Leo Swadling

Novel Adenovirus-Based Vaccines Induce Broad and Sustained T Cell Responses to HCV in Man

An adenoviral HCV vaccine induces antiviral T cell responses in human volunteers. Hepatitis Hide and Seek Like venture capitalists and Wall Street bankers, patients receiving results of their blood work don’t like surprises, and more than money is at stake. Because infections caused by the hepatitis C virus (HCV) frequently are asymptomatic, patients might not know they’ve been infected: Symptoms don’t usually appear until irreversible liver scarring has occurred, which may cause cirrhosis, liver failure, or cancer. Even if infection is caught early, current therapies to combat this stealth virus have serious side effects, and there is no vaccine to prevent or treat HCV infection. Now, Barnes et al. demonstrate that vaccines developed with adenoviral vectors can induce broad and sustained immune responses to HCV in humans. Adenoviral vectors have shown promise in vaccine trials in animal models; however, preexisting immunity to common serotypes in humans has limited their use. In a phase 1 clinical trial, Barnes et al. vaccinated healthy subjects with two rare serotype adenoviral vectors that expressed an HCV protein. Both the human and the chimp adenoviral vaccinations elicited HCV-specific immune responses in the recipients that responded to multiple HCV antigens, were sustained for at least a year with boost, and elicited memory responses. And the researchers got a surprise they liked: Vaccination primed T cells to respond to multiple HCV strains at a level consistent with protective immunity. Further trials will be needed to confirm protective or therapeutic roles in HCV-infected individuals. Currently, no vaccine exists for hepatitis C virus (HCV), a major pathogen thought to infect 170 million people globally. Many studies suggest that host T cell responses are critical for spontaneous resolution of disease, and preclinical studies have indicated a requirement for T cells in protection against challenge. We aimed to elicit HCV-specific T cells with the potential for protection using a recombinant adenoviral vector strategy in a phase 1 study of healthy human volunteers. Two adenoviral vectors expressing NS proteins from HCV genotype 1B were constructed based on rare serotypes [human adenovirus 6 (Ad6) and chimpanzee adenovirus 3 (ChAd3)]. Both vectors primed T cell responses against HCV proteins; these T cell responses targeted multiple proteins and were capable of recognizing heterologous strains (genotypes 1A and 3A). HCV-specific T cells consisted of both CD4+ and CD8+ T cell subsets; secreted interleukin-2, interferon-γ, and tumor necrosis factor–α; and could be sustained for at least a year after boosting with the heterologous adenoviral vector. Studies using major histocompatibility complex peptide tetramers revealed long-lived central and effector memory pools that retained polyfunctionality and proliferative capacity. These data indicate that an adenoviral vector strategy can induce sustained T cell responses of a magnitude and quality associated with protective immunity and open the way for studies of prophylactic and therapeutic vaccines for HCV.

A Human Vaccine Strategy Based On Chimpanzee Adenoviral and MVA Vectors That Primes, Boosts and Sustains Functional HCV Specific T-Cell Memory

A prime-boost HCV vaccine strategy induces durable and broad T cell responses, characteristic of those associated with viral control. An Ounce of HCV Prevention Chronic hepatitis C virus (HCV) infection causes liver inflammation that can lead to diminished liver function or liver failure. Recent approval of antiviral drugs for HCV affords health care providers with treatment options; however, these new therapies are expensive with limited availability, leaving the door open for preventative approaches such as vaccines. Swadling et al. report a first-in-human trial of a prime-boost vaccine strategy for HCV. They prime with a simian adenoviral vector followed by a modified vaccinia Ankara vector encoding HCV proteins, which induces a T cell response similar to that found in HCV control in natural infection. If this strategy can show efficacy in later-stage studies, this approach could be used in a preventative HCV vaccine. A protective vaccine against hepatitis C virus (HCV) remains an unmet clinical need. HCV infects millions of people worldwide and is a leading cause of liver cirrhosis and hepatocellular cancer. Animal challenge experiments, immunogenetics studies, and assessment of host immunity during acute infection highlight the critical role that effective T cell immunity plays in viral control. In this first-in-man study, we have induced antiviral immunity with functional characteristics analogous to those associated with viral control in natural infection, and improved upon a vaccine based on adenoviral vectors alone. We assessed a heterologous prime-boost vaccination strategy based on a replicative defective simian adenoviral vector (ChAd3) and modified vaccinia Ankara (MVA) vector encoding the NS3, NS4, NS5A, and NS5B proteins of HCV genotype 1b. Analysis used single-cell mass cytometry and human leukocyte antigen class I peptide tetramer technology in healthy human volunteers. We show that HCV-specific T cells induced by ChAd3 are optimally boosted with MVA, and generate very high levels of both CD8+ and CD4+ HCV-specific T cells targeting multiple HCV antigens. Sustained memory and effector T cell populations are generated, and T cell memory evolved over time with improvement of quality (proliferation and polyfunctionality) after heterologous MVA boost. We have developed an HCV vaccine strategy, with durable, broad, sustained, and balanced T cell responses, characteristic of those associated with viral control, paving the way for the first efficacy studies of a prophylactic HCV vaccine.

CD161 Defines a Transcriptional and Functional Phenotype across Distinct Human T Cell Lineages

Summary The C-type lectin CD161 is expressed by a large proportion of human T lymphocytes of all lineages, including a population known as mucosal-associated invariant T (MAIT) cells. To understand whether different T cell subsets expressing CD161 have similar properties, we examined these populations in parallel using mass cytometry and mRNA microarray approaches. The analysis identified a conserved CD161++/MAIT cell transcriptional signature enriched in CD161+CD8+ T cells, which can be extended to CD161+ CD4+ and CD161+TCRγδ+ T cells. Furthermore, this led to the identification of a shared innate-like, TCR-independent response to interleukin (IL)-12 plus IL-18 by different CD161-expressing T cell populations. This response was independent of regulation by CD161, which acted as a costimulatory molecule in the context of T cell receptor stimulation. Expression of CD161 hence identifies a transcriptional and functional phenotype, shared across human T lymphocytes and independent of both T cell receptor (TCR) expression and cell lineage.

Ever closer to a prophylactic vaccine for HCV

Introduction: With 3 – 4 million new infections occurring annually, hepatitis C virus (HCV) is a major global health problem. There is increasing evidence to suggest that HCV will be highly amenable to a vaccine approach, and despite advances in treatment, a vaccine remains the most cost-effective and realistic means to significantly reduce the worldwide mortality and morbidity associated with persistent HCV infection. Areas covered: In this review we discuss immune responses to HCV during natural infection, and describe how they may inform vaccine design. We introduce the current candidate vaccines for HCV and compare how these fare against the expected requirements of an effective prophylactic HCV vaccine in relation to the breadth, functionality, magnitude and phenotype of the vaccine-induced immune response. Expert opinion: Although the correlates of immune protection against HCV are not completely defined, we now have vaccine technologies capable of inducing HCV-specific adaptive immune responses to an order of magnitude that are associated with protection during natural infection. The challenge next is to i) establish well-characterised cohorts of people at risk of HCV infection for vaccine efficacy testing and ii) to better understand the correlates of protection in natural history studies. If these can be achieved, a vaccine against HCV appears a realistic goal.

CD161 int CD8+ T cells: a novel population of highly functional, memory CD8+ T cells enriched within the gut

The C-type lectin-like receptor CD161 is expressed by lymphocytes found in human gut and liver, as well as blood, especially natural killer (NK) cells, T helper 17 (Th17) cells, and a population of unconventional T cells known as mucosal-associated invariant T (MAIT) cells. The association of high CD161 expression with innate T-cell populations including MAIT cells is established. Here we show that CD161 is also expressed, at intermediate levels, on a prominent subset of polyclonal CD8+ T cells, including antiviral populations that display a memory phenotype. These memory CD161intCD8+ T cells are enriched within the colon and express both CD103 and CD69, markers associated with tissue residence. Furthermore, this population was characterized by enhanced polyfunctionality, increased levels of cytotoxic mediators, and high expression of the transcription factors T-bet and eomesodermin (EOMES). Such populations were induced by novel vaccine strategies based on adenoviral vectors, currently in trial against hepatitis C virus. Thus, intermediate CD161 expression marks potent polyclonal, polyfunctional tissue-homing CD8+ T-cell populations in humans. As induction of such responses represents a major aim of T-cell prophylactic and therapeutic vaccines in viral disease and cancer, analysis of these populations could be of value in the future.

Chronic hepatitis C viral infection subverts vaccine‐induced T‐cell immunity in humans

Adenoviral vectors encoding hepatitis C virus (HCV) nonstructural (NS) proteins induce multispecific, high‐magnitude, durable CD4+ and CD8+ T‐cell responses in healthy volunteers. We assessed the capacity of these vaccines to induce functional HCV‐specific immune responses and determine T‐cell cross‐reactivity to endogenous virus in patients with chronic HCV infection. HCV genotype 1‐infected patients were vaccinated using heterologous adenoviral vectors (ChAd3‐NSmut and Ad6‐NSmut) encoding HCV NS proteins in a dose escalation, prime‐boost regimen, with and without concomitant pegylated interferon‐α/ribavirin therapy. Analysis of immune responses ex vivo used human leukocyte antigen class I pentamers, intracellular cytokine staining, and fine mapping in interferon‐γ enzyme‐linked immunospot assays. Cross‐reactivity of T cells with population and endogenous viral variants was determined following viral sequence analysis. Compared to healthy volunteers, the magnitude of HCV‐specific T‐cell responses following vaccination was markedly reduced. CD8+ HCV‐specific T‐cell responses were detected in 15/24 patients at the highest dose, whereas CD4+ T‐cell responses were rarely detectable. Analysis of the host circulating viral sequence showed that T‐cell responses were rarely elicited when there was sequence homology between vaccine immunogen and endogenous virus. In contrast, T cells were induced in the context of genetic mismatch between vaccine immunogen and endogenous virus; however, these commonly failed to recognize circulating epitope variants and had a distinct partially functional phenotype. Vaccination was well tolerated but had no significant effect on HCV viral load. Conclusion: Vaccination with potent HCV adenoviral vectored vaccines fails to restore T‐cell immunity except where there is genetic mismatch between vaccine immunogen and endogenous virus; this highlights the major challenge of overcoming T‐cell exhaustion in the context of persistent antigen exposure with implications for cancer and other persistent infections. (Hepatology 2016;63:1455‐1470)

Adenoviral Vector Vaccination Induces a Conserved Program of CD8+ T Cell Memory Differentiation in Mouse and Man

Summary Following exposure to vaccines, antigen-specific CD8+ T cell responses develop as long-term memory pools. Vaccine strategies based on adenoviral vectors, e.g., those developed for HCV, are able to induce and sustain substantial CD8+ T cell populations. How such populations evolve following vaccination remains to be defined at a transcriptional level. We addressed the transcriptional regulation of divergent CD8+ T cell memory pools induced by an adenovector encoding a model antigen (beta-galactosidase). We observe transcriptional profiles that mimic those following infection with persistent pathogens, murine and human cytomegalovirus (CMV). Key transcriptional hallmarks include upregulation of homing receptors and anti-apoptotic pathways, driven by conserved networks of transcription factors, including T-bet. In humans, an adenovirus vaccine induced similar CMV-like phenotypes and transcription factor regulation. These data clarify the core features of CD8+ T cell memory following vaccination with adenovectors and indicate a conserved pathway for memory development shared with persistent herpesviruses.

Targeted reconstruction of T cell receptor sequence from single cell RNA-seq links CDR3 length to T cell differentiation state

Abstract The T cell compartment must contain diversity in both T cell receptor (TCR) repertoire and cell state to provide effective immunity against pathogens. However, it remains unclear how differences in the TCR contribute to heterogeneity in T cell state. Single cell RNA-sequencing (scRNA-seq) can allow simultaneous measurement of TCR sequence and global transcriptional profile from single cells. However, current methods for TCR inference from scRNA-seq are limited in their sensitivity and require long sequencing reads, thus increasing the cost and decreasing the number of cells that can be feasibly analyzed. Here we present TRAPeS, a publicly available tool that can efficiently extract TCR sequence information from short-read scRNA-seq libraries. We apply it to investigate heterogeneity in the CD8+ T cell response in humans and mice, and show that it is accurate and more sensitive than existing approaches. Coupling TRAPeS with transcriptome analysis of CD8+ T cells specific for a single epitope from Yellow Fever Virus (YFV), we show that the recently described ‘naive-like’ memory population have significantly longer CDR3 regions and greater divergence from germline sequence than do effector-memory phenotype cells. This suggests that TCR usage is associated with the differentiation state of the CD8+ T cell response to YFV.

Cross-reactivity of hepatitis C virus specific vaccine-induced T cells at immunodominant epitopes.

Viral diversity is a challenge to the development of a hepatitis C virus (HCV) vaccine. Following vaccination of humans with adenoviral vectors, we determined the capacity of T cells to target common viral variants at immundominant epitopes ex vivo. We identified two major variants for epitopes NS31073 and NS31446, and multiple variants for epitope NS31406 that occurred in >5% of genotype 1 and 3 sequences at a population level. Cross‐reactivity of vaccine‐induced T cells was determined using variant peptides in IFN‐γ ELISPOT assays. Vaccine‐induced T cells targeted approximately 90% of NS31073 genotype 1 sequences and 50% of NS31446 genotype 1 and 3 sequences. For NS31406, 62% of subtype‐1b sequences were targeted. Next, we assessed whether an in vitro priming system, using dendritic cells and T cells from healthy donors, could identify a variant of NS31406 that was maximally cross‐reactive. In vitro priming assays showed that of those tested the NS31406 vaccine variant was the most immunogenic. T cells primed with genotype 1 variants from subtype 1a or 1b were broadly cross‐reactive with other variants from the same subtype. We conclude that immunization with candidate HCV adenoviral vaccines generates cross‐reactive T cells at immunodominant epitopes. The degree of cross‐reactivity varies between epitopes and may be HCV‐subtype specific.

Characterisation of the specificity, functionality and durability of host T-cell responses against the full-length hepatitis E virus.

The interplay between host antiviral immunity and immunopathology during hepatitis E virus (HEV) infection determines important clinical outcomes. We characterized the specificity, functionality, and durability of host T‐cell responses against the full‐length HEV virus and assessed a novel “Quantiferon” assay for the rapid diagnosis of HEV infection. Eighty‐nine volunteers were recruited from Oxford, Truro (UK), and Toulouse (France), including 44 immune‐competent patients with acute HEV infection, 18 HEV‐exposed immunosuppressed organ‐transplant recipients (8 with chronic HEV), and 27 healthy volunteers. A genotype 3a peptide library (616 overlapping peptides spanning open reading frames [ORFs] 1‐3) was used in interferon‐gamma (IFN‐γ) T‐cell ELISpot assays. CD4+/CD8+ T‐cell subsets and polyfunctionality were defined using ICCS and SPICE analysis. Quantification of IFN‐γ used whole‐blood stimulation with recombinant HEV‐capsid protein in the QuantiFERON kit. HEV‐specific T‐cell responses were detected in 41/44 immune‐competent HEV exposed volunteers (median magnitude: 397 spot‐forming units/106 peripheral blood mononuclear cells), most frequently targeting ORF2. High‐magnitude, polyfunctional CD4 and CD8+ T cells were detected during acute disease and maintained to 12 years, but these declined over time, with CD8+ responses becoming more monofunctional. Low‐level responses were detectable in immunosuppressed patients. Twenty‐three novel HEV CD4+ and CD8+ T‐cell targets were mapped predominantly to conserved genomic regions. QuantiFERON testing demonstrated an inverse correlation between IFN‐γ production and the time from clinical presentation, providing 100% specificity, and 71% sensitivity (area under the receiver operator characteristic curve of 0.86) for HEV exposure at 0.3 IU/mL. Conclusion: Robust HEV‐specific T‐cell responses generated during acute disease predominantly target ORF2, but decline in magnitude and polyfunctionality over time. Defining HEV T‐cell targets will be important for the investigation of HEV‐associated autoimmune disease. (Hepatology 2016;64:1934‐1950).