Discontinuous sequence change of human immunodeficiency virus (HIV) type 1 env sequences in plasma viral and lymphocyte-associated proviral populations in vivo: implications for models of HIV pathogenesis.

Sequence change in different hypervariable regions of the external membrane glycoprotein (gp120) of human immunodeficiency virus type 1 (HIV-1) was studied. Viral RNA associated with cell-free virus particles circulating in plasma and proviral DNA present in HIV-infected peripheral blood mononuclear cells (PBMCs) were extracted from blood samples of two currently asymptomatic hemophiliac patients over a 5-year period. HIV sequences were amplified by polymerase chain reaction to allow analysis in the V3, V4, and V5 hypervariable regions of gp120. Rapid sequence change, consisting of regular replacements by a succession of distinct viral populations, was found in both plasma virus and PBMC provirus populations. Significant differences between the frequencies of sequence variants in DNA and RNA populations within the same sample were observed, indicating that at any one time point, the predominant plasma virus variants were antigenically distinct from viruses encoded by HIV DNA sequences in PBMCs. How these findings contribute to current models of HIV pathogenesis is discussed.     

The gut mucosal viral reservoir in HIV-infected patients is not the major source of rebound plasma viremia following interruption of highly active antiretroviral therapy

Interruption of suppressive highly active antiretroviral therapy (HAART) in HIV-infected patients leads to increased HIV replication and viral rebound in peripheral blood. Effects of therapy interruption on gut-associated lymphoid tissue (GALT) have not been well investigated. We evaluated longitudinal changes in viral replication and emergence of viral variants in the context of T cell homeostasis and gene expression in GALT of three HIV-positive patients who initiated HAART during primary HIV infection but opted to interrupt therapy thereafter. Longitudinal viral sequence analysis revealed that a stable proviral reservoir was established in GALT during primary HIV infection that persisted through early HAART and post-therapy interruption. Proviral variants in GALT and peripheral blood mononuclear cells (PBMCs) displayed low levels of genomic diversity at all times. A rapid increase in viral loads with a modest decline of CD4(+) T cells in peripheral blood was observed, while gut mucosal CD4(+) T cell loss was severe following HAART interruption. This was accompanied by increased mucosal gene expression regulating interferon (IFN)-mediated antiviral responses and immune activation, a profile similar to those found in HAART-naive HIV-infected patients. Sequence analysis of rebound virus suggested that GALT was not the major contributor to the postinterruption plasma viremia nor were GALT HIV reservoirs rapidly replaced by HIV rebound variants. Our data suggest an early establishment and persistence of viral reservoirs in GALT with minimal diversity. Early detection of and therapy for HIV infection may be beneficial in controlling viral evolution and limiting establishment of diverse viral reservoirs in the mucosal compartment.     

A human immunodeficiency virus prime-boost immunization regimen in humans induces antibodies that show interclade cross-reactivity and neutralize several X4-, R5-, and dualtropic clade B and C primary isolates

A human immunodeficiency virus (HIV) vaccine that will be useful in diverse geographic regions will need to induce a broad immune response characterized by cross-clade immunity. To test whether a clade B-based HIV candidate vaccine could induce interclade humoral responses, including neutralizing activity against primary HIV-1 isolates, sera were tested from recipients of a vaccine consisting of recombinant canarypox virus vCP205 and recombinant gp120(SF2). Serum antibodies exhibited strong immunochemical cross-reactivity with V3 peptides from clades B, C, and F, with weaker activity for several V3 peptides from clades A, D, G, and H; essentially no reactivity could be demonstrated with V3 peptides from clades E and O. Extensive cross-clade reactivity was also documented by enzyme-linked immunosorbent assay with all nine recombinant HIV envelope glycoproteins tested from clades B, D, and E. In addition, vaccinees' sera displayed significant neutralizing activity against 5 of 14 primary isolates tested, including one X4 virus and two dualtropic viruses (from clade B) and two R5 viruses (from clades B and C). This is the first demonstration of the induction by a candidate HIV vaccine constructed from clade B laboratory strains of HIV of neutralizing activity against R5 and clade C primary isolates. The data suggest that, by virtue of their ability to induce cross-clade immune responses, appropriately formulated HIV vaccines based on a finite number of HIV isolates may ultimately be able to protect against the wide range of HIV isolates affecting the populations of many geographic regions.     

Human immunodeficiency virus type 1 variants isolated from single plasma samples display a wide spectrum of neutralization sensitivity

Individuals infected with human immunodeficiency virus type 1 (HIV-1) harbor a mixture of viral variants with different sequences and in some instances with different phenotypic properties. Major and rapid fluctuations in the proportion of viral variants coexisting in an infected individual can be observed under strong pharmacological and immune selective pressure. Because of the short half-life of HIV-infected cells and of HIV virions in the blood, plasma virus populations are highly relevant to HIV evolution in the face of these selective pressures. Here we analyzed the sensitivity to antibody-mediated neutralization of viral variants coexisting in the plasma virus populations of two infected patients. For each patient, several replication-competent viral clones were constructed that carry primary envelope gene sequences obtained from a single plasma sample. Viral clones differed in their tropism and replicative capacity and in the number and positions of glycosylation sites in the envelope glycoproteins. Viruses were tested against heterologous and autologous sera obtained at different time points. Interestingly, we found that viral variants coexisting in each plasma sample were highly heterogeneous in terms of sensitivity to neutralization. The order of sensitivity depended on the serum used and was not associated with virus tropism. The neutralization potency of sera increased with the duration of the infection for both autologous and heterologous neutralization.     

Multiple V1/V2 env variants are frequently present during primary infection with human immunodeficiency virus type 1

Human immunodeficiency virus type 1 (HIV-1) exists as a complex population of multiple genotypic variants in persons with chronic infection. However, acute HIV-1 infection via sexual transmission is a low-probability event in which there is thought to be low genetic complexity in the initial inoculum. In order to assess the viral complexity present during primary HIV-1 infection, the V1/V2 and V3 variable regions of the env gene were examined by using a heteroduplex tracking assay (HTA) capable of resolving these genotypic variants. Blood plasma samples from 26 primary HIV-1-infected subjects were analyzed for their level of diversity. Half of the subjects had more than one V1/V2 viral variant during primary infection, indicating the frequent transmission of multiple variants. This observation is inconsistent with the idea of infrequent transmission based on a small transmitting inoculum of cell-free virus. In chronically infected subjects, the complexity of the viral populations was even greater in both the V1/V2 and the V3 regions than in acutely infected subjects, indicating that in spite of the presence of multiple variants in acute infection, the virus does pass through a genetic bottleneck during transmission. We also examined how well the infecting virus penetrated different anatomical compartments by using the HTA. Viral variants detected in blood plasma were compared to those detected in seminal plasma and/or cerebral spinal fluid of six individuals. The virus in each of these compartments was to a large extent identical to virus in blood plasma, a finding consistent with rapid penetration of the infecting variant(s). The low-probability transmission of multiple variants could be the result of transient periods of hyperinfectiousness or hypersusceptibility. Alternatively, the inefficient transfer of a multiply infected cell could account for both the low probability of transmission and the transfer of multiple variants.     

Quantitative Deep Sequencing Reveals Dynamic HIV-1 Escape and Large Population Shifts during CCR5 Antagonist Therapy In Vivo

High-throughput sequencing platforms provide an approach for detecting rare HIV-1 variants and documenting more fully quasispecies diversity. We applied this technology to the V3 loop-coding region of env in samples collected from 4 chronically HIV-infected subjects in whom CCR5 antagonist (vicriviroc [VVC]) therapy failed. Between 25,000–140,000 amplified sequences were obtained per sample. Profound baseline V3 loop sequence heterogeneity existed; predicted CXCR4-using populations were identified in a largely CCR5-using population. The V3 loop forms associated with subsequent virologic failure, either through CXCR4 use or the emergence of high-level VVC resistance, were present as minor variants at 0.8–2.8% of baseline samples. Extreme, rapid shifts in population frequencies toward these forms occurred, and deep sequencing provided a detailed view of the rapid evolutionary impact of VVC selection. Greater V3 diversity was observed post-selection. This previously unreported degree of V3 loop sequence diversity has implications for viral pathogenesis, vaccine design, and the optimal use of HIV-1 CCR5 antagonists.     

Cell-to-cell contact results in a selective translocation of maternal human immunodeficiency virus type 1 quasispecies across a trophoblastic barrier by both transcytosis and infection

Mother-to-child transmission can occur in utero, mainly intrapartum and postpartum in case of breastfeeding. In utero transmission is highly restricted and results in selection of viral variant from the mother to the child. We have developed an in vitro system that mimics the interaction between viruses, infected cells present in maternal blood, and the trophoblast, the first barrier protecting the fetus. Trophoblastic BeWo cells were grown as a tight polarized monolayer in a two-chamber system. Cell-free virions applied to the apical pole neither crossed the barrier nor productively infected BeWo cells. In contrast, apical contact with human immunodeficiency virus (HIV)-infected peripheral blood mononuclear cells (PBMCs) resulted in transcytosis of infectious virus across the trophoblastic monolayer and in productive infection correlating with the fusion of HIV-infected PBMCs with trophoblasts. We showed that viral variants are selected during these two steps and that in one case of in utero transmission, the predominant maternal viral variant characterized after transcytosis was phylogenetically indistinguishable from the predominant child's virus. Hence, the first steps of transmission of HIV-1 in utero appear to involve the interaction between HIV type 1-infected cells and the trophoblastic layer, resulting in the passage of infectious HIV by transcytosis and by fusion/infection, both leading to a selection of virus quasispecies.     

HIV-1 clinical isolates resistant to CCR5 antagonists exhibit delayed entry kinetics that are corrected in the presence of drug

HIV CCR5 antagonists select for env gene mutations that enable virus entry via drug-bound coreceptor. To investigate the mechanisms responsible for viral adaptation to drug-bound coreceptor-mediated entry, we studied viral isolates from three participants who developed CCR5 antagonist resistance during treatment with vicriviroc (VCV), an investigational small-molecule CCR5 antagonist. VCV-sensitive and -resistant viruses were isolated from one HIV subtype C- and two subtype B-infected participants; VCV-resistant isolates had mutations in the V3 loop of gp120 and were cross-resistant to TAK-779, an investigational antagonist, and maraviroc (MVC). All three resistant isolates contained a 306P mutation but had variable mutations elsewhere in the V3 stem. We used a virus-cell β-lactamase (BlaM) fusion assay to determine the entry kinetics of recombinant viruses that incorporated full-length VCV-sensitive and -resistant envelopes. VCV-resistant isolates exhibited delayed entry rates in the absence of drug, relative to pretherapy VCV-sensitive isolates. The addition of drug corrected these delays. These findings were generalizable across target cell types with a range of CD4 and CCR5 surface densities and were observed when either population-derived or clonal envelopes were used to construct recombinant viruses. V3 loop mutations alone were sufficient to restore virus entry in the presence of drug, and the accumulation of V3 mutations during VCV therapy led to progressively higher rates of viral entry. We propose that the restoration of pre-CCR5 antagonist therapy HIV entry kinetics drives the selection of V3 loop mutations and may represent a common mechanism that underlies the emergence of CCR5 antagonist resistance.     

In vivo distribution and cytopathology of variants of human immunodeficiency virus type 1 showing restricted sequence variability in the V3 loop.

The distribution, cell tropism, and cytopathology in vivo of human immunodeficiency virus (HIV) was investigated in postmortem tissue samples from a series of HIV-infected individuals who died either of complications associated with AIDS or for unrelated reasons while they were asymptomatic. Proviral sequences were detected at a high copy number in lymphoid tissue of both presymptomatic patients and patients with AIDS, whereas significant infection of nonlymphoid tissue such as that from brains, spinal cords, and lungs were confined to those with AIDS. V3 loop sequences from both groups showed highly restricted sequence variability and a low overall positive charge of the encoded amino acid sequence compared with those of standard laboratory isolates of HIV type 1 (HIV-1). The low charge and the restriction in sequence variability were comparable to those observed with isolates showing a non-syncytium-inducing (NSI) and macrophage-tropic phenotype in vitro. All patients were either exclusively infected (six of seven cases) or predominantly infected (one case) with variants with a predicted NSI/macrophage-tropic phenotype, irrespective of the degree of disease progression. p24 antigen was detected by immunocytochemical staining of paraffin-fixed sections in the germinal centers within lymphoid tissue, although little or no antigen was found in areas of lymph node or spleen containing T lymphocytes from either presymptomatic patients or patients with AIDS. The predominant p24 antigen-expressing cells in the lungs and brains of the patients with AIDS were macrophages and microglia (in brains), frequently forming multinucleated giant cells (syncytia) even though the V3 loop sequences of these variants resembled those of NSI isolates in vitro. These studies indicate that lack of syncytium-forming ability in established T-cell lines does not necessarily predict syncytium-forming ability in primary target cells in vivo. Furthermore, variants of HIV with V3 sequences characteristic of NSI/macrophage-tropic isolates form the predominant population in a range of lymphoid and nonlymphoid tissues in vivo, even in patients with AIDS.     

Cytopathic variants of an attenuated isolate of human immunodeficiency virus type 2 exhibit increased affinity for CD4.

Naturally occurring isolates of human immunodeficiency virus (HIV) have been described which are deficient in their ability to fuse with and kill CD4+ target cells. Although the molecular basis for their attenuation has not yet been defined, several lines of evidence point toward the viral envelope gene as a key determinant of viral pathogenicity. In the present article, we report the biological characterization of two highly cytopathic variants derived by repeated cell-free passage of an attenuated isolate of HIV type 2 (HIV-2), termed HIV-2/ST. Unlike the parental virus, the cytopathic variants were found to infect Sup-T1 cells with great efficiency and to induce both cell fusion and profound killing in these cultures. To determine whether changes in the viral envelope gene were responsible for the observed phenotypic differences, we examined the CD4 binding affinity of these viruses using a novel assay designed to quantitate the binding of fluoresceinated CD4 to viral envelope in its native configuration on the cell surface. The results demonstrated that the affinity of parental HIV-2/ST envelope for CD4 was 2 orders of magnitude reduced, while the cytopathic variants exhibited a high CD4 binding affinity, comparable to that of cytopathic HIV-1 and HIV-2 isolates. From these data, we conclude that the cytopathic potential of HIV depends, at least in part, on its receptor-binding affinity. In addition, our study documents strong selection pressures for viruses with increased CD4 affinity during propagation in immortalized T-cell lines, thus emphasizing the need to study HIV envelope biology in natural target cells.     

Human immunodeficiency virus (HIV) from experimentally infected chimpanzees: isolation and characterization

Human immunodeficiency virus-1 (HIV-1) isolates obtained from chimpanzees that had undergone various immunosuppressive treatments were characterized by growth on various primary cells and cell lines as well as by restriction endonuclease analysis. Viruses recovered from animals inoculated with uncloned HIV showed genetic variation from the original inoculum, whereas viruses isolated from an animal infected with a molecular clone of HIV did not. In some cases, virus recovery was possible only after enrichment for CD4+ cells by panning, inoculation with a chimpanzee cytomegalovirus, or a combination of these procedures. These findings indicate a role for viral and host cofactors in the control of virus replication and suggest explanations for the absence of clinical manifestations in HIV-infected chimpanzees.     

In-depth characterization of viral isolates from plasma and cells compared with plasma circulating quasispecies in early HIV-1 infection

Background

The use of in vitro models to unravel the phenotypic characteristics of circulating viral variants is key to understanding HIV-1 pathogenesis but limited by the availability of primary viral isolates from biological samples. However, overall in vivo genetic variability of HIV-1 within a subject may not be reflected in the viable viral population obtained after isolation. Although several studies have tried to determine whether viral populations expanded in vitro are representative of in vivo findings, the answer remains unclear due to the reduced number of clonal sequences analyzed or samples compared. In order to overcome previous experimental limitations, here we applied Deep Pyrosequencing (DPS) technology in combination with phenotypic experiments to analyze and compare with unprecedented detail the composition of viral isolates and in vivo quasispecies.

Methodology/Principal Findings

We amplified by DPS HIV-1 genomic regions covering gag, protease, integrase and env-V3 to characterize paired isolates from plasma and peripheral blood mononuclear cells and compare them with total plasma viral RNA in four recently HIV-1 infected subjects. Our study demonstrated the presence of unique haplotypes scattered between sample types with conservation of major variants. In addition, no differences in intra- and inter-population encoded protein variability were found between the different types of isolates or when these were compared to plasma viral RNA within subjects. Additionally, in vitro experiments demonstrated phenotypic similarities in terms of replicative capacity and co-receptor usage between viral isolates and plasma viral RNA.

Conclusion

This study is the first in-depth comparison and characterization of viral isolates from different sources and plasma circulating quasispecies using DPS in recently HIV-1 infected subjects. Our data supports the use of primary isolates regardless of their plasma or cellular origin to define genetic variability and biological traits of circulating HIV-1 quasispecies.     

Relationship between the V3 loop and the phenotypes of human immunodeficiency virus type 1 (HIV-1) isolates from children perinatally infected with HIV-1.

The third variable region (V3) of the envelope protein of human immunodeficiency virus type 1 (HIV-1) contains group- and type-specific epitopes for neutralizing antibodies and contains determinants involved in viral tropism and syncytium-inducing (SI) activity. We studied the in vivo relationship between V3 sequences and viral phenotypes in 24 perinatally HIV-1-infected children. To avoid in vitro selection of intrapatient minor variants, genetic studies were performed directly on uncultured peripheral blood mononuclear cells (PBMC), and the tropisms of HIV-1 isolates were evaluated by culturing patients' PBMC directly with monocyte-derived macrophages, lymphocytes, and MT-2 cells. According to their phenotypes, we could define five types of primary isolates: (i) non-syncytium-inducing (NSI) macrophagetropic, (ii) NSI macrophage-lymphotropic, (iii) NSI lymphotropic, (iv) SI lympho-T-cell line-tropic, and (v) SI pleiotropic. The SI viral phenotype was correlated with a more advanced status of disease. Genetic analysis of intrapatient molecular variants revealed that no relationship between the degree of intrapatient V3 variability and the pattern of viral tropism existed; moreover, within a single patient, the values for V3 variability between CD4+ lymphocytes and CD14+ monocytes were similar, thus suggesting that in vivo variability of the monocytotropic variants is more extensive than previously appreciated. A comparison between the intrapatient major variants and the phenotype of primary isolates disclosed that a negatively charged amino acid at residue site 25 was associated with an NSI macrophage- and macrophage-lymphotropic viral phenotype. Finally, by comparing the V3 sequences derived from our study population with those of several prototypes, we observed that the majority of isolates circulating in Italy are related to the North American subtype B macrophagetropic isolates.     

Genetic characterization of human immunodeficiency virus type 1 in blood and genital secretions: evidence for viral compartmentalization and selection during sexual transmission.

To explore the mechanism of sexual transmission of human immunodeficiency virus type 1 (HIV-1), we compared HIV-1 gp120 sequences in longitudinal samples from five acute seroconvertors with those from their corresponding sexual partners (transmitters). We used a quantitative homoduplex tracking assay to compare the overall genetic composition of HIV-1 quasispecies in each transmission pair and to track the transmitted viruses during the acute and asymptomatic stages of HIV-1 infection. In the chronically infected transmitters, HIV-1 variants in genital secretions differed from those in blood and variants in cells differed from those in cell-free plasma, indicating remarkable sequence heterogeneity in these subjects as well as compartmentalization of the virus in different bodily sites. Conversely, two of five seroconvertors had only a few related variants and three of five harbored only one viral population, indicating that in these subjects the transmitted viruses were typically homogeneous. Transmitted viruses were evident in the donor's seminal plasma (one of five cases) and even more so in their seminal cells (three of five cases), suggesting that both cell-associated and cell-free viruses can be transmitted. In every pair studied, the transmitted variant(s) represents only a minor population in the semen of the corresponding transmitter, thereby providing evidence that HIV-1 selection indeed occurs during sexual transmission.     

Origin and evolution of HIV-1 in breast milk determined by single-genome amplification and sequencing

HIV transmission via breastfeeding accounts for a considerable proportion of infant HIV acquisition. However, the origin and evolution of the virus population in breast milk, the likely reservoir of transmitted virus variants, are not well characterized. In this study, HIV envelope (env) genes were sequenced from virus variants amplified by single-genome amplification from plasmas and milk of 12 chronically HIV-infected, lactating Malawian women. Maximum likelihood trees and statistical tests of compartmentalization revealed interspersion of plasma and milk HIV env sequences in the majority of subjects, indicating limited or no compartmentalization of milk virus variants. However, phylogenetic tree analysis further revealed monotypic virus variants that were significantly more frequent in milk (median proportion of identical viruses, 29.5%; range, 0 to 61%) than in plasma (median proportion of identical viruses, 0%; range, 0 to 26%) (P = 0.002), suggesting local virus replication in the breast milk compartment. Moreover, clonally amplified virus env genes in milk produced functional virus Envs that were all CCR5 tropic. Milk and plasma virus Envs had similar predicted phenotypes and neutralization sensitivities to broadly neutralizing antibodies in both transmitting and nontransmitting mothers. Finally, phylogenetic comparison of longitudinal milk and plasma virus env sequences revealed synchronous virus evolution and new clonal amplification of evolved virus env genes in milk. The limited compartmentalization and the clonal amplification of evolving, functional viruses in milk indicate continual seeding of the mammary gland by blood virus variants, followed by transient local replication of these variants in the breast milk compartment.     

Detection of minor drug-resistant populations by parallel allele-specific sequencing

We developed a highly sensitive parallel allele-specific sequencing (PASS) assay to simultaneously analyze a large number of viral genomes and detect minor drug-resistant populations at approximately 0.1-0.01% levels. Using this assay on samples from individuals infected with human immunodeficiency viruses (HIV), we successfully detected and quantified minor populations of drug-resistant viruses and performed linkage analysis of multiple-drug resistance mutations. This assay may serve as a useful tool to study drug resistance in HIV and other infectious agents.     

West Nile virus genetic diversity is maintained during transmission by Culex pipiens quinquefasciatus mosquitoes

Due to error-prone replication, RNA viruses exist within hosts as a heterogeneous population of non-identical, but related viral variants. These populations may undergo bottlenecks during transmission that stochastically reduce variability leading to fitness declines. Such bottlenecks have been documented for several single-host RNA viruses, but their role in the population biology of obligate two-host viruses such as arthropod-borne viruses (arboviruses) in vivo is unclear, but of central importance in understanding arbovirus persistence and emergence. Therefore, we tracked the composition of West Nile virus (WNV; Flaviviridae, Flavivirus) populations during infection of the vector mosquito, Culex pipiens quinquefasciatus to determine whether WNV populations undergo bottlenecks during transmission by this host. Quantitative, qualitative and phylogenetic analyses of WNV sequences in mosquito midguts, hemolymph and saliva failed to document reductions in genetic diversity during mosquito infection. Further, migration analysis of individual viral variants revealed that while there was some evidence of compartmentalization, anatomical barriers do not impose genetic bottlenecks on WNV populations. Together, these data suggest that the complexity of WNV populations are not significantly diminished during the extrinsic incubation period of mosquitoes.     

Comparison of Major and Minor Viral SNPs Identified through Single Template Sequencing and Pyrosequencing in Acute HIV-1 Infection

Massively parallel sequencing (MPS) technologies, such as 454-pyrosequencing, allow for the identification of variants in sequence populations at lower levels than consensus sequencing and most single-template Sanger sequencing experiments. We sought to determine if the greater depth of population sampling attainable using MPS technology would allow detection of minor variants in HIV founder virus populations very early in infection in instances where Sanger sequencing detects only a single variant. We compared single nucleotide polymorphisms (SNPs) during acute HIV-1 infection from 32 subjects using both single template Sanger and 454-pyrosequencing. Pyrosequences from a median of 2400 viral templates per subject and encompassing 40% of the HIV-1 genome, were compared to a median of five individually amplified near full-length viral genomes sequenced using Sanger technology. There was no difference in the consensus nucleotide sequences over the 3.6kb compared in 84% of the subjects infected with single founders and 33% of subjects infected with multiple founder variants: among the subjects with disagreements, mismatches were found in less than 1% of the sites evaluated (of a total of nearly 117,000 sites across all subjects). The majority of the SNPs observed only in pyrosequences were present at less than 2% of the subject’s viral sequence population. These results demonstrate the utility of the Sanger approach for study of early HIV infection and provide guidance regarding the design, utility and limitations of population sequencing from variable template sources, and emphasize parameters for improving the interpretation of massively parallel sequencing data to address important questions regarding target sequence evolution.