The Poxvirus A35 Protein Promotes Virulence by Regulating the Host Adaptive Immune Response

ECU Author/Contributor (non-ECU co-authors, if there are any, appear on document)
Kristina Elizabeth Rehm (Creator)
East Carolina University (ECU )
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Abstract: Poxviruses are large double-stranded DNA viruses with a broad host range and worldwide distribution. The most infamous poxvirus is Variola virus, the causative agent of smallpox, which caused over 500 million deaths in the twentieth century before it was declared eradicated in 1980 by the World Health Organization. Nevertheless, more than 25 years after its eradication from nature, smallpox remains a threat, as there is now concern for its use in an act of bioterrorism. Other poxviruses have emerged in recent years. In 2003, there was a monkeypox outbreak in the United States, with nearly 80 cases. This disease is similar to smallpox, but can be spread not only from human to human, but also from animal to human, which emphasizes the fact that many poxviruses are a zoonotic threat. The current poxvirus vaccine (Vaccinia virus, VACV), while highly effective, is unsafe and its use is contraindicated for an estimated 25% of the population due to its virulence. The virulence of VACV is attributed to the production of numerous proteins that function to evade the host immune response, including inhibitors of interferons, regulators of apoptosis, and cytokine receptor homologs. Despite the large amount of work that has been done to characterize poxvirus virulence mechanisms, there are more than 25 highly conserved genes that remain uncharacterized. The poxvirus A35R gene is highly conserved in all sequenced mammalian tropic poxviruses and has little similarity to any non-pox protein, suggesting an important and novel function. The A35 protein was not required for viral replication in eight cell lines from various hosts, but removal of A35 from the viral genome resulted in a 1000-fold decrease in virulence in the mouse model. We therefore hypothesized that the poxvirus A35 gene product promotes virulence by affecting viral replication in certain cell types in vivo or by regulating aspects of the host adaptive immune response. We show that A35 is not required for viral replication in various cell types from six different mammalian hosts and also in mouse tissues early after infection. Using a model major histocompatibility complex (MHC) class II-restricted antigen presentation system, we have shown that VACV and A35 inhibit the amount of numerous cytokines that are produced from both the T cell and the antigen presenting cell (APC) as a result of specific antigen presentation, including IL-2 and nitric oxide. Further data implicated the APC as the viral target, as the T cells used in the assays were unaffected by VACV and A35. The A35 protein did not induce apoptosis of the APC or affect antigen-independent NO production. Furthermore, A35 localized to the endosomal compartments of the APC and decreased MHC class II expression and the amount of peptide presented in the cleft of MHC class II molecules. To determine if A35 would decrease the host adaptive immune response, mice were vaccinated with 500 plaque forming units (pfu) of either VACV (Western Reserve strain, WR) or A35 deletion mutant virus. Our data indicate that A35 decreased VACV- specific immune response including interferon- (IFN)-gamma production, cytotoxic T lymphocyte (CTL) killing, serum antibody, and neutralizing antibody. The decreased immune response was shown to be important as it resulted in increased viral replication in target organs. The A35 deletion mutant virus was also an effective vaccine, as it protected mice as well as WR from a lethal challenge. We also made a recombinant A35 deletion mutant virus in the Modified Vaccinia Ankara (MVA) replication-deficient vaccine strain, which has been shown to be safe when used in immunocompromised individuals. Results with MVA were similar to studies performed with WR: A35 was not required for virus replication and decreased VACV-specific immune responses. Furthermore, the MVA35 deletion mutant vaccine protected mice as well as MVA from a lethal respiratory challenge. Our data expands the knowledge of what is known regarding poxvirus virulence mechanisms and is applicable to the development of safer and highly immunogenic vaccines. Since poxviruses are now being used widely as a platform for a number of vaccines for various diseases, including human immunodeficiency virus (HIV) and cancer, our work with A35 is broadly applicable to vaccine development.  

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Language: English
Date: 2010
Biology, Virology

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