Investigation of CD8+ T cell priming by two commonly used Vaccinia virus strains

CD8+ T cells help to control virus infections and tumours. Two main pathways, namely direct and cross presentation, are involved in generating and presenting antigenic peptides to induce CD8+ T cell responses. In general, antigens that require minimal processing or are rapidly degraded allow better direct presentation while only stable antigens can be cross presented. Currently, several vaccinia virus (VACV) recombinants are being tested in clinical trials as vaccines to induce protective immunity, including CD8+ T cell responses, against heterologous pathogens and cancers. The current paradigm, based on work with the virulent VACV strain Western Reserve (WR), suggests an important role of direct presentation for CD8+ T cell priming. However, a study has reported that cross presentation is the major pathway for priming CD8+ T cells against the highly attenuated modified VACV Ankara (MVA), a candidate vaccine vector. Understanding this disparity between WR and MVA may improve the design of VACV-based recombinant vaccines. The priming pathway of native antigens expressed from WR and MVA was first examined in mice pre-treated with Toll-like receptor 9 ligand or cytochrome c. These methods have been reported to selectively inhibit cross presentation. However, results in this thesis demonstrate that these treatments also suppress direct presentation. Further investigations show that the extent of inhibition on CD8+ T cell priming by CpG is dependent on the immunisation route, the viral replicative ability and, more crucially, the immunisation dose. Similarly, the replicative ability of VACV influences the inhibition effect of cytochrome c treatment. Overall, these two strategies do not reveal how VACV antigens are presented. The immunogenicity of recombinant antigens expressed as minigenes from WR and MVA was then examined. The magnitude of CD8+ T cell responses induced by the minigenes, which can only be directly presented, are at least similar to, if not higher than, the full-length antigens when expressed from WR and MVA. Contrary to the published work, the model antigen ovalbumin expressed as a minigene can be more immunogenic than the full-length antigen when expressed from MVA. This suggests that MVA behaves similarly to WR to prime CD8+ T cells, i.e. by direct presentation. We further demonstrate that the location where the transgene encoding the foreign antigen is inserted in the MVA genome determines its immunogenicity. Two unexpected phenomena observed in this thesis were also explored. Firstly, a CD8+ T cell response to an immunogenic peptide of influenza A virus, known as NP-366, can only be induced if the peptide is processed from a full-length protein from WR, but not as a minigene. This contradicts our studies described above and most of the VACV literature. Several hypotheses were tested to identify the possible mechanisms responsible. Secondly, we found that completely inactivated VACV induces CD8+ T cell responses in vivo. Its application as a recombinant vaccine was examined. In summary, this thesis has expanded our understanding on how antigens expressed from VACV strains WR and MVA are presented for CD8+ T cell priming. The findings presented here provide important insights for VACV-based recombinant vaccine design

Surprisingly Effective Priming of CD8+ T Cells by Heat-Inactivated Vaccinia Virus Virions

Robust priming of CD8+ T cells by viruses is considered to require infection and de novo expression of viral antigens. A corollary of this is that inactivated viruses are thought of as being inevitably poor vaccines for eliciting these responses. In contrast to this dogma, we found that some antigens present in vaccinia virus (VACV) virions prime strong CD8+ T cell responses when the virus was rendered noninfectious by heat. More surprisingly, in some cases these responses were similar in magnitude to those primed by infectious virus administered at an equivalent dose. Next, we tested whether this was a special property of particular antigens and their epitopes and found that foreign epitopes tagged onto three different VACV virion proteins were able to elicit CD8+ T cell responses irrespective of whether the virus was viable or heat killed. Further, the polyfunctionality and cytotoxic ability of the CD8+ T cells primed by these VACVs was equivalent irrespective of whether they were administered to mice as inactivated or live viruses. Finally, we used these VACVs in prime-boost combinations of inactivated and live virus and found that priming with dead virus before a live booster was the most immunogenic regime. We conclude that VACV virions can be efficient vectors for targeting antigens to dendritic cells for effective priming of CD8+ T cells, even when rendered noninfectious and speculate that this might also be the case for other viruses.D.C.T. was funded by an NHMRC fellowship (APP1104329) and project grants (APP1084283 and APP1023141) and ANU Connect Ventures project DTF085

Direct priming of CD8+ T cells persists in the face of cowpox virus inhibitors of antigen presentation

Vaccinia virus (VACV) was the vaccine used to eradicate smallpox and is being repurposed as a vaccine vector. CD8+ T cells are key antiviral mediators but require priming to become effector or memory cells. Priming requires an interaction with dendritic cells that are either infected (direct priming) or have acquired virus proteins but remain uninfected (cross priming). To investigate CD8+ T cell priming pathways for VACV, we engineered the virus to express CPXV12 and CPXV203, two inhibitors of antigen presentation encoded by cowpox virus. These intracellular proteins would be expected to block direct but not cross priming. The inhibitors had diverse impacts on the size of anti-VACV CD8+ T cell responses across epitopes and by different infection routes in mice, superficially suggesting variable use of direct and cross priming. However, when we then tested a form of antigen that requires direct priming, we found, surprisingly, that CD8+ T cell responses were not diminished by coexpression with CPXV12 and CPXV203. We then directly quantified the impact of CPXV12 and CPXV203 on viral antigen presentation using mass spectrometry, which revealed strong but incomplete inhibition of antigen presentation by the CPXV proteins. Therefore, direct priming of CD8+ T cells by poxviruses is robust enough to withstand highly potent viral inhibitors of antigen presentation. This is a reminder of the limits of viral immune evasion and shows that viral inhibitors of antigen presentation cannot be assumed to dissect cleanly direct and cross priming of antiviral CD8+ T cells.This work was funded by NHMRC fellowships to D.C.T. (APP1104329) and A.W.P. (APP1137739) and NHMRC project grants to D.C.T., A.W.P., and N.P.C. (389819, APP1023141, and APP1084283

Kinetics of antigen expression and epitope presentation during virus infection

Current knowledge about the dynamics of antigen presentation to T cells during viral infection is very poor despite being of fundamental importance to our understanding of anti-viral immunity. Here we use an advanced mass spectrometry method to simultaneously quantify the presentation of eight vaccinia virus peptide-MHC complexes (epitopes) on infected cells and the amounts of their source antigens at multiple times after infection. The results show a startling 1000-fold range in abundance as well as strikingly different kinetics across the epitopes monitored. The tight correlation between onset of protein expression and epitope display for most antigens provides the strongest support to date that antigen presentation is largely linked to translation and not later degradation of antigens. Finally, we show a complete disconnect between the epitope abundance and immunodominance hierarchy of these eight epitopes. This study highlights the complexity of viral antigen presentation by the host and demonstrates the weakness of simple models that assume total protein levels are directly linked to epitope presentation and immunogenicity.NHMRC (National Health and Medical Research Council of Australia

Linear Fidelity in Quantification of Anti-Viral CD8+ T Cells

Enumeration of anti-viral CD8+ T cells to make comparisons between mice, viruses and vaccines is a frequently used approach, but controversy persists as to the most appropriate methods. Use of peptide-MHC tetramers (or variants) and intracellular staining for cytokines, in particular IFNγ, after a short ex vivo stimulation are now common, as are a variety of cytotoxicity assays, but few direct comparisons have been made. It has been argued that use of tetramers leads to the counting of non-functional T cells and that measurement of single cytokines will fail to identify cells with alternative functions. Further, the linear range of these methods has not been tested and this is required to give confidence that relative quantifications can be compared across samples. Here we show for two acute virus infections and CD8+ T cells activated in vitro that DimerX (a tetramer variant) and intracellular staining for IFNγ, alone or in combination with CD107 to detect degranulation, gave comparable results at the peak of the response. Importantly, these methods were highly linear over nearly two orders of magnitude. In contrast, in vitro and in vivo assays for cytotoxicity were not linear, suffering from high background killing, plateaus in maximal killing and substantial underestimation of differences in magnitude of responses

Association analyses of East Asian individuals and trans-ancestry analyses with European individuals reveal new loci associated with cholesterol and triglyceride levels

Large-scale meta-analyses of genome-wide association studies (GWAS) have identified >175 loci associated with fasting cholesterol levels, including total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), and triglycerides (TG). With differences in linkage disequilibrium (LD) structure and allele frequencies between ancestry groups, studies in additional large samples may detect new associations. We conducted staged GWAS meta-analyses in up to 69,414 East Asian individuals from 24 studies with participants from Japan, the Philippines, Korea, China, Singapore, and Taiwan. These meta-analyses identified (P < 5 × 10-8) three novel loci associated with HDL-C near CD163-APOBEC1 (P = 7.4 × 10-9), NCOA2 (P = 1.6 × 10-8), and NID2-PTGDR (P = 4.2 × 10-8), and one novel locus associated with TG near WDR11-FGFR2 (P = 2.7 × 10-10). Conditional analyses identified a second signal near CD163-APOBEC1. We then combined results from the East Asian meta-analysis with association results from up to 187,365 European individuals from the Global Lipids Genetics Consortium in a trans-ancestry meta-analysis. This analysis identified (log10Bayes Factor ≥6.1) eight additional novel lipid loci. Among the twelve total loci identified, the index variants at eight loci have demonstrated at least nominal significance with other metabolic traits in prior studies, and two loci exhibited coincident eQTLs (P < 1 × 10-5) in subcutaneous adipose tissue for BPTF and PDGFC. Taken together, these analyses identified multiple novel lipid loci, providing new potential therapeutic targets

Molecular control of HIV-1 postintegration latency: implications for the development of new therapeutic strategies

The persistence of HIV-1 latent reservoirs represents a major barrier to virus eradication in infected patients under HAART since interruption of the treatment inevitably leads to a rebound of plasma viremia. Latency establishes early after infection notably (but not only) in resting memory CD4+ T cells and involves numerous host and viral trans-acting proteins, as well as processes such as transcriptional interference, RNA silencing, epigenetic modifications and chromatin organization. In order to eliminate latent reservoirs, new strategies are envisaged and consist of reactivating HIV-1 transcription in latently-infected cells, while maintaining HAART in order to prevent de novo infection. The difficulty lies in the fact that a single residual latently-infected cell can in theory rekindle the infection. Here, we review our current understanding of the molecular mechanisms involved in the establishment and maintenance of HIV-1 latency and in the transcriptional reactivation from latency. We highlight the potential of new therapeutic strategies based on this understanding of latency. Combinations of various compounds used simultaneously allow for the targeting of transcriptional repression at multiple levels and can facilitate the escape from latency and the clearance of viral reservoirs. We describe the current advantages and limitations of immune T-cell activators, inducers of the NF-κB signaling pathway, and inhibitors of deacetylases and histone- and DNA- methyltransferases, used alone or in combinations. While a solution will not be achieved by tomorrow, the battle against HIV-1 latent reservoirs is well- underway

Analyzing CD8 T cells in mouse models of poxvirus infection

Mouse models of immunology are frequently used to study host responses to poxviruses or poxvirus-based recombinant vaccines. In this context, the magnitude of CD8+ T cell responses is often of interest. Methods to evaluate CD8+ T cell responses extend fr

Systemic toll-like receptor ligation and selective killing of dendritic cell subsets fail to dissect priming pathways for anti-vaccinia virus CD8+ T cells

CD8 T cell responses can be generated by direct or cross-priming mechanisms, and several mouse models have been used to reveal which of these is the most important pathway for various viruses. Among these models is systemic treatment of mice with a CpG-containing oligodeoxynucleotide (CpG) to mature all dendritic cells (DCs), rendering them incapable of cross-presentation. A second is the use of cytochrome c (cytc) as a selective poison of the subsets of DCs able to cross-present antigen. In this study, using two vaccinia virus (VACV) strains, namely, WR and MVA, we found that the CpG and cytc methods gave conflicting data. Moreover, we show for both strains of VACV that treatment of mice with CpG and cytc inhibited CD8 T cell responses to antigens designed to prime exclusively by direct presentation. Further investigation of the CpG method found that the extent to which priming is inhibited depends on the antigen examined, immunization route, replication ability of the virus, and, crucially, immunization dose. We suggest that greater caution is required when interpreting data using these methods and that priming pathways for antiviral CD8 T cells are not simply separated according to DC subsets or their maturation state