Truncation of gene F5L partially masks rescue of vaccinia virus strain MVA growth on mammalian cells by restricting plaque size

Modified vaccinia virus Ankara (MVA) is a candidate vaccine vector that is severely attenuated due to mutations acquired during several hundred rounds of serial passage in vitro. A previous study used marker rescue to produce a set of MVA recombinants with improved replication on mammalian cells. Here, we extended the characterization of these rescued MVA strains and identified vaccinia virus (VACV) gene F5L as a determinant of plaque morphology but not replication in vitro. F5 joins a growing group of VACV proteins that influence plaque formation more strongly than virus replication and which are disrupted in MVA. These defective genes in MVA confound the interpretation of marker rescue experiments designed to map mutations responsible for the attenuation of this important VACV strain.This work was funded by grants to DCT: NHMRC APP1023141 and ARC FT110100310

Strikingly poor CD8+ T-cell immunogenicity of vaccinia virus strain MVA in BALB/c mice

Vaccinia virus (VACV) strain MVA is a highly attenuated vector for vaccines that is being explored in clinical trials. We compared the CD8(+) T-cell immunogenicity of MVA with that of a virulent laboratory strain of VACV (strain WR) in BALB/c mice by examining epitope-specific responses as well as estimating the total number of activated CD8(+) T cells, irrespective of specificity. We found that MVA elicited total CD8(+) T-cell responses that were reduced by at least 20-fold compared with strain WR in BALB/c mice. In C57Bl/6 mice, we also found a substantial difference in immunogenicity between these VACV strains, but it was more modest at around fivefold. Of note, the size of responses to the virulent WR virus was similar in both strains of mice suggesting that BALB/c mice can mount robust CD8(+) T-cell responses to VACV. Although the data for total responses clearly showed that MVA overall is poorly immunogenic in BALB/c mice, we found one epitope for which strong responses were made irrespective of virus strain. Therefore, in the context of a vaccine, some recombinant epitopes may have similar immunogenicity when expressed from MVA and other strains of VACV, but we would expect these to be exceptions. These data show clearly the substantial difference in immunogenicity between MVA and virulent VACV strains and suggest that the impact of host genetics on responses to attenuated vaccine vectors like MVA requires more consideration.This work was funded by grants from the NIH (R01AI067401), NHMRC (APP1023141) and an ARC Future Fellowship (FT110100310) to DCT

Engineering herpes simplex viruses by infection–transfection methods including recombination site targeting by CRISPR/Cas9 nucleases

Herpes simplex viruses (HSVs) are frequent human pathogens and the ability to engineer these viruses underpins much research into their biology and pathogenesis. Often the ultimate aim is to produce a virus that has the desired phenotypic change and no additional alterations in characteristics. This requires methods that minimally disrupt the genome and, for insertions of foreign DNA, sites must be found that can be engineered without disrupting HSV gene function or expression. This study advances both of these requirements. Firstly, the use of homologous recombination between the virus genome and plasmids in mammalian cells is a reliable way to engineer HSV such that minimal genome changes are made. This has most frequently been achieved by cotransfection of plasmid and isolated viral genomic DNA, but an alternative is to supply the virus genome by infection in a transfection-infection method. Such approaches can also incorporate CRISPR/Cas9 genome engineering methods. Current descriptions of infection-transfection methods, either with or without the addition of CRISPR/Cas9 targeting, are limited in detail and the extent of optimization. In this study it was found that transfection efficiency and the length of homologous sequences improve the efficiency of recombination in these methods, but the targeting of the locus to be engineered by CRISPR/Cas9 nucleases has an overriding positive impact. Secondly, the intergenic space between UL26 and UL27 was reexamined as a site for the addition of foreign DNA and a position identified that allows insertions without compromising HSV growth in vitro or in vivo.This work was funded by NHMRC Project Grant APP1005846 and ARC Future Fellowship FT110100310

An intact signal peptide on dengue virus E protein enhances immunogenicity for CD8+ T cells and antibody when expressed from modified vaccinia Ankara

Dengue is a global public health concern and this is aggravated by a lack of vaccines or antiviral therapies. Despite the well-known role of CD8(+) T cells in the immunopathogenesis of Dengue virus (DENV), only recent studies have highlighted the importance of this arm of the immune response in protection against the disease. Thus, the majority of DENV vaccine candidates are designed to achieve protective titers of neutralizing antibodies, with less regard for cellular responses. Here, we used a mouse model to investigate CD8(+) T cell and humoral responses to a set of potential DENV vaccines based on recombinant modified vaccinia virus Ankara (rMVA). To enable this study, we identified two CD8(+) T cell epitopes in the DENV-3 E protein in C57BL/6 mice. Using these we found that all the rMVA vaccines elicited DENV-specific CD8(+) T cells that were cytotoxic in vivo and polyfunctional in vitro. Moreover, vaccines expressing the E protein with an intact signal peptide sequence elicited more DENV-specific CD8(+) T cells than those expressing E proteins in the cytoplasm. Significantly, it was these same ER-targeted E protein vaccines that elicited antibody responses. Our results support the further development of rMVA vaccines expressing DENV E proteins and add to the tools available for dengue vaccine development.Parts of this work were supported by the InstitutoNacional de Ciência e Tecnologia de Vacinas–INCTV (National Insti-tute of Science and Technology of Vaccines) and by a FAPEMIGPPM grant (CBB, PPM-00461-11). BRQ was a CAPES/PDSE fellow-ship recipient (8815-11-9). FGF is a CNPq fellowship recipient. DCTis an ARC Future Fellow (FT110100310)

Reduced Interleukin-4 Receptor α Expression on CD8+ T Cells Correlates with Higher Quality Anti-Viral Immunity

With the hope of understanding how interleukin (IL)-4 and IL-13 modulated quality of anti-viral CD8(+) T cells, we evaluated the expression of receptors for these cytokines following a range of viral infections (e.g. pox viruses and influenza virus). Results clearly indicated that unlike other IL-4/IL-13 receptor subunits, IL-4 receptor α (IL-4Rα) was significantly down-regulated on anti-viral CD8(+) T cells in a cognate antigen dependent manner. The infection of gene knockout mice and wild-type (WT) mice with vaccinia virus (VV) or VV expressing IL-4 confirmed that IL-4, IL-13 and signal transducer and activator of transcription 6 (STAT6) were required to increase IL-4Rα expression on CD8(+) T cells, but not interferon (IFN)-γ. STAT6 dependent elevation of IL-4Rα expression on CD8(+) T cells was a feature of poor quality anti-viral CD8(+) T cell immunity as measured by the production of IFN-γ and tumor necrosis factor α (TNF-α) in response to VV antigen stimulation in vitro. We propose that down-regulation of IL-4Rα, but not the other IL-4/IL-13 receptor subunits, is a mechanism by which CD8(+) T cells reduce responsiveness to IL-4 and IL-13. This can improve the quality of anti-viral CD8(+) T cell immunity. Our findings have important implications in understanding anti-viral CD8(+) T cell immunity and designing effective vaccines against chronic viral infections.This work was supported by the Australian National Health and Medical Research Council project grant award 525431 (CR) and development grant award APP1000703 (CR) and the Australian Centre for Hepatitis and HIV Virology EOI grant 2010 (CR). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Immunodomination during peripheral vaccinia virus infection

Immunodominance is a fundamental property of CD8+ T cell responses to viruses and vaccines. It had been observed that route of administration alters immunodominance after vaccinia virus (VACV) infection, but only a few epitopes were examined and no mechanism was provided. We re-visited this issue, examining a panel of 15 VACV epitopes and four routes, namely intradermal (i.d.), subcutaneous (s.c.), intraperitoneal (i.p.) and intravenous (i.v.) injection. We found that immunodominance is sharpened following peripheral routes of infection (i.d. and s.c.) compared with those that allow systemic virus dissemination (i.p. and i.v.). This increased immunodominance was demonstrated with native epitopes of VACV and with herpes simplex virus glycoprotein B when expressed from VACV. Responses to some subdominant epitopes were altered by as much as fourfold. Tracking of virus, examination of priming sites, and experiments restricting virus spread showed that priming of CD8+ T cells in the spleen was necessary, but not sufficient to broaden responses. Further, we directly demonstrated that immunodomination occurs more readily when priming is mainly in lymph nodes. Finally, we were able to reduce immunodominance after i.d., but not i.p. infection, using a VACV expressing the costimulators CD8+ (B7-1) and CD8+ (B7-2), which is notable because VACV-based vaccines incorporating these molecules are in clinical trials. Taken together, our data indicate that resources for CD8+ T cell priming are limiting in local draining lymph nodes, leading to greater immunodomination. Further, we provide evidence that costimulation can be a limiting factor that contributes to immunodomination. These results shed light on a possible mechanism of immunodomination and highlight the need to consider multiple epitopes across the spectrum of immunogenicities in studies aimed at understanding CD8+ T cell immunity to viruses.NHMRC (National Health and Medical Research Council of Australia

IL-4 and IL-13 mediated down-regulation of CD8 expression levels can dampen anti-viral CD8⁺ T cell avidity following HIV-1 recombinant pox viral vaccination

We have shown that mucosal HIV-1 recombinant pox viral vaccination can induce high, avidity HIV-specific CD8(+) T cells with reduced interleukin (IL)-4 and IL-13 expression compared to, systemic vaccine delivery. In the current study how these cytokines act to regulate anti-viral CD8(+) T, cell avidity following HIV-1 recombinant pox viral prime-boost vaccination was investigated. Out of a panel of T cell avidity markers tested, only CD8 expression levels were found to be enhanced on, KdGag197-205 (HIV)-specific CD8(+) T cells obtained from IL-13(-/-), IL-4(-/-) and signal transducer and, activator of transcription of 6 (STAT6)(-/-) mice compared to wild-type (WT) controls following, vaccination. Elevated CD8 expression levels in this instance also correlated with polyfunctionality, (interferon (IFN)-γ, tumour necorsis factor (TNF)-α and IL-2 production) and the avidity of HIVspecific CD8(+) T cells. Furthermore, mucosal vaccination and vaccination with the novel adjuvanted IL-13 inhibitor (i.e. IL-13Rα2) vaccines significantly enhanced CD8 expression levels on HIV-specific CD8(+), T cells, which correlated with avidity. Using anti-CD8 antibodies that blocked CD8 availability on CD8(+), T cells, it was established that CD8 played an important role in increasing HIV-specific CD8(+) T cell avidity and polyfunctionality in IL-4(-/-), IL-13(-/-) and STAT6(-/-) mice compared to WT controls, following vaccination. Collectively, our data demonstrate that IL-4 and IL-13 dampen CD8 expression levels on anti-viral CD8(+) T cells, which can down-regulate anti-viral CD8(+) T cell avidity and, polyfunctionality following HIV-1 recombinant pox viral vaccination. These findings can be exploited to, design more efficacious vaccines not only against HIV-1, but many chronic infections where high, avidity CD8(+) T cells help protection.This work was supported by the Australian National Health and Medical Research Council project grant award 525431 (CR) and development grant award APP1000703 (CR), the Australian Centre for Hepatitis and HIV Virology EOI grant 2010/12 (CR&RJ), and Bill and Melinda Gates Foundation GCE Phase I grant OPP1015149 (CR)

Herpes Simplex Virus Latency Is Noisier the Closer We Look

During herpes simplex virus (HSV) latency, the viral genome is harbored in peripheral neurons in the absence of infectious virus but with the potential to restart infection. Advances in epigenetics have helped explain how viral gene expression is largely inhibited during latency. Paradoxically, at the same time, the view that latency is entirely silent has been eroding. This low-level noise has implications for our understanding of HSV latency and should not be ignored

Evolutionary History and Attenuation of Myxoma Virus on Two Continents

The attenuation of myxoma virus (MYXV) following its introduction as a biological control into the European rabbit populations of Australia and Europe is the canonical study of the evolution of virulence. However, the evolutionary genetics of this profound change in host-pathogen relationship is unknown. We describe the genome-scale evolution of MYXV covering a range of virulence grades sampled over 49 years from the parallel Australian and European epidemics, including the high-virulence progenitor strains released in the early 1950s. MYXV evolved rapidly over the sampling period, exhibiting one of the highest nucleotide substitution rates ever reported for a double-stranded DNA virus, and indicative of a relatively high mutation rate and/or a continually changing selective environment. Our comparative sequence data reveal that changes in virulence involved multiple genes, likely losses of gene function due to insertion-deletion events, and no mutations common to specific virulence grades. Hence, despite the similarity in selection pressures there are multiple genetic routes to attain either highly virulent or attenuated phenotypes in MYXV, resulting in convergence for phenotype but not genotype. © 2012 Kerr et al