Crystal structures of H-2Db in complex with the LCMV-derived peptides GP92 and GP392 explain pleiotropic effects of glycosylation on antigen presentation and immunogenicity.

Post-translational modifications significantly broaden the epitope repertoire for major histocompatibility class I complexes (MHC-I) and may allow viruses to escape immune recognition. Lymphocytic choriomeningitis virus (LCMV) infection of H-2b mice generates CD8+ CTL responses directed towards several MHC-I-restricted epitopes including the peptides GP92 (CSANNSHHYI) and GP392 (WLVTNGSYL), both with a N-glycosylation site. Interestingly, glycosylation has different effects on the immunogenicity and association capacity of these two epitopes to H-2Db. To assess the structural bases underlying these functional results, we determined the crystal structures of H-2Db in complex with GP92 (CSANNSHHYI) and GP392 (WLVTNGSYL) to 2.4 and 2.5 Å resolution, respectively. The structures reveal that while glycosylation of GP392 most probably impairs binding, the glycosylation of the asparagine residue in GP92, which protrudes towards the solvent, possibly allows for immune escape and/or forms a neo-epitope that may select for a different set of CD8 T cells. Altogether, the presented results provide a structural platform underlying the effects of post-translational modifications on epitope binding and/or immunogenicity, resulting in viral immune escape.

Progression of benign prostatic hyperplasia is associated with pro-inflammatory mediators and chronic activation of prostate-infiltrating lymphocytes.

Benign prostatic hyperplasia (BPH) is a common chronic non-malignant condition whose prevalence substantially increases with age. Immune cell infiltration and pro-inflammatory mediators have been implicated in the pathogenesis. Here, we characterized 21 extracellular markers on prostate-infiltrating lymphocytes (PILs) and analyzed expression of 26 soluble proteins in prostate tissue obtained from BPH patients (n = 31). These data were correlated with clinical parameters and compared with peripheral blood mononuclear cells (PBMCs) (n = 10). Increased frequencies of T cells expressing co-inhibitory receptors LAG-3, PD-1, TIM-3 or CTLA-4, and co-stimulatory receptors CD28, OX40 or 4-1BB were observed in BPH tissue compared to PBMCs. These findings are consistent with chronic activation and possible functional exhaustion of PILs that may be further augmented by several identified pro-inflammatory factors, such as IL-8 and MCP-1, promoting inflammation and chemotaxis of immune cells to the prostate. Prostate size and plasma prostate-specific antigen levels positively correlated with IL-8 and MCP-1 concentrations, and frequencies of T cells expressing CTLA-4 and TIM-3. It remains to be established whether the link between inflammation and BPH progression supported by our findings reflects a progressive failure of the immune system leading to decreased immune surveillance and development of prostate cancer.

Baseline CD4+ T cell counts correlates with HIV-1 synonymous rate in HLA-B*5701 subjects with different risk of disease progression.

HLA-B*5701 is the host factor most strongly associated with slow HIV-1 disease progression, although risk of progression may vary among patients carrying this allele. The interplay between HIV-1 evolutionary rate variation and risk of progression to AIDS in HLA-B*5701 subjects was studied using longitudinal viral sequences from high-risk progressors (HRPs) and low-risk progressors (LRPs). Posterior distributions of HIV-1 genealogies assuming a Bayesian relaxed molecular clock were used to estimate the absolute rates of nonsynonymous and synonymous substitutions for different set of branches. Rates of viral evolution, as well as in vitro viral replication capacity assessed using a novel phenotypic assay, were correlated with various clinical parameters. HIV-1 synonymous substitution rates were significantly lower in LRPs than HRPs, especially for sets of internal branches. The viral population infecting LRPs was also characterized by a slower increase in synonymous divergence over time. This pattern did not correlate to differences in viral fitness, as measured by in vitro replication capacity, nor could be explained by differences among subjects in T cell activation or selection pressure. Interestingly, a significant inverse correlation was found between baseline CD4+ T cell counts and mean HIV-1 synonymous rate (which is proportional to the viral replication rate) along branches representing viral lineages successfully propagating through time up to the last sampled time point. The observed lower replication rate in HLA-B*5701 subjects with higher baseline CD4+ T cell counts provides a potential model to explain differences in risk of disease progression among individuals carrying this allele.

Novel method to characterize immune cells from human prostate tissue.

BACKGROUND: Benign prostatic hyperplasia (BPH) is the most common benign adenoma and prostate cancer is the most frequent malignancy in men over 50 years of age in the Western world, where it remains a significant health problem. Prostate lesions are known to contain immune cells, which may contribute to the immune control of tumor progression. However, due to their low numbers and restricted access to necessary material it is difficult to isolate immune cells from prostate tissue to characterize their immunological features. METHODS: An efficient and robust method was developed to process prostate tissue and isolate immune cells for phenotypic analysis by multicolor flow cytometry as downstream application. Fresh prostate tissue from 11 patients undergoing surgery for bladder outlet obstruction due to BPH was processed to evaluate the number, viability, yield, and frequency of various immune cell types. RESULTS: The presented method does not include enzymatic digestion nor incubation steps at 37 °C, increasing cellular viability and avoiding possible phenotypic modification. Various immune cell populations were detected in all patient samples and the median cellular viability was 90%. The number of detected events of individual cell populations varied between patients. The median frequency of different immune cell populations also varied, being 87% for the CD3- and 15% for the CD3+ cell population. CONCLUSIONS: This novel method will allow the phenotypic characterization of immune cell populations present in tumor tissue of prostate cancer patients and promote development of novel approaches to immunotherapy of the disease.

Functional avidity and IL-2/perforin production is linked to the emergence of mutations within HLA-B*5701-restricted epitopes and HIV-1 disease progression.

Viral escape from HIV-1-specific CD8(+) T cells has been demonstrated in numerous studies previously. However, the qualitative features driving the emergence of mutations within epitopes are still unclear. In this study, we aimed to distinguish whether specific functional characteristics of HLA-B*5701-restricted CD8(+) T cells influence the emergence of mutations in high-risk progressors (HRPs) versus low-risk progressors (LRPs). Single-genome sequencing was performed to detect viral mutations (variants) within seven HLA-B*5701-restricted epitopes in Gag (n = 4) and Nef (n = 3) in six untreated HLA-B*5701 subjects followed from early infection up to 7 y. Several well-characterized effector markers (IFN-γ, IL-2, MIP-1ß, TNF, CD107a, and perforin) were identified by flow cytometry following autologous (initial and emerging variant/s) epitope stimulations. This study demonstrates that specific functional attributes may facilitate the outgrowth of mutations within HLA-B*5701-restricted epitopes. A significantly lower fraction of IL-2-producing cells and a decrease in functional avidity and polyfunctional sensitivity were evident in emerging epitope variants compared with the initial autologous epitopes. Interestingly, the HRPs mainly drove these differences, whereas the LRPs maintained a directed and maintained functional response against emerging epitope variants. In addition, LRPs induced improved cell-cycle progression and perforin upregulation after autologous and emerging epitope variant stimulations in contrast to HRPs. The maintained quantitative and qualitative features of the CD8(+) T cell responses in LRPs toward emerging epitope variants provide insights into why HLA-B*5701 subjects have different risks of HIV-1 disease progression.

Combination of immune and viral factors distinguishes low-risk versus high-risk HIV-1 disease progression in HLA-B*5701 subjects.

HLA-B*5701 is the host factor most strongly associated with slow HIV-1 disease progression, although rates can vary within this group. Underlying mechanisms are not fully understood but likely involve both immunological and virological dynamics. The present study investigated HIV-1 in vivo evolution and epitope-specific CD8(+) T cell responses in six HLA-B*5701 patients who had not received antiretroviral treatment, monitored from early infection for up to 7 years. The subjects were classified as high-risk progressors (HRPs) or low-risk progressors (LRPs) based on baseline CD4(+) T cell counts. Dynamics of HIV-1 Gag p24 evolution and multifunctional CD8(+) T cell responses were evaluated by high-resolution phylogenetic analysis and polychromatic flow cytometry, respectively. In all subjects, substitutions occurred more frequently in flanking regions than in HLA-B*5701-restricted epitopes. In LRPs, p24 sequence diversity was significantly lower; sequences exhibited a higher degree of homoplasy and more constrained mutational patterns than HRPs. The HIV-1 intrahost evolutionary rate was also lower in LRPs and followed a strict molecular clock, suggesting neutral genetic drift rather than positive selection. Additionally, polyfunctional CD8(+) T cell responses, particularly to TW10 and QW9 epitopes, were more robust in LRPs, who also showed significantly higher interleukin-2 (IL-2) production in early infection. Overall, the findings indicate that HLA-B*5701 patients with higher CD4 counts at baseline have a lower risk of HIV-1 disease progression because of the interplay between specific HLA-linked immune responses and the rate and mode of viral evolution. The study highlights the power of a multidisciplinary approach, integrating high-resolution evolutionary and immunological data, to understand mechanisms underlying HIV-1 pathogenesis.

Characterization of HIV-specific CD4+ T cell responses against peptides selected with broad population and pathogen coverage.

CD4+ T cells orchestrate immunity against viral infections, but their importance in HIV infection remains controversial. Nevertheless, comprehensive studies have associated increase in breadth and functional characteristics of HIV-specific CD4+ T cells with decreased viral load. A major challenge for the identification of HIV-specific CD4+ T cells targeting broadly reactive epitopes in populations with diverse ethnic background stems from the vast genomic variation of HIV and the diversity of the host cellular immune system. Here, we describe a novel epitope selection strategy, PopCover, that aims to resolve this challenge, and identify a set of potential HLA class II-restricted HIV epitopes that in concert will provide optimal viral and host coverage. Using this selection strategy, we identified 64 putative epitopes (peptides) located in the Gag, Nef, Env, Pol and Tat protein regions of HIV. In total, 73% of the predicted peptides were found to induce HIV-specific CD4+ T cell responses. The Gag and Nef peptides induced most responses. The vast majority of the peptides (93%) had predicted restriction to the patient's HLA alleles. Interestingly, the viral load in viremic patients was inversely correlated to the number of targeted Gag peptides. In addition, the predicted Gag peptides were found to induce broader polyfunctional CD4+ T cell responses compared to the commonly used Gag-p55 peptide pool. These results demonstrate the power of the PopCover method for the identification of broadly recognized HLA class II-restricted epitopes. All together, selection strategies, such as PopCover, might with success be used for the evaluation of antigen-specific CD4+ T cell responses and design of future vaccines.

PhyloTempo: A Set of R Scripts for Assessing and Visualizing Temporal Clustering in Genealogies Inferred from Serially Sampled Viral Sequences.

Serially-sampled nucleotide sequences can be used to infer demographic history of evolving viral populations. The shape of a phylogenetic tree often reflects the interplay between evolutionary and ecological processes. Several approaches exist to analyze the topology and traits of a phylogenetic tree, by means of tree balance, branching patterns and comparative properties. The temporal clustering (TC) statistic is a new topological measure, based on ancestral character reconstruction, which characterizes the temporal structure of a phylogeny. Here, PhyloTempo is the first implementation of the TC in the R language, integrating several other topological measures in a user-friendly graphical framework. The comparison of the TC statistic with other measures provides multifaceted insights on the dynamic processes shaping the evolution of pathogenic viruses. The features and applicability of PhyloTempo were tested on serially-sampled intra-host human and simian immunodeficiency virus population data sets. PhyloTempo is distributed under the GNU general public license at https://sourceforge.net/projects/phylotempo/.

Towards a new paradigm linking virus molecular evolution and pathogenesis: experimental design and phylodynamic inference.

Phylogenetic analysis has become a powerful tool for the investigation of evolution at a molecular level. During the last three decades, statistical phylogenetics has increasingly been applied to the study of microbial pathogens. The new field of phylodynamics was formally introduced in 2004 and encompasses the interaction between evolutionary and ecological processes that shape the spatiotemporal and phylogenetic patterns of infectious disease dynamics. This novel framework has significantly enhanced the study of measurable evolving pathogen populations, in particular RNA viruses and retroviruses. One of the major challenges in phylodynamic studies, however, is the generation of data in the form of dense coverage in sequence sampling coupled with high quality epidemiological and/or accurate clinical information. This review focuses specifically on experimental and data assembling strategies that are required to test multi-level phylodynamic hypotheses, ranging from intra-host viral evolution to population dynamics of infectious disease pandemics. Ultimately, bridging the gap between rational experimental design and phylodynamic inference will prove to be essential to take full advantage of this new exciting area of research.

Reduction of the HIV-1 reservoir in resting CD4+ T-lymphocytes by high dosage intravenous immunoglobulin treatment: a proof-of-concept study.

BACKGROUND: The latency of HIV-1 in resting CD4+ T-lymphocytes constitutes a major obstacle for the eradication of virus in patients on antiretroviral therapy (ART). As yet, no approach to reduce this viral reservoir has proven effective. METHODS: Nine subjects on effective ART were included in the study and treated with high dosage intravenous immunoglobulin (IVIG) for five consecutive days. Seven of those had detectable levels of replication-competent virus in the latent reservoir and were thus possible to evaluate. Highly purified resting memory CD4+ T-cells were activated and cells containing replication-competent HIV-1 were quantified. HIV-1 from plasma and activated memory CD4+ T-cells were compared with single genome sequencing (SGS) of the gag region. T-lymphocyte activation markers and serum interleukins were measured. RESULTS: The latent HIV-1 pool decreased with in median 68% after IVIG was added to effective ART. The reservoir decreased in five, whereas no decrease was found in two subjects with detectable virus. Plasma HIV-1 RNA >or= 2 copies/mL was detected in five of seven subjects at baseline, but in only one at follow-up after 8-12 weeks. The decrease of the latent HIV-1 pool and the residual plasma viremia was preceded by a transitory low-level increase in plasma HIV-1 RNA and serum interleukin 7 (IL-7) levels, and followed by an expansion of T regulatory cells. The magnitude of the viral increase in plasma correlated to the size of the latent HIV-1 pool and SGS of the gag region showed that viral clones from plasma clustered together with virus from activated memory T-cells, pointing to the latent reservoir as the source of HIV-1 RNA in plasma. CONCLUSION: The findings from this uncontrolled proof-of-concept study suggest that the reservoir became accessible by IVIG treatment through activation of HIV-1 gene expression in latently-infected resting CD4+ T-cells. We propose that IVIG should be further evaluated as an adjuvant to effective ART.

Broadly immunogenic HLA class I supertype-restricted elite CTL epitopes recognized in a diverse population infected with different HIV-1 subtypes.

The genetic variations of the HIV-1 virus and its human host constitute major obstacles for obtaining potent HIV-1-specific CTL responses in individuals of diverse ethnic backgrounds infected with different HIV-1 variants. In this study, we developed and used a novel algorithm to select 184 predicted epitopes representing seven different HLA class I supertypes that together constitute a broad coverage of the different HIV-1 strains as well as the human HLA alleles. Of the tested 184 HLA class I-restricted epitopes, 114 were recognized by at least one study subject, and 45 were novel epitopes, not previously described in the HIV-1 immunology database. In addition, we identified 21 "elite" epitopes that induced CTL responses in at least 4 of the 31 patients. A majority (27 of 31) of the study population recognized one or more of these highly immunogenic epitopes. We also found a limited set of 9 epitopes that together induced HIV-1-specific CTL responses in all HIV-1-responsive patients in this study. Our results have important implications for the validation of potent CTL responses and show that the goal for a vaccine candidate in inducing broadly reactive CTL immune responses is attainable.