Definition of the viral targets of protective HIV-1-specific T cell responses

  • Beatriz Mothe1, 2, 3,

    Affiliated with

    • Anuska Llano1,

      Affiliated with

      • Javier Ibarrondo1,

        Affiliated with

        • Marcus Daniels4,

          Affiliated with

          • Cristina Miranda2,

            Affiliated with

            • Jennifer Zamarreño1,

              Affiliated with

              • Vanessa Bach1,

                Affiliated with

                • Rosario Zuniga5,

                  Affiliated with

                  • Susana Pérez-Álvarez1, 6,

                    Affiliated with

                    • Christoph T Berger7,

                      Affiliated with

                      • Maria C Puertas1,

                        Affiliated with

                        • Javier Martinez-Picado1, 8,

                          Affiliated with

                          • Morgane Rolland9,

                            Affiliated with

                            • Marilu Farfan5,

                              Affiliated with

                              • James J Szinger4,

                                Affiliated with

                                • William H Hildebrand10,

                                  Affiliated with

                                  • Otto O Yang11,

                                    Affiliated with

                                    • Victor Sanchez-Merino12,

                                      Affiliated with

                                      • Chanson J Brumme13,

                                        Affiliated with

                                        • Zabrina L Brumme13, 14,

                                          Affiliated with

                                          • David Heckerman15,

                                            Affiliated with

                                            • Todd M Allen7,

                                              Affiliated with

                                              • James I Mullins16,

                                                Affiliated with

                                                • Guadalupe Gómez16,

                                                  Affiliated with

                                                  • Philip J Goulder17, 18,

                                                    Affiliated with

                                                    • Bruce D Walker7, 18, 19,

                                                      Affiliated with

                                                      • Jose M Gatell12,

                                                        Affiliated with

                                                        • Bonaventura Clotet1, 2,

                                                          Affiliated with

                                                          • Bette T Korber4, 20,

                                                            Affiliated with

                                                            • Jorge Sanchez5 and

                                                              Affiliated with

                                                              • Christian Brander1, 8Email author

                                                                Affiliated with

                                                                Journal of Translational Medicine20119:208

                                                                DOI: 10.1186/1479-5876-9-208

                                                                Received: 14 September 2011

                                                                Accepted: 7 December 2011

                                                                Published: 7 December 2011

                                                                Abstract

                                                                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.

                                                                Keywords

                                                                HIV specific CTL clade B clade C HLA vaccine immunogen design functional avidity epitope entropy immune correlate

                                                                Background

                                                                HIV-1 infection induces strong and broadly directed HLA class I restricted T cell responses for which specific epitopes and restricting HLA class I alleles have been associated with relative in vivo viral control [1]. The bulk of the anti-viral CTL response appears to be disproportionately HLA-B restricted, but the relative contribution of targeted viral regions and restricting HLA molecules on the effectiveness of these responses remains unclear [25]. In addition, the impact of HIV-1 sequence diversity on the effectiveness of virus-specific T cell immunity in vivo is unclear, as functional constraints of escape variants, codon-usage at individual protein positions, T cell receptor (TCR) plasticity and functional avidity and cross-reactivity potential may all contribute to the overall antiviral activity of a specific T cell response [613]. Of note, T cell responses to Gag have most consistently been associated with reduced viral loads in both clade B and clade C infected cohorts [1416]; however, the specific regions in Gag responsible for this effective control remain poorly defined. In addition, it is unclear whether the relative benefit of Gag is due to any other specific characteristic of this protein, such as rapid antigen-representation upon infection, protein expression levels, amino acid composition and/or inherently greater processability and immunogenicity, particularly in the context of selected HLA class I alleles [17, 18]. Thus, concerns remain that a purely Gag-based vaccine might mainly benefit those people with a particular HLA genotype and will not take advantage of potentially beneficial targets outside of Gag [4, 16, 17, 19]. In addition, CTL escape and viral fitness studies have focused largely on Gag-derived epitopes presented in the context of protective HLA class I alleles such as HLA-B27 and -B57 [7, 20, 21], yielding results that may not be generalizable to the genetically diverse majority of the human population. Furthermore, many studies have focused on immunodominant targets only, despite some studies in HIV-1 and SIV infection demonstrating a crucial contribution of sub-dominant responses to targets outside of Gag to the effective in-vivo viral control [4, 22]. Thus, the current view on what may constitute a protective cellular immune response to HIV-1 is likely biased towards a immunodominant responses and those restricted by frequent HLA class I alleles and HLA alleles associated with superior disease outcome.

                                                                To overcome these potential limitations, the design of an effective and broadly applicable HIV-1 vaccine should to be based on information gained through comprehensive analyses that extend across large portions of the population's HLA class I heterogeneity. Here we focus on three cohorts totaling more than 950 untreated, chronically HIV-1 infected individuals with clade B and C infections, from which responses to certain regions of the viral genome and specific T cell response patterns emerge as correlates of viral control. Importantly, the analyses identify functional properties unique to these responses and control for the impact of HLA class I alleles known to be associated with superior control of HIV-1 infection, thus providing vaccine immunogen sequence candidates with potential usefulness in a broadly applicable HIV-1 vaccine.

                                                                Methods

                                                                Cohorts

                                                                A HIV clade B infected cohort of 223 chronically infected, treatment naïve individuals was recruited and tested at IMPACTA in Lima, Peru. The majority (78%) of enrollees were male and all recruited individuals considered themselves to be of a mixed Amerindian ethnicity [14]. The cohort had a median viral load 37,237 copies/ml (range < 50- > 750,000) and a median CD4 count of 385 cell/ul (range170-1151). A second clade B infected cohort was established at the HIV-1 outpatient clinic "Lluita contra la SIDA" at Hospital Germans Trias i Pujol in Badalona (Barcelona, Spain) consisting of 48 treatment-naïve subjects with viral loads below 10,000 and CD4 cell counts > 350 cells/mm3 ("controllers", n = 24) or above 50,000 copies/ml and CD4 cell counts < 350 cells/mm3 ("non-controllers", n = 24). The HIV-1 clade C infected cohort has been described in the past and consisted of 631 treatment naïve South African with a median viral load of 37,900 copies/ml (range < 50-> 750,000) and a median CD4 count of 393 cells/ul (range 1-1378) [16]. An additional 78 from a recently published cohort in Boston were included in the analyses of functional avidities [2329]. HLA typing was performed as previously described using SSP-PCR [30]. For Hepitope and FASS analyses, 4digit typing was used for the Lima cohort and 2-digit typing for the Durban cohort. Protocols were approved in Lima by the IMPACTA Human Research Committee, in Durban by the Ethical Committee of the Nelson R. Mandela School of Medicine at the University of KwaZulu-Natal and in Barcelona by the Human Research Committee at Hospital Germans Trias i Pujol. All subjects provided written informed consent.

                                                                Peptide test set and ELISpot assay: Previously described peptide sets matching HLA-clade B and C consensus sequences were used in all experiments for which the OLP-specific entropies have been calculated in the past, based on available sequence datasets [3133] and http://​www.​hiv.​lanl.​gov/​content/​immunology/​hlatem/​index.​html. The peptides were clade-specific sets of adapted 18mers, overlapping by 11 residues designed using the PeptGen tool available at the Los Alamos HIV database http://​www.​hiv.​lanl.​gov/​content/​sequence/​PEPTGEN/​peptgen.​html. The individual OLP in the peptide sets for clade B and clade C had all the same starting and ending position relative to the source protein and follow the same numbering across the entire viral proteome for both clades. Peripheral blood mononuclear cells (PBMCs) were separated from whole blood by density centrifugation and used directly to test for CD8+ T cell responses in vitro. IFN-γ ELISpot assays were performed as described previously, using Mabtech antibodies (Mabtech, Stockholm, Sweden) and a matrix format that allowed simultaneous testing of all 410 overlapping (OLP) peptides in the respective test set [14]. Thresholds for positive responses were defined as: exceeding 5 spots (50 SFC/106) per well and exceeding the mean of negative wells plus 3 standard deviation or three times the mean of negative wells, whichever was higher. Stimulation with PHA was used as a positive control in all ELISpot assays.

                                                                Definition of functional avidity

                                                                Responses targeting 18 mer OLP in HIV-1 Gag p24 were assessed for their functional avidity using OLP-specific sets of 10 mer peptides overlapping by 9 residues that span the 18 mer peptide sequence. Functional avidity was defined as the peptide concentration needed to elicit half maximal response rates in the ELISpot assay and was calculated as a sigmoidal dose response curve fit using GraphPad Prism software [13].

                                                                In vitro viral replication inhibition assay

                                                                A double mutant virus containing a Nef M20A and Integrase G140S/Q148H Raltegravir (integrase inhibitor) resistance mutations was tested for replication in CD4 T cells in the presence or absence of autologous T cell lines targeting protective or non-protective OLP. Use of the Raltegravir-resistant virus allows to prevent potential replication of autologous virus in the inhibition assays [28], excludes potential negative impacts on antigen processing or CTL functions attributed to protease inhibitors [34] and avoids overlap between the resistance mutations sites (i.e. G140S/Q148H) and location of beneficial and non-beneficial OLP sequences. In brief, the p83-10 plasmid containing mutations for a methionine to alanine substitution at position 20 of the Nef protein and the p83-2 plasmid engineered to contain the G140S and Q148H mutations in the integrase were combined to produce a virus that is replication competent, highly resistant to Raltegravir and does not downregulate HLA class I in infected cells [35, 36]. Although not entirely physiological, this approach was chosen to potentially increase the signal in the in vitro inhibition assay, even when responses were restricted by Nef-sensitive HLA class I alleles. Plasmids were co-transfected into MT4 cells and virus was harvested after 7 days [35, 37, 38]. Autologous CD4 cells were enriched by magnetic beads isolation (Miltenyi) and expanded for 3 days using a bi-specific anti-CD3/8 antibody and IL-2 containing medium (50 IU r-IL2) before infecting them at multiplicities of infection (MOI) between 0.01 and 1. Effector cells were obtained by stimulating PBMC with either beneficial or non-beneficial OLP for 12 days before isolating specific OLP-reactive cells by IFN-γ capture assay according to manufacturers' instructions (Miltenyi, Bergisch Gladbach, Germany). The effector T cells were analyzed by flow cytometry for the specificity to their respective targets after capture assay and quantified to adjust effector-to-target ratios. Since the NL4-3 backbone sequence differed in several positions in beneficial and non-beneficial OLP, the epitope specificity was predicted based on the HLA class I genotype of the tested individual and responses confirmed to efficiently recognize variant sequences in the NL4-3 backbone sequence. Culture supernatant was harvested and replaced by Raltegravir containing medium 0.05 μg/ml after 72 h. Levels of Gagp24 in the culture supernatant were determined by ELISA as described [39].

                                                                Statistical Analyses

                                                                Statistical analyses were performed using Prism Version 5 and R Statistical Language [40]. Results are presented as median values unless otherwise stated. Tests included ANOVA, non-parametric Mann-Whitney test (two-tailed) and Spearman rank test. The significance of differences in viral load distribution between OLP-responders and OLP-non-responders was assessed by a two-sided Student's T Test with multiple tests addressed using, instead of a Bonferroni correction, a q-value approach to compensate for multiple comparisons [39]. The multivariate analysis was based on a novel multivariate combined regression method known as FASS, a forward selection method combined with all-subsets regression [4143]. Briefly, the FASS approach works by iteratively performing the following procedure: Let 'V' be the set of all variables and 'M' be the set of variables included in a model. In the first step, those variables that are not already in the model are divided into equal-sized blocks of variables (the last block may have less than 'g' variables). Then, for each block of variables, 'm' is a new estimated and evaluated model using the Bayesian Information Criterion (BIC). The best model 'm' according to its BIC is retained and the procedure starts all over again until in one step or more the model is not improved.

                                                                Results

                                                                HIV-1-specific T cell responses targeting conserved regions are associated with lower viral loads

                                                                In a first analysis, HIV-1-specific T cell responses were assessed in a cohort of 223 HIV-1 clade B infected individuals recruited in Lima, Peru using IFNg ELISpot assays and a previously described set of 410 clade B overlapping peptides (OLP) [14, 31]. For each OLP, a protective ratio (PR) was calculated as the ratio of the median viral loads between OLP non-responders and OLP responders, such that OLP with PR > 1 were reflective of OLP predominantly targeted by individuals with reduced viral loads. OLP-specific PR were a) compared between OLP spanning the different viral proteins and b) correlated with the viral sequence heterogeneity in the region covered by the OLP. The data showed highest median PR values for OLP spanning the Gag protein sequence, whereas Nef, Env and Tat had the lowest median PR values (Figure 1A, p < 0.0001, ANOVA). A protein-subunit-breakdown of PR values showed the p15 subunit of Gag and RT in Pol to score less favorable than the remainder of the respective proteins (Figure 1B, p = 0.0032 and p = 0.0025, respectively). While these data confirm the association between HIV-1 Gag-specific responses and lower viral loads, it is important to note that all proteins contained OLP with PR > 1, suggesting that some beneficial responses can be located outside of Gag; data that has not emerged from any of the previous studies linking Gag responses to relative viral control. At the same time, all proteins contained OLP with PR < 1, indicating that proteins considered overall beneficial may contain non-beneficial regions as well. In addition, when the OLP-specific PR was compared to the sequence entropy of the region spanned by the individual OLP, a significant negative correlation between PR and entropy was observed (p = 0.0028, r = -0.15; Figure 1C). Although rarely targeted OLP may have introduced statistically less robust data points in this comparison and caused a wide scatter of data points, the results show a relative absence of OLP with high entropy and high PR values, suggesting that responses to more variable regions are less effective in mediating in vivo viral control.
                                                                http://static-content.springer.com/image/art%3A10.1186%2F1479-5876-9-208/MediaObjects/12967_2011_987_Fig1_HTML.jpg
                                                                Figure 1

                                                                Localization and conservation of beneficial and non-beneficial OLP in HIV-1 clade B and C cohorts. Total HIV-1-specific T cell responses were assessed in a cohort of 223 chronically HIV clade B infected, untreated individuals in Lima, Peru (graphs A-C) and in 631 chronically HIV clade C infected, untreated individuals in Durban, South Africa (graphs D-F) using peptide test sets of 410 18 mer overlapping peptides (OLP) spanning the consensus B and C sequences, respectively [2, 31]. For each OLP, the protective ratio (PR, defined as "the ratio of the log median viral load in OLP non-responders divided by log median viral load in OLP responders") was determined. Each symbol represents an individual OLP, grouped either by (A, D) proteins or (B, E) protein-subunits for OLP located in Gag, Pol and Env (p-values in A, D based on ANOVA, in B, E on Mann-Whitney by pariwise comparing the different protein subunits, red lines indicating median PR values). In (C and F), the OLP-specific entropy (a measure of the viral diversity in the region the OLP spans) is compared to the OLP-specific PR and shows an inverse association between the sequence conservation and PR (Spearman rank).

                                                                To assess whether the above observations would also hold true outside of clade B infection, the same analyses were conducted in a cohort of 631 clade C HIV-1 infected subjects enrolled in Durban, South Africa and tested for responses against a clade C consensus OLP sequence as described previously [33]. As in clade B infection, the OLP specific PR values were highest for OLP spanning Gag without any significant differences between the Gag and Pol protein subunits (Figure 1D and 1E). As in the clade B cohort, the PR values were negatively correlated with the OLP-specific entropy (p = 0.0323, Figure 1F), confirming the findings in the clade B cohort and further pointing towards the importance of targeting conserved segments of the viral proteome for effective in vivo viral control.

                                                                Identification of individual beneficial OLP sequences in clade B and C infection

                                                                In order to identify individual OLP that were significantly more frequently targeted in individuals with relative viral control and to compare the beneficial OLP in clade B and C infection, the viral load distribution in OLP-responders and non-responders was analyzed individually for each OLP. For the clade B cohort in Peru, the analyses yielded 43 OLP sequences for which the median viral load differed between the two groups with an uncorrected p-value of < 0.05. Of these 43 OLP, 26 were OLP with a PR > 1 (referred to as "beneficial" OLP), and 17 OLP with a PR < 1 ("non-beneficial" OLP, Table 1). The distribution of OLP with PR > 1 among viral proteins was biased towards Gag and Pol, while Env produced exclusively OLP with PR < 1 (Figure 2A).
                                                                Table 1

                                                                Beneficial and non-beneficial OLP identified in Lima clade B cohort (p < 0.05)

                                                                OLP #

                                                                Protein

                                                                Sub-unit

                                                                OLP clade B

                                                                consensus

                                                                sequence

                                                                Median viral

                                                                load in OLP

                                                                responders

                                                                Median viral

                                                                load in OLP

                                                                non-responders

                                                                Protective Ratio

                                                                (PR)*

                                                                p-value

                                                                3

                                                                Gag

                                                                p17

                                                                EKIRLRPGGKKKYKLKHI

                                                                22947

                                                                39014

                                                                1.053

                                                                0.037

                                                                6

                                                                Gag

                                                                p17

                                                                ASRELERFAVNPGLL

                                                                15380

                                                                43189

                                                                1.107

                                                                0.001

                                                                7

                                                                Gag

                                                                p17

                                                                ERFAVNPGLLETSEGCR

                                                                25939

                                                                38974

                                                                1.040

                                                                0.049

                                                                10

                                                                Gag

                                                                p17

                                                                QLQPSLQTGSEELRSLY

                                                                16285

                                                                37237

                                                                1.085

                                                                0.031

                                                                12

                                                                Gag

                                                                p17

                                                                SLYNTVATLYCVHQRIEV

                                                                23855

                                                                37113

                                                                1.044

                                                                0.037

                                                                23

                                                                Gag

                                                                p24

                                                                AFSPEVIPMFSALSEGA

                                                                22947

                                                                37113

                                                                1.048

                                                                0.036

                                                                31

                                                                Gag

                                                                p24

                                                                IAPGQMREPRGSDIA

                                                                3563

                                                                35483

                                                                1.281

                                                                0.028

                                                                34

                                                                Gag

                                                                p24

                                                                STLQEQIGWMTNNPPIPV

                                                                6127

                                                                37360

                                                                1.207

                                                                0.002

                                                                48

                                                                Gag

                                                                p24

                                                                ACQGVGGPGHKARVLAEA

                                                                12975

                                                                35755

                                                                1.107

                                                                0.041

                                                                60

                                                                Gag

                                                                p15

                                                                GKIWPSHKGRPGNFLQSR

                                                                16266

                                                                36434

                                                                1.083

                                                                0.044

                                                                75

                                                                Nef

                                                                -

                                                                WLEAQEEEEVGFPVRPQV

                                                                13407

                                                                37360

                                                                1.108

                                                                0.026

                                                                76

                                                                Nef

                                                                -

                                                                EVGFPVRPQVPLRPMTYK

                                                                59618

                                                                29855

                                                                0.937

                                                                0.001

                                                                84

                                                                Nef

                                                                -

                                                                NYTPGPGIRYPLTFGWCF

                                                                55402

                                                                30538

                                                                0.945

                                                                0.006

                                                                85

                                                                Nef

                                                                -

                                                                RYPLTFGWCFKLVPV

                                                                69890

                                                                29903

                                                                0.924

                                                                0.002

                                                                90

                                                                Nef

                                                                -

                                                                SLHGMDDPEKEVLVWKF

                                                                89687

                                                                32650

                                                                0.911

                                                                0.042

                                                                159

                                                                Pol

                                                                Pro

                                                                KMIGGIGGFIKVRQYDQI

                                                                14736

                                                                36434

                                                                1.094

                                                                0.020

                                                                160

                                                                Pol

                                                                Pro

                                                                FIKVRQYDQILIEICGHK

                                                                3682

                                                                35755

                                                                1.277

                                                                0.031

                                                                161

                                                                Pol

                                                                Pro

                                                                QILIEICGHKAIGTVLV

                                                                9117

                                                                35483

                                                                1.149

                                                                0.050

                                                                163

                                                                Pol

                                                                Pro

                                                                LVGPTPVNIIGRNLLTQI

                                                                25965

                                                                45637

                                                                1.055

                                                                0.007

                                                                171

                                                                Pol

                                                                RT

                                                                LVEICTEMEKEGKISKI

                                                                1865

                                                                35483

                                                                1.391

                                                                0.014

                                                                181

                                                                Pol

                                                                RT

                                                                LDVGDAYFSVPLDKDFRK

                                                                65858

                                                                32871

                                                                0.937

                                                                0.041

                                                                195

                                                                Pol

                                                                RT

                                                                LRWGFTTPDKKHQKEPPF

                                                                5624

                                                                37113

                                                                1.219

                                                                0.006

                                                                196

                                                                Pol

                                                                RT

                                                                DKKHQKEPPFLWMGYELH

                                                                10103

                                                                35483

                                                                1.136

                                                                0.044

                                                                210

                                                                Pol

                                                                RT

                                                                EIQKQGQGQWTYQIY

                                                                18155

                                                                35483

                                                                1.068

                                                                0.045

                                                                222

                                                                Pol

                                                                RT

                                                                PPLVKLWYQLEKEPIVGA

                                                                412599

                                                                34640

                                                                0.808

                                                                0.030

                                                                230

                                                                Pol

                                                                RT

                                                                IHLALQDSGLEVNIV

                                                                85102

                                                                34117

                                                                0.919

                                                                0.030

                                                                237

                                                                Pol

                                                                RT

                                                                VYLAWVPAHKGIGGNEQV

                                                                85102

                                                                34117

                                                                0.919

                                                                0.029

                                                                240

                                                                Pol

                                                                RT

                                                                SAGIRKVLFLDGIDKA

                                                                116902

                                                                32761

                                                                0.891

                                                                0.019

                                                                269

                                                                Pol

                                                                Int

                                                                TKELQKQITKIQNFRVYY

                                                                6629

                                                                35755

                                                                1.192

                                                                0.030

                                                                270

                                                                Pol

                                                                Int

                                                                TKIQNFRVYYRDSRDPLW

                                                                18171

                                                                37360

                                                                1.073

                                                                0.019

                                                                271

                                                                Pol

                                                                Int

                                                                YYRDSRDPLWKGPAKLLW

                                                                25939

                                                                35755

                                                                1.032

                                                                0.043

                                                                276

                                                                Pol

                                                                Int

                                                                KIIRDYGKQMAGDDCVA

                                                                6629

                                                                35755

                                                                1.192

                                                                0.021

                                                                279

                                                                Vpr

                                                                -

                                                                GPQREPYNEWTLELLEEL

                                                                60222

                                                                32650

                                                                0.944

                                                                0.042

                                                                307

                                                                Env

                                                                Gp120

                                                                DLNNNTNTTSSSGEKMEK

                                                                179419

                                                                34117

                                                                0.863

                                                                0.044

                                                                311

                                                                Env

                                                                Gp120

                                                                IRDKVQKEYALFYKLDVV

                                                                179419

                                                                32871

                                                                0.860

                                                                0.008

                                                                314

                                                                Env

                                                                Gp120

                                                                YRLISCNTSVITQACPKV

                                                                58206

                                                                31273

                                                                0.943

                                                                0.008

                                                                315

                                                                Env

                                                                Gp120

                                                                SVITQACPKVSFEPIPIH

                                                                61011

                                                                32871

                                                                0.944

                                                                0.034

                                                                320

                                                                Env

                                                                Gp120

                                                                TNVSTVQCTHGIRPVV

                                                                341587

                                                                34640

                                                                0.820

                                                                0.034

                                                                355

                                                                Env

                                                                Gp120

                                                                VAPTKAKRRVVQREKRAV

                                                                161602

                                                                34117

                                                                0.870

                                                                0.042

                                                                399

                                                                Env

                                                                Gp41

                                                                VIEVVQRACRAILHIPRR

                                                                388089

                                                                34640

                                                                0.812

                                                                0.026

                                                                405

                                                                Vif

                                                                -

                                                                VKHHMYISGKAKGWFYRH

                                                                16458

                                                                37237

                                                                1.084

                                                                0.021

                                                                406

                                                                Vif

                                                                -

                                                                GKAKGWFYRHHYESTHPR

                                                                16458

                                                                37237

                                                                1.084

                                                                0.022

                                                                424

                                                                Vif

                                                                -

                                                                TKLTEDRWNKPQKTKGHR

                                                                10319

                                                                36434

                                                                1.137

                                                                0.014

                                                                * PR values in bold indicate PR > 1, i.e. OLP-responses seen more frequently in individuals with reduced viral loads

                                                                http://static-content.springer.com/image/art%3A10.1186%2F1479-5876-9-208/MediaObjects/12967_2011_987_Fig2_HTML.jpg
                                                                Figure 2

                                                                Genome distribution, entropy and RT localization of OLP with significant impact on viral loads in HIV-1 clade B and C infection: The distribution of OLP with significantly elevated or reduced PR across the viral proteome is shown in A) for clade B infection (cut-off uncorrected p-value of p < 0.05) and in B) for clade C infection (cut-off q < 0.2). The entropy of beneficial and non-beneficial clade B OLP is compared in C) while in D), the entropy of beneficial OLP in HIV clade C Gag is compared to the remainder of Gag OLP (p-values based on Mann Whitney, red lines indicating median sequence entropies). In E and F, protein structures for HIV-1 reverse transcriptase (Protein databank structure ID 3IG1) were loaded into the Los Alamos HIV Database "protein feature accent" tool http://​www.​hiv.​lanl.​gov/​content/​sequence/​PROTVIS/​html/​protvis.​html and locations of beneficial RT OLP identified in clade B (Table 1) and in clade C (Table 2) marked by red highlights.

                                                                The same analyses were repeated for the clade C cohort in Durban, which due to its larger size allowed to apply more stringent statistical criteria to identify beneficial and non-beneficial OLP. To compensate for multiple statistical comparisons, we employed a previously described false-discovery rate approach [39], resulting in the identification of 33 clade C OLP with q-values of < 0.2 (i.e. OLP with significantly different viral load distributions between OLP-responders and non-responders with a false positive discovery rate (q-value) of 20%). The 33 OLP identified were comprised of 22 beneficial OLP and 11 non-beneficial OLP, with the beneficial OLP being again located in Gag, Pol and Vif, similar to what was seen in the clade B cohort (Figure 2B).

                                                                In both cohorts, the total breadth and magnitude of responses did not correlate with viral loads as reported for parts of these cohorts in the past [14, 16]. The OLP with significant differences in median viral loads (43 OLP in clade B and 33 OLP in clade C, Tables 1 and 2, respectively, i.e. "scoring OLP"), were more often targeted in their respective cohort than OLP that did not score with a significant difference in viral loads (p = 0.0015 Lima; p < 0.0001 Durban). However, beneficial and non-beneficial OLP were equally frequently targeted in either cohort. Also, there was no difference in the median magnitude of the OLP-specific responses, regardless whether it was a beneficial, non-beneficial or not-scoring OLP (all p > 0.7, data not shown). Finally, there was no correlation between the number of total OLP responses (against all 410 OLP) and the magnitude of responses to beneficial OLP in either cohort, indicating that the strength of beneficial OLP responses was not diminished by other responses to the rest of the viral proteome.
                                                                Table 2

                                                                Beneficial and non-beneficial OLP identified in Durban clade C cohort (q < 0.2)

                                                                OLP #

                                                                Protein

                                                                Sub-unit

                                                                OLP clade C

                                                                consensus

                                                                sequence

                                                                Median viral

                                                                load in OLP

                                                                responders

                                                                Median viral

                                                                load in OLP

                                                                non-responders

                                                                Protective Ratio

                                                                (PR)*

                                                                p-value

                                                                Q-value

                                                                3

                                                                Gag

                                                                p17

                                                                EKIRLRPGGKKHYMLKHL

                                                                18,700

                                                                45,100

                                                                1.09

                                                                0.0002

                                                                0.0006

                                                                6

                                                                Gag

                                                                p17

                                                                ASRELERFALNPGLL

                                                                6,570

                                                                44,100

                                                                1.22

                                                                0.0000

                                                                0.0000

                                                                7

                                                                Gag

                                                                p17

                                                                ERFALNPGLLETSEGCK

                                                                5,270

                                                                43,900

                                                                1.25

                                                                0.0000

                                                                0.0000

                                                                22

                                                                Gag

                                                                p24

                                                                WVKVIEEKAFSPEVIPMF

                                                                8,360

                                                                42,850

                                                                1.18

                                                                0.0000

                                                                0.0000

                                                                25

                                                                Gag

                                                                p24

                                                                GATPQDLNTMLNTVGGH

                                                                24,450

                                                                45,200

                                                                1.06

                                                                0.0021

                                                                0.0263

                                                                26

                                                                Gag

                                                                p24

                                                                NTMLNTVGGHQAAMQMLK

                                                                5,310

                                                                39,600

                                                                1.23

                                                                0.0061

                                                                0.0766

                                                                27

                                                                Gag

                                                                p24

                                                                GGHQAAMQMLKDTINEEA

                                                                9,715

                                                                42,100

                                                                1.16

                                                                0.0015

                                                                0.0170

                                                                29

                                                                Gag

                                                                p24

                                                                AAEWDRLHPVHAGPIA

                                                                19,700

                                                                40,900

                                                                1.07

                                                                0.0045

                                                                0.0544

                                                                31

                                                                Gag

                                                                p24

                                                                IAPGQMREPRGSDIA

                                                                6,480

                                                                38,950

                                                                1.20

                                                                0.0146

                                                                0.1478

                                                                33

                                                                Gag

                                                                p24

                                                                SDIAGTTSTLQEQIAWM

                                                                11,650

                                                                40,900

                                                                1.13

                                                                0.0025

                                                                0.0318

                                                                37

                                                                Gag

                                                                p24

                                                                WIILGLNKIVRMYSPVSI

                                                                9,360

                                                                44,100

                                                                1.17

                                                                0.0004

                                                                0.0018

                                                                39

                                                                Gag

                                                                p24

                                                                SILDIKQGPKEPFRDYV

                                                                2,630

                                                                38,250

                                                                1.34

                                                                0.0182

                                                                0.1838

                                                                41

                                                                Gag

                                                                p24

                                                                YVDRFFKTLRAEQATQDV

                                                                22,150

                                                                44,100

                                                                1.07

                                                                0.0020

                                                                0.0263

                                                                42

                                                                Gag

                                                                p24

                                                                LRAEQATQDVKNWMTDTL

                                                                16,480

                                                                40,900

                                                                1.09

                                                                0.0078

                                                                0.0935

                                                                55

                                                                Gag

                                                                p15

                                                                HIARNCRAPRKKGCWK

                                                                7,550

                                                                39,700

                                                                1.19

                                                                0.0092

                                                                0.1047

                                                                59

                                                                Gag

                                                                p15

                                                                RQANFLGKIWPSHKGR

                                                                9,840

                                                                42,200

                                                                1.16

                                                                0.0046

                                                                0.0539

                                                                60

                                                                Gag

                                                                p15

                                                                GKIWPSHKGRPGNFLQSR

                                                                6,130

                                                                39,700

                                                                1.21

                                                                0.0066

                                                                0.0799

                                                                63

                                                                Gag

                                                                p15

                                                                TAPPAESFRFEETTPAPK

                                                                6,040

                                                                38,950

                                                                1.21

                                                                0.0093

                                                                0.1020

                                                                116

                                                                Tat

                                                                Tat

                                                                TKGLGISYGRKKRRQRRS

                                                                109,000

                                                                36,700

                                                                0.91

                                                                0.0033

                                                                0.0410

                                                                178

                                                                Pol

                                                                RT

                                                                FWEVQLGIPHPAGLKKKK

                                                                258,000

                                                                37,300

                                                                0.84

                                                                0.0033

                                                                0.0384

                                                                181

                                                                Pol

                                                                RT

                                                                LDVGDAYFSVPLDEDFRK

                                                                7,100

                                                                38,950

                                                                1.19

                                                                0.0186

                                                                0.1832

                                                                190

                                                                Pol

                                                                RT

                                                                RAQNPEIVIYQYMDDLYV

                                                                84,900

                                                                34,700

                                                                0.92

                                                                0.0043

                                                                0.0555

                                                                199

                                                                Pol

                                                                RT

                                                                TVQPIQLPEKDSWTVNDI

                                                                6,700

                                                                38,300

                                                                1.20

                                                                0.0198

                                                                0.1926

                                                                216

                                                                Pol

                                                                RT

                                                                QKIAMESIVIWGKTPKFR

                                                                18,150

                                                                43,000

                                                                1.09

                                                                0.0026

                                                                0.0317

                                                                239

                                                                Pol

                                                                RT

                                                                QVDKLVSSGIRKVLFL

                                                                373,200

                                                                37,700

                                                                0.82

                                                                0.0205

                                                                0.1937

                                                                253

                                                                Pol

                                                                Int

                                                                PAETGQETAYFILKLAGR

                                                                92,800

                                                                35,400

                                                                0.92

                                                                0.0082

                                                                0.0954

                                                                265

                                                                Pol

                                                                Int

                                                                AVFIHNFKRKGGIGGYSA

                                                                63,650

                                                                33,800

                                                                0.94

                                                                0.0178

                                                                0.1826

                                                                283

                                                                Vpr

                                                                -

                                                                GLGQYIYETYGDTWTGV

                                                                78,000

                                                                35,600

                                                                0.93

                                                                0.0126

                                                                0.1302

                                                                284

                                                                Vpr

                                                                -

                                                                ETYGDTWTGVEALIRIL

                                                                85,050

                                                                35,200

                                                                0.92

                                                                0.0099

                                                                0.1034

                                                                312

                                                                Env

                                                                Gp120

                                                                YALFYRLDIVPLNENNSSEY

                                                                270,000

                                                                37,700

                                                                0.84

                                                                0.0208

                                                                0.1915

                                                                365

                                                                Env

                                                                Gp41

                                                                GIKQLQTRVLAIERYLK

                                                                151,000

                                                                34,700

                                                                0.88

                                                                0.0001

                                                                0.0002

                                                                393

                                                                Env

                                                                Gp41

                                                                LLGRSSLRGLQRGWEALKYL

                                                                750,000

                                                                37,450

                                                                0.78

                                                                0.0007

                                                                0.0041

                                                                417

                                                                Vif

                                                                -

                                                                CFADSAIRKAILGHIV

                                                                1,110

                                                                38,200

                                                                1.50

                                                                0.0178

                                                                0.1891

                                                                * PR values in bold indicate PR > 1, i.e. OLP-responses seen more frequently in individuals with reduced viral loads

                                                                In the clade B cohort, the 26 beneficial and 17 non-beneficial OLP showed a significant difference in their median entropy (p = 0.0327, Figure 2C), in line with the overall negative association between higher PR and lower sequence entropy seen in the comprehensive screening including the entire 410 OLP set (Figure 1C). While this comparison was not significant in clade C infection, a detailed look at Gag showed that beneficial Gag clade C OLP had a lower entropy values than the rest of the Gag OLP, suggesting that targeting of the most conserved regions even in Gag provided particular benefits for viral control (Figure 2D, p = 0.0172). These beneficial OLP were also more frequently targeted (median of 36 responders) compared to the rest of Gag OLP (median 12 responders, p = 0.0099), likely reflecting the high epitope density in these regions [33, 44].

                                                                Finally, the two cohorts showed a partial overlap in the targeted beneficial and non-beneficial OLP, despite the vastly different HLA genetics in these two populations [4, 31, 45, 46]. As Gag was enriched in beneficial OLP scattered throughout the entire protein sequence, we used the available reverse transcriptase (RT) protein structure to assess whether beneficial responses were targeting structurally related regions of the protein, even though the linear position of beneficial OLP did not precisely match between the two clades. Indeed, superimposing the locations of beneficial OLP in the RT protein indicates that in both clades, beneficial OLP fell in structurally related domains of the RT protein (Figure 2E and 2F). This suggests that despite differences in response patterns between ethnicities and clades, viruses from both clades may be vulnerable to responses targeting the same structural regions of at least some of their viral proteins.

                                                                Increased breadth of responses against beneficial OLP is associated with decreasing viral loads, independent of Gag-specificity or the presence of protective HLA class I alleles

                                                                To assess whether individuals targeting more than one beneficial OLP profit from a greater breadth of responses to these targets, subjects in both cohorts were stratified by the number of responses to beneficial OLP and their viral loads compared. In both cohorts, negative correlations between the number of responses to beneficial OLP and viral loads were observed (p < 0.0001, r = -0.33 for Lima; p < 0.0001, r = 0.-25 for Durban; data not shown), suggesting that there is a cumulative benefit of responses to these particularly effective targets. Similarly, when individuals in the clade C cohort were grouped based on mounting 1-2, 3-4 or five and more beneficial OLP responses, a gradual reduction in median viral loads was seen. This reduction was close to 20-fold when 5 or more of the 22 beneficial OLP were targeted (median viral load 5,210 copies/ml) compared to individuals without a response (98,800 copies/ml, Figure 3A). Importantly, this observation was not driven only by individuals expressing HLA class I alleles associated with relative control of viral replication (including HLA-B27, -B57, -B*5801, -B63 and -B81) as their exclusion still showed a strong association between increased breadth of responses to beneficial OLP and a gradual suppression of viremia (Figure 3B). This was further supported when translating the clade B data from Peru to a second clade B infected cohort in Barcelona, Spain where HIV-1 controllers also mounted a significantly greater proportion of their responses to the beneficial Peruvian OLP compared to the HIV-1 non-controllers (61% vs. 29%, p = 0.0011; Figure 3C); this despite the fact that the Barcelona cohort was genetically different and excluded individuals expressing HLA-B27, -B57, -B58 and B63. Thus, despite the frequent targeting of Gag and the inclusion of individuals expressing HLA alleles such as HLA-B*5701 and -B*5801 in the two larger clade B and C cohorts, the present data identify regions of the viral genome that serve as the targets of an effective host T cell response, largely independent of the presence of HLA alleles known to influence HIV-1 viral replication.
                                                                http://static-content.springer.com/image/art%3A10.1186%2F1479-5876-9-208/MediaObjects/12967_2011_987_Fig3_HTML.jpg
                                                                Figure 3

                                                                Increased breadth of responses to beneficial OLP results in gradually reduced viral loads and is independent of cohort and HLA-B27, -57, -B58, -B81 and -B63. (A) The number of responses to beneficial OLP in the clade C cohort in Durban was determined for each individual and compared to viral loads. An increased breadth of responses to the 22 beneficial OLP was associated with reduced viral loads (ANOVA, p < 0.0001). (B) This association remained equally stable after removing all individuals expressing known beneficial HLA allele (HLA-B27, -B57, -B5801, -B63, -B81) from the analysis (ANOVA, p < 0.0001). (C) The set of 26 beneficial and 17 non-beneficial OLP identified in the clade B infected cohort in Lima, Peru was tested in a second clade B infected cohort in Barcelona. HIV controllers showed a significantly higher focus of responses on the 22 beneficial OLP (61% of all responses to the 43 OLP) while non-controllers reacted predominantly with the non-beneficial OLP (only 29% of all responses targeting beneficial OLP). The Barcelona cohort did not included subject expressing any HLA allele previously associated with relative control of HIV-1 (p = 0.0011, Mann Whitney).

                                                                PR-values are mediated by individuals with broad HLA heterogeneity

                                                                To further assess the contribution of specific HLA class I alleles on the PR of individual OLP, the statistically significant OLP in the clade C cohort were further analyzed. In a first step, median viral loads in the OLP-responder and non-responder groups were compared after excluding individuals with specific HLA class I alleles. If the statistical significance of the comparison was lost, the excluded HLA class I allele was assumed to have significantly contributed to the initially observed elevated or reduced PR value and to restrict a potential CTL epitope in that OLP. In a second step, a "Hepitope" analyses http://​www.​hiv.​lanl.​gov/​content/​immunology/​hepitopes was conducted to identify HLA class I alleles overrepresented in the OLP responder group; providing an alternative approach to identify specific epitopes that may contribute to relative viral control. Together, the two strategies permit to estimate the HLA diversity in the OLP responders and to identify the most likely alleles that restrict the epitope-specific responses to the OLP. Both are important measures when determining the relative usefulness of a selected beneficial OLP in a potential immunogen sequence as it should provide broad HLA coverage. The data from these analyses are summarized for beneficial and non-beneficial OLP in Table 3 and 3, respectively. The results demonstrate that with a few exceptions, for each OLP, several HLA alleles appeared to be mediating the observed effects as their removal caused the statistical significance to be lost. However, for the most frequent HLA class I alleles, the loss of significance may be due to a reduction in sample size rather than the actual allele, since the exclusion of many allele carriers could reduce the number of OLP responders (and non-responders) sufficiently to lose statistical power. The "Hepitope" analysis controlled for this effect and confirmed the obtained results, strongly indicating that responses to beneficial OLP were mediated by responder populations with heterogeneous HLA allele distributions.
                                                                Table 3

                                                                Impact of HLA alleles on the statistical significance of observed PR values (clade C OLP)

                                                                A) Beneficial OLP (PR > 1)

                                                                  

                                                                OLP

                                                                Protein

                                                                PR

                                                                Removed HLA allele(s) abolishing statistical significance 1

                                                                Alleles over-represented in the OLP responder group 2

                                                                3

                                                                Gag

                                                                1.09

                                                                A30, B42, C17

                                                                A30, B08, A03, A74, C17, A43, B42, B07

                                                                6

                                                                Gag

                                                                1.22

                                                                B15

                                                                B49, B82, C14

                                                                7

                                                                Gag

                                                                1.25

                                                                -

                                                                B42, C17, B49, A30

                                                                22

                                                                Gag

                                                                1.18

                                                                B57, C07

                                                                B57, A74, B45, C07, C16, B13

                                                                25

                                                                Gag

                                                                1.06

                                                                A30, B15, C04, C07

                                                                B42, C17, B81, B39, A01, C12, C18, A30, B67

                                                                26

                                                                Gag

                                                                1.23

                                                                A02, A23, A68, B07, B14, B58, C07, C08

                                                                C03, B15, A68

                                                                27

                                                                Gag

                                                                1.16

                                                                B15, C07

                                                                B15, A68, C03, C08

                                                                29

                                                                Gag

                                                                1.07

                                                                A68, B15, B58, C02, C03, C06, C12

                                                                B35, B39, C12, B40, B07, C04

                                                                31

                                                                Gag

                                                                1.2

                                                                A02, A11, A23, A29, A32, A34, A68, B07,

                                                                B13, A29, C06, A11

                                                                   

                                                                B13, B15, B42, B44, B58, C04, C06, C07, C17

                                                                 

                                                                33

                                                                Gag

                                                                1.13

                                                                A02, A23, B44, B57, B58, C07

                                                                B58, B57, A02, C07, C03, A68

                                                                37

                                                                Gag

                                                                1.17

                                                                A30, B42, B58, C17

                                                                C18, B42, C17, A01, B81

                                                                39

                                                                Gag

                                                                1.34

                                                                A02, A03, A23, A29, A30, A68, A74, B08, B15,

                                                                A02

                                                                   

                                                                B18, B42, B45, B53, B57, B58, C02, C03, C06,

                                                                 
                                                                   

                                                                C07, C08, C16, C17

                                                                 

                                                                41

                                                                Gag

                                                                1.07

                                                                A23, C06

                                                                C03, B14, A68, C08, B15

                                                                42

                                                                Gag

                                                                1.09

                                                                A23, A30, B08, B15, B42, B53, B58, C03, C04, C07

                                                                B53, C03

                                                                55

                                                                Gag

                                                                1.19

                                                                A02, A24, A29, A30, B07, B15, B39, B42, B44,

                                                                B42, B08, C17

                                                                   

                                                                B58, C02, C06, C07, C17

                                                                 

                                                                59

                                                                Gag

                                                                1.16

                                                                A02, A30, B08, B42, B44, B58, C04, C07, C17

                                                                A02, B13, A29

                                                                60

                                                                Gag

                                                                1.21

                                                                A02, A30, B42, B58, C06, C07, C17

                                                                A02, B41, C07, C17

                                                                63

                                                                Gag

                                                                1.21

                                                                A02, A23, A29, A30, A68, B08, B15, B44, B58,

                                                                A23

                                                                   

                                                                C02, C03, C06, C07

                                                                 

                                                                181

                                                                Pol

                                                                1.19

                                                                A01, A23, A29, A30, A34, A68, A74, B14, B15,

                                                                B57, C18

                                                                   

                                                                B18, B35, B44, B45, B57, B58, C02, C03, C04,

                                                                 
                                                                   

                                                                C06, C07, C08, C16

                                                                 

                                                                199

                                                                Pol

                                                                1.2

                                                                A02, A03, A23, A24, A26, A30, A31, A34, A36, A66, A68,

                                                                B53, A23, C04

                                                                   

                                                                A80, B08, B13, B15, B18, B35, B40, B41, B42, B44, B45,

                                                                 
                                                                   

                                                                B49, B50, B51, B53, B57, B58, B81, C01, C02, C03, C04,

                                                                 
                                                                   

                                                                C05, C06, C07, C08, C15, C16, C17

                                                                 

                                                                216

                                                                Pol

                                                                1.09

                                                                A02, A30, B58, C07, C17

                                                                B53, B58, C07, B57

                                                                417

                                                                Vif

                                                                1.5

                                                                A03, A23, A30, A34, A36, A68, B08, B14, B15,

                                                                B14, C08, A36

                                                                   

                                                                B44, B53, B58, C03, C04, C06, C08

                                                                 

                                                                B) Non-beneficial OLP (PR < 1)

                                                                    

                                                                116

                                                                Tat

                                                                0.91

                                                                A02, A34, B15, C04

                                                                B15, C02

                                                                178

                                                                Pol

                                                                0.84

                                                                A03, A68, B15, B58, C04, C06, C07

                                                                A68, C06, B58, B82, A03

                                                                190

                                                                Pol

                                                                0.92

                                                                A03, A30, A66, B18, B42, B45, B58, C06, C07

                                                                A02, B18, B35, C05, C16, B45, A80, C12, B67, B39

                                                                239

                                                                Pol

                                                                0.82

                                                                -

                                                                C05, A03

                                                                253

                                                                Pol

                                                                0.92

                                                                A03, A68, B15, B39, B42, B44, B58, C02, C04, C06, C08,

                                                                A68, C03, B15, B07, C15, B41

                                                                   

                                                                C17, C18

                                                                 

                                                                265

                                                                Pol

                                                                0.94

                                                                A02, A03, A23, A24, A26, A29, A30, A31, A33,

                                                                B15, C02, A43, A74

                                                                   

                                                                A34, A66, A68, B07, B08, B14, B15, B27, B40, B41, B42,

                                                                 
                                                                   

                                                                B44, B53, B54, B55, B57, B81, C01, C02, C04, C06, C07,

                                                                 
                                                                   

                                                                C12, C17, C18

                                                                 

                                                                283

                                                                Vpr

                                                                0.93

                                                                A02, A03, A23, A30, A66, A68, A74, B07, B14, B18, B39,

                                                                A68, C03, B07, C17, B41

                                                                   

                                                                B41, B42, B45, B57, C02, C04, C07, C08, C15, C17

                                                                 

                                                                284

                                                                Vpr

                                                                0.92

                                                                A03, A23, A30, A66, A68, A74, B07, B14, B18, B39, B42,

                                                                A68, C03

                                                                   

                                                                B45, B57, C07, C08, C15, C17

                                                                 

                                                                312

                                                                Env

                                                                0.84

                                                                -

                                                                B08, C07

                                                                365

                                                                Env

                                                                0.88

                                                                B58, C06

                                                                C06, B58, A43, B45, C16, A66

                                                                393

                                                                Env

                                                                0.78

                                                                A30, B58, C06

                                                                A31, C06, B45

                                                                1) in italics HLA alleles that do not emerge from the Hepitope analysis 2) cut-off in Hepitope analyses for p < 0.05, alleles sorted according to strength of association

                                                                Effects of T cell specificity on in vivo viral load are at least as strong as those associated with host HLA genetics

                                                                To assess whether specific response patterns and/or HLA combinations could be identified that mediated synergistic or superior control of viral infection in clades B and C, multivariate combined regression analysis was conducted on either OLP only, HLA only or the combination of OLP and HLA variables [4143]. The OLP-only analysis for Lima identified 7 OLP of which 4 were associated with lower median viral loads and 3 with increases in viral loads, respectively (Table 4). Targeting at least one of these beneficial clade B OLP was associated with significantly reduced viral loads (median 11, 079 copies/ml) compared to the subjects who did not target any of these four OLP (median 52, 178 copies/ml; p < 0.0001, Figure 4A). As seen in the univariate analysis (Figure 2C), the four beneficial OLP emerging from the Lima FASS analysis were more conserved than the rest of the OLP (median entropy 0.0759 vs. 0.1649, p = 0.0267) or the three non-beneficial OLP (0.0759 vs. 0.1228, p = 0.0571, data not shown). In contrast to OLP-only FASS analysis, only one HLA allele (HLA-C04) emerged from the HLA-only multivariate analysis. The analysis for the combined variables (OLP and HLA) controlled for the potential bias in this result due to more OLP variables (n = 389) than HLA (n = 146) being included in the statistical tests; yet still identified more OLP variables (n = 9) than HLA class I alleles (n = 3). In addition, the relative co-efficients of these associations were stronger for the OLP than the HLA variables, suggesting that T cell specificity influenced viral loads to at least the same degree as host HLA class I genetics. Of note, the identified OLP and HLA variables did not reflect responses to known optimal CTL epitopes, as none of the OLP contained described epitope(s) restricted by any of the identified HLA alleles [44].
                                                                Table 4

                                                                Multivariate analysis of OLP and HLA variables for clade B and C cohorts

                                                                OLP variables only (Lima, clade B)

                                                                OLP variables only (Durban, clade C)

                                                                 

                                                                change viral load (co-efficient) *

                                                                p-value

                                                                 

                                                                change viral load (co-efficient) *

                                                                p-value

                                                                Beneficial

                                                                  

                                                                Beneficial

                                                                  

                                                                OLP.6

                                                                -0.4591

                                                                0.0008

                                                                OLP.7

                                                                -0.6256

                                                                0

                                                                OLP.31

                                                                -1.4055

                                                                0.0002

                                                                OLP.21

                                                                -0.6663

                                                                0

                                                                OLP.171

                                                                -2.5981

                                                                0

                                                                OLP.22

                                                                -0.4926

                                                                0.0006

                                                                OLP.276

                                                                -1.127

                                                                0.0007

                                                                OLP.25

                                                                -0.2822

                                                                0.0002

                                                                   

                                                                OLP.27

                                                                -0.4719

                                                                0.0053

                                                                Non-beneficial

                                                                  

                                                                OLP.33

                                                                -0.3396

                                                                0.0024

                                                                OLP.76

                                                                0.2486

                                                                0.0067

                                                                OLP.398

                                                                -1.8179

                                                                0.0027

                                                                OLP.306

                                                                3.2968

                                                                0.0001

                                                                OLP.417

                                                                -1.6535

                                                                0.0008

                                                                OLP.411

                                                                1.3329

                                                                0.012

                                                                   
                                                                   

                                                                Non-beneficial

                                                                  
                                                                   

                                                                OLP.38

                                                                0.9045

                                                                0.0022

                                                                   

                                                                OLP.84

                                                                0.1947

                                                                0.0091

                                                                   

                                                                OLP.116

                                                                0.6156

                                                                0.0039

                                                                   

                                                                OLP.183

                                                                0.661

                                                                0.0013

                                                                   

                                                                OLP.224

                                                                0.2508

                                                                0.0036

                                                                   

                                                                OLP.265

                                                                0.5782

                                                                0

                                                                   

                                                                OLP.365

                                                                0.4911

                                                                0.0009

                                                                   

                                                                OLP.393

                                                                1.2624

                                                                0.0013

                                                                HLA variables only (Lima, clade B)

                                                                HLA variables only (Durban, clade C)

                                                                Non-beneficial

                                                                 

                                                                Beneficial

                                                                  

                                                                HLA-C0401

                                                                0.35652

                                                                0.00024

                                                                HLA-A74

                                                                -0.3553

                                                                0.0025

                                                                   

                                                                HLA-B13

                                                                -0.6443

                                                                0.0004

                                                                   

                                                                HLA-B57

                                                                -0.5195

                                                                0.0007

                                                                   

                                                                HLA-B81

                                                                -0.3619

                                                                0.0015

                                                                   

                                                                HLA-C12

                                                                -0.6544

                                                                0.0001

                                                                   

                                                                Non-beneficial

                                                                 
                                                                   

                                                                HLA.B.15

                                                                0.2506

                                                                0.0012

                                                                   

                                                                HLA.B.18

                                                                0.5521

                                                                0.0005

                                                                   

                                                                HLA.C.6

                                                                0.3958

                                                                0

                                                                HLA and OLP variables together (Lima)

                                                                HLA and OLP variables together (Durban)

                                                                Beneficial

                                                                  

                                                                Beneficial

                                                                  

                                                                OLP.6

                                                                -0.5792

                                                                0

                                                                OLP.6

                                                                -0.4798

                                                                0.0023

                                                                OLP.31

                                                                -1.1607

                                                                0.0005

                                                                OLP.7

                                                                -0.4528

                                                                0.0015

                                                                OLP.171

                                                                -2.7948

                                                                0

                                                                OLP.27

                                                                -0.4676

                                                                0.0049

                                                                OLP.276

                                                                -0.9609

                                                                0.0011

                                                                OLP.59

                                                                -0.4196

                                                                0.0115

                                                                   

                                                                OLP.417

                                                                -1.387

                                                                0.0041

                                                                Non-beneficial

                                                                 

                                                                Non-beneficial

                                                                 

                                                                OLP.2

                                                                0.3945

                                                                0.018

                                                                OLP.148

                                                                2.5215

                                                                0.0029

                                                                OLP.237

                                                                0.7211

                                                                0.0035

                                                                OLP.183

                                                                0.6108

                                                                0.0023

                                                                OLP.288

                                                                1.5537

                                                                0.0016

                                                                OLP.393

                                                                1.1442

                                                                0.0023

                                                                OLP.311

                                                                0.7197

                                                                0.0091

                                                                   

                                                                OLP.411

                                                                1.5306

                                                                0.0024

                                                                Beneficial

                                                                  
                                                                   

                                                                HLA-A74

                                                                -0.3744

                                                                0.0007

                                                                Beneficial

                                                                  

                                                                HLA-B57

                                                                -0.4887

                                                                0.0007

                                                                HLA-B1502

                                                                -1.4688

                                                                0.0164

                                                                HLA-B81

                                                                -0.3859

                                                                0.0004

                                                                   

                                                                HLA-C12

                                                                -0.6003

                                                                0.0002

                                                                Non-beneficial

                                                                  

                                                                Non-beneficial

                                                                  

                                                                HLA-B0801

                                                                0.66

                                                                0.0049

                                                                HLA-B15

                                                                0.2797

                                                                0.0001

                                                                HLA-C0401

                                                                0.2894

                                                                0.0006

                                                                HLA-B18

                                                                0.5316

                                                                0.0003

                                                                   

                                                                HLA-B49

                                                                0.9713

                                                                0.0007

                                                                * negative co-efficient values indicate reduction in median viral loads

                                                                http://static-content.springer.com/image/art%3A10.1186%2F1479-5876-9-208/MediaObjects/12967_2011_987_Fig4_HTML.jpg
                                                                Figure 4

                                                                Responses to OLP identified in multi-variate analysis are associated with reduced viral loads: Response patterns and HLA class I genetics in the clade B cohort in Lima and clade C cohort in Durban were subjected to FASS multivariat analysis [4143]. Viral loads in individuals mounting zero vs. at least one response to beneficial OLP identified by the FASS multi-variate analysis were compared for (A) the Lima clade B cohort and the (B) Durban clade C cohort. The larger data set for the clade C cohort allowed for a further stratification of the responder group by increasing numbers of targeted OLP emerging from the FASS analysis (C). A gradually declining median viral load in relation to an increasing breadth of these responses was seen (ANOVA, p < 0.0001).

                                                                Results from the clade C cohort in Durban confirmed the clade B findings in Lima as the FASS analyses identified 16 OLP but only 8 HLA variables that had an impact on the individual viral loads. As in Lima, the impact of OLP specificity was at least as strong than HLA genotype (trend for higher coefficients for OLP than HLA; data not shown, p > 0.05). In addition, targeting at least one of the eight beneficial OLP in Durban was associated with strongly reduced viral loads (p < 0.0001, Figure 4B). This effect was, as in the univariate analysis, additive for more than one response (p < 0.0001, Figure 4C) and included OLP that were, aside from Gag, located in Pol and Vif. Also, the combined (OLP and HLA) analysis suggests the effect of OLP specificity on viral loads to be at least as strong as HLA genetics as 8 OLP and 7 HLA variables were identified. This especially since among the 7 HLA alleles, two (HLA-B57 and HLA-A74) are expressed in linkage disequilibrium [47], further reducing the number of HLA variables with a significant impact on viral loads.

                                                                Responses to beneficial OLP are of higher functional avidity and suppress viral replication in vitro more effective than responses to non-beneficial OLP

                                                                Functional avidity and the ability to suppress in vitro viral replication have emerged as two potentially crucial parameters of an effective CTL response against HIV-1 [2329]. To assess this potential functional characteristic of beneficial CTL populations, we determined the functional avidity of responses to the four beneficial OLP located in Gag p24, a region that has been most consistently associated with eliciting relatively protective CTL responses. As 18 mer peptides are suboptimal test peptides to determine functional avidity, 10 mer overlapping peptide sets were synthesized to cover the four beneficial OLP and all detected responses were titrated. The SD50% was determined for a comparable numbers of responses detected in controllers (n = 21 responses) and non-controllers (n = 24 responses) and showed a statistically significant difference between the two groups (median 3, 448 ng/ml vs. 25, 924 ng/ml, p = 0.0051, Figure 5A). This reduced avidity in HIV non-controllers to beneficial OLP could possibly explain why HIV-1 non-controllers did not control their in vivo viral replication despite targeting these regions in some instances and with responses of comparable magnitude as HIV controllers (278 SFC vs 305 SFC/106 PBMC, p = 0.55, data not shown).
                                                                http://static-content.springer.com/image/art%3A10.1186%2F1479-5876-9-208/MediaObjects/12967_2011_987_Fig5_HTML.jpg
                                                                Figure 5

                                                                Responses to beneficial OLP are of higher functional avidity and suppress in vitro viral replication more effectively. (A) Responses to the four beneficial OLP located in HIV-1 clade B Gag p24 were retested using a peptide set of 10 mers overlapping by 9 residues. A total of 21 responses in HIV-1 controllers and 24 responses in HIV-1 non-controllers were titrated and the SD50% compared between the two groups, showing a significantly higher functional avidity in the controllers (p = 0.0051, Mann Whitney). (B) Responses to 17 different optimally defined CTL epitopes located in beneficial, neutral and non-beneficial OLP were titrated in samples from 78 HIV infected individuals with variable viral load and disease status. The median SD50% (ng/ml) was defined for each epitope and compared to the OLP-specific protective ratio (Spearman Rank test, p = 0.0020). (C) Ten individuals who mounted responses to well-defined optimal CTL epitopes located in beneficial as well as in non-beneficial clade B OLP were identified and their responses titrated. The SD50% for responses detected in the same individual were compared (Wilcoxon matched pairs test, p = 0.0039). (D) In-vitro viral replication inhibition assays [48] were performed using a Nef modified and Raltegravir resistant test virus and purified CTL effector populations from the same individual targeting beneficial and non-beneficial OLP. One representative experiment of three assays conducted in different individuals is show. Levels of Gag p24 were determined after 4 days of co-culture of effector cells and auologous CD4 T cells used as target cells. Target cells were stimulated 3 days prior with dual-specific anti-CD3/8 mAb and infected at a MOI of 0.1. The negative control contained wells with target cells only ("no CD8").

                                                                To more directly assess whether responses to beneficial OLP were of particularly high functional avidity, regardless of HIV controller status, we determined SD50% of responses to 17 optimal epitopes from beneficial, neutral and non-beneficial OLP (Figure 5B). Median epitope-specific SD50% were determined from an average of 7 titrations per epitope and compared to the OLP specific PR. A strongly significant, negative association between the PR and the SD50% was noted (p = 0.002, r = -0.69), indicating that beneficial OLP are targeted by high-avidity responses. To control for inter-individual differences due to disease status and viral load, we identified 10 individuals who targeted optimal epitopes in beneficial and non-beneficial OLP and determined their functional avidity. As in the cross-sectional analysis before, this matched comparisons showed in all cases a higher functional avidity for the epitopes located in the beneficial OLP compared to the responses targeting non-beneficial OLP (Figure 5C, p = 0.0020). Lastly, to relate the higher functional avidity to potential superior anti-viral effects in vivo, the ability to inhibit in vitro viral replication was assessed in three individuals who mounted robust responses against both beneficial and non-beneficial OLP. The in vitro inhibition assay first developed by Yang et al [48], was modified so that the NL4-3 based test virus contained a single nucleotide mutation in Nef (M20A) that blocks the Nef-mediated down-regulation of HLA class I molecules as well as two mutations in the integrase gene that mediate Raltegravir-resistance to permit the suppression of potentially replicating autologous virus in the assay. Indeed, CTL specific for the beneficial OLP(s) were up to 2 logs more effective inhibiting viral replication than CTL targeting non-beneficial OLP (Figure 5D), in line with recent data demonstrating different suppressive ability of HIV-1 specific CTL populations targeting Gag and Env-derived epitopes [24]. Although the in vitro inhibition assays were limited to few individuals with suitable response patterns, these data together with the results from the extensive titration assays in Figure 5B and 5C indicate that responses to beneficial OLP are of particularly high functional avidity and inhibit in vitro viral replication more effectively than responses to non-beneficial OLP. Of note, higher avidity responses to beneficial OLP compared to non-beneficial OLP were seen in all 10 tested individuals, ruling out that inter-individual variability in viral loads, duration of infection and HIV disease status could have biased the analyses.

                                                                Conclusions

                                                                Defining functional correlates of HIV-1 immune control is critical to the design of effective immunogens. T cell responses to specific HIV-1 proteins and protein-subunits have been associated before with relatively superior viral control in vivo [14, 16, 49], but evidence from recent clinical trials suggests that including maximal immunogen content into various vectors does not necessarily induce more effective CTL responses [50, 51]. In fact, it has been argued that the existence of potential "decoy" epitopes may divert an effective CTL response towards variable and possibly less effective targets in the viral genome [52]. Thus, the definition of a minimal yet sufficient immunogen sequence that can elicit CTL responses in a broad HLA context is urgently needed. Thereby, focusing vaccine responses on conserved regions could help induce responses towards mutationally constrained targets and provide the basis for protection from heterologous viral challenge.

                                                                We present here the results of an extensive analysis that included more than 950 HIV-1 infected individuals with diverse HLA genotypes, from three different continents and including clade B and C infections. In both, the analysis in clade B in Lima and clade C in Durban, individual OLP were identified that are predominantly targeted by individuals with reduced or elevated viral loads, although the different size of the cohorts required different statistical approaches for their identification. In general, most of these OLP were among the more frequent targets in the HIV proteome, possibly due to both, the need for sizable responder groups to achieve statistical significance in the viral loads comparison as well as the high epitope density in these OLP. The identified OLP were frequently located in HIV-1 Gag and Pol, but rarely in the more variable proteins such as Env and Nef. With one exception, Nef and Env featured only non-beneficial OLP, thus arguing against their inclusion, at least as full proteins, in a CTL immunogen sequence [16]. In addition, in both cohorts, the Vif protein yielded few, yet exclusively beneficial OLP, which may warrant a renewed look at the inclusion of regulatory proteins in vaccine design [53, 54]. Also common to both clades, (and despite the wide scatter possibly due to the inclusion of less-frequently targeted OLP), an negative correlation between sequence entropy and PR was observed providing strong rationale for vaccine approaches that focus on conserved viral regions where T cell escape may be complicated by structural constrains [55]. This was particularly evident in the clade C cohort, where even within the relatively conserved Gag protein, a lower entropy was seen for the beneficial OLP compared to the remainder of the OLP spanning the protein. On the other hand, while beneficial and non-beneficial OLP showed a significant difference in their median entropy in the clade B cohort, this comparison was not significant in the clade C cohort. It is possible that the immunogen sequence, designed in 2001, did not optimally cover the circulating viral population in Durban throughout the enrollment period (until 2006), leading to missed responses particularly in the more variable segments of the virus [32, 56]. The study may have thus failed to identify beneficial as well as non-beneficial OLP in the more variable genes of HIV. This should have preferentially affected highly variable OLP due to a more frequent mismatch between autologous viral sequence and in vitro test set in these regions. However, even if scoring as beneficial OLP, such high-entropy OLP may from an immunogen-design point of view be of less interest as they would possible contribute only little to protection from heterologous viral challenge. It needs however also to be considered that the OLP-specific entropy values are based on variable numbers of sequences in the Los Alamos HIV database covering the different OLP, introducing potential further bias into these analyses, particularly for less covered proteins such as Vpu and other viral protein products. Such differences between autologous viral sequences and in vitro test sets may also have impacted the assessment of functional avidities. These determinations included responses in the same individual towards epitopes located in beneficial and non-beneficial OLP; with the former overall being more conserved. Thus, the higher functional avidity towards epitopes located in beneficial OLP could be biased by the higher chance that these epitopes matched the autologous viral sequence compared to epitopes located in non-beneficial OLP and which may thus have induced a more robust, avid response. Apart from covering autologous sequences, future studies will ideally also include comparable analyses in individuals identified and tested in acute infection that go on to control the infection at undetectable levels of viral replication (i.e. elite-controllers) so that the selective early emergence of responses to beneficial OLP could be linked to relative control of viral replication in chronic infection. As is, the identified beneficial responses may be particularly important to maintain low viral replication in chronic stages of infection, which in theory could be different (for instance due to more accelerated intra-individual viral evolution in variable genes) from responses determining viral set point during acute infection. However, the existing HLA bias in such cohorts and the small number of responses identified during earliest stages of infection may make such analyses a formidable undertaking that will require large numbers of individuals to be tested longitudinally.

                                                                A broadly applicable T cell immunogen sequence should include T cell targets restricted by a wide array of HLA class I alleles. Although broad representation of HLA-B alleles may be particularly important in this regard, emerging data on the effects HLA-C alleles in these cohorts may warrant a broad HLA-C representation as well [2, 47, 57]. In the present study, the 26 beneficial OLP from Lima and the 22 beneficial OLP from Durban covered 26 described, optimally defined CTL epitopes restricted by 20 different HLA alleles for the clade B cohort and 33 epitopes presented by 34 alleles for the clade C cohort, respectively [44]. As this is likely to be an underestimate of the true diversity in HLA restriction (Table 2 and ref [58]), it is reasonable to predict that the inclusion of identified beneficial OLP, or even a subset thereof, could evoke potential responses in a widely diverse HLA context. This could also provide the basis for the induction of poly-specific T cell responses with increased breath, which the present data clearly associates with progressively lower viral loads and which emerge as a potentially important parameter from several recent vaccine studies showing superior protection from SIV challenge in animals with a broad vaccine induced responses to Gag p17 [59, 60].

                                                                Recent studies have suggested a global adaptation of HIV-1 to its various host ethnicities [4, 46]. The consequence of such adaptation has led in some cases to the elimination of protective CTL targets, causing a profound absence of responses to these epitopes and detrimentally changing the association between HLA allele and HIV-1 disease outcome [4]. It is thus not surprising that the two main cohorts tested here yielded only partially overlapping sets of beneficial OLP as the impact of host genetics and viral evolution in the studied populations cannot readily be overcome. In fact, given studies by Frahm et al [4], the past and current adaptation of HIV-1 to common HLA class I alleles will likely still call for somewhat population tailored vaccine approaches, especially if the immunogen sequences should be kept short to avoid regions of potentially reduced immunological value [52]. Such approaches will also profit from more extensive structural analyses that may identify specific domains of viral proteins that are or are not enriched in valuable T cell targets; of which the latter could possibly be ignored for the design of T cell immunogen sequences. Additional analyses in other genetically unrelated cohorts of HIV-1 infected individuals and studies in SIV infection may further help to guide such selective immunogen design and to understand the factors defining the effectiveness of different epitopes in mediating relative HIV-1 control. Of note, the beneficial OLP identified here, 24 in clade B and 22 in clade C infection matched other immunogen design based on conserved elements in some parts as well, i.e. of the 14 conserved elements proposed by Hanke et al, eight (57%) overlapped at least partly with beneficial OLP identified here [61]. Similarly, among the highly conserved elements proposed by Rolland et al [52], 35% (5/14) were covered by our beneficial OLP in clade B infection. These differences possibly emerge because the present analysis is based on functional T cell data rather than viral sequence alignments, which may not take into consideration epitope density and processing preferences of certain regions. Nevertheless, the partial overlap with these other immunogen design support the focus on conserved regions and offers the opportunity for alternative or combined vaccine approach that elicit responses to regions where the virus is and possibly remains vulnerable [4, 46, 55, 62].

                                                                Finally, we used the extensive data set available to approach the question of relative effects of host genetics (i.e. HLA) and CTL specificity on HIV-1 control. While the two factors cannot be entirely disentangled, our data suggest that CTL specificity has an at least equal if not stronger effect on viral control than HLA class I allele expression. These findings are also in line with data by Mothe et al [63] showing that targeting key regions in p24 surrounding the dominant epitopes restricted by known protective alleles (KK10 for HLA-B27 and TW10 for HLA-B57/58) in HLA-B27, -57 or B58 negative individuals is associated with significantly reduced viral loads. In addition, the presence of individuals not expressing known beneficial alleles in HIV-1 elite controller cohorts [64], further indicates that HIV-1 control is not necessarily bound to a few specific HLA class I alleles. A detailed study of the total HIV-1-specific CTL response of subjects not expressing these alleles yet effectively controlling HIV-1 can be expected to provide further and crucially needed insight into the importance of targeting specific (conserved) regions of the viral genome for HIV-1 control. Similarly, the characterization of functional attributes of these responses, including functional avidity and the ability to suppress in vitro viral replication will need to be further assessed in such individuals. Building on experimentally derived and potentially promising immunogen sequences as defined here may thus provide a suitable basis for further immunogen design and iterative clinical trials in the human setting.

                                                                Declarations

                                                                Acknowledgements

                                                                This work was supported by NIH contracts N01-AI-30024 and N01-AI-15422 (CB, TA, BDW, BTK, JS), NIH-NIDCR R01 DE018925-04 (CB), grant MTM2008--06747--C02-00 (GG) from the Ministerio de Ciencia y Tecnología, a grant from the Instituto de Salud Carlos III (FIS PS09/00283, AL) and a grant from the Fundacio para la Investigacion y Prevencion del SIDA en España (FIPSE) # 360737/09, CB), Spain as well as a grant from the European Community FP7 ("CUTHIVAC"). BM holds a research fellowship grant from the FIS (Rio Hortega, CM08/00020), Madrid, Spain. ZB is supported by a New Investigator Award from the Canadian Institutes of Health Research (CIHR). CB and JMP are ICREA (Institució Catalana de Recerca i Estudis Avançats) Senior Research Professors. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

                                                                Authors’ Affiliations

                                                                (1)
                                                                Irsicaixa AIDS Research Institute-HIVACAT
                                                                (2)
                                                                ‘Lluita contra la SIDA’ Foundation, Hospital Germans Trias i Pujol
                                                                (3)
                                                                Universitat Autònoma de Barcelona
                                                                (4)
                                                                Los Alamos National Laboratory
                                                                (5)
                                                                Asociación Civil IMPACTA Salud y Educacion
                                                                (6)
                                                                Dept. Estadística i Investigació Operativa, Universitat Politècnica de Catalunya
                                                                (7)
                                                                Ragon Institute of MGH, Harvard and MIT
                                                                (8)
                                                                Institucio Catalana de Recerca i Estudis Avançats (ICREA)
                                                                (9)
                                                                MHRP
                                                                (10)
                                                                University of Oklahoma Medical Center
                                                                (11)
                                                                University of California
                                                                (12)
                                                                Services of Immunology and Institut d’Investigacions Biomediques August Pi i Sunyer (IDIBAPS)-AIDS Research Group-HIVACAT, Hospital Clinic
                                                                (13)
                                                                British Columbia Centre for Excellence in HIV/AIDS
                                                                (14)
                                                                Simon Fraser University
                                                                (15)
                                                                Miscrosoft Research
                                                                (16)
                                                                Department of Microbiology, University of Washington
                                                                (17)
                                                                Department of Paediatrics, Nuffield Department of Medicine
                                                                (18)
                                                                University of KwaZulu-Natal, HIV Pathogenesis Program, DDMRI
                                                                (19)
                                                                Howard Hughes Medical Institute
                                                                (20)
                                                                Santa Fe Institute

                                                                References

                                                                1. Brander C, Frahm N, Walker B: Host immunity and viral diversity in HIV vaccine design. Current Opinion Immunol 2006, 18:1–8.View Article
                                                                2. Kiepiela P, Leslie AJ, Honeyborne I, Ramduth D, Thobakgale C, Chetty S, Rathnavalu P, Moore C, Pfafferott KJ, Hilton L, Zimbwa P, Moore S, Allen T, Brander C, Addo MM, Altfeld M, James I, Mallal S, Bunce M, Barber LD, Szinger J, Day C, Klenerman P, Mullins J, Korber B, Coovadia HM, Walker BD, Goulder PJ: Dominant influence of HLA-B in mediating the potential co-evolution of HIV and HLA. Nature 2004,432(7018):769–775.PubMedView Article
                                                                3. Frahm N, Adams S, Kiepiela P, Linde CH, Hewitt HS, Lichterfeld M, Sango K, Brown NV, Pae E, Wurcel AG, Altfeld M, Feeney ME, Allen TM, Roach T, St John MA, Daar ES, Rosenberg E, Korber B, Marincola F, Walker BD, Goulder PJ, Brander C: HLA-B63 presents HLA-B57/B58-restricted cytotoxic T-lymphocyte epitopes and is associated with low human immunodeficiency virus load. J Virol 2005,79(16):10218–10225.PubMedView Article
                                                                4. Frahm N, Kiepiela P, Adams S, Linde CH, Hewitt HS, Sango K, Feeney ME, Addo MM, Lichterfeld M, Lahaie MP, Pae E, Wurcel AG, Roach T, St John MA, Altfeld M, Marincola FM, Moore C, Mallal S, Carrington M, Heckerman D, Allen TM, Mullins JI, Korber BT, Goulder PJ, Walker BD, Brander C: Control of human immunodeficiency virus replication by cytotoxic T lymphocytes targeting subdominant epitopes. Nat Immunol 2006,7(2):173–178.PubMedView Article
                                                                5. Ngumbela KC, Day CL, Mncube Z, Nair K, Ramduth D, Thobakgale C, Moodley E, Reddy S, de Pierres C, Mkhwanazi N, Bishop K, van der Stok M, Ismail N, Honeyborne I, Crawford H, Kavanagh DG, Rousseau C, Nickle D, Mullins J, Heckerman D, Korber B, Coovadia H, Kiepiela P, Goulder PJ, Walker BD: Targeting of a CD8 T cell env epitope presented by HLA-B*5802 is associated with markers of HIV disease progression and lack of selection pressure. AIDS Res Hum Retroviruses 2008,24(1):72–82.PubMedView Article
                                                                6. Brockman MA, Schneidewind A, Lahaie M, Schmidt A, Miura T, Desouza I, Ryvkin F, Derdeyn CA, Allen S, Hunter E, Mulenga J, Goepfert PA, Walker BD, Allen TM: Escape and compensation from early HLA-B57-mediated cytotoxic T-lymphocyte pressure on human immunodeficiency virus type 1 Gag alter capsid interactions with cyclophilin A. J Virol 2007,81(22):12608–12618.PubMedView Article
                                                                7. Schneidewind A, Brockman MA, Yang R, Adam RI, Li B, Le Gall S, Rinaldo CR, Craggs SL, Allgaier RL, Power KA, Kuntzen T, Tung CS, LaBute MX, Mueller SM, Harrer T, McMichael AJ, Goulder PJ, Aiken C, Brander C, Kelleher AD, Allen TM: Escape from the dominant HLA-B27-restricted cytotoxic T-lymphocyte response in Gag is associated with a dramatic reduction in human immunodeficiency virus type 1 replication. J Virol 2007,81(22):12382–12393.PubMedView Article
                                                                8. Leslie A, Price DA, Mkhize P, Bishop K, Rathod A, Day C, Crawford H, Honeyborne I, Asher TE, Luzzi G, Edwards A, Rousseau CM, Mullins JI, Tudor-Williams G, Novelli V, Brander C, Douek DC, Kiepiela P, Walker BD, Goulder PJ: Differential selection pressure exerted on HIV by CTL targeting identical epitopes but restricted by distinct HLA alleles from the same HLA supertype. J Immunol 2006,177(7):4699–4708.PubMed
                                                                9. Iversen AK, Stewart-Jones G, Learn GH, Christie N, Sylvester-Hviid C, Armitage AE, Kaul R, Beattie T, Lee JK, Li Y, Chotiyarnwong P, Dong T, Xu X, Luscher MA, MacDonald K, Ullum H, Klarlund-Pedersen B, Skinhoj P, Fugger L, Buus S, Mullins JI, Jones EY, van der Merwe PA, McMichael AJ: Conflicting selective forces affect T cell receptor contacts in an immunodominant human immunodeficiency virus epitope. Nat Immunol 2006,7(2):179–189.PubMedView Article
                                                                10. Kijak GH, Currier JR, Tovanabutra S, Cox JH, Michael NL, Wegner SA, Birx DL, McCutchan FE: Lost in translation: implications of HIV-1 codon usage for immune escape and drug resistance. AIDS Rev 2004,6(1):54–60.PubMed
                                                                11. Price DA, West SM, Betts MR, Ruff LE, Brenchley JM, Ambrozak DR, Edghill-Smith Y, Kuroda MJ, Bogdan D, Kunstman K, Letvin NL, Franchini G, Wolinsky SM, Koup RA, Douek D: T cell receptor recognition motifs govern immune escape patterns in acute SIV infection. Immunity 2004.
                                                                12. Bihl F, Frahm N, Di Giammarino L, Sidney J, John M, Yusim K, Woodberry T, Sango K, Hewitt HS, Henry L, Linde CH, Chisholm JV, Zaman TM, Pae E, Mallal S, Walker BD, Sette A, Korber BT, Heckerman D, Brander C: Impact of HLA-B alleles, epitope binding affinity, functional avidity, and viral coinfection on the immunodominance of virus-specific CTL responses. J Immunol 2006,176(7):4094–4101.PubMed
                                                                13. Yerly D, Heckerman D, Allen TM, Chisholm JV, Faircloth K, Linde CH, Frahm N, Timm J, Pichler WJ, Cerny A, Brander C: Increased cytotoxic T-lymphocyte epitope variant cross-recognition and functional avidity are associated with hepatitis C virus clearance. J Virol 2008,82(6):3147–3153.PubMedView Article
                                                                14. Zuñiga R, Lucchetti A, Galvan P, Sanchez S, Sanchez C, Hernandez A, Sanchez H, Frahm N, Linde C, Hewitt HS, Hildebrand WH, Altfeld M, Allen T, Walker B, Korber B, Leitner T, Sanchez J, Brander C: Relative dominance of Gag p24 specific CTL is associated with HIV control. J Virology 2006, 80:3122–3125.PubMedView Article
                                                                15. Masemola A, Mashishi T, Khoury G, Mohube P, Mokgotho P, Vardas E, Colvin M, Zijenah L, Katzenstein D, Musonda R, Allen S, Kumwenda N, Taha T, Gray G, McIntyre J, Karim SA, Sheppard HW, Gray CM: Hierarchical targeting of subtype C human immunodeficiency virus type 1 proteins by CD8+ T cells: correlation with viral load. J Virol 2004,78(7):3233–3243.PubMedView Article
                                                                16. Kiepiela P, Ngumbela K, Thobakgale C, Ramduth D, Honeyborne I, Moodley E, Reddy S, de Pierres C, Mncube Z, Mkhwanazi N, Bishop K, van der Stok M, Nair K, Khan N, Crawford H, Payne R, Leslie A, Prado J, Prendergast A, Frater J, McCarthy N, Brander C, Learn GH, Nickle D, Rousseau C, Coovadia H, Mullins JI, Heckerman D, Walker BD, Goulder P: CD8+ T-cell responses to different HIV proteins have discordant associations with viral load. Nat Med 2007,13(1):46–53.PubMedView Article
                                                                17. Borghans JA, Molgaard A, de Boer RJ, Kesmir C: HLA alleles associated with slow progression to AIDS truly prefer to present HIV-1 p24. PLoS ONE 2007,2(9):e920.PubMedView Article
                                                                18. Sacha JB, Chung C, Rakasz EG, Spencer SP, Jonas AK, Bean AT, Lee W, Burwitz BJ, Stephany JJ, Loffredo JT, Allison DB, Adnan S, Hoji A, Wilson NA, Friedrich TC, Lifson JD, Yang OO, Watkins DI: Gag-specific CD8+ T lymphocytes recognize infected cells before AIDS-virus integration and viral protein expression. J Immunol 2007,178(5):2746–2754.PubMed
                                                                19. Honeyborne I, Prendergast A, Pereyra F, Leslie A, Crawford H, Payne R, Reddy S, Bishop K, Moodley E, Nair K, van der Stok M, McCarthy N, Rousseau CM, Addo M, Mullins JI, Brander C, Kiepiela P, Walker BD, Goulder PJ: Control of human immunodeficiency virus type 1 is associated with HLA-B*13 and targeting of multiple gag-specific CD8+ T-cell epitopes. J Virol 2007,81(7):3667–3672.PubMedView Article
                                                                20. Leslie AJ, Pfafferott KJ, Chetty P, Draenert R, Addo MM, Feeney M, Tang Y, Holmes EC, Allen T, Prado JG, Altfeld M, Brander C, Dixon C, Ramduth D, Jeena P, Thomas SA, St John A, Roach TA, Kupfer B, Luzzi G, Edwards A, Taylor G, Lyall H, Tudor-Williams G, Novelli V, Martinez-Picado J, Kiepiela P, Walker BD, Goulder PJ: HIV evolution: CTL escape mutation and reversion after transmission. Nat Med 2004,10(3):282–289.PubMedView Article
                                                                21. Martinez-Picado J, Prado JG, Fry EE, Pfafferott K, Leslie A, Chetty S, Thobakgale C, Honeyborne I, Crawford H, Matthews P, Pillay T, Rousseau C, Mullins JI, Brander C, Walker BD, Stuart DI, Kiepiela P, Goulder P: Fitness cost of escape mutations in p24 Gag in association with control of human immunodeficiency virus type 1. J Virol 2006,80(7):3617–3623.PubMedView Article
                                                                22. Friedrich TC, Valentine LE, Yant LJ, Rakasz EG, Piaskowski SM, Furlott JR, Weisgrau KL, Burwitz B, May GE, Leon EJ, Soma T, Napoe G, Capuano SV, Wilson NA, Watkins DI: Subdominant CD8+ T-cell responses are involved in durable control of AIDS virus replication. J Virol 2007,81(7):3465–3476.PubMedView Article
                                                                23. Bennett MS, Joseph A, Ng HL, Goldstein H, Yang OO: Fine-tuning of T-cell receptor avidity to increase HIV epitope variant recognition by cytotoxic T lymphocytes. Aids 2010.
                                                                24. Chen H, Piechocka-Trocha A, Miura T, Brockman MA, Julg BD, Baker BM, Rothchild AC, Block BL, Schneidewind A, Koibuchi T, Pereyra F, Allen TM, Walker BD: Differential neutralization of human immunodeficiency virus (HIV) replication in autologous CD4 T cells by HIV-specific cytotoxic T lymphocytes. J Virol 2009,83(7):3138–3149.PubMedView Article
                                                                25. Almeida JR, Price DA, Papagno L, Arkoub ZA, Sauce D, Bornstein E, Asher TE, Samri A, Schnuriger A, Theodorou I, Costagliola D, Rouzioux C, Agut H, Marcelin AG, Douek D, Autran B, Appay V: Superior control of HIV-1 replication by CD8+ T cells is reflected by their avidity, polyfunctionality, and clonal turnover. J Exp Med 2007,204(10):2473–2485.PubMedView Article
                                                                26. Almeida JR, Sauce D, Price DA, Papagno L, Shin SY, Moris A, Larsen M, Pancino G, Douek DC, Autran B, Saez-Cirion A, Appay V: Antigen sensitivity is a major determinant of CD8+ T-cell polyfunctionality and HIV-suppressive activity. Blood 2009,113(25):6351–6360.PubMedView Article
                                                                27. Saez-Cirion A, Lacabaratz C, Lambotte O, Versmisse P, Urrutia A, Boufassa F, Barre-Sinoussi F, Delfraissy JF, Sinet M, Pancino G, Venet A: HIV controllers exhibit potent CD8 T cell capacity to suppress HIV infection ex vivo and peculiar cytotoxic T lymphocyte activation phenotype. Proc Natl Acad Sci USA 2007,104(16):6776–6781.PubMedView Article
                                                                28. Julg B, Williams KL, Reddy S, Bishop K, Qi Y, Carrington M, Goulder PJ, Ndung'u T, Walker BD: Enhanced anti-HIV functional activity associated with Gag-specific CD8 T-cell responses. J Virol 2010,84(11):5540–5549.PubMedView Article
                                                                29. Berger C, Frahm N, Price D, Mothe B, Ghebremichael M, Hartman K, Henry L, Brenchley J, Ruff L, Venturi V, Pereyra F, Sidney J, Sette A, Douek D, Walker B, Kaufmann D, Brander C: High functional avidity CTL responses to HLA-B-restricted Gag-derived epitopes associate with relative HIV control. J Virology 2011,85(18):9334–9345.PubMedView Article
                                                                30. Bunce M, O'Neill CM, Barnardo MC, Krausa P, Browning MJ, Morris PJ, Welsh KI: Phototyping: comprehensive DNA typing for HLA-A, B, C, DRB1, DRB3, DRB4, DRB5 & DQB1 by PCR with 144 primer mixes utilizing sequence- specific primers (PCR-SSP). Tissue Antigens 1995,46(5):355–367.PubMedView Article
                                                                31. Frahm N, Korber BT, Adams CM, Szinger JJ, Draenert R, Addo MM, Feeney ME, Yusim K, Sango K, Brown NV, SenGupta D, Piechocka-Trocha A, Simonis T, Marincola FM, Wurcel AG, Stone DR, Russell CJ, Adolf P, Cohen D, Roach T, StJohn A, Khatri A, Davis K, Mullins J, Goulder PJ, Walker BD, Brander C: Consistent cytotoxic-T-lymphocyte targeting of immunodominant regions in human immunodeficiency virus across multiple ethnicities. J Virol 2004,78(5):2187–2200.PubMedView Article
                                                                32. Frahm N, Nickle DC, Linde CH, Cohen DE, Zuniga R, Lucchetti A, Roach T, Walker BD, Allen TM, Korber BT, Mullins JI, Brander C: Increased detection of HIV-specific T cell responses by combination of central sequences with comparable immunogenicity. Aids 2008,22(4):447–456.PubMedView Article
                                                                33. Kiepiela P, Leslie AJ, Honeyborne I, Ramduth D, Thobakgale C, Chetty S, Rathnavalu P, Moore C, Pfafferott KJ, Hilton L, Zimbwa P, Moore S, Allen T, Brander C, Addo MM, Altfeld M, James I, Mallal S, Bunce M, Barber LD, Szinger J, Day C, Klenerman P, Mullins J, Korber B, Coovadia HM, Walker BD, Goulder PJ: Coevolutionary influences of HIV and HLA: the dominant role of HLA-B. Nature 2004, 432:769–774.PubMedView Article
                                                                34. Andre P, Groettrup M, Klenerman P, de Giuli R, Booth BL, Cerundolo V, Bonneville M, Jotereau F, Zinkernagel RM, Lotteau V: An inhibitor of HIV-1 protease modulates proteasome activity, antigen presentation, and T cell responses. Proc Natl Acad Sci 1998,95(22):13120–13124.PubMedView Article
                                                                35. Ali A, Jamieson BD, Yang OO: Half-genome human immunodeficiency virus type 1 constructs for rapid production of reporter viruses. J Virol Methods 2003,110(2):137–142.PubMedView Article
                                                                36. Hu Z, Kuritzkes DR: Effect of raltegravir resistance mutations in HIV-1 integrase on viral fitness. J Acquir Immune Defic Syndr 2010,55(2):148–155.PubMedView Article
                                                                37. Adnan S, Balamurugan A, Trocha A, Bennett MS, Ng HL, Ali A, Brander C, Yang OO: Nef interference with HIV-1-specific CTL antiviral activity is epitope specific. Blood 2006,108(10):3414–3419.PubMedView Article
                                                                38. Gibbs JS, Regier DA, Desrosiers RC: Construction and in vitro properties of HIV-1 mutants with deletions in "nonessential" genes. AIDS Res Hum Retroviruses 1994,10(4):343–350.PubMedView Article
                                                                39. Berger CT, Carlson JM, Brumme CJ, Hartman KL, Brumme ZL, Henry LM, Rosato PC, Piechocka-Trocha A, Brockman MA, Harrigan PR, Heckerman D, Kaufmann DE, Brander C: Viral adaptation to immune selection pressure by HLA class I-restricted CTL responses targeting epitopes in HIV frameshift sequences. J Exp Med 2010,207(1):61–75.PubMedView Article
                                                                40. Team RDC: R: A Language and Environment for Statistical Computing. [http://​www.​R-project.​org] R Foundation for Statistical Computing; 2010.
                                                                41. Liebminger A, Seyfang L, Filzmoser P, Varmuza K: A New Variable Selection Method Based on All Subsets Regression. 10th SSC 2007, Scandinavian Symposium on Chemometrics: 2007.
                                                                42. Seyfang L: Heuristiken zur Variablenselektion bei hochdimensionalen Daten. Institut für Statistik und Wahrscheinlichkeitstheorie, Technische Universität Wien, Vienna 2008.
                                                                43. Pérez-Álvarez S, Gomez G, Pérez-Álvarez N, Brander C: Technical Report: Use of comprehensive cohort data to identify host genetic and immunological and virological factors associated with relative in vivo control of HIV. Deparment of Statistics and Operational Research, Universitat Politècnica de Catalunya (UPC), Barcelona 2011.
                                                                44. Llano A, Frahm N, Brander C: How to Optimally Define Optimal Cytotoxic T Lymphocyte Epitopes in HIV Infection? In HIV Molecular immunology database. Edited by: Korber CB B, Walker B, Koup R, Moore J, Haynes B, Meyers G. Los Alamos, NM, USA: Los Alamos National Laboratory: Theoretical Biology and Biophysics; 2009.
                                                                45. Goulder PJ, Brander C, Annamalai K, Mngqundaniso N, Govender U, Tang Y, He S, Hartman KE, O'Callaghan CA, Ogg GS, Altfeld MA, Rosenberg ES, Cao H, Kalams SA, Hammond M, Bunce M, Pelton SI, Burchett SA, McIntosh K, Coovadia HM, Walker BD: Differential narrow focusing of immunodominant human immunodeficiency virus gag-specific cytotoxic T-lymphocyte responses in infected african and caucasoid adults and children. J Virol 2000,74(12):5679–5690.PubMedView Article
                                                                46. Kawashima Y, Pfafferott K, Frater J, Matthews P, Payne R, Addo M, Gatanaga H, Fujiwara M, Hachiya A, Koizumi H, Kuse N, Oka S, Duda A, Prendergast A, Crawford H, Leslie A, Brumme Z, Brumme C, Allen T, Brander C, Kaslow R, Tang J, Hunter E, Allen S, Mulenga J, Branch S, Roach T, John M, Mallal S, Ogwu A, et al.: Adaptation of HIV-1 to human leukocyte antigen class I. Nature 2009,458(7238):641–645.PubMedView Article
                                                                47. Leslie A, Matthews PC, Listgarten J, Carlson JM, Kadie C, Ndung'u T, Brander C, Coovadia H, Walker BD, Heckerman D, Goulder PJ: Additive contribution of HLA class I alleles in the immune control of HIV-1 infection. J Virol 2010,84(19):9879–9888.PubMedView Article
                                                                48. Yang OO, Kalams SA, Trocha A, Cao H, Luster A, Johnson RP, Walker BD: Suppression of human immunodeficiency virus type 1 replication by CD8+ cells: evidence for HLA class 1-restricted triggering of cytolytic and noncytolytic mechanisms. J Virol 1997,71(4):3120–3128.PubMed
                                                                49. Rolland M, Heckerman D, Deng W, Rousseau CM, Coovadia H, Bishop K, Goulder PJ, Walker BD, Brander C, Mullins JI: Broad and Gag-biased HIV-1 epitope repertoires are associated with lower viral loads. PLoS ONE 2008,3(1):e1424.PubMedView Article
                                                                50. Buchbinder SP, Mehrotra DV, Duerr A, Fitzgerald DW, Mogg R, Li D, Gilbert PB, Lama JR, Marmor M, Del Rio C, McElrath MJ, Casimiro DR, Gottesdiener KM, Chodakewitz JA, Corey L, Robertson MN: Efficacy assessment of a cell-mediated immunity HIV-1 vaccine (the Step Study): a double-blind, randomised, placebo-controlled, test-of-concept trial. Lancet 2008,372(9653):1881–1893.PubMedView Article
                                                                51. Rerks-Ngarm S, Pitisuttithum P, Nitayaphan S, Kaewkungwal J, Chiu J, Paris R, Premsri N, Namwat C, de Souza M, Adams E, Benenson M, Gurunathan S, Tartaglia J, McNeil JG, Francis DP, Stablein D, Birx DL, Chunsuttiwat S, Khamboonruang C, Thongcharoen P, Robb ML, Michael NL, Kunasol P, Kim JH: Vaccination with ALVAC and AIDSVAX to prevent HIV-1 infection in Thailand. N Engl J Med 2009,361(23):2209–2220.PubMedView Article
                                                                52. Rolland M, Nickle DC, Mullins JI: HIV-1 Group M Conserved Elements Vaccine. PLOS Pathogen 2007,3(11):e157.View Article
                                                                53. Addo M, Altfeld M, Rosenberg E, Eldridge B, Philips M, Habeeb K, Khatri A, Brander C, Robbins G, Mazzara G, Goulder P, Walker B: The HIV-1 regulatory proteins Tat and Rev are freqently targeted by cytotoxic T lymphocyte (CTL) derived from HIV infected individuals. Proc Nat Acad Sci 2001, 98:1781–1786.PubMedView Article
                                                                54. Kloverpris H, Karlsson I, Bonde J, Thorn M, Vinner L, Pedersen AE, Hentze JL, Andresen BS, Svane IM, Gerstoft J, Kronborg G, Fomsgaard A: Induction of novel CD8+ T-cell responses during chronic untreated HIV-1 infection by immunization with subdominant cytotoxic T-lymphocyte epitopes. Aids 2009,23(11):1329–1340.PubMedView Article
                                                                55. Altfeld M, Allen TM: Hitting HIV where it hurts: an alternative approach to HIV vaccine design. Trends Immunol 2006,27(11):504–510.PubMedView Article
                                                                56. Frahm N, Kaufmann DE, Yusim K, Muldoon M, Kesmir C, Linde CH, Fischer W, Allen TM, Li B, McMahon BH, Faircloth KL, Hewitt HS, Mackey EW, Miura T, Khatri A, Wolinsky S, McMichael A, Funkhouser RK, Walker BD, Brander C, Korber BT: Increased sequence diversity coverage improves detection of HIV-specific T cell responses. J Immunol 2007,179(10):6638–6650.PubMed
                                                                57. Ibarrondo J, Zuniga R, Farfan M, Suarez J, Mothe B, Llano A, Szinger J, Hildebrand W, Sanchez J, Korber B, Brander C: Crucial contribution of sub-dominant HLA-C allele restricted CTL responses to the control of HIV. AIDs Vaccine 2009: 2009; Paris
                                                                58. Frahm N, Yusim K, Adams S, Sidney J, Hraber P, Hewitt HS, Linde CH, Woodberry T, Henry L, Listgarten J, Kadie C, Jojic N, Sango K, Brown NV, Pae E, Zaman T, John M, Mallal S, Marincola F, Walker BD, Sette A, Heckerman D, Korber BT, Brander C: Extensive HLA class I allele promiscuity among viral cytotoxic T lymphocyte (CTL) epitopes. Europ J Immunol 2007, 37:2419–2433.View Article
                                                                59. Liu J, O'Brien KL, Lynch DM, Simmons NL, La Porte A, Riggs AM, Abbink P, Coffey RT, Grandpre LE, Seaman MS, Landucci G, Forthal DN, Montefiori DC, Carville A, Mansfield KG, Havenga MJ, Pau MG, Goudsmit J, Barouch DH: Immune control of an SIV challenge by a T-cell-based vaccine in rhesus monkeys. Nature 2009,457(7225):87–91.PubMedView Article
                                                                60. Reece JC, Loh L, Alcantara S, Fernandez CS, Stambas J, Sexton A, De Rose R, Petravic J, Davenport MP, Kent SJ: Timing of immune escape linked to success or failure of vaccination. PLoS One 2010.,5(9):
                                                                61. Letourneau S, Im EJ, Mashishi T, Brereton C, Bridgeman A, Yang H, Dorrell L, Dong T, Korber B, McMichael AJ, Hanke T: Design and pre-clinical evaluation of a universal HIV-1 vaccine. PLoS One 2007,2(10):e984.PubMedView Article
                                                                62. Koga M, Kawana-Tachikawa A, Heckerman D, Odawara T, Nakamura H, Koibuchi T, Fujii T, Miura T, Iwamoto A: Changes in impact of HLA class I allele expression on HIV-1 plasma virus loads at a population level over time. Microbiol Immunol 2010,54(4):196–205.PubMedView Article
                                                                63. Mullins J: A global AIDS vaccine based on conserved elements of the viral proteome. AIDS Vaccine 2010: 2010; Atlanta, Georgia, USA;
                                                                64. Pereyra F, Addo MM, Kaufmann DE, Liu Y, Miura T, Rathod A, Baker B, Trocha A, Rosenberg R, Mackey E, Ueda P, Lu Z, Cohen D, Wrin T, Petropoulos CJ, Rosenberg ES, Walker BD: Genetic and immunologic heterogeneity among persons who control HIV infection in the absence of therapy. J Infect Dis 2008,197(4):563–571.PubMedView Article

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                                                                This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://​creativecommons.​org/​licenses/​by/​2.​0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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