in an MSM subject who had a particularly well-documented virus exposure history 3–6 days before symptom onset and 14–17 days before peak plasma viremia (47,600,000 vRNA molecules/ml). All 239 sequences coalesced to a single transmitted/founder virus genome in a time frame consistent with the clinical history, and a molecular clone of this genome encoded replication competent virus in accord with model predictions. Higher multiplicity of HIV-1 infection in MSM compared with HSX is consistent with the demonstrably higher epidemiological risk of virus acquisition in MSM and could indicate a greater challenge for HIV-1 vaccines than previously recognized., Author Summary Understanding the biology of sexual transmission of HIV-1 could contribute importantly to the development of effective prevention measures. However, different routes of virus transmission (vaginal, rectal, penile or oral) and inaccessibility of tissues at or near the time of virus transmission make this goal elusive. Here, we apply single genome amplification and sequencing of plasma HIV-1 and a model of random virus evolution to a cohort of acutely infected men who have sex with men (MSM) and find that MSM are twice as likely as heterosexuals to become infected by multiple viruses as opposed to a single virus. Some MSM subjects were infected by as many as 7 to 10 or more genetically distinct viruses as a consequence of a single exposure event. We go on to molecularly clone the first full-length transmitted/founder subtype B HIV-1 virus and show that it is highly replicative in human CD4+ T-cells but not macrophages. Our study provides the first comparative, quantitative analysis of the multiplicity of HIV-1 infection in the two primary risk groups—MSM and heterosexuals—driving the global pandemic, and we discuss the implications of the findings to HIV-1 vaccine development and prevention research.

Motivation: Existing coalescent models and phylogenetic tools based on them are not designed for studying the genealogy of sequences like those of HIV, since in HIV recombinants with multiple cross-over points between the parental strains frequently arise. Hence, ambiguous cases in the classification of HIV sequences into subtypes and circulating recombinant forms (CRFs) have been treated with ad hoc methods in lack of tools based on a comprehensive coalescent model accounting for complex recombination patterns. Results: We developed the program ARGUS that scores classifications of sequences into subtypes and recombinant forms. It reconstructs ancestral recombination graphs (ARGs) that reflect the genealogy of the input sequences given a classification hypothesis. An ARG with maximal probability is approximated using a Markov chain Monte Carlo approach. ARGUS was able to distinguish the correct classification with a low error rate from plausible alternative classifications in simulation studies with realistic parameters. We applied our algorithm to decide between two recently debated alternatives in the classification of CRF02 of HIV-1 and find that CRF02 is indeed a recombinant of Subtypes A and G. Availability: ARGUS is implemented in C++ and the source code is available at http://gobics.de/software Contact: ibulla@uni-goettingen.de Supplementary Information: Supplementary data are available at Bioinformatics online.

Signature pattern analysis identifies particular sites in amino acid or nucleic acid alignments of variable sequences that are distinctly representative of a query set of sequences relative to a background set. We explore the merits of using signature patterns for analysis of HIV-1 (human immunodeficiency virus type 1) sequences in cases of epidemiological linkage and potential superinfection. For these purposes, query sets are viral sequences that are all derived from one HIV-1 infected individual, hence the signature pattern is the array of sites that are characteristic of the range of viral variants obtained from that person. Once a signature pattern has been objectively defined, it can be used to examine other viral sequences from other individuals for evidence of genetic relatedness. A computer program to facilitate this analysis, VESPA, is described and applied to sequence data gathered during the investigation of HIV-1 transmission in a dental practice. The implications of signature polymorphisms seen within an infected individual, and shared polymorphisms between linked individuals, are also considered. VESPA may also be applied to the molecular analysis of biological phenotypes.

To examine the sequence diversity of human immunodeficiency virus type 1 (HIV-1) between known transmission sets, sequences from the V3 and V4-V5 region of the envelope gene from four mother-infant pairs were analyzed. The mean interpatient sequence variation between isolates from linked mother-infant pairs was comparable to the sequence diversity found between isolates from other close contacts. The mean intrapatient variation was significantly less in the infants' isolates then the isolates from both their mothers and other characterized intrapatient sequence sets. In addition, a distinct and characteristic difference in the glycosylation pattern preceding the V3 loop was found between each linked transmission pair. These findings indicate that selection of specific genotypic variants, which may play a role in some direct transmission sets, and the duration of infection are important factors in the degree of diversity seen between the sequence sets.

The restricted neutralization breadth of vaccine-elicited antibodies is a major limitation of current human immunodeficiency virus-1 (HIV-1) candidate vaccines. In order to permit the efficient identification of vaccines with enhanced capacity for eliciting cross-reactive neutralizing antibodies (NAbs) and to assess the overall breadth and potency of vaccine-elicited NAb reactivity, we assembled a panel of 109 molecularly cloned HIV-1 Env pseudoviruses representing a broad range of genetic and geographic diversity. Viral isolates from all major circulating genetic subtypes were included, as were viruses derived shortly after transmission and during the early and chronic stages of infection. We assembled a panel of genetically diverse HIV-1-positive (HIV-1 + ) plasma pools to assess the neutralization sensitivities of the entire virus panel. When the viruses were rank ordered according to the average sensitivity to neutralization by the HIV-1 + plasmas, a continuum of average sensitivity was observed. Clustering analysis of the patterns of sensitivity defined four subgroups of viruses: those having very high (tier 1A), above-average (tier 1B), moderate (tier 2), or low (tier 3) sensitivity to antibody-mediated neutralization. We also investigated potential associations between characteristics of the viral isolates (clade, stage of infection, and source of virus) and sensitivity to NAb. In particular, higher levels of NAb activity were observed when the virus and plasma pool were matched in clade. These data provide the first systematic assessment of the overall neutralization sensitivities of a genetically and geographically diverse panel of circulating HIV-1 strains. These reference viruses can facilitate the systematic characterization of NAb responses elicited by candidate vaccine immunogens.

Induction of antibodies that neutralize a broad range of human immunodeficiency virus type 1 (HIV-1) isolates is a major goal of vaccine development. To study natural examples of broad neutralization, we analyzed sera from 103 HIV-1-infected subjects. Among progressor patients, 20% of sera neutralized more than 75% of a panel of 20 diverse viral isolates. Little activity was observed in sera from long-term nonprogressors (elite controllers). Breadth of neutralization was correlated with viral load, but not with CD4 count, history of past antiretroviral use, age, gender, race/ethnicity, or route of exposure. Clustering analysis of sera by a novel method identified a statistically robust subgrouping of sera that demonstrated broad and potent neutralization activity.