M. Tschochner

High-avidity, high-IFNγ-producing CD8 T-cell responses following immune selection during HIV-1 infection

HIV‐1 mutations, which reduce or abolish CTL responses against virus‐infected cells, are frequently selected in acute and chronic HIV infection. Among population HIV‐1 sequences, immune selection is evident as human leukocyte antigen (HLA) allele‐associated substitutions of amino acids within or near CD8 T‐cell epitopes. In these cases, the non‐adapted epitope is susceptible to immune recognition until an escape mutation renders the epitope less immunogenic. However, several population‐based studies have independently identified HLA‐associated viral changes, which lead to the formation of a new T‐cell epitope, suggesting that the immune responses that these variants or ‘neo‐epitopes’ elicit provide an evolutionary advantage to the virus rather than the host. Here, we examined the functional characteristics of eight CD8 T‐cell responses that result from viral adaptation in 125 HLA‐genotyped individuals with chronic HIV‐1 infection. Neo‐epitopes included well‐characterized immunodominant epitopes restricted by common HLA alleles, and in most cases the T‐cell responses against the neo‐epitope showed significantly greater functional avidity and higher IFNγ production than T cells for non‐adapted epitopes, but were not more cytotoxic. Neo‐epitope formation and emergence of cognate T‐cell response coincident with a rise in viral load was then observed in vivo in an acutely infected individual. These findings show that HIV‐1 adaptation not only abrogates the immune recognition of early targeted epitopes, but may also increase immune recognition to other epitopes, which elicit immunodominant but non‐protective T‐cell responses. These data have implications for immunodominance associated with polyvalent vaccines based on the diversity of chronic HIV‐1 sequences.

Contributions of vitamin D response elements and HLA promoters to multiple sclerosis risk

Objective: The identification of a vitamin D–responsive (VDRE) motif within the HLA-DRB1*15:01 promoter region provides an attractive explanation for the combined effects of HLA-DR inheritance and vitamin D exposure on multiple sclerosis (MS) risk. We therefore sought to incorporate HLA-DRB1 promoter variation, including the VDRE motif, in an assessment of HLA-DRB1–associated MS risk. Methods: We utilized 32 homozygous HLA cell lines (covering 17 DRB1 alleles) and 53 heterozygote MS samples (20 DRB1 alleles) for HLA-DRB1 promoter sequencing. The influence of HLA-DRB1 variation on MS risk was then assessed among 466 MS cases and 498 controls. Results: The majority of HLA*DRB1 alleles (including HLA-DRB1*15:01) express the functional VDRE motif, apart from HLA-DRB1*04, *07, and *09 alleles that comprise the HLA-DR53 serologic group. Allele-specific variation within functional X-box and Y-box motifs was also associated with serologically defined HLA-DR haplotypes. Incorporating these results in an analysis of MS risk, we identified a strong protective effect of HLA-DRB1*04, *07, and *09 (DR53) alleles (p = 10−12) and elevated risk associated with DRB1*15 and *16 (DR51) and *08 (DR8) alleles (p < 10−18). Conclusions: HLA-DRB1 groups corresponding to serologic HLA-DR profiles as well as promoter polymorphism haplotypes effectively stratified MS risk over an 11-fold range, suggesting functional relationships between risk-modifying HLA-DRB1 alleles. An independent contribution of VDRE motif variation to increase MS risk was not discernible, although vitamin D–dependent regulation of HLA-DR expression may still play an important role given that HLA-DRB1*04/*07/*09 (DR53) alleles that express the “nonresponsive” VDRE motif were associated with significantly reduced risk of MS.

No evidence for persistence of multidrug-resistant viral strains after a 7-month treatment interruption in an HIV-1-infected individual.

The number of HIV-1-infected patients harboring multidrug-resistant viruses is increasing. Since new antiretroviral drugs with favorable resistance profiles are limited, innovative strategies are urgently needed. Treatment interruptions can lead to a loss in HIV resistance followed by improved response to reinitiated therapy. The authors report the case of a patient with sustained antiretroviral response for 3.5 years after a 7-month treatment interruption. Concomitant with an increase in replication capacity, multidrug-resistant viruses gradually disappeared during treatment interruption. Resistance to protease inhibitors (PI) was completely lost, and resistance to reverse transcriptase inhibitors was still present when therapy was reinitiated. PI-resistant variants were not detected at four time points after treatment reinitiation. The alignment of the nucleic acid sequences from all different time points suggested that the viruses obtained after treatment reinitiation evolved from less-resistant variants prior to treatment interruption. This was supported by in vitro propagation of the viral plasma population and an individual clone derived from the time point of treatment interruption. This is consistent with a model favoring reversible binding of HIV-1 to reservoirs, as has recently been proposed for follicular dendritic cells. Understanding of this process could help to exploit the reduced fitness of drug-resistant viruses for treatment interruptions.

Combining HLA-DR risk alleles and anti-Epstein-Barr virus antibody profiles to stratify multiple sclerosis risk.

Background: Risk factors for multiple sclerosis (MS) include human leukocyte antigen (HLA)-DR and Epstein-Barr virus (EBV)-specific antibody responses, including an epitope within EBV nuclear antigen 1 (EBNA-1) that is of recent interest. Objective: The objective of this paper is to assess case-control associations between MS risk and anti-EBV antibody levels as well as HLA-DR profiles, gender and age in a population-based cohort. Methods: Serological responses to EBV were measured in 426 MS patients and 186 healthy controls. HLA-DR typing was performed using sequence-based methods. Results: MS patients had significantly higher levels of antibodies against epitope-specific and polyspecific EBNA-1 and viral capsid antigen (VCA), compared with controls (all p < 10−15). In regression analyses, anti-EBNA-1 and anti-VCA antibody levels, protective HLA-DR*04/07/09 alleles and gender (all p < 0.003) contributed independently to a model that classified cases and controls with an odds ratio > 20 (sensitivity 92%, specificity 64%). Notably, the strong influence of high-risk HLA-DR alleles was abrogated after inclusion of EBV serology results. Conclusions: The ability to discriminate MS cases and controls can be substantially enhanced by including anti-EBV serology as well as HLA-DR risk profiles. These findings support the relevance of EBV-specific immunity in MS pathogenesis, and implicate both HLA-dependent and HLA-independent immune responses against EBNA-1 as prominent disease risk factors.

Identifying patient-specific Epstein-Barr Nuclear Antigen-1 genetic variation and potential autoreactive targets relevant to multiple sclerosis pathogenesis

Background Epstein-Barr virus (EBV) infection represents a major environmental risk factor for multiple sclerosis (MS), with evidence of selective expansion of Epstein-Barr Nuclear Antigen-1 (EBNA1)-specific CD4+ T cells that cross-recognize MS-associated myelin antigens in MS patients. HLA-DRB1*15-restricted antigen presentation also appears to determine susceptibility given its role as a dominant risk allele. In this study, we have utilised standard and next-generation sequencing techniques to investigate EBNA-1 sequence variation and its relationship to HLA-DR15 binding affinity, as well as examining potential cross-reactive immune targets within the central nervous system proteome. Methods Sanger sequencing was performed on DNA isolated from peripheral blood samples from 73 Western Australian MS cases, without requirement for primary culture, with additional FLX 454 Roche sequencing in 23 samples to identify low-frequency variants. Patient-derived viral sequences were used to predict HLA-DRB1*1501 epitopes (NetMHCII, NetMHCIIpan) and candidates were evaluated for cross recognition with human brain proteins. Results EBNA-1 sequence variation was limited, with no evidence of multiple viral strains and only low levels of variation identified by FLX technology (8.3% nucleotide positions at a 1% cut-off). In silico epitope mapping revealed two known HLA-DRB1*1501-restricted epitopes (‘AEG’: aa 481–496 and ‘MVF’: aa 562–577), and two putative epitopes between positions 502–543. We identified potential cross-reactive targets involving a number of major myelin antigens including experimentally confirmed HLA-DRB1*15-restricted epitopes as well as novel candidate antigens within myelin and paranodal assembly proteins that may be relevant to MS pathogenesis. Conclusions This study demonstrates the feasibility of obtaining autologous EBNA-1 sequences directly from buffy coat samples, and confirms divergence of these sequences from standard laboratory strains. This approach has identified a number of immunogenic regions of EBNA-1 as well as known and novel targets for autoreactive HLA-DRB1*15-restricted T cells within the central nervous system that could arise as a result of cross-reactivity with EBNA-1-specific immune responses.

Only Slight Impact of Predicted Replicative Capacity for Therapy Response Prediction

Background Replication capacity (RC) of specific HIV isolates is occasionally blamed for unexpected treatment responses. However, the role of viral RC in response to antiretroviral therapy is not yet fully understood. Materials and Methods We developed a method for predicting RC from genotype using support vector machines (SVMs) trained on about 300 genotype-RC pairs. Next, we studied the impact of predicted viral RC (pRC) on the change of viral load (VL) and CD4+ T-cell count (CD4) during the course of therapy on about 3,000 treatment change episodes (TCEs) extracted from the EuResist integrated database. Specifically, linear regression models using either treatment activity scores (TAS), the drug combination, or pRC or any combination of these covariates were trained to predict change in VL and CD4, respectively. Results The SVM models achieved a Spearman correlation (ρ) of 0.54 between measured RC and pRC. The prediction of change in VL (CD4) was best at 180 (360) days, reaching a correlation of ρ = 0.45 (ρ = 0.27). In general, pRC was inversely correlated to drug resistance at treatment start (on average ρ = −0.38). Inclusion of pRC in the linear regression models significantly improved prediction of virological response to treatment based either on the drug combination or on the TAS (t-test; p-values range from 0.0247 to 4 10−6) but not for the model using both TAS and drug combination. For predicting the change in CD4 the improvement derived from inclusion of pRC was not significant. Conclusion Viral RC could be predicted from genotype with moderate accuracy and could slightly improve prediction of virological treatment response. However, the observed improvement could simply be a consequence of the significant correlation between pRC and drug resistance.

Effects of HIV type-1 immune selection on susceptability to integrase inhibitor resistance

BACKGROUND All site-specific interactions between HIV type-1 (HIV-1) subtype, human leukocyte antigen (HLA)-associated immune selection and integrase inhibitor resistance are not completely understood. We examined naturally occurring polymorphisms in HIV-1 integrase sequences from 342 antiretroviral-naive individuals from the Western Australian HIV Cohort Study and the Swiss HIV Cohort Study. METHODS Standard bulk sequencing and sequence-based typing were used to generate integrase sequences and high-resolution HLA genotypes, respectively. Viral residues were examined with respect to drug resistance mutations and CD8(+) T-cell escape mutations. RESULTS In both predominantly subtype B cohorts, 12 of 38 sites that mediate integrase inhibitor resistance mutations were absolutely conserved, and these included the primary resistance mutations. There were 18 codons with non-primary drug resistance-associated substitutions at rates of up to 58.8% and eight sites with alternative polymorphisms. Five viral residues were potentially subject to dual-drug and HLA-associated immune selection in which both selective pressures either drove the same amino acid substitution (codons 72, 157 and 163) or HLA alleles were associated with an alternative polymorphism that would alter the genetic barrier to resistance (codons 125 and 193). The common polymorphism T125A, which was characteristic of non-subtype B and was also associated with carriage of HLA-B*57/*5801, increased the mutational barrier to the resistance mutation T125K. CONCLUSIONS Primary integrase inhibitor resistance mutations were not detected in the absence of drug exposure in keeping with sites of high constraint. Viral polymorphisms caused by immune selection and/or associated with non-subtype B might alter the genetic barrier to some non-primary resistance-associated mutations.