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Large datasets including an extensive number of covariates are generated these days in many different situations, for instance, in detailed genetic studies of outbreed human populations or in complex analyses of immune responses to different infections. Aiming at informing clinical interventions or vaccine design, methods for variable selection identifying those variables with the optimal prediction performance for a specific outcome are crucial. However, testing for all potential subsets of variables is not feasible and alternatives to existing methods are needed. Here, we describe a new method to handle such complex datasets, referred to as FARMS, that combines forward and all subsets regression for model selection. We apply FARMS to a host genetic and immunological dataset of over 800 individuals from Lima (Peru) and Durban (South Africa) who were HIV infected and tested for antiviral immune responses. This dataset includes more than 500 explanatory variables: around 400 variables with information on HIV immune reactivity and around 100 individual genetic characteristics. We have implemented FARMS in R statistical language and we showed that FARMS is fast and outcompetes other comparable commonly used approaches, thus providing a new tool for the thorough analysis of complex datasets without the need for massive computational infrastructure., Peer Reviewed, Postprint (published version)

Objective: The objective of this study is to identify human leukocyte antigen (HLA) class I and killer-cell immunoglobulin-like receptor (KIR) genotypes associated with different risks for HIV acquisition and HIV disease progression.; Design: A cross-sectional study of a cohort of 468 high-risk individuals (246 HIV-positive and 222 HIV-negative) from outpatient clinics in Lima (Peru).; Methods: The cohort was high-resolution HLA and KIR-typed and analysed for potential differences in single-allele frequencies and allele combinations between HIV-positive and HIV-negative individuals and for associations with HIV viral load and CD4(+) cell counts in infected individuals.; Results: HLA class I alleles associated with a lack of viral control had a significantly higher population frequency than relatively protective alleles (P = 0.0093), in line with a rare allele advantage. HLA-A*02 : 01 and HLA-C*04 : 01 were both associated with high viral loads (P = 0.0313 and 0.0001, respectively) and low CD4(+) cell counts (P = 0.0008 and 0.0087, respectively). Importantly, the association between HLA-C*04 : 01 and poor viral control was not due to its linkage disequilibrium with other HLA alleles. Rather, the coexpression of its putative KIR ligand KIR2DS4f was critically linked to elevated viral loads.; Conclusion: These results highlight the impact of population allele frequency on viral control and identify a novel association between HLA-C*04 : 01 in combination with KIR2DS4f and uncontrolled HIV infection. Our data further support the importance of the interplay of markers of the adaptive and innate immune system in viral control. Copyright (C) 2015 Wolters Kluwer Health, Inc. All rights reserved., Postprint (author's final draft)

Pathogen-induced immune responses prevent the establishment of transplantation tolerance in experimental animal models. Whether this occurs in humans as well remains unclear. The development of operational tolerance in liver transplant recipients with chronic hepatitis C virus (HCV) infection allows us to address this question. We conducted a clinical trial of immunosuppression withdrawal in HCV-infected adult liver recipients to elucidate (i) the mechanisms through which allograft tolerance can be established in the presence of an ongoing inflammatory response and (ii) whether anti-HCV heterologous immune responses influence this phenomenon. Of 34 enrolled liver recipients, drug withdrawal was successful in 17 patients (50%). Tolerance was associated with intrahepatic overexpression of type I interferon and immunoregulatory genes and with an expansion of exhausted PD1/CTLA4/2B4-positive HCV-specific circulating CD8(+) T cells. These findings were already present before immunosuppression was discontinued and were specific for HCV infection. In contrast, the magnitude of HCV-induced proinflammatory gene expression and the breadth of anti-HCV effector T cell responses did not influence drug withdrawal outcome. Our data suggest that in humans, persistent viral infections exert immunoregulatory effects that could contribute to the restraining of alloimmune responses, and do not necessarily preclude the development of allograft tolerance.

Genetic polymorphisms within the MHC encoding region have the strongest impact on HIV disease progression of any in the human genome and provide important clues to the mechanisms of HIV immune control. Few analyses have been undertaken of HLA alleles associated with rapid disease progression. HLA-B*07:02 is an HLA class I molecule that is prevalent in most populations worldwide and that has previously been consistently linked to accelerated disease progression in B-clade infection. This study investigates the observation that HLA-B*07:02 is not associated with a high viral setpoint in C-clade infection. We examine the hypothesis that this clade-specific difference in association with disease outcome may be related to distinct targeting of CD8(+) T cell epitopes. We observed that C-clade-infected individuals with HLA-B*07:02 target a broader range of Gag epitopes, and to higher magnitudes, than do individuals infected with B-clade infection. In particular, a novel p17-Gag (Gag22-30, RPGGKKHYM) epitope is targeted in >50% of HLA-B*07:02-positive C-clade-infected individuals but clade-specific differences in this epitope result in nonimmunogenicity in B-clade infection. Only the C-clade p24-Gag "GL9" (Gag355-363, GPSHKARVL) epitope-specific CD8(+) T cell response out of 16 studied was associated with a low viral setpoint. Although this epitope was also targeted in B-clade infection, the escape mutant S357S is present at higher frequency in B-clade infection than in C-clade infection (70% versus 43% in HLA-B*07:02-negative subjects). These data support earlier studies suggesting that increased breadth of the Gag-specific CD8(+) T cell response may contribute to improved HIV immune control irrespective of the particular HLA molecules expressed.

Aquesta tesi doctoral té com a objectiu caracteritzar millor les respostes VIH específiques de les cèl·lules T (CTL), identificar les dianes virals implicades en el control del virus i desenvolupar com aquesta informació s'ha incorporat en el disseny d'un inmunogen per a una futura vacuna contra el VIH. S'inicia amb una introducció actualitzada i àmpliament referenciada sobre el VIH/sida, inclosos aspectes històrics i epidemiològics de l'epidèmia, així com una visió des de la virologia i la immunologia de les dificultats en el desenvolupament de vacunes eficaces contra el VIH. En el primer capítol, es revisa la interacció entre factors virals, característiques genètiques de l'hoste i mecanismes immunològics implicats en el control del VIH. S'han incorporat les troballes més recents sobre aspectes immunològics -especialment en CTL- correlacionats amb l'evolució de la malaltia. A més, s'exposen els nous coneixements sobre com l'evolució viral és modula per diferències en la freqüència de característiques genètiques de l'hoste en les diferents regions del món. Es desenvolupa alhora la importància de discernir millor entre causa i efecte dels marcadors immunològics associats amb el control relatiu de la infecció pel VIH, doncs els factors que són un mer reflex de la replicació continguda del virus per altres motius poden induir a errors de concepte que s'incorporin al disseny de noves vacunes. Al llarg del segon capítol, s'exposa la identificació de 26 dianes virals (OLP) del VIH més potents per induir respostes CTL associades amb un millor control de la replicació viral en tres cohorts molt àmplies de pacients infectats. Es demostra que l'efecte dels OLP identificats en les càrregues virals dels pacients és tan fort com l'associat a la influència de l' HLA individual. Alhora, les dianes identificades com a beneficioses van resultar trobar-se en regions més conservades dins del genoma viral, fet que està en relació amb treballs d'altres grups enfocats en els elements co, This doctoral thesis aims to better characterize HIV specific T cell responses, define the viral targets most implicated in mediating viral control and discuss how this information has been incorporated into a rational immunogen design for a future HIV vaccine. A well-researched and referenced introduction to HIV, including historical and epidemiological aspects of the infection as well as virological and immunological overview of the vaccine development field challenges is firstly provided. In the first Chapter, a review of the important interplay between viral factors, host genetic and immune characteristics in HIV control is given. Some novel insights on immune correlates –specially on CTLs- elucidated over the last years to complete the work compiled in Chapter 1 are discussed. Also, new data is presenting regarding how viral evolution is shaped by differences in the frequency of different host genetic markers within different regions. New concepts to better discern between cause and effect of immune parameters associated with relatively controlled HIV infection will be discussed, as the factors that are a mere reflection of otherwise contained replication can mislead vaccine design. Throughout the second Chapter, the most potent viral targets of the virus-specific T cell responses in controlling viral replication were identified in 26 regions over the HIV proteome. The relative effects of host genetics (i.e. HLA) and CTL specificity on HIV-1 control were assessed in large cohorts of HIV infected individuals. Importantly, the effect of T cell specificity towards the identified regions on viral loads was shown to be at least as strong as the associated with host HLA genetics. These preferred targets turned out to be in the most conserved regions of the viral genome, which is in accordance with other investigators work developing novel vaccine concepts focused on conserved elements. An important contribution of our work is the incorporation of a significant amount

The strongest genetic influence on immune control in HIV-1 infection is the HLA class I genotype. Rapid disease progression in B-clade infection has been linked to HLA-B*35 expression, in particular to the less common HLA-B*3502 and HLA-B*3503 subtypes but also to the most prevalent subtype, HLA-B*3501. In these studies we first demonstrated that whereas HLA-B*3501 is associated with a high viral set point in two further B-clade-infected cohorts, in Japan and Mexico, this association does not hold in two large C-clade-infected African cohorts. We tested the hypothesis that clade-specific differences in HLA associations with disease outcomes may be related to distinct targeting of critical CD8(+) T-cell epitopes. We observed that only one epitope was significantly targeted differentially, namely, the Gag-specific epitope NPPIPVGDIY (NY10, Gag positions 253 to 262) (P = 2 × 10(-5)). In common with two other HLA-B*3501-restricted epitopes, in Gag and Nef, that were not targeted differentially, a response toward NY10 was associated with a significantly lower viral set point. Nonimmunogenicity of NY10 in B-clade-infected subjects derives from the Gag-D260E polymorphism present in ∼90% of B-clade sequences, which critically reduces recognition of the Gag NY10 epitope. These data suggest that in spite of any inherent HLA-linked T-cell receptor repertoire differences that may exist, maximizing the breadth of the Gag-specific CD8(+) T-cell response, by the addition of even a single epitope, may be of overriding importance in achieving immune control of HIV infection. This distinction is of direct relevance to development of vaccines designed to optimize the anti-HIV CD8(+) T-cell response in all individuals, irrespective of HLA type.

Deep sequencing technologies have the potential to transform the study of highly variable viral pathogens by providing a rapid and cost-effective approach to sensitively characterize rapidly evolving viral quasispecies. Here, we report on a high-throughput whole HIV-1 genome deep sequencing platform that combines 454 pyrosequencing with novel assembly and variant detection algorithms. In one subject we combined these genetic data with detailed immunological analyses to comprehensively evaluate viral evolution and immune escape during the acute phase of HIV-1 infection. The majority of early, low frequency mutations represented viral adaptation to host CD8+ T cell responses, evidence of strong immune selection pressure occurring during the early decline from peak viremia. CD8+ T cell responses capable of recognizing these low frequency escape variants coincided with the selection and evolution of more effective secondary HLA-anchor escape mutations. Frequent, and in some cases rapid, reversion of transmitted mutations was also observed across the viral genome. When located within restricted CD8 epitopes these low frequency reverting mutations were sufficient to prime de novo responses to these epitopes, again illustrating the capacity of the immune response to recognize and respond to low frequency variants. More importantly, rapid viral escape from the most immunodominant CD8+ T cell responses coincided with plateauing of the initial viral load decline in this subject, suggestive of a potential link between maintenance of effective, dominant CD8 responses and the degree of early viremia reduction. We conclude that the early control of HIV-1 replication by immunodominant CD8+ T cell responses may be substantially influenced by rapid, low frequency viral adaptations not detected by conventional sequencing approaches, which warrants further investigation. These data support the critical need for vaccine-induced CD8+ T cell responses to target more highly constrained re

Background The efficacy of the CTL component of a future HIV-1 vaccine will depend on the induction of responses with the most potent antiviral activity and broad HLA class I restriction. However, current HIV vaccine designs are largely based on viral sequence alignments only, not incorporating experimental data on T cell function and specificity. Methods Here, 950 untreated HIV-1 clade B or -C infected individuals were tested for responses to sets of 410 overlapping peptides (OLP) spanning the entire HIV-1 proteome. For each OLP, a "protective ratio" (PR) was calculated as the ratio of median viral loads (VL) between OLP non-responders and responders. Results For both clades, there was a negative relationship between the PR and the entropy of the OLP sequence. There was also a significant additive effect of multiple responses to beneficial OLP. Responses to beneficial OLP were of significantly higher functional avidity than responses to non-beneficial OLP. They also had superior in-vitro antiviral activities and, importantly, were at least as predictive of individuals' viral loads than their HLA class I genotypes. Conclusions The data thus identify immunogen sequence candidates for HIV and provide an approach for T cell immunogen design applicable to other viral infections.

Background Despite high potential for HIV-1 genetic variation, the emergence of some mutations is constrained by fitness costs, and may be associated with compensatory amino acid (AA) co-variation. To characterize the interplay between Cytotoxic T Lymphocyte (CTL)-mediated pressure and HIV-1 evolutionary pathways, we investigated AA co-variation in Gag sequences obtained from 449 South African individuals chronically infected with HIV-1 subtype C. Methodology/Principal Findings Individuals with CTL responses biased toward Gag presented lower viral loads than individuals with under-represented Gag-specific CTL responses. Using methods that account for founder effects and HLA linkage disequilibrium, we identified 35 AA sites under Human Leukocyte Antigen (HLA)-restricted CTL selection pressure and 534 AA-to-AA interactions. Analysis of two-dimensional distances between co-varying residues revealed local stabilization mechanisms since 40% of associations involved neighboring residues. Key features of our co-variation analysis included sites with a high number of co-varying partners, such as HLA-associated sites, which had on average 55% more connections than other co-varying sites. Conclusions/Significance Clusters of co-varying AA around HLA-associated sites (especially at typically conserved sites) suggested that cooperative interactions act to preserve the local structural stability and protein function when CTL escape mutations occur. These results expose HLA-imprinted HIV-1 polymorphisms and their interlinked mutational paths in Gag that are likely due to opposite selective pressures from host CTL-mediated responses and viral fitness constraints., Bill & Melinda Gates Foundation (#43437), American Foundation for AIDS Research (#107005-43-RFNT), Center for AIDS Research (CFAR), United States. Public Health Service (AI057005)

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-pathog, National Institute of Allergy and Infectious Diseases (Award Number U01AI068636 and AI068634)

Immunodominance is variably used to describe either the most frequently detectable response among tested individuals or the strongest response within a single individual, yet factors determining either inter- or intraindividual immunodominance are still poorly understood. More than 90 individuals were tested against 184 HIV- and 92 EBV-derived, previously defined CTL epitopes. The data show that HLA-B-restricted epitopes were significantly more frequently recognized than HLA-A- or HLA-C-restricted epitopes. HLA-B-restricted epitopes also induced responses of higher magnitude than did either HLA-A- or HLA-C-restricted epitopes, although this comparison only reached statistical significance for EBV epitopes. For both viruses, the magnitude and frequency of recognition were correlated with each other, but not with the epitope binding affinity to the restricting HLA allele. The presence or absence of HIV coinfection did not impact EBV epitope immunodominance patterns significantly. Peptide titration studies showed that the magnitude of responses was associated with high functional avidity, requiring low concentration of cognate peptide to respond in in vitro assays. The data support the important role of HLA-B alleles in antiviral immunity and afford a better understanding of the factors contributing to inter- and intraindividual immunodominance