Association of HIV diversity and survival in HIV-infected Ugandan infants

Background

The level of viral diversity in an HIV-infected individual can change during the course of HIV infection, reflecting mutagenesis during viral replication and selection of viral variants by immune and other selective pressures. Differences in the level of viral diversity in HIV-infected infants may reflect differences in viral dynamics, immune responses, or other factors that may also influence HIV disease progression. We used a novel high resolution melting (HRM) assay to measure HIV diversity in Ugandan infants and examined the relationship between diversity and survival through 5 years of age.

Methods

Plasma samples were obtained from 31 HIV-infected infants (HIVNET 012 trial). The HRM assay was used to measure diversity in two regions in the gag gene (Gag1 and Gag2) and one region in the pol gene (Pol).

Results

HRM scores in all three regions increased with age from 6–8 weeks to 12–18 months (for Gag1: P = 0.005; for Gag2: P = 0.006; for Pol: P = 0.016). Higher HRM scores at 6–8 weeks of age (scores above the 75^th percentile) were associated with an increased risk of death by 5 years of age (for Pol: P = 0.005; for Gag1/Gag2 (mean of two scores): P = 0.003; for Gag1/Gag2/Pol (mean of three scores): P = 0.002). We did not find an association between HRM scores and other clinical and laboratory variables.

Conclusions

Genetic diversity in HIV gag and pol measured using the HRM assay was typically low near birth and increased over time. Higher HIV diversity in these regions at 6–8 weeks of age was associated with a significantly increased risk of death by 5 years of age.     

HIV-1 Tropism Dynamics and Phylogenetic Analysis from Longitudinal Ultra-Deep Sequencing Data of CCR5- and CXCR4-Using Variants

Objective

Coreceptor switch from CCR5 to CXCR4 is associated with HIV disease progression. The molecular and evolutionary mechanisms underlying the CCR5 to CXCR4 switch are the focus of intense recent research. We studied the HIV-1 tropism dynamics in relation to coreceptor usage, the nature of quasispecies from ultra deep sequencing (UDPS) data and their phylogenetic relationships.

Methods

Here, we characterized C2-V3-C3 sequences of HIV obtained from 19 patients followed up for 54 to 114 months using UDPS, with further genotyping and phylogenetic analysis for coreceptor usage. HIV quasispecies diversity and variability as well as HIV plasma viral load were measured longitudinally and their relationship with the HIV coreceptor usage was analyzed. The longitudinal UDPS data were submitted to phylogenetic analysis and sampling times and coreceptor usage were mapped onto the trees obtained.

Results

Although a temporal viral genetic structuring was evident, the persistence of several viral lineages evolving independently along the infection was statistically supported, indicating a complex scenario for the evolution of viral quasispecies. HIV X4-using variants were present in most of our patients, exhibiting a dissimilar inter- and intra-patient predominance as the component of quasispecies even on antiretroviral therapy. The viral populations from some of the patients studied displayed evidences of the evolution of X4 variants through fitness valleys, whereas for other patients the data favored a gradual mode of emergence.

Conclusions

CXCR4 usage can emerge independently, in multiple lineages, along the course of HIV infection. The mode of emergence, i.e. gradual or through fitness valleys seems to depend on both virus and patient factors. Furthermore, our analyses suggest that, besides becoming dominant after population-level switches, minor proportions of X4 viruses might exist along the infection, perhaps even at early stages of it. The fate of these minor variants might depend on both viral and host factors.     

Development of a low bias method for characterizing viral populations using next generation sequencing technology

Background

With an estimated 38 million people worldwide currently infected with human immunodeficiency virus (HIV), and an additional 4.1 million people becoming infected each year, it is important to understand how this virus mutates and develops resistance in order to design successful therapies.

Methodology/Principal Findings

We report a novel experimental method for amplifying full-length HIV genomes without the use of sequence-specific primers for high throughput DNA sequencing, followed by assembly of full length viral genome sequences from the resulting large dataset. Illumina was chosen for sequencing due to its ability to provide greater coverage of the HIV genome compared to prior methods, allowing for more comprehensive characterization of the heterogeneity present in the HIV samples analyzed. Our novel amplification method in combination with Illumina sequencing was used to analyze two HIV populations: a homogenous HIV population based on the canonical NL4-3 strain and a heterogeneous viral population obtained from a HIV patient''s infected T cells. In addition, the resulting sequence was analyzed using a new computational approach to obtain a consensus sequence and several metrics of diversity.

Significance

This study demonstrates how a lower bias amplification method in combination with next generation DNA sequencing provides in-depth, complete coverage of the HIV genome, enabling a stronger characterization of the quasispecies present in a clinically relevant HIV population as well as future study of how HIV mutates in response to a selective pressure.     

Insertion of Vaccinia Virus C7L Host Range Gene into NYVAC-B Genome Potentiates Immune Responses against HIV-1 Antigens

Background

The highly attenuated vaccinia virus strain NYVAC expressing HIV-1 components has been evaluated as a vaccine candidate in preclinical and clinical trials with encouraging results. We have previously described that the presence of C7L in the NYVAC genome prevents the induction of apoptosis and renders the vector capable of replication in human and murine cell lines while maintaining an attenuated phenotype in mice.

Methodology/Principal Findings

In an effort to improve the immunogenicity of NYVAC, we have developed a novel poxvirus vector by inserting the VACV host-range C7L gene into the genome of NYVAC-B, a recombinant virus that expresses four HIV-1 antigens from clade B (Env, Gag, Pol and Nef) (referred as NYVAC-B-C7L). In the present study, we have compared the in vitro and in vivo behavior of NYVAC-B and NYVAC-B-C7L. In cultured cells, NYVAC-B-C7L expresses higher levels of heterologous antigen than NYVAC-B as determined by Western blot and fluorescent-activated cell sorting to score Gag expressing cells. In a DNA prime/poxvirus boost approach with BALB/c mice, both recombinants elicited robust, broad and multifunctional antigen-specific T-cell responses to the HIV-1 immunogens expressed from the vectors. However, the use of NYVAC-B-C7L as booster significantly enhanced the magnitude of the T cell responses, and induced a more balanced cellular immune response to the HIV-1 antigens in comparison to that elicited in animals boosted with NYVAC-B.

Conclusions/Significance

These findings demonstrate the possibility to enhance the immunogenicity of the highly attenuated NYVAC vector by the insertion of the host-range gene C7L and suggest the use of this modified vector as an improved vaccine candidate against HIV/AIDS.     

A comparison of parallel pyrosequencing and sanger clone-based sequencing and its impact on the characterization of the genetic diversity of HIV-1

Background

Pyrosequencing technology has the potential to rapidly sequence HIV-1 viral quasispecies without requiring the traditional approach of cloning. In this study, we investigated the utility of ultra-deep pyrosequencing to characterize genetic diversity of the HIV-1 gag quasispecies and assessed the possible contribution of pyrosequencing technology in studying HIV-1 biology and evolution.

Methodology/Principal Findings

HIV-1 gag gene was amplified from 96 patients using nested PCR. The PCR products were cloned and sequenced using capillary based Sanger fluorescent dideoxy termination sequencing. The same PCR products were also directly sequenced using the 454 pyrosequencing technology. The two sequencing methods were evaluated for their ability to characterize quasispecies variation, and to reveal sites under host immune pressure for their putative functional significance. A total of 14,034 variations were identified by 454 pyrosequencing versus 3,632 variations by Sanger clone-based (SCB) sequencing. 11,050 of these variations were detected only by pyrosequencing. These undetected variations were located in the HIV-1 Gag region which is known to contain putative cytotoxic T lymphocyte (CTL) and neutralizing antibody epitopes, and sites related to virus assembly and packaging. Analysis of the positively selected sites derived by the two sequencing methods identified several differences. All of them were located within the CTL epitope regions.

Conclusions/Significance

Ultra-deep pyrosequencing has proven to be a powerful tool for characterization of HIV-1 genetic diversity with enhanced sensitivity, efficiency, and accuracy. It also improved reliability of downstream evolutionary and functional analysis of HIV-1 quasispecies.     

Non-Immunogenicity of Overlapping Gag Peptides Pulsed on Autologous Cells after Vaccination of HIV Infected Individuals

Background

HIV Gag-specific CD4+ and CD8+ T-cell responses are important for HIV immune control. Pulsing overlapping Gag peptides on autologous lymphocytes (OPAL) has proven immunogenic and effective in reducing viral loads in multiple pigtail macaque studies, warranting clinical evaluation.

Methodology

We performed a phase I, single centre, placebo-controlled, double-blinded and dose-escalating study to evaluate the safety and preliminary immunogenicity of a novel therapeutic vaccine approach ‘OPAL-HIV-Gag(c)’. This vaccine is comprised of 120 15mer peptides, overlapping by 11 amino acids, spanning the HIV Gag C clade sequence proteome, pulsed on white blood cells enriched from whole blood using a closed system, followed by intravenous reinfusion. Patients with undetectable HIV viral loads (<50 copies/ml plasma) on HAART received four administrations at week 0, 4, 8 and 12, and were followed up for 12 weeks post-treatment. Twenty-three people were enrolled in four groups: 12 mg (n = 6), 24 mg (n = 7), 48 mg (n = 2) or matching placebo (n = 8) with 18 immunologically evaluable. T-cell immunogenicity was assessed by IFNγ ELIspot and intracellular cytokine staining (ICS).

Results

The OPAL-HIV-Gag(c) peptides were antigenic in vitro in 17/17 subjects. After vaccination with OPAL-HIV-Gag(c), 1/6 subjects at 12 mg and 1/6 subjects at 24 mg dose groups had a 2- and 3-fold increase in ELIspot magnitudes from baseline, respectively, of Gag-specific CD8+ T-cells at week 14, compared to 0/6 subjects in the placebo group. No Gag-specific CD4+ T-cell responses or overall change in Rev, Nef, Tat and CMV specific responses were detected. Marked, transient and self-limiting lymphopenia was observed immediately post-vaccination (4 hours) in OPAL-HIV-Gag(c) but not in placebo recipients, with median fall from 1.72 to 0.67 million lymphocytes/mL for active groups (P<0.001), compared to post-placebo from 1.70 to 1.56 lymphocytes/ml (P = 0.16).

Conclusion/Significance

Despite strong immunogenicity observed in several Macaca nemestrina studies using this approach, OPAL-HIV-Gag(c) was not significantly immunogenic in humans and improved methods of generating high-frequency Gag-specific T-cell responses are required.

Name of Registry

ClinicalTrials.gov, Registry number: {"type":"clinical-trial","attrs":{"text":"NCT01123915","term_id":"NCT01123915"}}NCT01123915, URL trial registry database: http://www.clinicaltrials.gov/ct2/results?term=OPAL-HIV-1001&Search=Search     

Analysis of the genetic diversity of influenza A viruses using next-generation DNA sequencing

Background

Influenza viruses exist as a large group of closely related viral genomes, also called quasispecies. The composition of this influenza viral quasispecies can be determined by an accurate and sensitive sequencing technique and data analysis pipeline. We compared the suitability of two benchtop next-generation sequencers for whole genome influenza A quasispecies analysis: the Illumina MiSeq sequencing-by-synthesis and the Ion Torrent PGM semiconductor sequencing technique.

Results

We first compared the accuracy and sensitivity of both sequencers using plasmid DNA and different ratios of wild type and mutant plasmid. Illumina MiSeq sequencing reads were one and a half times more accurate than those of the Ion Torrent PGM. The majority of sequencing errors were substitutions on the Illumina MiSeq and insertions and deletions, mostly in homopolymer regions, on the Ion Torrent PGM. To evaluate the suitability of the two techniques for determining the genome diversity of influenza A virus, we generated plasmid-derived PR8 virus and grew this virus in vitro. We also optimized an RT-PCR protocol to obtain uniform coverage of all eight genomic RNA segments. The sequencing reads obtained with both sequencers could successfully be assembled de novo into the segmented influenza virus genome. After mapping of the reads to the reference genome, we found that the detection limit for reliable recognition of variants in the viral genome required a frequency of 0.5% or higher. This threshold exceeds the background error rate resulting from the RT-PCR reaction and the sequencing method. Most of the variants in the PR8 virus genome were present in hemagglutinin, and these mutations were detected by both sequencers.

Conclusions

Our approach underlines the power and limitations of two commonly used next-generation sequencers for the analysis of influenza virus gene diversity. We conclude that the Illumina MiSeq platform is better suited for detecting variant sequences whereas the Ion Torrent PGM platform has a shorter turnaround time. The data analysis pipeline that we propose here will also help to standardize variant calling in small RNA genomes based on next-generation sequencing data.     

Optimal antigens for HIV vaccines based on CD8+ T response,protein length,and sequence variability

The identification of optimal antigens to include in an HIV vaccine designed to elicit a cellular immune response requires careful consideration of protein length, variability, and immunogenicity. Here, we have examined the relationship of these parameters in a cohort of HIV-infected subjects. We find that HIV Gag and Nef represent optimal antigens for the CD8+ T cell response, based on size, variability, and immunogenicity. Although the Env and Pol proteins have a poorer response to length (Pol) or variability (Env) ratio than Gag or Nef, they are still strong candidates for inclusion into an HIV vaccine, based on overall response frequency in the cohort. The accessory proteins Tat, Rev, Vif, Vpr, and Vpu in general all elicit very low CD8+ T cell responses, and this, in combination with their high variability, makes them less attractive as vaccine antigen.     

Improving the MVA vaccine potential by deleting the viral gene coding for the IL-18 binding protein

Background

Modified Vaccinia Ankara (MVA) is an attenuated strain of Vaccinia virus (VACV) currently employed in many clinical trials against HIV/AIDS and other diseases. MVA still retains genes involved in host immune response evasion, enabling its optimization by removing some of them. The aim of this study was to evaluate cellular immune responses (CIR) induced by an IL-18 binding protein gene (C12L) deleted vector (MVAΔC12L).

Methodology/Principal Findings

BALB/c and C57BL/6 mice were immunized with different doses of MVAΔC12L or MVA wild type (MVAwt), then CIR to VACV epitopes in immunogenic proteins were evaluated in spleen and draining lymph nodes at acute and memory phases (7 and 40 days post-immunization respectively). Compared with parental MVAwt, MVAΔC12L immunization induced a significant increase of two to three-fold in CD8⁺ and CD4⁺ T-cell responses to different VACV epitopes, with increased percentage of anti-VACV cytotoxic CD8⁺ T-cells (CD107a/b⁺) during the acute phase of the response. Importantly, the immunogenicity enhancement was also observed after MVAΔC12L inoculation with different viral doses and by distinct routes (systemic and mucosal). Potentiation of MVA''s CIR was also observed during the memory phase, in correlation with a higher protection against an intranasal challenge with VACV WR. Of note, we could also show a significant increase in the CIR against HIV antigens such as Env, Gag, Pol and Nef from different subtypes expressed from two recombinants of MVAΔC12L during heterologous DNA prime/MVA boost vaccination regimens.

Conclusions/Significance

This study demonstrates the relevance of IL-18 bp contribution in the immune response evasion during MVA infection. Our findings clearly show that the deletion of the viral IL-18 bp gene is an effective approach to increase MVA vaccine efficacy, as immunogenicity improvements were observed against vector antigens and more importantly to HIV antigens.     

Use of RT-Defective HIV Virions: New Tool to Evaluate Specific Response in Chronic Asymptomatic HIV-Infected Individuals

Background

Generation of new reagents that can be used to screen or monitor HIV-1-specific responses constituted an interesting field in the development of HIV vaccines to improve their efficacy.

Methods

We have evaluated the specific T cell response against different types of NL4-3 virions (including NL4-3 aldrithiol-2 treated, NL4-3/ΔRT and R5 envelopes: NL4-3/ΔRT/ΔEnv[AC10] and NL4-3/ΔRT/ΔEnv[Bal]) and against pools of overlapping peptides (15 mer) encompassing the HIV-1 Gag and Nef regions. Cryopreserved PBMC from a subset of 69 chronic asymptomatic HIV positive individuals have been employed using different techniques including IFN-γ ELISPOT assay, surface activation markers and intracellular cytokine staining (ICS) by flow cytometry.

Results

The differential response obtained against NL4-3 aldrithiol-2 treated and NL4-3/ΔRT virions (25% vs 55%, respectively) allow us to divide the population in three groups: “full-responders” (positive response against both viral particles), “partial-responders” (positive response only against NL4-3/ΔRT virions) and “non-responders” (negative responses). There was no difference between X4 and R5 envelopes. The magnitude of the total responses was higher against NL4-3/ΔRT and was positively correlated with gender and inverse correlated with viral load. On the contrary CD4+ T cell count was not associated with this response. In any case responses to the viruses tended to be lower in magnitude than those detected by the overlapping peptides tested. Finally we have found an increased frequency of HLA-B27 allele (23% vs 9%) and a significant reduction in some activation markers (CD69 and CD38) on T cells surface in responders vs non-responders individuals.

Conclusions

In summary these virions could be considered as alternative and useful reagents for screening HIV-1-specific T cell responses in HIV exposed uninfected people, HIV infected patients and to assess immunogenicity of new prototypes both in vitro and in vaccine trials, by a feasible, simply, effective and low cost assay.     

A Flow Cytometry-Based FRET Assay to Identify and Analyse Protein-Protein Interactions in Living Cells

Background

Försters resonance energy transfer (FRET) microscopy is widely used for the analysis of protein interactions in intact cells. However, FRET microscopy is technically challenging and does not allow assessing interactions in large cell numbers. To overcome these limitations we developed a flow cytometry-based FRET assay and analysed interactions of human and simian immunodeficiency virus (HIV and SIV) Nef and Vpu proteins with cellular factors, as well as HIV Rev multimer-formation.

Results

Amongst others, we characterize the interaction of Vpu with CD317 (also termed Bst-2 or tetherin), a host restriction factor that inhibits HIV release from infected cells and demonstrate that the direct binding of both is mediated by the Vpu membrane-spanning region. Furthermore, we adapted our assay to allow the identification of novel protein interaction partners in a high-throughput format.

Conclusion

The presented combination of FRET and FACS offers the precious possibility to discover and define protein interactions in living cells and is expected to contribute to the identification of novel therapeutic targets for treatment of human diseases.     

Sexually-Transmitted/Founder HIV-1 Cannot Be Directly Predicted from Plasma or PBMC-Derived Viral Quasispecies in the Transmitting Partner

Objective

Characterization of HIV-1 sequences in newly infected individuals is important for elucidating the mechanisms of viral sexual transmission. We report the identification of transmitted/founder viruses in eight pairs of HIV-1 sexually-infected patients enrolled at the time of primary infection (“recipients”) and their transmitting partners (“donors”).

Methods

Using a single genome-amplification approach, we compared quasispecies in donors and recipients on the basis of 316 and 376 C2V5 env sequences amplified from plasma viral RNA and PBMC-associated DNA, respectively.

Results

Both DNA and RNA sequences indicated very homogeneous viral populations in all recipients, suggesting transmission of a single variant, even in cases of recent sexually transmitted infections (STIs) in donors (n = 2) or recipients (n = 3). In all pairs, the transmitted/founder virus was derived from an infrequent variant population within the blood of the donor. The donor variant sequences most closely related to the recipient sequences were found in plasma samples in 3/8 cases and/or in PBMC samples in 6/8 cases. Although donors were exclusively (n = 4) or predominantly (n = 4) infected by CCR5-tropic (R5) strains, two recipients were infected with highly homogeneous CXCR4/dual-mixed-tropic (X4/DM) viral populations, identified in both DNA and RNA. The proportion of X4/DM quasispecies in donors was higher in cases of X4/DM than R5 HIV transmission (16.7–22.0% versus 0–2.6%), suggesting that X4/DM transmission may be associated with a threshold population of X4/DM circulating quasispecies in donors.

Conclusions

These suggest that a severe genetic bottleneck occurs during subtype B HIV-1 heterosexual and homosexual transmission. Sexually-transmitted/founder virus cannot be directly predicted by analysis of the donor’s quasispecies in plasma and/or PBMC. Additional studies are required to fully understand the traits that confer the capacity to transmit and establish infection, and determine the role of concomitant STIs in mitigating the genetic bottleneck in mucosal HIV transmission.     

Trans-cellular introduction of HIV-1 protein Nef induces pathogenic response in Caenorhabditis elegans

Background

Caenorhabditis elegans has emerged as a very powerful model for studying the host pathogen interactions. Despite the absence of a naturally occurring viral infection for C. elegans, the model is now being exploited experimentally to study the basic aspects of virus-host interplay. The data generated from recent studies suggests that the virus that infects mammalian cells does infect, replicate and accumulate in C. elegans.

Methodology/Principal Findings

We took advantage of the easy-to-achieve protein introduction in C. elegans and employing the methodology, we administered HIV-1 protein Nef into live worms. Nef is known to be an important protein for exacerbating HIV-1 pathogenesis in host by enhancing viral replication. The deletion of nef from the viral genome has been reported to inhibit its replication in the host, thereby leading to delayed pathogenesis. Our studies, employing Nef introduction into C. elegans, led to creation of an in-vivo model that allowed us to study, whether or not, the protein induces effect in the whole organism. We observed a marked lipodystrophy, effect on neuromuscular function, impaired fertility and reduced longevity in the worms exposed to Nef. The observed effects resemble to those observed in Nef transgenic mice and most interestingly the effects also relate to some of the pathogenic aspects exhibited by human AIDS patients.

Conclusions/Significance

Our studies underline the importance of this in vivo model for studying the interactions of Nef with host proteins, which could further be used for identifying possible inhibitors of such interactions.     

Presence of IgG Anti-gp160/120 Antibodies Confers Higher HIV Capture Capacity to Erythrocytes from HIV-Positive Individuals

Background

HIV binding has been demonstrated in erythrocytes from HIV-positive and HIV-negative individuals. However, the presence of immunoglobulins G anti-HIV (IgG anti-HIV) in erythrocytes from HIV-positive individuals is still to be elucidated. Moreover, the capacity of erythrocytes from HIV-positive individuals to capture an additional amount of HIV has not been studied. Indeed, it is unknown if HIV binding to erythrocytes in HIV-positive persons could have consequences on the cell-free infectious virus available.

Methodology/Principal Findings

IgGs anti-HIV associated to erythrocytes were found in 77.3% (58/75) of the HIV-positive individuals studied and the IgGs anti-gp160 and anti-p24 were the most frequently found. We found a positive association between detectable plasma viral load (pVL) and presence of IgGs anti-HIV associated to erythrocyte (p<0.005), though the anti-p24/160 were present with or without detectable pVL. The HIV capture capacity was higher in erythrocytes from HIV-positive than HIV-negative individuals (p<0.0001). Furthermore, among the HIV-positive individuals the higher viral capture capacity was associated with the presence of anti-gp160/gp120 on erythrocytes. Moreover, the viral capture by erythrocytes was independent of pVL (rho = 0.022, p = 0.8817). Additionally, reduction of cell-free infectious virus and available viral load was observed in the presence of erythrocytes from HIV-positive individuals.

Conclusions/Significance

Results suggest that in HIV-positive individuals, erythrocytes are capable of capturing high amounts of HIV by the presence of IgGs anti-gp160/120 on their membranes and this may produce a reduction in the available free virus. Finally, the current measurement of pVL would underestimate the real viral quantity due to the HIV binding through specific antibodies to erythrocytes.     

Transient Nature of Long-Term Nonprogression and Broad Virus-Specific Proliferative T-Cell Responses with Sustained Thymic Output in HIV-1 Controllers

Background

HIV-1⁺ individuals who, without therapy, conserve cellular anti-HIV-1 responses, present with high, stable CD4⁺ T-cell numbers, and control viral replication, facilitate analysis of atypical viro-immunopathology. In the absence of universal definition, immune function in such HIV controllers remains an indication of non-progression.

Methodology/Principal Findings

CD4 T-cell responses to a number of HIV-1 proteins and peptide pools were assessed by IFN-γ ELISpot and lymphoproliferative assays in HIV controllers and chronic progressors. Thymic output was assessed by sjTRECs levels. Follow-up of 41 HIV-1⁺ individuals originally identified as “Long-term non-progressors” in 1996 according to clinical criteria, and longitudinal analysis of two HIV controllers over 22 years, was also performed. HIV controllers exhibited substantial IFN-γ producing and proliferative HIV-1-specific CD4 T-cell responses to both recombinant proteins and peptide pools of Tat, Rev, Nef, Gag and Env, demonstrating functional processing and presentation. Conversely, HIV-specific T-cell responses were limited to IFN-γ production in chronic progressors. Additionally, thymic output was approximately 19 fold higher in HIV controllers than in age-matched chronic progressors. Follow-up of 41 HIV-1⁺ patients identified as LTNP in 1996 revealed the transitory characteristics of this status. IFN-γ production and proliferative T-cell function also declines in 2 HIV controllers over 22 years.

Conclusions

Although increased thymic output and anti-HIV-1 T-cell responses are observed in HIV controllers compared to chronic progressors, the nature of nonprogressor/controller status appears to be transitory.     

HIV-1 Nef induces proinflammatory state in macrophages through its acidic cluster domain: involvement of TNF alpha receptor associated factor 2

Background

HIV-1 Nef is a virulence factor that plays multiple roles during HIV replication. Recently, it has been described that Nef intersects the CD40 signalling in macrophages, leading to modification in the pattern of secreted factors that appear able to recruit, activate and render T lymphocytes susceptible to HIV infection. The engagement of CD40 by CD40L induces the activation of different signalling cascades that require the recruitment of specific tumor necrosis factor receptor-associated factors (i.e. TRAFs). We hypothesized that TRAFs might be involved in the rapid activation of NF-κB, MAPKs and IRF-3 that were previously described in Nef-treated macrophages to induce the synthesis and secretion of proinflammatory cytokines, chemokines and IFNβ to activate STAT1, -2 and -3.

Methodology/Principal Findings

Searching for possible TRAF binding sites on Nef, we found a TRAF2 consensus binding site in the AQEEEE sequence encompassing the conserved four-glutamate acidic cluster. Here we show that all the signalling effects we observed in Nef treated macrophages depend on the integrity of the acidic cluster. In addition, Nef was able to interact in vitro with TRAF2, but not TRAF6, and this interaction involved the acidic cluster. Finally silencing experiments in THP-1 monocytic cells indicate that both TRAF2 and, surprisingly, TRAF6 are required for the Nef-induced tyrosine phosphorylation of STAT1 and STAT2.

Conclusions

Results reported here revealed TRAF2 as a new possible cellular interactor of Nef and highlighted that in monocytes/macrophages this viral protein is able to manipulate both the TRAF/NF-κB and TRAF/IRF-3 signalling axes, thereby inducing the synthesis of proinflammatory cytokines and chemokines as well as IFNβ.     

Broad and Gag-biased HIV-1 epitope repertoires are associated with lower viral loads

Background

HLA class-I alleles differ in their ability to control HIV replication through cell-mediated immune responses. No consistent associations have been found between the breadth of Cytotoxic T Lymphocytes (CTL) responses and the control of HIV-1, and it is unknown whether the size or distribution of the viral proteome-wide epitope repertoire, i.e., the intrinsic ability to present fewer, more or specific viral epitopes, could affect clinical markers of disease progression.

Methodology/Principal Findings

We used an epitope prediction model to identify all epitope motifs in a set of 302 HIV-1 full-length proteomes according to each individual''s HLA (Human Leukocyte Antigen) genotype. The epitope repertoire, i.e., the number of predicted epitopes per HIV-1 proteome, varied considerably between HLA alleles and thus among individual proteomes. In a subgroup of 270 chronically infected individuals, we found that lower viral loads and higher CD4 counts were associated with a larger predicted epitope repertoire. Additionally, in Gag and Rev only, more epitopes were restricted by alleles associated with low viral loads than by alleles associated with higher viral loads.

Conclusions/Significance

This comprehensive analysis puts forth the epitope repertoire as a mechanistic component of the multi-faceted HIV-specific CTL response. The favorable impact on markers of disease status of the propensity to present more HLA binding peptides and specific proteins gives impetus to vaccine design strategies that seek to elicit responses to a broad array of HIV-1 epitopes, and suggest a particular focus on Gag.     

HLA-Associated Immune Escape Pathways in HIV-1 Subtype B Gag,Pol and Nef Proteins

Background

Despite the extensive genetic diversity of HIV-1, viral evolution in response to immune selective pressures follows broadly predictable mutational patterns. Sites and pathways of Human Leukocyte-Antigen (HLA)-associated polymorphisms in HIV-1 have been identified through the analysis of population-level data, but the full extent of immune escape pathways remains incompletely characterized. Here, in the largest analysis of HIV-1 subtype B sequences undertaken to date, we identify HLA-associated polymorphisms in the three HIV-1 proteins most commonly considered in cellular-based vaccine strategies. Results are organized into protein-wide escape maps illustrating the sites and pathways of HLA-driven viral evolution.

Methodology/Principal Findings

HLA-associated polymorphisms were identified in HIV-1 Gag, Pol and Nef in a multicenter cohort of >1500 chronically subtype-B infected, treatment-naïve individuals from established cohorts in Canada, the USA and Western Australia. At q≤0.05, 282 codons commonly mutating under HLA-associated immune pressures were identified in these three proteins. The greatest density of associations was observed in Nef (where close to 40% of codons exhibited a significant HLA association), followed by Gag then Pol (where ∼15–20% of codons exhibited HLA associations), confirming the extensive impact of immune selection on HIV evolution and diversity. Analysis of HIV codon covariation patterns identified over 2000 codon-codon interactions at q≤0.05, illustrating the dense and complex networks of linked escape and secondary/compensatory mutations.

Conclusions/Significance

The immune escape maps and associated data are intended to serve as a user-friendly guide to the locations of common escape mutations and covarying codons in HIV-1 subtype B, and as a resource facilitating the systematic identification and classification of immune escape mutations. These resources should facilitate research in HIV epitope discovery and host-pathogen co-evolution, and are relevant to the continued search for an effective CTL-based AIDS vaccine.