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Background The importance of events in acute HIV-1 infection (AHI) in determining the subsequent disease course prompts a need to understand the virus-immune system interactions in this phase of infection that impact on concurrent and ensuing viral replication and pathogenesis. The speed with which innate responses can be mobilized following pathogen exposure suggests they may play critical roles in AHI. We sought to characterise the innate responses activated during AHI and identify components of the response that contribute to control of viremia or conversely promote immune activation and virus replication.

Background Intrapartum administration of single-dose nevirapine (sdNVP) reduces perinatal HIV-1 transmission in resource-limiting settings by half. Yet this strategy has limited effect on subsequent breast milk transmission, making the case for new treatment approaches to extend maternal/infant antiretroviral prophylaxis through the period of lactation. Maternal and transmitted infant HIV-1 variants frequently develop NVP resistance mutations following sdNVP, complicating subsequent treatment/prophylaxis regimens. However, it is not clear whether NVP-resistant viruses are transmitted via breastfeeding or arise de novo in the infant. Findings We performed a detailed HIV genetic analysis using single genome sequencing to identify the origin of drug-resistant variants in an sdNVP-treated postnatally-transmitting mother-infant pair. Phylogenetic analysis of HIV sequences from the child revealed low-diversity variants indicating infection by a subtype C single transmitted/founder virus that shared full-length sequence identity with a clonally-amplified maternal breast milk virus variant harboring the K103N NVP resistance mutation. Conclusion In this mother/child pair, clonal amplification of maternal NVP-resistant HIV variants present in systemic and mammary gland compartments following intrapartum sdNVP represents one source of transmitted NVP-resistant variants that is responsible for the acquisition of drug resistant virus by the breastfeeding infant. This finding emphasizes the need for combination antiretroviral prophylaxis to prevent mother-to-child HIV transmission.

Background Breastfeeding is a leading cause of infant HIV-1 infection in the developing world, yet only a minority of infants exposed to HIV-1 via breastfeeding become infected. As a genetic bottleneck severely restricts the number of postnatally-transmitted variants, genetic or phenotypic properties of the virus Envelope (Env) could be important for the establishment of infant infection. We examined the efficiency of virologic functions required for initiation of infection in the gastrointestinal tract and the neutralization sensitivity of HIV-1 Env variants isolated from milk of three postnatally-transmitting mothers (n=13 viruses), five clinically-matched nontransmitting mothers (n=16 viruses), and seven postnatally-infected infants (n = 7 postnatally-transmitted/founder (T/F) viruses). Results There was no difference in the efficiency of epithelial cell interactions between Env virus variants from the breast milk of transmitting and nontransmitting mothers. Moreover, there was similar efficiency of DC-mediated trans-infection, CCR5-usage, target cell fusion, and infectivity between HIV-1 Env-pseudoviruses from nontransmitting mothers and postnatal T/F viruses. Milk Env-pseudoviruses were generally sensitive to neutralization by autologous maternal plasma and resistant to breast milk neutralization. Infant T/F Env-pseudoviruses were equally sensitive to neutralization by broadly-neutralizing monoclonal and polyclonal antibodies as compared to nontransmitted breast milk Env variants. Conclusion Postnatally-T/F Env variants do not appear to possess a superior ability to interact with and cross a mucosal barrier or an exceptional resistance to neutralization that define their capability to initiate infection across the infant gastrointestinal tract in the setting of preexisting maternal antibodies.

Background RV217/ECHO aims at capturing HIV-1 incident cases during the very earliest stages of acute HIV-1 infection (AHI). Limitations in single genome amplification(SGA) sampling depth have the potential to confound the study of multiplicity of infection and viral evolution during AHI. Here we combined SGA and deep, next-generation sequencing(NGS) to investigate the prevalence of minor viral variants during pre-peak viremia and characterize subsequent viral evolution.

Background A modest change in HIV-1 fitness can have a significant impact on viral quasispecies evolution and viral pathogenesis, transmission and disease progression. To determine the impact of immune escape mutations selected by cytotoxic T lymphocytes (CTL) on viral fitness in the context of the cognate transmitted/founder (T/F) genome, we developed a new competitive fitness assay using molecular clones of T/F genomes lacking exogenous genetic markers and a highly sensitive and precise parallel allele-specific sequencing (PASS) method. Results The T/F and mutant viruses were competed in CD4+ T-cell enriched cultures, relative proportions of viruses were assayed after repeated cell-free passage, and fitness costs were estimated by mathematical modeling. Naturally occurring HLA B57-restricted mutations involving the TW10 epitope in Gag and two epitopes in Tat/Rev and Env were assessed independently and together. Compensatory mutations which restored viral replication fitness were also assessed. A principal TW10 escape mutation, T242N, led to a 42% reduction in replication fitness but V247I and G248A mutations in the same epitope restored fitness to wild-type levels. No fitness difference was observed between the T/F and a naturally selected variant carrying the early CTL escape mutation (R355K) in Env and a reversion mutation in the Tat/Rev overlapping region. Conclusions These findings reveal a broad spectrum of fitness costs to CTL escape mutations in T/F viral genomes, similar to recent findings reported for neutralizing antibody escape mutations, and highlight the extraordinary plasticity and adaptive potential of the HIV-1 genome. Analysis of T/F genomes and their evolved progeny is a powerful approach for assessing the impact of composite mutational events on viral fitness.

Background HIV transmission most commonly occurs at mucosal surfaces, and vaccine development and other preventative strategies should be directed at the transmitted viruses that are responsible for establishing productive infection. The simian immunodeficiency virus (SIV) models of HIV infection use virus stocks for inoculation of monkeys at defined times. This greatly simplifies the process of identifying the transmitted or early founder virus populations, the variants evolving after transmission that establish systemic infection and the role of viral selection during sexual transmission.

Background Identification of the transmitted/founder virus in HIV-1 infection allows a unique opportunity to explore the earliest processes of immune pressure and escape. Through single genome amplification (SGA), we can identify the transmitted envelope (env), characterize the diversity of plasma viral quasispecies at subsequent time points, and characterize neutralizing antibody (Nab) responses.

Background Autologous neutralizing antibody responses against HIV1 have been shown to emerge a number of months after primary infection. It is not known why neutralizing antibodies against HIV-1 are relatively slow to appear. We use the SIV/rhesus macaque model to characterize the kinetics of autologous neutralizing antibody responses and envelope sequence evolution in 4 rhesus monkeys infected intrarectally with SIVmac251.

Background We have recently shown that transmitted/founder virus(es) can be identified unambiguously using single genome amplification (SGA) (Keele, PNAS, 2008; Salazar, JEM, 2009) and that in heterosexual transmissions approximately 80% of patients are infected by a single virus or infected cell (Keele, PNAS, 2008; Haaland, PLoS Pathogens, 2009; Abrahams, JVirol, 2009). Here we explore the characteristics of virus transmission in men who have sex with men (MSM) and injection drug users (IDU).

Background Previously, we employed site-directed mutagenesis to introduce HIV-1 4E10 and 2F5 epitopes into the corresponding region of a functional HIV-2 envelope glycoproteins (AIDS Vaccine 2005, abstract #A210). The resulting "chimeric" viruses were used to screen HIV-1 infected human plasma for 4E10 or 2F5-like neutralizing antibodies (Nabs). Among 177 subjects infected with HIV-1 representing ten different subtypes or circulating recombinant forms, none had significant Nab titers directed toward these epitopes.

Background Following mucosal human immunodeficiency virus type 1 (HIV-1) transmission, type 1 interferons (IFNs) are rapidly induced at sites of initial virus replication in the mucosa and draining lymph nodes. However, the role played by IFN-stimulated antiviral activity in restricting HIV-1 replication during the initial stages of infection is not clear. We hypothesized that if type 1 IFNs exert selective pressure on HIV-1 replication in the earliest stages of infection, the founder viruses that succeed in establishing systemic infection would be more IFN-resistant than viruses replicating during chronic infection, when type 1 IFNs are produced at much lower levels. To address this hypothesis, the relative resistance of virus isolates derived from HIV-1-infected individuals during acute and chronic infection to control by type 1 IFNs was analysed. Results The replication of plasma virus isolates generated from subjects acutely infected with HIV-1 and molecularly cloned founder HIV-1 strains could be reduced but not fully suppressed by type 1 IFNs in vitro. The mean IC50 value for IFNα2 (22 U/ml) was lower than that for IFNβ (346 U/ml), although at maximally-inhibitory concentrations both IFN subtypes inhibited virus replication to similar extents. Individual virus isolates exhibited differential susceptibility to inhibition by IFNα2 and IFNβ, likely reflecting variation in resistance to differentially up-regulated IFN-stimulated genes. Virus isolates from subjects acutely infected with HIV-1 were significantly more resistant to in vitro control by IFNα than virus isolates generated from the same individuals during chronic, asymptomatic infection. Viral IFN resistance declined rapidly after the acute phase of infection: in five subjects, viruses derived from six-month consensus molecular clones were significantly more sensitive to the antiviral effects of IFNs than the corresponding founder viruses. Conclusions The establishment of systemic HIV-1 infection by relatively IFNα-resistant founder viruses lends strong support to the hypothesis that IFNα plays an important role in the control of HIV-1 replication during the earliest stages of infection, prior to systemic viral spread. These findings suggest that it may be possible to harness the antiviral activity of type 1 IFNs in prophylactic and potentially also therapeutic strategies to combat HIV-1 infection.

Address: 1Los Alamos National Laboratory, Los Alamos, NM, USA, 2Dept. of Medicine, Univ. of Alabama at Birmingham, Birmingham, AL, USA, 3Group T-2, Los Alamos National Laboratory, Los Alamos, NM, USA, 4Group B-6, Los Alamos National Laboratory, Los Alamos, NM, USA, 5Depts. of Medicine and Microbiology, Univ. of Alabama at Birmingham, Birmingham, AL, USA, 6Division of Hematology/Oncology, Univ. of Alabama at Birmingham, Birmingham, AL, USA and 7Duke Human Vaccine Institute, Duke University Medical Center, Durham, NC, USA * Corresponding author

Address: 1Centers for Disease Control and Prevention, Atlanta, GA, USA, 2Zambia Blood Transfusion Service, Lusaka, Zambia, 3Public Health Research Institute, UMDNJ-New Jersey Medical School, Newark, NJ, USA, 4University of Alabama at Birmingham, Birmingham, USA, 5Los Alamos National Laboratory, Los Alamos, USA, 6Tulane University, New Orleans, LA, USA and 7Pathology and Laboratory Medicine, Emory University, Atlanta, GA, USA * Corresponding author

Methods Blood PCs from three AHI subjects obtained approximately 17, 20 and 30 days after HIV-1 transmission were sorted into 96-well plates for amplification of VH and VL genes by RT/PCR. The isolated VH and VL genes were expressed as recombinant IgG1 mAbs in 293T cells by transfection using linear Ig expression cassettes, The specificity of produced mAbs was determined by ELISA and luminex bead immunoassays against a panel of HIV-1 and non-HIV-1 antigens.