Lian Ni Lee

Protein arginine methyltransferase (PRMT) 5 is over-expressed in a variety of cancers and the master transcription regulator E2F1 is an important methylation target. We have explored the role of PRMT5 and E2F1 in regulating the non-coding genome and report here a striking effect on long non-coding (lnc) RNA gene expression. Moreover, many MHC class I protein-associated peptides were derived from small open reading frames in the lncRNA genes. Pharmacological inhibition of PRMT5 or adjusting E2F1 levels qualitatively altered the repertoire of lncRNA-derived peptide antigens displayed by tumour cells. When presented to the immune system as either ex vivo-loaded dendritic cells or expressed from a viral vector, lncRNA-derived peptides drove a potent antigen-specific CD8 T lymphocyte response, which translated into a significant delay in tumour growth. Thus, lncRNA genes encode immunogenic peptides that can be deployed as a cancer vaccine.

Protein acetylation plays a key role in regulating cellular processes and is subject to aberrant control in diverse pathologies. Although histone deacetylase (HDAC) inhibitors are approved drugs for certain cancers, it is not known whether they can be deployed in other therapeutic contexts. We have explored the clinical HDAC inhibitor, zabadinostat/CXD101, and found that it is a stand-alone regulator of the adaptive immune response. Zabadinostat treatment increased expression of MHC class I and II genes in a variety of cells, including dendritic cells (DCs) and healthy tissue. Remarkably, zabadinostat enhanced the activity of DCs, and CD4 and CD8 T lymphocytes. Using an antigenic peptide presented to the immune system by MHC class I, zabadinostat caused an increase in antigen-specific CD8 T lymphocytes. Further, mice immunised with covid19 spike protein and treated with zabadinostat exhibit enhanced covid19 neutralising antibodies and an increased level of T lymphocytes. The enhanced humoral response reflected increased activity of T follicular helper (Tfh) cells and germinal centre (GC) B cells. Our results argue strongly that zabadinostat has potential to augment diverse therapeutic agents that act through the immune system.

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.

It is unclear whether prior endemic coronavirus infections affect COVID-19 severity. Here, we show that in cases of fatal COVID-19, antibody responses to the SARS-COV-2 spike are directed against epitopes shared with endemic beta-coronaviruses in the S2 subunit of the SARS-CoV-2 spike protein. This immune response is associated with the compromised production of a de novo SARS-CoV-2 spike response among individuals with fatal COVID-19 outcomes. Funding: This work was supported by the Georg and Emily von Opel Foundation, the National Institute for Health Research (NIHR) [award CO-­‐CIN-­‐01], the Medical Research Council [grant MC_PC_19059], ME was supported by The Leona M. and Harry B. Helmsley Charitable Trust on May 31, 2021 for the project titled, "ICARUS –IBD: International study of COVID-­‐19 Antibody Response Under Sustained immune suppression in Inflammatory Bowel Disease. RP also supported by funds provided under Professor RW Snow’s Wellcome Trust Principal Fellowship (# 212176). Meso Scale Diagnostics (USA) provided loan of equipment, reagents and technical support. JSB was supported by funding from the Biotechnology and Biological Sciences Research Council (BBSRC) [grant number BB/M011224/1]. CPT was funded by an ERC research grant UNIFLUVAC’ and two MRC CiC grants (Ref: BR00140). ALM is funded by a NIHR Research Capability Funding grant. HJK is supported by The Future of Humanity Institute at the University of Oxford DPhil Scholarship program. DE is funded by the Robertson Foundation. The funders played no role in the design, execution or reporting of the study. Declaration of Interest: DE declares lecture fees from Gilead. CPT and SG hold funding from Blue Water Vaccines. All others have nothing to disclose. Ethical Approval: Ethical approval was obtained for the Scottish National Blood Transfusion Service (SNBTS) anonymous archive -­‐ IRAS project number 18005. SNBTS blood donors gave fully informed consent to virological testing, donation was made under the SNBTS Blood Establishment Authorisation and the study was approved by the SNBTS Research and Sample Governance Committee.

Background: The progression and geographical distribution of SARS coronavirus 2 (SARS-CoV-2) infection in the UK and elsewhere is unknown because typically only symptomatic individuals are diagnosed. We performed a serological study of blood donors in Scotland between the 17 ^th of March and the 18 ^th of May to detect neutralising antibodies to SARS-CoV-2 as a marker of past infection and epidemic progression. Aim: To determine if sera from blood bank donors can be used to track the emergence and progression of the SARS-CoV-2 epidemic. Methods: A pseudotyped SARS-CoV-2 virus microneutralisation assay was used to detect neutralising antibodies to SARS-CoV-2. The study group comprised samples from 3,500 blood donors collected in Scotland between the 17 ^th of March and 19 ^th of May, 2020. Controls were collected from 100 donors in Scotland during 2019. Results: All samples collected on the 17 ^th March, 2020 (n=500) were negative in the pseudotyped SARS-CoV-2 virus microneutralisation assay. Neutralising antibodies were detected in 6/500 donors from the 23 ^th -26 ^th of March. The number of samples containing neutralising antibodies did not significantly rise after the 5 ^th -6 ^th April until the end of the study on the 18 ^th of May. We find that infections are concentrated in certain postcodes indicating that outbreaks of infection are extremely localised. In contrast, other areas remain comparatively untouched by the epidemic. Conclusion: These data indicate that sero-surveys of blood banks can serve as a useful tool for tracking the emergence and progression of an epidemic like the current SARS-CoV-2 outbreak.

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.

Background: Persistent viruses such as murine cytomegalovirus (MCMV) and adenovirus-based vaccines induce strong, sustained CD8+ T-cell responses, described as memory “inflation”. These retain functionality, home to peripheral organs and are associated with a distinct transcriptional program. Methods: To further define the nature of the transcriptional mechanisms underpinning memory inflation at different sites we used single-cell RNA sequencing of tetramer-sorted cells from MCMV-infected mice, analyzing transcriptional networks in virus-specific populations in the spleen and gut intra-epithelial lymphocytes (IEL). Results: We provide a transcriptional map of T-cell memory and define a module of gene expression, which distinguishes memory inflation in spleen from resident memory T-cells (TRM) in the gut. Conclusions: These data indicate that CD8+ T-cell memory in the gut epithelium induced by persistent viruses and vaccines has a distinct quality from both conventional memory and “inflationary” memory which may be relevant to protection against mucosal infections.

For many years, the focus of prophylactic vaccines was to elicit neutralizing antibodies, but it has become increasingly evident that T cell-mediated immunity plays a central role in controlling persistent viral infections such as with human immunodeficiency virus, cytomegalovirus, and hepatitis C virus. Currently, various promising prophylactic vaccines, capable of inducing substantial vaccine-specific T cell responses, are investigated in preclinical and clinical studies. There is compelling evidence that protection by T cells is related to the magnitude and breadth of the T cell response, the type and homing properties of the memory T cell subsets, and their cytokine polyfunctionality and metabolic fitness. In this review, we evaluated these key factors that determine the qualitative and quantitative properties of CD4⁺ and CD8⁺ T cell responses in the context of chronic viral disease and prophylactic vaccine development. Elucidation of the mechanisms underlying T cell-mediated protection against chronic viral pathogens will facilitate the development of more potent, durable and safe prophylactic T cell-based vaccines.

The efficacies of many new T cell vaccines rely on generating large populations of long-lived pathogen-specific effector memory CD8 T cells. However, it is now increasingly recognized that prior infection history impacts on the host immune response. Additionally, the order in which these infections are acquired could have a major effect. Exploiting the ability to generate large sustained effector memory (i.e. inflationary) T cell populations from murine cytomegalovirus (MCMV) and human Adenovirus-subtype (AdHu5) 5-beta-galactosidase (Ad-lacZ) vector, the impact of new infections on pre-existing memory and the capacity of the host’s memory compartment to accommodate multiple inflationary populations from unrelated pathogens was investigated in a murine model. Simultaneous and sequential infections, first with MCMV followed by Ad-lacZ, generated inflationary populations towards both viruses with similar kinetics and magnitude to mono-infected groups. However, in Ad-lacZ immune mice, subsequent acute MCMV infection led to a rapid decline of the pre-existing Ad-LacZ-specific inflating population, associated with bystander activation of Fas-dependent apoptotic pathways. However, responses were maintained long-term and boosting with Ad-lacZ led to rapid re-expansion of the inflating population. These data indicate firstly that multiple specificities of inflating memory cells can be acquired at different times and stably co-exist. Some acute infections may also deplete pre-existing memory populations, thus revealing the importance of the order of infection acquisition. Importantly, immunization with an AdHu5 vector did not alter the size of the pre-existing memory. These phenomena are relevant to the development of adenoviral vectors as novel vaccination strategies for diverse infections and cancers. (241 words) Many natural pathogens elicit large antigen-specific long-lived effector memory CD8 T cell responses that contribute to pathogen protection and control. This strategy is also employed by novel CD8 T cell vaccine vectors. In the real world, the host is likely to encounter a number of such pathogens at different times but the effect of pre-existing effector memory pools on the development of new CD8 T cell responses against unrelated infections, and vice versa, is still unclear. We address this through mouse models of sequential infection with MCMV and an Ad-Hu5 vaccine vector. We find that the host is able to accommodate and accumulate multiple new specificities of inflating effector memory populations at different times but certain infections, such as acute MCMV, appear to rapidly deplete existing memory populations via a Fas-dependent pathway. Additionally, immunization with vectors based on human adenovirus subtype 5 do not appear to detrimentally affect the host’s pre-existing effector memory pool. (154 words)