Optimising T cell (re)boosting strategies for adenoviral and modified vaccinia Ankara vaccine regimens in humans.

Simian adenoviral and modified vaccinia Ankara (MVA) viral vectors used in heterologous prime-boost strategies are potent inducers of T cells against encoded antigens and are in advanced testing as vaccine carriers for a wide range of infectious agents and cancers. It is unclear if these responses can be further enhanced or sustained with reboosting strategies. Furthermore, despite the challenges involved in MVA manufacture dose de-escalation has not been performed in humans. In this study, healthy volunteers received chimpanzee-derived adenovirus-3 and MVA vaccines encoding the non-structural region of hepatitis C virus (ChAd3-NSmut/MVA-NSmut) 8 weeks apart. Volunteers were then reboosted with a second round of ChAd3-NSmut/MVA-NSmut or MVA-NSmut vaccines 8 weeks or 1-year later. We also determined the capacity of reduced doses of MVA-NSmut to boost ChAd3-NSmut primed T cells. Reboosting was safe, with no enhanced reactogenicity. Reboosting after an 8-week interval led to minimal re-expansion of transgene-specific T cells. However, after a longer interval, T cell responses expanded efficiently and memory responses were enhanced. The 8-week interval regimen induced a higher percentage of terminally differentiated and effector memory T cells. Reboosting with MVA-NSmut alone was as effective as with ChAd3-NSmut/MVA-NSmut. A ten-fold lower dose of MVA (2 × 107pfu) induced high-magnitude, sustained, broad, and functional Hepatitis C virus (HCV)-specific T cell responses, equivalent to standard doses (2 × 108 pfu). Overall, we show that following Ad/MVA prime-boost vaccination reboosting is most effective after a prolonged interval and is productive with MVA alone. Importantly, we also show that a ten-fold lower dose of MVA is as potent in humans as the standard dose.

Characterizing Hepatitis C Virus-Specific CD4+ T Cells Following Viral-Vectored Vaccination, Directly Acting Antivirals, and Spontaneous Viral Cure.

BACKGROUND AND AIMS: Induction of functional helper CD4+ T cells is the hallmark of a protective immune response against hepatitis C virus (HCV), associated with spontaneous viral clearance. Heterologous prime/boost viral vectored vaccination has demonstrated induction of broad and polyfunctional HCV-specific CD8+ T cells in healthy volunteers; however, much less is known about CD4+ T-cell subsets following vaccination. APPROACH AND RESULTS: We analyzed HCV-specific CD4+ T-cell populations using major histocompatibility complex class II tetramers in volunteers undergoing HCV vaccination with recombinant HCV adenoviral/modified vaccinia Ankara viral vectors. Peptide-specific T-cell responses were tracked over time, and functional (proliferation and cytokine secretion) and phenotypic (cell surface and intranuclear) markers were assessed using flow cytometry. These were compared to CD4+ responses in 10 human leukocyte antigen-matched persons with HCV spontaneous resolution and 21 chronically infected patients treated with directly acting antiviral (DAA) therapy. Vaccination induced tetramer-positive CD4+ T cells that were highest 1-4 weeks after boosting (mean, 0.06%). Similar frequencies were obtained for those tracked following spontaneous resolution of disease (mean, 0.04%). In addition, the cell-surface phenotype (CD28, CD127) memory subset markers and intranuclear transcription factors, as well as functional capacity of peptide-specific CD4+ T-cell responses characterized after vaccination, are comparable to those following spontaneous viral resolution. In contrast, helper responses in chronic infection were infrequently detected and poorly functional and did not consistently recover following HCV cure. CONCLUSIONS: Helper CD4+ T-cell phenotype and function following HCV viral vectored vaccination resembles "protective memory" that is observed following spontaneous clearance of HCV. DAA cure does not promote resurrection of exhausted CD4+ T-cell memory in chronic infection.

A Novel Vaccine Strategy Employing Serologically Different Chimpanzee Adenoviral Vectors for the Prevention of HIV-1 and HCV Coinfection.

Background: Nearly 3 million people worldwide are coinfected with HIV and HCV. Affordable strategies for prevention are needed. We developed a novel vaccination regimen involving replication-defective and serologically distinct chimpanzee adenovirus (ChAd3, ChAd63) vector priming followed by modified vaccinia Ankara (MVA) boosts, for simultaneous delivery of HCV non-structural (NSmut) and HIV-1 conserved (HIVconsv) region immunogens. Methods: We conducted a phase I trial in which 33 healthy volunteers were sequentially enrolled and vaccinated via the intramuscular route as follows: 9 received ChAd3-NSmut [2.5 × 1010 vp] and MVA-NSmut [2 × 108 pfu] at weeks 0 and 8, respectively; 8 received ChAdV63.HIVconsv [5 × 1010 vp] and MVA.HIVconsv [2 × 108 pfu] at the same interval; 16 were co-primed with ChAd3-NSmut [2.5 × 1010 vp] and ChAdV63.HIVconsv [5 × 1010 vp] followed at week 8 by MVA-NSmut and MVA.HIVconsv [both 1 × 108 pfu]. Immunogenicity was assessed using peptide pools in ex vivo ELISpot and intracellular cytokine assays. Vaccine-induced whole blood transcriptome changes were assessed by microarray analysis. Results: All vaccines were well tolerated and no vaccine-related serious adverse events occurred. Co-administration of the prime-boost vaccine regimens induced high magnitude and broad T cell responses that were similar to those observed following immunization with either regimen alone. Median (interquartile range, IQR) peak responses to NSmut were 3,480 (2,728-4,464) and 3,405 (2,307-7,804) spot-forming cells (SFC)/106 PBMC for single and combined HCV vaccinations, respectively (p = 0.8). Median (IQR) peak responses to HIVconsv were 1,305 (1,095-4,967) and 1,005 (169-2,482) SFC/106 PBMC for single and combined HIV-1 vaccinations, respectively (p = 0.5). Responses were maintained above baseline to 34 weeks post-vaccination. Intracellular cytokine analysis indicated that the responding populations comprised polyfunctional CD4+ and CD8+ T cells. Canonical pathway analysis showed that in the single and combined vaccination groups, pathways associated with antiviral and innate immune responses were enriched for upregulated interferon-stimulated genes 24 h after priming and boosting vaccinations. Conclusions: Serologically distinct adenoviral vectors encoding HCV and HIV-1 immunogens can be safely co-administered without reducing the immunogenicity of either vaccine. This provides a novel strategy for targeting these viruses simultaneously and for other pathogens that affect the same populations. Clinical trial registration: https://clinicaltrials.gov, identifier: NCT02362217.

Highly-Immunogenic Virally-Vectored T-cell Vaccines Cannot Overcome Subversion of the T-cell Response by HCV during Chronic Infection.

An effective therapeutic vaccine for the treatment of chronic hepatitis C virus (HCV) infection, as an adjunct to newly developed directly-acting antivirals (DAA), or for the prevention of reinfection, would significantly reduce the global burden of disease associated with chronic HCV infection. A recombinant chimpanzee adenoviral (ChAd3) vector and a modified vaccinia Ankara (MVA), encoding the non-structural proteins of HCV (NSmut), used in a heterologous prime/boost regimen induced multi-specific, high-magnitude, durable HCV-specific CD4+ and CD8+ T-cell responses in healthy volunteers, and was more immunogenic than a heterologous Ad regimen. We now assess the immunogenicity of this vaccine regimen in HCV infected patients (including patients with a low viral load suppressed with interferon/ribavirin therapy), determine T-cell cross-reactivity to endogenous virus, and compare immunogenicity with that observed previously in both healthy volunteers and in HCV infected patients vaccinated with the heterologous Ad regimen. Vaccination of HCV infected patients with ChAd3-NSmut/MVA-NSmut was well tolerated. Vaccine-induced HCV-specific T-cell responses were detected in 8/12 patients; however, CD4+ T-cell responses were rarely detected, and the overall magnitude of HCV-specific T-cell responses was markedly reduced when compared to vaccinated healthy volunteers. Furthermore, HCV-specific cells had a distinct partially-functional phenotype (lower expression of activation markers, granzyme B, and TNFα production, weaker in vitro proliferation, and higher Tim3 expression, with comparable Tbet and Eomes expression) compared to healthy volunteers. Robust anti-vector T-cells and antibodies were induced, showing that there is no global defect in immunity. The level of viremia at the time of vaccination did not correlate with the magnitude of the vaccine-induced T-cell response. Full-length, next-generation sequencing of the circulating virus demonstrated that T-cells were only induced by vaccination when there was a sequence mismatch between the autologous virus and the vaccine immunogen. However, these T-cells were not cross-reactive with the endogenous viral variant epitopes. Conversely, when there was complete homology between the immunogen and circulating virus at a given epitope T-cells were not induced. T-cell induction following vaccination had no significant impact on HCV viral load. In vitro T-cell culture experiments identified the presence of T-cells at baseline that could be expanded by vaccination; thus, HCV-specific T-cells may have been expanded from pre-existing low-level memory T-cell populations that had been exposed to HCV antigens during natural infection, explaining the partial T-cell dysfunction. In conclusion, vaccination with ChAd3-NSmut and MVA-NSmut prime/boost, a potent vaccine regimen previously optimized in healthy volunteers was unable to reconstitute HCV-specific T-cell immunity in HCV infected patients. This highlights the major challenge of overcoming T-cell exhaustion in the context of persistent antigen exposure.

A Monovalent Chimpanzee Adenovirus Ebola Vaccine Boosted with MVA.

BACKGROUND: The West African outbreak of Ebola virus disease that peaked in 2014 has caused more than 11,000 deaths. The development of an effective Ebola vaccine is a priority for control of a future outbreak. METHODS: In this phase 1 study, we administered a single dose of the chimpanzee adenovirus 3 (ChAd3) vaccine encoding the surface glycoprotein of Zaire ebolavirus (ZEBOV) to 60 healthy adult volunteers in Oxford, United Kingdom. The vaccine was administered in three dose levels--1×10(10) viral particles, 2.5×10(10) viral particles, and 5×10(10) viral particles--with 20 participants in each group. We then assessed the effect of adding a booster dose of a modified vaccinia Ankara (MVA) strain, encoding the same Ebola virus glycoprotein, in 30 of the 60 participants and evaluated a reduced prime-boost interval in another 16 participants. We also compared antibody responses to inactivated whole Ebola virus virions and neutralizing antibody activity with those observed in phase 1 studies of a recombinant vesicular stomatitis virus-based vaccine expressing a ZEBOV glycoprotein (rVSV-ZEBOV) to determine relative potency and assess durability. RESULTS: No safety concerns were identified at any of the dose levels studied. Four weeks after immunization with the ChAd3 vaccine, ZEBOV-specific antibody responses were similar to those induced by rVSV-ZEBOV vaccination, with a geometric mean titer of 752 and 921, respectively. ZEBOV neutralization activity was also similar with the two vaccines (geometric mean titer, 14.9 and 22.2, respectively). Boosting with the MVA vector increased virus-specific antibodies by a factor of 12 (geometric mean titer, 9007) and increased glycoprotein-specific CD8+ T cells by a factor of 5. Significant increases in neutralizing antibodies were seen after boosting in all 30 participants (geometric mean titer, 139; P<0.001). Virus-specific antibody responses in participants primed with ChAd3 remained positive 6 months after vaccination (geometric mean titer, 758) but were significantly higher in those who had received the MVA booster (geometric mean titer, 1750; P<0.001). CONCLUSIONS: The ChAd3 vaccine boosted with MVA elicited B-cell and T-cell immune responses to ZEBOV that were superior to those induced by the ChAd3 vaccine alone. (Funded by the Wellcome Trust and others; ClinicalTrials.gov number, NCT02240875.).

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

UNLABELLED: 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.