Eric J. Gowans

Natural Regulatory T Cells and Persistent Viral Infection

Suppressor T cell is an old term, originally found in the 1970s literature (49, 50), but it was short-lived because advances in molecular biology soon afterward proved that the gene locus, thought to be associated with suppression, was nonexistent. Our recent understanding started with the finding that a small proportion of CD4+ T cells in mice constitutively expressed the high-affinity interleukin-2 (IL-2) receptor α-chain, CD25, and depletion of these cells (now designated natural regulatory T cells [Treg]) caused autoimmune disease and enhanced responses to foreign antigens (112). This study resulted in a rebound of intense interest in suppressor T cells, and similar cells in humans were identified shortly afterward (78, 98; reviewed in reference 9). It is now well established that natural Treg suppress a diverse range of immune responses in a contact-dependent manner in vitro and in vivo, in response to T-cell receptor (TCR)-mediated stimulation (reviewed in references 43 and 136). Human Treg are less well defined than their murine counterparts and less well studied in general, although the two have features in common. Differences between human and murine Treg, which may complicate the interpretation of human data, have been noted. For instance, in naive inbred pathogen-free mice, natural Treg can be reliably isolated based on their CD25 expression; however, this population in adult outbred humans is inevitably a mixture of Treg and recently activated T effector cells, with the latter expected particularly during an ongoing infection. Opinions are divided over whether Treg play a pathogenic role in chronic viral infection in humans, especially in infections for which the development of a vaccine has so far failed, such as in the case of human immunodeficiency virus (HIV) and hepatitis C virus (HCV). This review considers the key findings in Treg biology and discusses the current position for Treg in viral infection, with particular emphasis on the key aspects of persistent viral infections in humans. Other types of suppressor cells, Tr1 and Th3, that suppress via soluble factors independent of cell-to-cell contact have been reviewed recently (106, 107).

Analysis of FOXP3+ regulatory T cells that display apparent viral antigen specificity during chronic hepatitis C virus infection.

We reported previously that a proportion of natural CD25(+) cells isolated from the PBMC of HCV patients can further upregulate CD25 expression in response to HCV peptide stimulation in vitro, and proposed that virus-specific regulatory T cells (Treg) were primed and expanded during the disease. Here we describe epigenetic analysis of the FOXP3 locus in HCV-responsive natural CD25(+) cells and show that these cells are not activated conventional T cells expressing FOXP3, but hard-wired Treg with a stable FOXP3 phenotype and function. Of approximately 46,000 genes analyzed in genome wide transcription profiling, about 1% were differentially expressed between HCV-responsive Treg, HCV-non-responsive natural CD25(+) cells and conventional T cells. Expression profiles, including cell death, activation, proliferation and transcriptional regulation, suggest a survival advantage of HCV-responsive Treg over the other cell populations. Since no Treg-specific activation marker is known, we tested 97 NS3-derived peptides for their ability to elicit CD25 response (assuming it is a surrogate marker), accompanied by high resolution HLA typing of the patients. Some reactive peptides overlapped with previously described effector T cell epitopes. Our data offers new insights into HCV immune evasion and tolerance, and highlights the non-self specific nature of Treg during infection.

IMGT/HighV QUEST paradigm for T cell receptor IMGT clonotype diversity and next generation repertoire immunoprofiling.

T cell repertoire diversity and clonotype follow-up in vaccination, cancer, infectious and immune diseases represent a major challenge owing to the enormous complexity of the data generated. Here we describe a next generation methodology, which combines 5′RACE PCR, 454 sequencing and, for analysis, IMGT, the international ImMunoGeneTics information system (IMGT), IMGT/HighV-QUEST web portal and IMGT-ONTOLOGY concepts. The approach is validated in a human case study of T cell receptor beta (TRB) repertoire, by chronologically tracking the effects of influenza vaccination on conventional and regulatory T cell subpopulations. The IMGT/HighV-QUEST paradigm defines standards for genotype/haplotype analysis and characterization of IMGT clonotypes for clonal diversity and expression and achieves a degree of resolution for next generation sequencing verifiable by the user at the sequence level, while providing a normalized reference immunoprofile for human TRB.

Characterization of RNA synthesis during a one-step growth curve and of the replication mechanism of bovine viral diarrhoea virus.

The noncytopathic Australian bovine viral diarrhoea virus (BVDV) Trangie isolate was used to establish a one-step growth curve and to investigate previously uncharacterized aspects of pestivirus replication. The eclipse phase was found to be approximately 8 to 10 h and the first appearance of viral antigen assayed by immunofluorescence occurred around 6 h post-infection (p.i.). Both positive- and negative-sense virus RNAs were first detected at 4 h p.i. by Northern blot hybridization using strand-specific RNA probes generated by in vitro transcription from cDNA cloned from the NS3 region. The ratio of positive- to negative-sense virus RNA changed from 2:1 at 4 h p.i. to 10:1 at 12 h p.i. and thereafter. The kinetics of synthesis showed that the rate of synthesis of positive-strand viral RNA increased rapidly from 6 h p.i., whereas the rate of synthesis of the negative-strand remained constant. The copy number of genomic RNA determined by Northern blot hybridization analysis was estimated to be 1.5 x 10(4) copies per cell, 16 to 24 h p.i. Viral RNA species that were thought to represent replicative intermediate (RI) and replicative forms (RF) were detected after electrophoretic separation by urea-PAGE. Confirmation of the identity of the RI and RF was obtained using LiCl precipitation and RNase A digestion of [3H]uridine-labelled RNA. Pulse-chase labelling of BVDV-infected cells was consistent with synthesis of nascent BVDV RNA through an RI derived by strand displacement from an RF template and thus the synthesis of BVDV RNA is likely to be similar to the model proposed for flavivirus replication.

Antigenicity and Immunogenicity of Novel Chimeric Hepatitis B Surface Antigen Particles with Exposed Hepatitis C Virus Epitopes

ABSTRACT The small envelope protein of hepatitis B virus (HBsAg-S) can self-assemble into highly organized virus like particles (VLPs) and induce an effective immune response. In this study, a restriction enzyme site was engineered into the cDNA of HBsAg-S at a position corresponding to the exposed site within the hydrophilic a determinant region (amino acid [aa] 127–128) to create a novel HBsAg vaccine vector allowing surface orientation of the inserted sequence. We inserted sequences of various lengths from hypervariable region 1 (HVR1) of the hepatitis C virus (HCV) E2 protein containing immunodominant epitopes and demonstrated secretion of the recombinant HBsAg VLPs from transfected mammalian cells. A number of different recombinant proteins were synthesized, and HBsAg VLPs containing inserts up to 36 aa were secreted with an efficiency similar to that of wild-type HBsAg. The HVR1 region exposed on the particles retained an antigenic structure similar to that recognized immunologically during natural infection. VLPs containing epitopes from either HCV-1a or -1b strains were produced that induced strain-specific antibody responses in immunized mice. Injection of a combination of these VLPs induced antibodies against both HVR1 epitopes that resulted in higher titers than were achieved by vaccination with the individual VLPs, suggesting a synergistic effect. This may lead to the development of recombinant particles which are able to induce a broad anti-HCV immune response against the HCV quasispecies or other quasispecies-like infectious agents.

Hepatitis δ antigen is necessary for access of hepatitis s virus rna to the cell transcriptional machinery but is not part of the transcriptional complex

Abstract To examine the role of hepatitis S virus antigen in the replication of hepatitis δ virus RNA, we have transfected a stable HDAg-positive cell line (A3) and the parental HDAg-negative line (HepG2) with HDV RNA produced in vitro ; synthesis of complementary HDV RNA was only detected in HDAg-positive cultures. In contrast, nuclear homogenates from both HDAg-positive and -negative cells synthesized comparable levels of complementary RNA from exogenous HDV RNA. These findings indicate that HDAg is not a necessary component of the transcriptional complex and suggest with other evidence, that a major role for HDAg is likely to be transport of HDV RNA from cytoplasm to nucleus. Transcription of HDV RNA by intact nuclei was sensitive to 1 μ/ml a-amanatin providing firm evidence that, like viroids, this function is performed by host RNA polymerase II.

Defining target antigens for CD25+FOXP3+IFN-γ- regulatory T cells in chronic hepatitis C virus infection

The mechanism behind the apparent lack of effective antiviral immune responses in chronic hepatitis C virus (HCV) patients is poorly understood. It remains unclear if natural regulatory T cells (Treg) contribute to the induction and maintenance of HCV persistence. We herein report for the first time that CD25highIFN‐γ−FOXP3high Tregs can be rapidly induced by culturing peripheral blood mononuclear cells (PBMCs) of HCV‐positive patients with HCV protein‐derived peptides. The HCV‐specific Tregs, generally CD4+CD45RO+, did not proliferate in response to HCV peptide and failed to produce interferon (IFN)‐γ, in distinct contrast to antiviral effector cells. Stimulation of healthy donor PBMCs with HCV peptides did not result in CD25 and FOXP3 upregulation above non‐antigen background. To further investigate the antigen specificity of these potentially disease‐associated natural Tregs, CD25+ cells were isolated from PBMCs, labeled with carboxyfluorescein diacetate succinimidylester and added back to CD25‐depleted PBMCs, and the co‐cultures were then stimulated with individual peptides derived from the HCV core protein. We found that the actual peptide that can stimulate Treg varied between patients, but within any given subject only a small number of the peptides were able to stimulate Treg, suggesting the existence of dominant Treg epitopes. Although functional experiments for these peptides are ongoing in our laboratory, data presented here suggests that HCV‐specific natural Tregs are abundant in infected individuals, in contrast to the extremely low frequency of anti‐HCV effector T cells, supporting the view that natural Treg may be implicated in host immune tolerance during HCV infection.

Hepatitis delta virus RNA, protein synthesis and associated cytotoxicity in a stably transfected cell line

A trimer of hepatitis delta virus (HDV) cDNA in a retrovirus expression vector was transfected into subclone of the PLC/PRF/5 human hepatoma cell line, and a stable cell line (H1 delta 9) was clonally selected that supported the synthesis of both genomic and antigenomic sense HDV RNA. The H1 delta 9 cell line also expressed hepatitis delta antigen (HDAg) in cell nuclei in three distinct morphological patterns, including patterns typically seen in HDV-infected livers. HDAg expression was restricted to the smaller (p24) of the two HDAg-associated polypeptides in early passages of the H1 delta 9 cell line, but continuous passage of the cells resulted in increasing of expression of the larger (p27) HDAg-specific polypeptide. Passage of the H1 delta 9 cells also led to sustained expression of monomeric HDV RNA and a reduction in the levels of dimeric- and trimeric-HDV RNA. This was accompanied by an attenuation of virus-related cytotoxicity which was a feature of early cell passage numbers. HDV RNA replication in these cells was resistant to actinomycin D suggesting that replication was not dependent on continued expression from the transfected HDV cDNA and thus was likely to be self-sustaining.

A phase I clinical trial of dendritic cell immunotherapy in HCV-infected individuals.

BACKGROUND & AIMS HCV patients who fail conventional interferon-based therapy have limited treatment options. Dendritic cells are central to the priming and development of antigen-specific CD4(+) and CD8(+) T cell immunity, necessary to elicit effective viral clearance. The aim of the study was to investigate the safety and efficacy of vaccination with autologous dendritic cells loaded with HCV-specific cytotoxic T cell epitopes. METHODS We examined the potential of autologous monocyte-derived dendritic cells (MoDC), presenting HCV-specific HLA A2.1-restricted cytotoxic T cell epitopes, to influence the course of infection in six patients who failed conventional therapy. Dendritic cells were loaded and activated ex vivo with lipopeptides. In this phase 1 dose escalation study, all patients received a standard dose of cells by the intradermal route while sequential patients received an increased dose by the intravenous route. RESULTS No patient showed a severe adverse reaction although all experienced transient minor side effects. HCV-specific CD8(+) T cell responses were enumerated in PBMC by ELIspot for interferon-gamma. Patients generated de novo responses, not only to peptides presented by the cellular vaccine but also to additional viral epitopes not represented in the lipopeptides, suggestive of epitope spreading. Despite this, no increases in ALT levels were observed. However, the responses were not sustained and failed to influence the viral load, the anti-HCV core antibody response and the level of circulating cytokines. CONCLUSIONS Immunotherapy using autologous MoDC pulsed with lipopeptides was safe, but was unable to generate sustained responses or alter the outcome of the infection. Alternative dosing regimens or vaccination routes may need to be considered to achieve therapeutic benefit.

A self-adjuvanting lipopeptide-based vaccine candidate for the treatment of hepatitis C virus infection ☆

Effective CD8(+) T cell responses have been induced using totally synthetic self-adjuvanting lipopeptides containing the dipalmitoyl-S-glyceryl cysteine lipid moiety, which is a ligand for Toll-like receptor 2 (TLR2) on dendritic cells (DC). In this study, we evaluated the use of lipopeptide vaccine candidates containing HLA-A2-restricted epitopes for DC-based immunotherapy of HCV infection. Lipopeptides were able to induce specific CD8(+) T cell responses in HLA-A2 transgenic mice and consistently activated human monocyte-derived DC from both healthy individuals and HCV infected patients. Lipopeptide-pulsed human DC were also found to secrete the pro-inflammatory cytokine IL-12p70 and were able to activate antigen-specific IFN-gamma production by autologous CD8(+) T cells obtained from a hepatitis C patient. These results show that DC from HCV patients can be matured and antigen loaded with TLR2-targeting lipopeptides for effective presentation of CD8(+) T cell epitopes; the use of autologous lipopeptide-pulsed DC or direct lipopeptide vaccination may be successful approaches for the priming or boosting of anti-HCV CD8(+) T cell responses to aid in the clearance of the virus in chronically infected individuals.