Lichen Jing

Merkel Cell Polyomavirus-Specific CD8+ and CD4+ T-cell Responses Identified in Merkel Cell Carcinomas and Blood

Purpose: Merkel cell polyomavirus (MCPyV) is prevalent in the general population, integrates into most Merkel cell carcinomas (MCC), and encodes oncoproteins required for MCC tumor growth. We sought to characterize T-cell responses directed against viral proteins that drive this cancer as a step toward immunotherapy. Experimental Design: Intracellular cytokine cytometry, IFN-γ enzyme-linked immunospot (ELISPOT) assay, and a novel HLA-A*2402–restricted MCPyV tetramer were used to identify and characterize T-cell responses against MCPyV oncoproteins in tumors and blood of MCC patients and control subjects. Results: We isolated virus-reactive CD8 or CD4 T cells from MCPyV-positive MCC tumors (2 of 6) but not from virus-negative tumors (0 of 4). MCPyV-specific T-cell responses were also detected in the blood of MCC patients (14 of 27) and control subjects (5 of 13). These T cells recognized a broad range of peptides derived from capsid proteins (2 epitopes) and oncoproteins (24 epitopes). HLA-A*2402–restricted MCPyV oncoprotein processing and presentation by mammalian cells led to CD8-mediated cytotoxicity. Virus-specific CD8 T cells were markedly enriched among tumor infiltrating lymphocytes as compared with blood, implying intact T-cell trafficking into the tumor. Although tetramer-positive CD8 T cells were detected in the blood of 2 of 5 HLA-matched MCC patients, these cells failed to produce IFN-γ when challenged ex vivo with peptide. Conclusions: Our findings suggest that MCC tumors often develop despite the presence of T cells specific for MCPyV T-Ag oncoproteins. The identified epitopes may be candidates for peptide-specific vaccines and tumor- or virus-specific adoptive immunotherapies to overcome immune evasion mechanisms in MCC patients. Clin Cancer Res; 17(21); 6671–80. ©2011 AACR.

Diversity in the Acute CD8 T Cell Response to Vaccinia Virus in Humans

Orthopoxviruses have complex proteomes. Infection provokes a brisk CD8 response, which is required in some systems for recovery from primary infection. Little is known concerning the Ags and epitopes recognized by CD8 T cells. We examined the fine specificity of cloned and bulk human vaccinia-specific CD8 CTL by expressing polypeptide fragments from a library of vaccinia genomic DNA. This epitope discovery method emphasizes virus-specific biological activity, as the responder cells are all reactive with whole vaccinia virus. Sixteen novel epitopes, restricted by several HLA A and B alleles, were defined to the nomamer peptide level in diverse vaccinia open reading frames. An additional seven epitope were mapped to short regions of vaccinia proteins. Targets of the CD8 response included proteins assigned to structural, enzymatic, transcription factor, and immune evasion functions, and included members of all viral kinetic classes. Most epitopes were conserved in other orthopoxviruses. Responses to at least 18 epitopes were detected within a single blood sample, revealing a surprising degree of diversity. These epitopes will be useful in natural history studies of CD8 responses to vaccinia, a nonpersisting virus with long-term memory, and in the design and evaluation of attenuated and replication-incompetent vaccinia strains being tested for variola and monkeypox prevention and for the delivery of heterologous Ags.

Cross-presentation and genome-wide screening reveal candidate T cells antigens for a herpes simplex virus type 1 vaccine

Herpes simplex virus type 1 (HSV-1) not only causes painful recurrent oral-labial infections, it can also cause permanent brain damage and blindness. There is currently no HSV-1 vaccine. An effective vaccine must stimulate coordinated T cell responses, but the large size of the genome and the low frequency of HSV-1-specific T cells have hampered the search for the most effective T cell antigens for inclusion in a candidate vaccine. We have now developed what we believe to be novel methods to efficiently generate a genome-wide map of the responsiveness of HSV-1-specific T cells, and demonstrate the applicability of these methods to a second complex microbe, vaccinia virus. We used cross-presentation and CD137 activation-based FACS to enrich for polyclonal CD8+ T effector T cells. The HSV-1 proteome was prepared in a flexible format for analyzing both CD8+ and CD4+ T cells from study participants. Scans with participant-specific panels of artificial APCs identified an oligospecific response in each individual. Parallel CD137-based CD4+ T cell research showed discrete oligospecific recognition of HSV-1 antigens. Unexpectedly, the two HSV-1 proteins not previously considered as vaccine candidates elicited both CD8+ and CD4+ T cell responses in most HSV-1-infected individuals. In this era of microbial genomics, our methods - also demonstrated in principle for vaccinia virus for both CD8+ and CD4+ T cells - should be broadly applicable to the selection of T cell antigens for inclusion in candidate vaccines for many pathogens.

An extremely diverse CD4 response to vaccinia virus in humans is revealed by proteome-wide T-cell profiling

ABSTRACT CD4 T cells are required for the maintenance and recall of antiviral CD8 T cells and for antibody responses. Little is known concerning the overall architecture of the CD4 response to complex microbial pathogens. In a whole-proteome approach, 180 predicted open reading frames (ORFs) in the vaccinia virus genome were expressed and tested using responder cells from 20 blood samples from 11 vaccinees. Validation assays established the sensitivity and specificity of the system. Overall, CD4 responses were detected for 122 ORFs (68%). A mean of 39 ORFs were recognized per person (range, 13 to 63). The most frequently recognized ORFS were present in virions, including A3L and A10L (core proteins), WR148 (a fragmented homolog of an orthopoxvirus protein that forms inclusions in cells), H3L (a membrane protein), D13L (a membrane scaffold protein), and L4R (a nucleic acid binding protein). Serum immunoglobulin G profiling by proteome microarray detected responses to 45 (25%) of the ORFs and confirmed recent studies showing a diverse response directed to membrane and nonmembrane antigens. Our results provide the first empirical whole-proteome data set regarding the global CD4 response to full-length proteins in a complex virus and are consistent with the theory that abundant structural proteins are immunodominant.

Dominance and Diversity in the Primary Human CD4 T Cell Response to Replication-Competent Vaccinia Virus

Vaccination with replication-competent vaccinia protects against heterologous orthopoxvirus challenge. CD4 T cells have essential roles helping functionally important Ab and CD8 antiviral responses, and contribute to the durability of vaccinia-specific memory. Little is known about the specificity, diversity, or dominance hierarchy of orthopoxvirus-specific CD4 T cell responses. We interrogated vaccinia-reactive CD4 in vitro T cell lines with vaccinia protein fragments expressed from an unbiased genomic library, and also with a panel of membrane proteins. CD4 T cells from three primary vaccinees reacted with 44 separate antigenic regions in 35 vaccinia proteins, recognizing 8 to 20 proteins per person. The integrated responses to the Ags that we defined accounted for 49 to 81% of the CD4 reactivity to whole vaccinia Ag. Individual dominant Ags drove up to 30% of the total response. The gene F11L-encoded protein was immunodominant in two of three subjects and is fragmented in a replication-incompetent vaccine candidate. The presence of protein in virions was strongly associated with CD4 antigenicity. These findings are consistent with models in which exogenous Ag drives CD4 immunodominance, and provides tools to investigate the relationship between Ab and CD4 T cell specificity for complex pathogens.

Genome Sequencing and Analysis of Geographically Diverse Clinical Isolates of Herpes Simplex Virus 2

ABSTRACT Herpes simplex virus 2 (HSV-2), the principal causative agent of recurrent genital herpes, is a highly prevalent viral infection worldwide. Limited information is available on the amount of genomic DNA variation between HSV-2 strains because only two genomes have been determined, the HG52 laboratory strain and the newly sequenced SD90e low-passage-number clinical isolate strain, each from a different geographical area. In this study, we report the nearly complete genome sequences of 34 HSV-2 low-passage-number and laboratory strains, 14 of which were collected in Uganda, 1 in South Africa, 11 in the United States, and 8 in Japan. Our analyses of these genomes demonstrated remarkable sequence conservation, regardless of geographic origin, with the maximum nucleotide divergence between strains being 0.4% across the genome. In contrast, prior studies indicated that HSV-1 genomes exhibit more sequence diversity, as well as geographical clustering. Additionally, unlike HSV-1, little viral recombination between HSV-2 strains could be substantiated. These results are interpreted in light of HSV-2 evolution, epidemiology, and pathogenesis. Finally, the newly generated sequences more closely resemble the low-passage-number SD90e than HG52, supporting the use of the former as the new reference genome of HSV-2. IMPORTANCE Herpes simplex virus 2 (HSV-2) is a causative agent of genital and neonatal herpes. Therefore, knowledge of its DNA genome and genetic variability is central to preventing and treating genital herpes. However, only two full-length HSV-2 genomes have been reported. In this study, we sequenced 34 additional HSV-2 low-passage-number and laboratory viral genomes and initiated analysis of the genetic diversity of HSV-2 strains from around the world. The analysis of these genomes will facilitate research aimed at vaccine development, diagnosis, and the evaluation of clinical manifestations and transmission of HSV-2. This information will also contribute to our understanding of HSV evolution.

Virologic and Immunologic Evidence of Multifocal Genital Herpes Simplex Virus 2 Infection

ABSTRACT Genital herpes simplex virus (HSV) reactivation is thought to be anatomically and temporally localized, coincident with limited ganglionic infection. Short, subclinical shedding episodes are the most common form of HSV-2 reactivation, with host clearance mechanisms leading to rapid containment. The anatomic distribution of shedding episodes has not been characterized. To precisely define patterns of anatomic reactivation, we divided the genital tract into a 22-region grid and obtained daily swabs for 20 days from each region in 28 immunocompetent, HSV-2-seropositive persons. HSV was detected via PCR, and sites of asymptomatic HSV shedding were subjected to a biopsy procedure within 24 h. CD4+ and CD8+ T cells were quantified by immunofluorescence, and HSV-specific CD4+ T cells were identified by intracellular cytokine cytometry. HSV was detected in 868 (7%) of 11,603 genital swabs at a median of 12 sites per person (range, 0 to 22). Bilateral HSV detection occurred on 83 (67%) days with shedding, and the median quantity of virus detected/day was associated with the number of sites positive (P < 0.001). In biopsy specimens of asymptomatic shedding sites, we found increased numbers of CD8+ T cells compared to control tissue (27 versus 13 cells/mm2, P = 0.03) and identified HSV-specific CD4+ T cells. HSV reactivations emanate from widely separated anatomic regions of the genital tract and are associated with a localized cellular infiltrate that was demonstrated to be HSV specific in 3 cases. These data provide evidence that asymptomatic HSV-2 shedding contributes to chronic inflammation throughout the genital tract. IMPORTANCE This detailed report of the anatomic patterns of genital HSV-2 shedding demonstrates that HSV-2 reactivation can be detected at multiple bilateral sites in the genital tract, suggesting that HSV establishes latency throughout the sacral ganglia. In addition, genital biopsy specimens from sites of asymptomatic HSV shedding have increased numbers of CD8+ T cells compared to control tissue, and HSV-specific CD4+ T cells are found at sites of asymptomatic shedding. These findings suggest that widespread asymptomatic genital HSV-2 shedding is associated with a targeted host immune response and contributes to chronic inflammation throughout the genital tract.

ORFeome approach to the clonal, HLA allele-specific CD4 T-cell response to a complex pathogen in humans

The CD4 T-cell response to vaccinia promotes antibody and long-term CD8 responses. HLA class II molecules present microbial epitopes to CD4 T-cells. In humans, at least 3 loci encode cell-surface peptide-binding HLA class II heterodimers. Using intracellular cytokine cytometry (ICC) assays, we determined that HLA DR had the strongest contribution to vaccinia antigen presentation. Among panels of vaccinia-restricted T-cell clones, most were DR-restricted but rare DQ-restricted clones were also recovered. Vaccinia has over 200 open reading frames (ORFs), providing a significant bottleneck to assigning fine specificity. To overcome this, we expressed each predicted vaccinia ORF using in vitro transcription and translation. Array-based pool proteins were used to rapidly assign fine specificity to each DQ-restricted clone and to a sample of HLA DR-restricted clones. Reactivity was confirmed using synthetic peptides for selected CD4 T-cell clones. This method should be broadly applicable to the study of large-genome, sequenced pathogens, and could also be used to investigate T-cell responses to cDNAs expressed in neoplastic and autoimmune disorders in which CD4 responses might be adaptive or harmful.

CD4 T-Cell Memory Responses to Viral Infections of Humans Show Pronounced Immunodominance Independent of Duration or Viral Persistence

ABSTRACT Little is known concerning immunodominance within the CD4 T-cell response to viral infections and its persistence into long-term memory. We tested CD4 T-cell reactivity against each viral protein in persons immunized with vaccinia virus (VV), either recently or more than 40 years ago, as a model self-limited viral infection. Similar tests were done with persons with herpes simplex virus 1 (HSV-1) infection as a model chronic infection. We used an indirect method capable of counting the CD4 T cells in blood reactive with each individual viral protein. Each person had a clear CD4 T-cell dominance hierarchy. The top four open reading frames accounted for about 40% of CD4 virus-specific T cells. Early and long-term memory CD4 T-cell responses to vaccinia virus were mathematically indistinguishable for antigen breadth and immunodominance. Despite the chronic intermittent presence of HSV-1 antigen, the CD4 T-cell dominance and diversity patterns for HSV-1 were identical to those observed for vaccinia virus. The immunodominant CD4 T-cell antigens included both long proteins abundantly present in virions and shorter, nonstructural proteins. Limited epitope level and direct ex vivo data were also consistent with pronounced CD4 T-cell immunodominance. We conclude that human memory CD4 T-cell responses show a pattern of pronounced immunodominance for both chronic and self-limited viral infections and that this pattern can persist over several decades in the absence of antigen.

Tumor-infiltrating merkel cell polyomavirus-specific T cells are diverse and associated with improved patient survival

Merkel cell carcinoma patients had improved survival if their tumors contained a greater frequency or diversity of T cells that were specific for a prevalent Merkel cell polyomavirus epitope. Thus, transgenic T-cell receptor therapy could potentially benefit patients. Tumor-infiltrating CD8+ T cells are associated with improved survival of patients with Merkel cell carcinoma (MCC), an aggressive skin cancer causally linked to Merkel cell polyomavirus (MCPyV). However, CD8+ T-cell infiltration is robust in only 4% to 18% of MCC tumors. We characterized the T-cell receptor (TCR) repertoire restricted to one prominent epitope of MCPyV (KLLEIAPNC, “KLL”) and assessed whether TCR diversity, tumor infiltration, or T-cell avidity correlated with clinical outcome. HLA-A*02:01/KLL tetramer+ CD8+ T cells from MCC patient peripheral blood mononuclear cells (PBMC) and tumor-infiltrating lymphocytes (TIL) were isolated via flow cytometry. TCRβ (TRB) sequencing was performed on tetramer+ cells from PBMCs or TILs (n = 14) and matched tumors (n = 12). Functional avidity of T-cell clones was determined by IFNγ production. We identified KLL tetramer+ T cells in 14% of PBMC and 21% of TIL from MCC patients. TRB repertoires were strikingly diverse (397 unique TRBs were identified from 12 patients) and mostly private (only one TCRb clonotype shared between two patients). An increased fraction of KLL-specific TIL (>1.9%) was associated with significantly increased MCC-specific survival P = 0.0009). T-cell cloning from four patients identified 42 distinct KLL-specific TCRa/b pairs. T-cell clones from patients with improved MCC-specific outcomes were more avid (P < 0.05) and recognized an HLA-appropriate MCC cell line. T cells specific for a single MCPyV epitope display marked TCR diversity within and between patients. Intratumoral infiltration by MCPyV-specific T cells was associated with significantly improved MCC-specific survival, suggesting that augmenting the number or avidity of virus-specific T cells may have therapeutic benefit. Cancer Immunol Res; 5(2); 137–47. ©2017 AACR.