A pan-genotype hepatitis C virus viral vector vaccine generates T cells and neutralizing antibodies in mice.

BACKGROUND AND AIMS: A prophylactic vaccine targeting multiple HCV genotypes (gt) is urgently required to meet World Health Organization elimination targets. Neutralizing antibodies (nAbs) and CD4+ and CD8+ T cells are associated with spontaneous clearance of HCV, and each may contribute to protective immunity. However, current vaccine candidates generate either nAbs or T cells targeting genetically variable epitopes and have failed to show efficacy in human trials. We have previously shown that a simian adenovirus vector (ChAdOx1) encoding conserved sequences across gt1-6 (ChAd-Gt1-6), and separately gt-1a E2 protein with variable regions deleted (E2Δ123HMW ), generates pan-genotypic T cells and nAbs, respectively. We now aim to develop a vaccine to generate both viral-specific B- and T-cell responses concurrently. APPROACH AND RESULTS: We show that vaccinating with ChAd-Gt1-6 and E2Δ123HMW sequentially in mice generates T-cell and antibody (Ab) responses comparable to either vaccine given alone. We encoded E2Δ123 in ChAdOx1 (ChAd-E2Δ123) and show that this, given with an E2Δ123HMW protein boost, induces greater CD81-E2 inhibitory and HCV-pseudoparticle nAb titers compared to the E2Δ123HMW prime boost. We developed bivalent viral vector vaccines (ChAdOx1 and modified vaccinia Ankara [MVA]) encoding both Gt1-6 and E2Δ123 immunogens (Gt1-6-E2Δ123) generating polyfunctional CD4+ and CD8+ T cells and nAb titers in prime/boost strategies. This approach generated nAb responses comparable to monovalent E2Δ123 ChAd/MVA vaccines and superior to three doses of recombinant E2Δ123HMW protein, while also generating high-magnitude T-cell responses. CONCLUSIONS: These data are an important step forward for the development of a pan-genotype HCV vaccine to elicit T cells and nAbs for future assessment in humans.

T cell assays differentiate clinical and subclinical SARS-CoV-2 infections from cross-reactive antiviral responses.

Identification of protective T cell responses against SARS-CoV-2 requires distinguishing people infected with SARS-CoV-2 from those with cross-reactive immunity to other coronaviruses. Here we show a range of T cell assays that differentially capture immune function to characterise SARS-CoV-2 responses. Strong ex vivo ELISpot and proliferation responses to multiple antigens (including M, NP and ORF3) are found in 168 PCR-confirmed SARS-CoV-2 infected volunteers, but are rare in 119 uninfected volunteers. Highly exposed seronegative healthcare workers with recent COVID-19-compatible illness show T cell response patterns characteristic of infection. By contrast, >90% of convalescent or unexposed people show proliferation and cellular lactate responses to spike subunits S1/S2, indicating pre-existing cross-reactive T cell populations. The detection of T cell responses to SARS-CoV-2 is therefore critically dependent on assay and antigen selection. Memory responses to specific non-spike proteins provide a method to distinguish recent infection from pre-existing immunity in exposed populations.

MHC class II invariant chain-adjuvanted viral vectored vaccines enhances T cell responses in humans.

Strategies to enhance the induction of high magnitude T cell responses through vaccination are urgently needed. Major histocompatibility complex (MHC) class II-associated invariant chain (Ii) plays a critical role in antigen presentation, forming MHC class II peptide complexes for the generation of CD4+ T cell responses. Preclinical studies evaluating the fusion of Ii to antigens encoded in vector delivery systems have shown that this strategy may enhance T cell immune responses to the encoded antigen. We now assess this strategy in humans, using chimpanzee adenovirus 3 and modified vaccinia Ankara vectors encoding human Ii fused to the nonstructural (NS) antigens of hepatitis C virus (HCV) in a heterologous prime/boost regimen. Vaccination was well tolerated and enhanced the peak magnitude, breadth, and proliferative capacity of anti-HCV T cell responses compared to non-Ii vaccines in humans. Very high frequencies of HCV-specific T cells were elicited in humans. Polyfunctional HCV-specific CD8+ and CD4+ responses were induced with up to 30% of CD3+CD8+ cells targeting single HCV epitopes; these were mostly effector memory cells with a high proportion expressing T cell activation and cytolytic markers. No volunteers developed anti-Ii T cell or antibody responses. Using a mouse model and in vitro experiments, we show that Ii fused to NS increases HCV immune responses through enhanced ubiquitination and proteasomal degradation. This strategy could be used to develop more potent HCV vaccines that may contribute to the HCV elimination targets and paves the way for developing class II Ii vaccines against cancer and other infections.

Viral vectored hepatitis C virus vaccines generate pan-genotypic T cell responses to conserved subdominant epitopes.

BACKGROUND: Viral genetic variability presents a major challenge to the development of a prophylactic hepatitis C virus (HCV) vaccine. A promising HCV vaccine using chimpanzee adenoviral vectors (ChAd) encoding a genotype (gt) 1b non-structural protein (ChAd-Gt1b-NS) generated high magnitude T cell responses. However, these T cells showed reduced cross-recognition of dominant epitope variants and the vaccine has recently been shown to be ineffective at preventing chronic HCV. To address the challenge of viral diversity, we developed ChAd vaccines encoding HCV genomic sequences that are conserved between all major HCV genotypes and adjuvanted by truncated shark invariant chain (sIitr). METHODS: Age-matched female mice were immunised intramuscularly with ChAd (108 infectious units) encoding gt-1 and -3 (ChAd-Gt1/3) or gt-1 to -6 (ChAd-Gt1-6) conserved segments spanning the HCV proteome, or gt-1b (ChAd-Gt1b-NS control), with immunogenicity assessed 14-days post-vaccination. RESULTS: Conserved segment vaccines, ChAd-Gt1/3 and ChAd-Gt1-6, generated high-magnitude, broad, and functional CD4+ and CD8+ T cell responses. Compared to the ChAd-Gt1b-NS vaccine, these vaccines generated significantly greater responses against conserved non-gt-1 antigens, including conserved subdominant epitopes that were not targeted by ChAd-Gt1b-NS. Epitopes targeted by the conserved segment HCV vaccine induced T cells, displayed 96.6% mean sequence homology between all HCV subtypes (100% sequence homology for the majority of genotype-1, -2, -4 sequences and 94% sequence homology for gt-3, -6, -7, and -8) in contrast to 85.1% mean sequence homology for epitopes targeted by ChAd-Gt1b-NS induced T cells. The addition of truncated shark invariant chain (sIitr) increased the magnitude, breadth, and cross-reactivity of the T cell response. CONCLUSIONS: We have demonstrated that genetically adjuvanted ChAd vectored HCV T cell vaccines encoding genetic sequences conserved between genotypes are immunogenic, activating T cells that target subdominant conserved HCV epitopes. These pre-clinical studies support the use of conserved segment HCV T cell vaccines in human clinical trials.

The Design and Development of a Multi-HBV Antigen Encoded in Chimpanzee Adenoviral and Modified Vaccinia Ankara Viral Vectors; A Novel Therapeutic Vaccine Strategy against HBV.

Chronic hepatitis B virus (HBV) infection affects 257 million people globally. Current therapies suppress HBV but viral rebound occurs on cessation of therapy; novel therapeutic strategies are urgently required. To develop a therapeutic HBV vaccine that can induce high magnitude T cells to all major HBV antigens, we have developed a novel HBV vaccine using chimpanzee adenovirus (ChAd) and modified vaccinia Ankara (MVA) viral vectors encoding multiple HBV antigens. ChAd vaccine alone generated very high magnitude HBV specific T cell responses to all HBV major antigens. The inclusion of a shark Invariant (SIi) chain genetic adjuvant significantly enhanced the magnitude of T-cells against HBV antigens. Compared to ChAd alone vaccination, ChAd-prime followed by MVA-boost vaccination further enhanced the magnitude and breadth of the vaccine induced T cell response. Intra-cellular cytokine staining study showed that HBV specific CD8+ and CD4+ T cells were polyfunctional, producing combinations of IFNγ, TNF-α, and IL-2. In summary, we have generated genetically adjuvanted ChAd and MVA vectored HBV vaccines with the potential to induce high-magnitude T cell responses through a prime-boost therapeutic vaccination approach. These pre-clinical studies pave the way for new studies of HBV therapeutic vaccination in humans with chronic hepatitis B infection.

The generation of a simian adenoviral vectored HCV vaccine encoding genetically conserved gene segments to target multiple HCV genotypes.

BACKGROUND: Hepatitis C virus (HCV) genomic variability is a major challenge to the generation of a prophylactic vaccine. We have previously shown that HCV specific T-cell responses induced by a potent T-cell vaccine encoding a single strain subtype-1b immunogen target epitopes dominant in natural infection. However, corresponding viral regions are highly variable at a population level, with a reduction in T-cell reactivity to these variants. We therefore designed and manufactured second generation simian adenovirus vaccines encoding genomic segments, conserved between viral genotypes and assessed these for immunogenicity. METHODS: We developed a computer algorithm to identify HCV genomic regions that were conserved between viral subtypes. Conserved segments below a pre-defined diversity threshold spanning the entire HCV genome were combined to create novel immunogens (1000-1500 amino-acids), covering variation in HCV subtypes 1a and 1b, genotypes 1 and 3, and genotypes 1-6 inclusive. Simian adenoviral vaccine vectors (ChAdOx) encoding HCV conserved immunogens were constructed. Immunogenicity was evaluated in C57BL6 mice using panels of genotype-specific peptide pools in ex-vivo IFN-ϒ ELISpot and intracellular cytokine assays. RESULTS: ChAdOx1 conserved segment HCV vaccines primed high-magnitude, broad, cross-reactive T-cell responses; the mean magnitude of total HCV specific T-cell responses was 1174 SFU/106 splenocytes for ChAdOx1-GT1-6 in C57BL6 mice targeting multiple genomic regions, with mean responses of 935, 1474 and 1112 SFU/106 against genotype 1a, 1b and 3a peptide panels, respectively. Functional assays demonstrated IFNg and TNFa production by vaccine-induced CD4 and CD8 T-cells. In silico analysis shows that conserved immunogens contain multiple epitopes, with many described in natural HCV infection, predicting immunogenicity in humans. CONCLUSIONS: Simian adenoviral vectored vaccines encoding genetic segments that are conserved between all major HCV genotypes contain multiple T-cell epitopes and are highly immunogenic in pre-clinical models. These studies pave the way for the assessment of multi-genotypic HCV T-cell vaccines in humans.

Normal radial migration and lamination are maintained in dyslexia-susceptibility candidate gene homolog Kiaa0319 knockout mice.

Developmental dyslexia is a common disorder with a strong genetic component, but the underlying molecular mechanisms are still unknown. Several candidate dyslexia-susceptibility genes, including KIAA0319, DYX1C1, and DCDC2, have been identified in humans. RNA interference experiments targeting these genes in rat embryos have shown impairments in neuronal migration, suggesting that defects in radial cortical migration could be involved in the disease mechanism of dyslexia. Here we present the first characterisation of a Kiaa0319 knockout mouse line. Animals lacking KIAA0319 protein do not show anatomical abnormalities in any of the layered structures of the brain. Neurogenesis and radial migration of cortical projection neurons are not altered, and the intrinsic electrophysiological properties of Kiaa0319-deficient neurons do not differ from those of wild-type neurons. Kiaa0319 overexpression in cortex delays radial migration, but does not affect final neuronal position. However, knockout animals show subtle differences suggesting possible alterations in anxiety-related behaviour and in sensorimotor gating. Our results do not reveal a migration disorder in the mouse model, adding to the body of evidence available for Dcdc2 and Dyx1c1 that, unlike in the rat in utero knockdown models, the dyslexia-susceptibility candidate mouse homolog genes do not play an evident role in neuronal migration. However, KIAA0319 protein expression seems to be restricted to the brain, not only in early developmental stages but also in adult mice, indicative of a role of this protein in brain function. The constitutive and conditional knockout lines reported here will be useful tools for further functional analyses of Kiaa0319.