NK cells limit therapeutic vaccine-induced CD8+T cell immunity in a PD-L1-dependent manner.

A better understanding of mechanisms that regulate CD8+T cell responses to therapeutic vaccines is needed to develop approaches to enhance vaccine efficacy for chronic viral infections and cancers. We show here that NK cell depletion enhanced antigen-specific T cell responses to chimp adenoviral vector (ChAdOx) vaccination in a mouse model of chronic HBV infection (CHB). Probing the mechanism underlying this negative regulation, we observed that CHB drove parallel up-regulation of programmed cell death ligand 1 (PD-L1) on liver-resident NK cells and programmed cell death 1 (PD-1) on intrahepatic T cells. PD-L1-expressing liver-resident NK cells suppressed PD-1hiCD8+T cells enriched within the HBV-specific response to therapeutic vaccination. Cytokine activation of NK cells also induced PD-L1, and combining cytokine activation with PD-L1 blockade resulted in conversion of NK cells into efficient helpers that boosted HBV-specific CD8+T cell responses to therapeutic vaccination in mice and to chronic infection in humans. Our findings delineate an immunotherapeutic combination that can boost the response to therapeutic vaccination in CHB and highlight the broader importance of PD-L1-dependent regulation of T cells by cytokine-activated NK cells.

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.

Antibody testing for COVID-19: A report from the National COVID Scientific Advisory Panel.

Background: The COVID-19 pandemic caused >1 million infections during January-March 2020. There is an urgent need for reliable antibody detection approaches to support diagnosis, vaccine development, safe release of individuals from quarantine, and population lock-down exit strategies. We set out to evaluate the performance of ELISA and lateral flow immunoassay (LFIA) devices. Methods: We tested plasma for COVID (severe acute respiratory syndrome coronavirus 2; SARS-CoV-2) IgM and IgG antibodies by ELISA and using nine different LFIA devices. We used a panel of plasma samples from individuals who have had confirmed COVID infection based on a PCR result (n=40), and pre-pandemic negative control samples banked in the UK prior to December-2019 (n=142). Results: ELISA detected IgM or IgG in 34/40 individuals with a confirmed history of COVID infection (sensitivity 85%, 95%CI 70-94%), vs. 0/50 pre-pandemic controls (specificity 100% [95%CI 93-100%]). IgG levels were detected in 31/31 COVID-positive individuals tested ≥10 days after symptom onset (sensitivity 100%, 95%CI 89-100%). IgG titres rose during the 3 weeks post symptom onset and began to fall by 8 weeks, but remained above the detection threshold. Point estimates for the sensitivity of LFIA devices ranged from 55-70% versus RT-PCR and 65-85% versus ELISA, with specificity 95-100% and 93-100% respectively. Within the limits of the study size, the performance of most LFIA devices was similar. Conclusions: Currently available commercial LFIA devices do not perform sufficiently well for individual patient applications. However, ELISA can be calibrated to be specific for detecting and quantifying SARS-CoV-2 IgM and IgG and is highly sensitive for IgG from 10 days following first symptoms.

Enhancing cellular immunogenicity of MVA-vectored vaccines by utilizing the F11L endogenous promoter.

Modified vaccinia virus Ankara (MVA)-vectored vaccines against malaria, influenza, tuberculosis and recently Ebola virus are in clinical development. Although this vector is safe and immunogenic in humans, efforts remain on-going to enhance immunogenicity through various approaches such as using stronger promoters to boost transgene expression. We previously reported that endogenous MVA promoters such as pB8 and pF11 increased transgene expression and immunogenicity, as compared to the conventional p7.5 promoter. Here, we show that both promoters also rivalled the mH5 promoter in enhancing MVA immunogenicity. We investigated the mechanisms behind this improved immunogenicity and show that it was a result of strong early transgene expression in vivo, rather than in vitro as would normally be assessed. Moreover, keeping the TK gene intact resulted in a modest improvement in immunogenicity. Utilizing pB8 or pF11 as ectopic promoters at the TK locus instead of their natural loci also increased transgene expression and immunogenicity. In addition to a reporter antigen, the pF11 promoter was tested with the expression of two vaccine antigens for which cellular immunogenicity was significantly increased as compared to the p7.5 promoter. Our data support the use of the pF11 and pB8 promoters for improved immunogenicity in future MVA-vectored candidate vaccines.

Investigation of IRES Insertion into the Genome of Recombinant MVA as a Translation Enhancer in the Context of Transcript Decapping.

Recombinant modified vaccinia virus Ankara (MVA) has been used to deliver vaccine candidate antigens against infectious diseases and cancer. MVA is a potent viral vector for inducing high magnitudes of antigen-specific CD8+ T cells; however the cellular immune responses to a recombinant antigen in MVA could be further enhanced by increasing transgene expression. Previous reports showed the importance of utilizing an early poxviral promoter for increasing transgene expression and therefore enhancing cellular immune responses. However, the vaccinia D10 decapping enzyme is reported to target and decap vaccinia virus early transcripts - a mechanism that could limit the usefulness of early promoters in MVA viral vectors if this enzyme shows the same activity in this closely related virus. Therefore, we attempted to increase transgene expression in recombinant MVA by inserting the encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) upstream of a transgene sequence that is controlled by the B8R early promoter, and assessed D10 enzyme decapping activity in MVA. The aim of the IRES element was to initiate translation of the transgene transcript (after the removal of the cap structure by the D10 decapping protein) in a cap-independent manner. Here, we report that overexpression of the D10 decapping protein, in trans, in MVA reduced growth and transgene expression; however, the IRES element was not able to compensate for the negative effect of the D10 decapping protein. Recombinant MVA with EMCV IRES induced levels of both gene expression and transcription that were similar to the control recombinant MVA, encoding the same transgene but without the IRES element. Both viruses were tested in BALB/c mice and induced similar magnitudes of epitope-specific CD8+ T cells. This work indicates that the MVA version of the D10 decapping enzyme, overexpressed using a plasmid, is functional, but its negative effect on transgene expression by recombinant MVA cannot be overcome by the use of the EMCV IRES inserted upstream of the transgene initiation codon.

Different functions of the common P/V/W and V-specific domains of rinderpest virus V protein in blocking IFN signalling.

The V proteins of paramyxoviruses are composed of two evolutionarily distinct domains, the N-terminal 75 % being common to the viral P, V and W proteins, and not highly conserved between viruses, whilst the remaining 25 % consists of a cysteine-rich V-specific domain, which is conserved across almost all paramyxoviruses. There is evidence supporting a number of different functions of the V proteins of morbilliviruses in blocking the signalling pathways of type I and II IFNs, but it is not clear which domains of V are responsible for which activities and whether all these activities are required for effective blockade of IFN signalling. We have shown here that the two domains of rinderpest virus V protein have distinct functions: the N-terminal domain acted to bind STAT1, whilst the C-terminal V-specific domain interacted with the IFN receptor-associated kinases Jak1 and Tyk2. Effective blockade of IFN signalling required the intact V protein.

Morbillivirus v proteins exhibit multiple mechanisms to block type 1 and type 2 interferon signalling pathways.

Morbilliviruses form a closely related group of pathogenic viruses which encode three non-structural proteins V, W and C in their P gene. Previous studies with rinderpest virus (RPV) and measles virus (MeV) have demonstrated that these non-structural proteins play a crucial role in blocking type I (IFNα/ß) and type II (IFNγ) interferon action, and various mechanisms have been proposed for these effects. We have directly compared four important morbilliviruses, rinderpest (RPV), measles virus (MeV), peste des petits ruminants virus (PPRV) and canine distemper virus (CDV). These viruses and their V proteins could all block type I IFN action. However, the viruses and their V proteins had varying abilities to block type II IFN action. The ability to block type II IFN-induced gene transcription correlated with co-precipitation of STAT1 with the respective V protein, but there was no correlation between co-precipitation of either STAT1 or STAT2 and the abilities of the V proteins to block type I IFN-induced gene transcription or the creation of the antiviral state. Further study revealed that the V proteins of RPV, MeV, PPRV and CDV could all interfere with phosphorylation of the interferon-receptor-associated kinase Tyk2, and the V protein of highly virulent RPV could also block the phosphorylation of another such kinase, Jak1. Co-precipitation studies showed that morbillivirus V proteins all form a complex containing Tyk2 and Jak1. This study highlights the ability of morbillivirus V proteins to target multiple components of the IFN signalling pathways to control both type I and type II IFN action.