Melissa-Rose Abrahams

Quantitating the Multiplicity of Infection with Human Immunodeficiency Virus Type 1 Subtype C Reveals a Non-Poisson Distribution of Transmitted Variants

ABSTRACT Identifying the specific genetic characteristics of successfully transmitted variants may prove central to the development of effective vaccine and microbicide interventions. Although human immunodeficiency virus transmission is associated with a population bottleneck, the extent to which different factors influence the diversity of transmitted viruses is unclear. We estimate here the number of transmitted variants in 69 heterosexual men and women with primary subtype C infections. From 1,505 env sequences obtained using a single genome amplification approach we show that 78% of infections involved single variant transmission and 22% involved multiple variant transmissions (median of 3). We found evidence for mutations selected for cytotoxic-T-lymphocyte or antibody escape and a high prevalence of recombination in individuals infected with multiple variants representing another potential escape pathway in these individuals. In a combined analysis of 171 subtype B and C transmission events, we found that infection with more than one variant does not follow a Poisson distribution, indicating that transmission of individual virions cannot be seen as independent events, each occurring with low probability. While most transmissions resulted from a single infectious unit, multiple variant transmissions represent a significant fraction of transmission events, suggesting that there may be important mechanistic differences between these groups that are not yet understood.

Limited Neutralizing Antibody Specificities Drive Neutralization Escape in Early HIV-1 Subtype C Infection.

We previously showed that HIV-1 subtype C viruses elicit potent but highly type-specific neutralizing antibodies (nAb) within the first year of infection. In order to determine the specificity and evolution of these autologous nAbs, we examined neutralization escape in four individuals whose responses against the earliest envelope differed in magnitude and potency. Neutralization escape occurred in all participants, with later viruses showing decreased sensitivity to contemporaneous sera, although they retained sensitivity to new nAb responses. Early nAb responses were very restricted, occurring sequentially and targeting only two regions of the envelope. In V1V2, limited amino acid changes often involving indels or glycans, mediated partial or complete escape, with nAbs targeting the V1V2 region directly in 2 cases. The alpha-2 helix of C3 was also a nAb target, with neutralization escape associated with changes to positively charged residues. In one individual, relatively high titers of anti-C3 nAbs were required to drive genetic escape, taking up to 7 weeks for the resistant variant to predominate. Thereafter titers waned but were still measurable. Development of this single anti-C3 nAb specificity was associated with a 7-fold drop in HIV-1 viral load and a 4-fold rebound as the escape mutation emerged. Overall, our data suggest the development of a very limited number of neutralizing antibody specificities during the early stages of HIV-1 subtype C infection, with temporal fluctuations in specificities as escape occurs. While the mechanism of neutralization escape appears to vary between individuals, the involvement of limited regions suggests there might be common vulnerabilities in the HIV-1 subtype C transmitted envelope.

Genital Tract Inflammation During Early HIV-1 Infection Predicts Higher Plasma Viral Load Set Point in Women

BACKGROUND The biggest challenge in human immunodeficiency virus type 1 (HIV-1) prevention in Africa is the high HIV-1 burden in young women. In macaques, proinflammatory cytokine production in the genital tract is necessary for target cell recruitment and establishment of simian immunodeficiency virus (SIV) infection following vaginal inoculation. The purpose of this study was to assess if genital inflammation during early HIV-1 infection predisposes women to rapid disease progression. METHODS Inflammatory cytokine concentrations were measured in cervicovaginal lavage (CVL) from 49 women 6, 17, 30, and 55 weeks after HIV-1 infection and from 22 of these women before infection. Associations between genital inflammation and viral load set point and blood CD4 cell counts 12 months after infection were investigated. RESULTS Elevated genital cytokine concentrations 6 and 17 weeks after HIV-1 infection were associated with higher viral load set points and, to a lesser extent, with CD4 depletion. CVL cytokine concentrations during early infection did not differ relative to preinfection but were elevated in women who had vaginal discharge, detectable HIV-1 RNA in their genital tracts, and lower blood CD4 counts. CONCLUSION Genital inflammation during early HIV-1 infection was associated with higher viral load set point and CD4 depletion, which are markers of rapid disease progression. Strategies aimed at reducing genital inflammation during early HIV-1 infection may slow disease progression.

Growing Poxviruses and Determining Virus Titer

Poxviruses are cell-associated viruses that can be grown in adherent- or suspension-cell cultures or chorioallantoic membranes of embryonated hens eggs. The main principle of isolating the virus is to mechanically lyse infected cells. The virus can then be semipurified by centrifugation through a sucrose cushion. Further purification can be achieved with a sucrose gradient. This chapter describes methods for the preparation of large-scale growth and purification of the virus.

Increased Memory Differentiation Is Associated with Decreased Polyfunctionality for HIV but Not for Cytomegalovirus-Specific CD8+ T Cells

The generation of polyfunctional CD8+ T cells, in response to vaccination or natural infection, has been associated with improved protective immunity. However, it is unclear whether the maintenance of polyfunctionality is related to particular cellular phenotypic characteristics. To determine whether the cytokine expression profile is linked to the memory differentiation stage, we analyzed the degree of polyfunctionality of HIV-specific CD8+ T cells within different memory subpopulations in 20 antiretroviral therapy-naive HIV-1–infected individuals at ∼34 wk postinfection. These profiles were compared with CMV-specific CD8+ T cell responses in HIV-uninfected control subjects and in individuals chronically infected with HIV. Our results showed that the polyfunctional abilities of HIV-specific CD8+ T cells differed according to their memory phenotype. Early-differentiated cells (CD45RO+CD27+) exhibited a higher proportion of cells positive for three or four functions (p < 0.001), and a lower proportion of monofunctional cells (p < 0.001) compared with terminally differentiated (TD; CD45RO−CD27−) HIV-specific CD8+ T cells. The majority of TD HIV-specific CD8+ T cells were monofunctional (median 69% [interquartile range: 57–83]), producing predominantly CD107a or MIP1β. Moreover, proportions of HIV-specific monofunctional CD8+ T cells positively associated with proportions of TD HIV-specific CD8+ T cells (p = 0.019, r = 0.54). In contrast, CMV-specific CD8+ T cell polyfunctional capacities were similar across all memory subpopulations, with terminally and early-differentiated cells endowed with comparable polyfunctionality. Overall, these data show that the polyfunctional abilities of HIV-specific CD8+ T cells are influenced by the stage of memory differentiation, which is not the case for CMV-specific responses.

Rapid, complex adaptation of transmitted HIV-1 full-length genomes in subtype C-infected individuals with differing disease progression

Objective(s):There is limited information on full-length genome sequences and the early evolution of transmitted HIV-1 subtype C viruses, which constitute the majority of viruses spread in Africa. The purpose of this study was to characterize the earliest changes across the genome of subtype C viruses following transmission, to better understand early control of viremia. Design:We derived the near full-length genome sequence responsible for clinical infection from five HIV subtype C-infected individuals with different disease progression profiles and tracked adaptation to immune responses in the first 6 months of infection. Methods:Near full-length genomes were generated by single genome amplification and direct sequencing. Sequences were analyzed for amino acid mutations associated with cytotoxic T lymphocyte (CTL) or antibody-mediated immune pressure, and for reversion. Results:Fifty-five sequence changes associated with adaptation to the new host were identified, with 38% attributed to CTL pressure, 35% to antibody pressure, 16% to reversions and the remainder were unclassified. Mutations in CTL epitopes were most frequent in the first 5 weeks of infection, with the frequency declining over time with the decline in viral load. CTL escape predominantly occurred in nef, followed by pol and env. Shuffling/toggling of mutations was identified in 81% of CTL epitopes, with only 7% reaching fixation within the 6-month period. Conclusion:There was rapid virus adaptation following transmission, predominantly driven by CTL pressure, with most changes occurring during high viremia. Rapid escape and complex escape pathways provide further challenges for vaccine protection.

The Vaccinia Virus N1L ORF May Encode a Multifunctional Protein Possibly Targeting Different Kinases, One of Which Influences ATP Levels in Vivo

As the single‐most potent virulence factor of the vaccinia virus, the 13.8‐kDa protein enhances viral replication in the brain by an unknown mechanism. Due to the high energy demands of the brain and the at times inadequate energy supply and small energy reserves to support physiologic activity, the ability of this organ to support energy requirements for replication of a virus is unlikely. We investigated the possible role of the 13.8‐kDa protein in the enhancement of adenosine triphosphate (ATP) utilization in the brain to sustain viral replication. In vitro and in vivo monitoring and comparison of ATP levels in mouse brain tissue infected with a wild‐type vaccinia virus or a 13.8‐kDa deletion strain (vGK5) revealed differences in ATP utilization and a significant difference in ATP levels in vivo after 5 days of infection. Because of poor replication of the wild‐type Lister vaccinia virus in the brain, a role for the 13.8‐kDa protein in the modulation of ATP levels to support viral replication in the brain could not be conclusively implicated. Evaluation of the amino acid sequence and predicted secondary structure of the 13.8‐kDa protein and sequence and structural homologs thereof provided evidence of putative dimerization and adenine binding sites and a possible kinase‐related function for this protein.

Near full-length HIV type 1M genomic sequences from Cameroon Evidence of early diverging under-sampled lineages in the country

Background: Cameroon is the country in which HIV-1 group M (HIV-1M) likely originated and is today a major hotspot of HIV-1M genetic diversity. It remains unclear, however, whether the highly divergent HIV-1M lineages found in this country arose during the earliest phases of the global HIV-1M epidemic, or whether they arose more recently as a result of recombination events between globally circulating HIV-1M lineages. Methodology: To differentiate between these two possibilities, we performed phylogenetic analyses of the near full genome sequences of nine newly sequenced divergent HIV-1M isolates and 15 previously identified, apparently unique recombinant forms (URFs) from Cameroon. Results: Although two of the new genome sequences were clearly classifiable within subtype G, the remaining seven were highly divergent and phylogenetically branched either outside of, or very near the bases of clades containing the well characterised globally circulating viral lineages that they were most closely related to. Recombination analyses further revealed that these divergent viruses were likely complex URFs. We show, however that substantial portions (>1 Kb) of three of the new genome sequences and 15 of the previously characterised Cameroonian URFs have apparently been derived from divergent parental viruses that branch phylogenetically near the bases of the major HIV-1M clades. Conclusions and implications: Our analyses indicate the presence in Cameroon of contemporary descendants of numerous early-diverging HIV-1M lineages. Further efforts to sample and sequence viruses from such lineages could be crucial both for retracing the earliest evolutionary steps during the emergence of HIV-1M in humans, and accurately reconstructing the ancestral sequences of the major globally circulating HIV-1M lineages.

REPLICATION CAPACITY OF VIRUSES FROM ACUTE INFECTION DRIVES HIV-1 DISEASE PROGRESSION

ABSTRACT The viral genotype has been shown to play an important role in HIV pathogenesis following transmission. However, the viral phenotypic properties that contribute to disease progression remain unclear. Most studies have been limited to the evaluation of Gag function in the context of a recombinant virus backbone. Using this approach, important biological information may be lost, making the evaluation of viruses obtained during acute infection, representing the transmitted virus, a more biologically relevant model. Here, we evaluate the roles of viral infectivity and the replication capacity of viruses from acute infection in disease progression in women who seroconverted in the CAPRISA 004 tenofovir microbicide trial. We show that viral replication capacity, but not viral infectivity, correlates with the set point viral load (Spearman r = 0.346; P = 0.045) and that replication capacity (hazard ratio [HR] = 4.52; P = 0.01) can predict CD4 decline independently of the viral load (HR = 2.9; P = 0.004) or protective HLA alleles (HR = 0.61; P = 0.36). We further demonstrate that Gag-Pro is not the main driver of this association, suggesting that additional properties of the transmitted virus play a role in disease progression. Finally, we find that although viruses from the tenofovir arm were 2-fold less infectious, they replicated at rates similar to those of viruses from the placebo arm. This indicates that the use of tenofovir gel did not select for viral variants with higher replication capacity. Overall, this study supports a strong influence of the replication capacity in acute infection on disease progression, potentially driven by interaction of multiple genes rather than a dominant role of the major structural gene gag. IMPORTANCE HIV disease progression is known to differ between individuals, and defining which fraction of this variation can be attributed to the virus is important both clinically and epidemiologically. In this study, we show that the replication capacity of viruses isolated during acute infection predicts subsequent disease progression and drives CD4 decline independently of the viral load. This provides further support for the hypothesis that the replication capacity of the transmitted virus determines the initial damage to the immune system, setting the pace for later disease progression. However, we did not find evidence that the major structural gene gag drives this correlation, highlighting the importance of other genes in determining disease progression.

Increased differentiation associates with decreased polyfunctionality for HIV but not CMV-specific CD8+ T cell responses

HIV-specific early-differentiated cells of a central memorylike phenotype (CD45RO+CD27+) exhibited a higher proportion of cells positive for three or four functions (p<0.001) and a lower proportion of mono-functional cells (median 27% [IQR: 16-38], p<0.001) compared to terminally-differentiated cells. In contrast, CMV-specific CD8+ T cell polyfunctional capacities were similar across all memory subpopulations, with terminally and early-differentiated cells endowed with comparable polyfunctionality. Conclusion Overall, these data show that for HIV-specific responses, the memory differentiation stage can influence the polyfunctional properties of CD8+ T cells, suggesting that terminal differentiation of HIV-specific CD8+ T cells might be detrimental for viral control. These results may help in understanding phenotypic attributes related to effective T cell responses against HIV-1.