Accurately Measuring Recombination between Closely Related HIV-1 Genomes

Retroviral recombination is thought to play an important role in the generation of immune escape and multiple drug resistance by shuffling pre-existing mutations in the viral population. Current estimates of HIV-1 recombination rates are derived from measurements within reporter gene sequences or genetically divergent HIV sequences. These measurements do not mimic the recombination occurring in vivo, between closely related genomes. Additionally, the methods used to measure recombination make a variety of assumptions about the underlying process, and often fail to account adequately for issues such as co-infection of cells or the possibility of multiple template switches between recombination sites. We have developed a HIV-1 marker system by making a small number of codon modifications in gag which allow recombination to be measured over various lengths between closely related viral genomes. We have developed statistical tools to measure recombination rates that can compensate for the possibility of multiple template switches. Our results show that when multiple template switches are ignored the error is substantial, particularly when recombination rates are high, or the genomic distance is large. We demonstrate that this system is applicable to other studies to accurately measure the recombination rate and show that recombination does not occur randomly within the HIV genome

Prediction of Co-Receptor Usage of HIV-1 from Genotype

Human Immunodeficiency Virus 1 uses for entry into host cells a receptor (CD4) and one of two co-receptors (CCR5 or CXCR4). Recently, a new class of antiretroviral drugs has entered clinical practice that specifically bind to the co-receptor CCR5, and thus inhibit virus entry. Accurate prediction of the co-receptor used by the virus in the patient is important as it allows for personalized selection of effective drugs and prognosis of disease progression. We have investigated whether it is possible to predict co-receptor usage accurately by analyzing the amino acid sequence of the main determinant of co-receptor usage, i.e., the third variable loop V3 of the gp120 protein. We developed a two-level machine learning approach that in the first level considers two different properties important for protein-protein binding derived from structural models of V3 and V3 sequences. The second level combines the two predictions of the first level. The two-level method predicts usage of CXCR4 co-receptor for new V3 sequences within seconds, with an area under the ROC curve of 0.937±0.004. Moreover, it is relatively robust against insertions and deletions, which frequently occur in V3. The approach could help clinicians to find optimal personalized treatments, and it offers new insights into the molecular basis of co-receptor usage. For instance, it quantifies the importance for co-receptor usage of a pocket that probably is responsible for binding sulfated tyrosine

A reflection on HIV/AIDS research after 25 years

Dr. Robert C. Gallo provides a personal reflection on the 25 year history of AIDS

Autonomous Targeting of Infectious Superspreaders Using Engineered Transmissible Therapies

Infectious disease treatments, both pharmaceutical and vaccine, face three universal challenges: the difficulty of targeting treatments to high-risk ‘superspreader’ populations who drive the great majority of disease spread, behavioral barriers in the host population (such as poor compliance and risk disinhibition), and the evolution of pathogen resistance. Here, we describe a proposed intervention that would overcome these challenges by capitalizing upon Therapeutic Interfering Particles (TIPs) that are engineered to replicate conditionally in the presence of the pathogen and spread between individuals — analogous to ‘transmissible immunization’ that occurs with live-attenuated vaccines (but without the potential for reversion to virulence). Building on analyses of HIV field data from sub-Saharan Africa, we construct a multi-scale model, beginning at the single-cell level, to predict the effect of TIPs on individual patient viral loads and ultimately population-level disease prevalence. Our results show that a TIP, engineered with properties based on a recent HIV gene-therapy trial, could stably lower HIV/AIDS prevalence by ∼30-fold within 50 years and could complement current therapies. In contrast, optimistic antiretroviral therapy or vaccination campaigns alone could only lower HIV/AIDS prevalence by <2-fold over 50 years. The TIP's efficacy arises from its exploitation of the same risk factors as the pathogen, allowing it to autonomously penetrate superspreader populations, maintain efficacy despite behavioral disinhibition, and limit viral resistance. While demonstrated here for HIV, the TIP concept could apply broadly to many viral infectious diseases and would represent a new paradigm for disease control, away from pathogen eradication but toward robust disease suppression

Complexity of the Inoculum Determines the Rate of Reversion of SIV Gag CD8 T Cell Mutant Virus and Outcome of Infection

Escape mutant (EM) virus that evades CD8+ T cell recognition is frequently observed following infection with HIV-1 or SIV. This EM virus is often less replicatively “fit” compared to wild-type (WT) virus, as demonstrated by reversion to WT upon transmission of HIV to a naïve host and the association of EM virus with lower viral load in vivo in HIV-1 infection. The rate and timing of reversion is, however, highly variable. We quantified reversion to WT of a series of SIV and SHIV viruses containing minor amounts of WT virus in pigtail macaques using a sensitive PCR assay. Infection with mixes of EM and WT virus containing ≥10% WT virus results in immediate and rapid outgrowth of WT virus at SIV Gag CD8 T cell epitopes within 7 days of infection of pigtail macaques with SHIV or SIV. In contrast, infection with biologically passaged SHIVmn229 viruses with much smaller proportions of WT sequence, or a molecular clone of pure EM SIVmac239, demonstrated a delayed or slow pattern of reversion. WT virus was not detectable until ≥8 days after inoculation and took ≥8 weeks to become the dominant quasispecies. A delayed pattern of reversion was associated with significantly lower viral loads. The diversity of the infecting inoculum determines the timing of reversion to WT virus, which in turn predicts the outcome of infection. The delay in reversion of fitness-reducing CD8 T cell escape mutations in some scenarios suggests opportunities to reduce the pathogenicity of HIV during very early infection

Faster HIV-1 Disease Progression among Brazilian Individuals Recently Infected with CXCR4-Utilizing Strains

Introduction: Primary HIV infection is usually caused by R5 viruses, and there is an association between the emergence of CCXR4-utilizing strains and faster disease progression. We characterized HIV-1 from a cohort of recently infected individuals in Brazil, predicted the virus's co-receptor use based on the env genotype and attempted to correlate virus profiles with disease progression. Methods: A total of 72 recently infected HIV patients were recruited based on the Serologic Testing Algorithm for Recent HIV Seroconversion and were followed every three to four months for up to 78 weeks. The HIV-1 V3 region was characterized by sequencing nine to twelve weeks after enrollment. Disease progression was characterized by CD4+ T-cell count decline to levels consistently below 350 cells/mu L. Results: Twelve out of 72 individuals (17%) were predicted to harbor CXCR4-utilizing strains; a baseline CD4,350 was more frequent among these individuals (p = 0.03). Fifty-seven individuals that were predicted to have CCR5-utilizing viruses and 10 individuals having CXCR4-utilizing strains presented with baseline CD4.350; after 78 weeks, 33 individuals with CCR5 strains and one individual with CXCR4 strains had CD4.350 (p = 0.001). There was no association between CD4 decline and demographic characteristics or HIV-1 subtype. Conclusions: Our findings confirm the presence of strains with higher in vitro pathogenicity during early HIV infection, suggesting that even among recently infected individuals, rapid progression may be a consequence of the early emergence of CXCR4-utilizing strains. Characterizing the HIV-1 V3 region by sequencing may be useful in predicting disease progression and guiding treatment initiation decisions.Brazilian Program for STD and AIDSBrazilian Program for STD and AIDSMinistry of Health [914/BRA/3014-UNESCO/Kallas]Ministry of HealthSao Paulo City Health DepartmentSao Paulo City Health Department [2004-0.168.922-7/Kallas]Fundacao de Amparo a Pesquisa do Estado de Sao PauloFundacao de Amparo a Pesquisa do Estado de Sao Paulo [04/15856-9/Diaz]Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES)Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES)Brazilian Ministry of EducationBrazilian Ministry of Educatio

Sequential Bottlenecks Drive Viral Evolution in Early Acute Hepatitis C Virus Infection

Hepatitis C is a pandemic human RNA virus, which commonly causes chronic infection and liver disease. The characterization of viral populations that successfully initiate infection, and also those that drive progression to chronicity is instrumental for understanding pathogenesis and vaccine design. A comprehensive and longitudinal analysis of the viral population was conducted in four subjects followed from very early acute infection to resolution of disease outcome. By means of next generation sequencing (NGS) and standard cloning/Sanger sequencing, genetic diversity and viral variants were quantified over the course of the infection at frequencies as low as 0.1%. Phylogenetic analysis of reassembled viral variants revealed acute infection was dominated by two sequential bottleneck events, irrespective of subsequent chronicity or clearance. The first bottleneck was associated with transmission, with one to two viral variants successfully establishing infection. The second occurred approximately 100 days post-infection, and was characterized by a decline in viral diversity. In the two subjects who developed chronic infection, this second bottleneck was followed by the emergence of a new viral population, which evolved from the founder variants via a selective sweep with fixation in a small number of mutated sites. The diversity at sites with non-synonymous mutation was higher in predicted cytotoxic T cell epitopes, suggesting immune-driven evolution. These results provide the first detailed analysis of early within-host evolution of HCV, indicating strong selective forces limit viral evolution in the acute phase of infection

Timing Constraints of In Vivo Gag Mutations during Primary HIV-1 Subtype C Infection

Background: Aiming to answer the broad question “When does mutation occur?” this study examined the time of appearance, dominance, and completeness of in vivo Gag mutations in primary HIV-1 subtype C infection. Methods: A primary HIV-1C infection cohort comprised of 8 acutely and 34 recently infected subjects were followed frequently up to 500 days post-seroconversion (p/s). Gag mutations were analyzed by employing single-genome amplification and direct sequencing. Gag mutations were determined in relation to the estimated time of seroconversion. Time of appearance, dominance, and completeness was compared for different types of in vivo Gag mutations. Results: Reverse mutations to the wild type appeared at a median (IQR) of 62 (44;139) days p/s, while escape mutations from the wild type appeared at 234 (169;326) days p/s (p&lt;0.001). Within the subset of mutations that became dominant, reverse and escape mutations appeared at 54 (30;78) days p/s and 104 (47;198) days p/s, respectively (p&lt;0.001). Among the mutations that reached completeness, reverse and escape mutations appeared at 54 (30;78) days p/s and 90 (44;196) days p/s, respectively (p = 0.006). Time of dominance for reverse mutations to and escape mutations from the wild type was 58 (44;105) days p/s and 219 (90;326) days p/s, respectively (p&lt;0.001). Time of completeness for reverse and escape mutations was 152 (100;176) days p/s and 243 (101;370) days p/s, respectively (p = 0.001). Fitting a Cox proportional hazards model with frailties confirmed a significantly earlier time of appearance (hazard ratio (HR): 2.6; 95% CI: 2.3–3.0), dominance (4.8 (3.4–6.8)), and completeness (3.6 (2.3–5.5)) of reverse mutations to the wild type Gag than escape mutations from the wild type. Some complex mutational pathways in Gag included sequential series of reversions and escapes. Conclusions: The study identified the timing of different types of in vivo Gag mutations in primary HIV-1 subtype C infection in relation to the estimated time of seroconversion. Overall, the in vivo reverse mutations to the wild type occurred significantly earlier than escape mutations from the wild type. This shorter time to incidence of reverse mutations remained in the subsets of in vivo Gag mutations that reached dominance or completeness

Role of CCL3L1-CCR5 Genotypes in the Epidemic Spread of HIV-1 and Evaluation of Vaccine Efficacy

Polymorphisms in CCR5, the major coreceptor for HIV, and CCL3L1, a potent CCR5 ligand and HIV-suppressive chemokine, are determinants of HIV-AIDS susceptibility. Here, we mathematically modeled the potential impact of these genetic factors on the epidemic spread of HIV, as well as on its prevention.Ro, the basic reproductive number, is a fundamental concept in explaining the emergence and persistence of epidemics. By modeling sexual transmission among HIV+/HIV- partner pairs, we find that Ro estimates, and concordantly, the temporal and spatial patterns of HIV outgrowth are highly dependent on the infecting partners' CCL3L1-CCR5 genotype. Ro was least and highest when the infected partner possessed protective and detrimental CCL3L1-CCR5 genotypes, respectively. The modeling data indicate that in populations such as Pygmies with a high CCL3L1 gene dose and protective CCR5 genotypes, the spread of HIV might be minimal. Additionally, Pc, the critical vaccination proportion, an estimate of the fraction of the population that must be vaccinated successfully to eradicate an epidemic was <1 only when the infected partner had a protective CCL3L1-CCR5 genotype. Since in practice Pc cannot be >1, to prevent epidemic spread, population groups defined by specific CCL3L1-CCR5 genotypes might require repeated vaccination, or as our models suggest, a vaccine with an efficacy of >70%. Further, failure to account for CCL3L1-CCR5-based genetic risk might confound estimates of vaccine efficacy. For example, in a modeled trial of 500 subjects, misallocation of CCL3L1-CCR5 genotype of only 25 (5%) subjects between placebo and vaccine arms results in a relative error of approximately 12% from the true vaccine efficacy.CCL3L1-CCR5 genotypes may impact on the dynamics of the HIV epidemic and, consequently, the observed heterogeneous global distribution of HIV infection. As Ro is lowest when the infecting partner has beneficial CCL3L1-CCR5 genotypes, we infer that therapeutic vaccines directed towards reducing the infectivity of the host may play a role in halting epidemic spread. Further, CCL3L1-CCR5 genotype may provide critical guidance for optimizing the design and evaluation of HIV-1 vaccine trials and prevention programs

Innate immunity against HIV: a priority target for HIV prevention research

This review summarizes recent advances and current gaps in understanding of innate immunity to human immunodeficiency virus (HIV) infection, and identifies key scientific priorities to enable application of this knowledge to the development of novel prevention strategies (vaccines and microbicides). It builds on productive discussion and new data arising out of a workshop on innate immunity against HIV held at the European Commission in Brussels, together with recent observations from the literature