By recognizing foreign peptides (epitopes) in combination with Major Histocompatibility Complex class II molecules (MHCII), CD4+ T lymphocytes (TCD4+) play a critical role in the adaptive responses to most viruses, including the poxviruses, which continue to pose a considerable threat to humans worldwide. According to a convention developed with nominal protein antigens, MHCII-bound peptides are derived from internalized material that is catabolized in the endocytic network and loaded onto nascent MHCII in the late endosome, the complex subsequently transiting to the plasma membrane. Previous and ongoing work in the Eisenlohr lab has demonstrated that this classical scheme contributes minimally to the activation of influenza (flu)-specific TCD4+. Catalysis of whole virions is inefficient and, instead, viral proteins synthesized within the infected antigen- presenting cell (APC) are the primary source of peptides, generated via a network of endogenous processing pathways and directly presented to flu-specific TCD4+. We have recently extended the work to poxviruses in order to explore the generality of these findings. Investigation of vaccinia (VACV) and ectromelia (ECTV) reveals the landscape to be far more complex. As with flu, exogenously provided poxvirions are also resistant to processing, necessitating biosynthesis of viral proteins. However, in contrast to flu, TCD4+ activation appears to depend primarily upon MHCII cross-presentation, in which the biosynthesized antigen is transferred from an infected cell to an uninfected APC. This is attributable to profound inhibition of direct presentation by the infected cell, particularly in the case of ECTV, a natural mouse pathogen. While direct disruption of MHC class I-restricted antigen processing and presentation is well known, it has rarely been reported for MHCII, and the degree and specificity of inhibition by poxviruses is unprecedented. Established work by the Roper lab has identified the A35 gene product of both VACV and ECTV as a major mediator of the inhibition, and preliminary data from the Hersperger lab implicates the B22 gene product as a potent co-conspirator. This Co-PI RO1 application, which combines expertise in antigen processing and presentation with that in poxvirus biology and pathogenesis, will extend these preliminary findings as follows: 1) Rigorously test the hypothesis both in vitro (Aim 1) and in vivo (Aim 2) that MHCII cross-presentation is the principal driver of TCD4+ responses to VACV- and ECTV-specific TCD4+ responses, 2) Determine the contributions of A35 and B22 to the block in direct presentation and identify other poxviral products that might provide complementary activity (Aim 3). 3) Identify the mechanisms underlying A35 and B22 inhibitory activities (Aim 4). These studies bring together three areas of high impact - TCD4+ recognition of viral antigens, poxvirus virulence, and viral subversion of MHCII antigen processing and presentation. Achievement of the aims will substantially enhance understanding of poxvirus pathogenesis and rational vaccine design, and could lead to novel experimental and clinical immunomodulatory strategies.

Public Health Relevance

This project focuses upon the dynamic relationship between CD4+ T cells, critical for protection against most viruses, and two prototypical poxviruses, both of which have developed strategies to avoid CD4+ T cell recognition. The proposed work seeks to reveal those strategies and gain insight into how the immune system circumvents them. Execution of the research plan could point to novel anti-poxviral therapeutics enhanced rational vaccine design, and the development of novel strategies to treat autoimmune diseases.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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Immunity and Host Defense (IHD)
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Gondre-Lewis, Timothy A
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Children's Hospital of Philadelphia
United States
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Frey, Tiffany R; Forsyth, Katherine S; Sheehan, Maura M et al. (2018) Ectromelia virus lacking the E3L ortholog is replication-defective and nonpathogenic but does induce protective immunity in a mouse strain susceptible to lethal mousepox. Virology 518:335-348
Veerappan Ganesan, Asha Purnima; Eisenlohr, Laurence C (2017) The elucidation of non-classical MHC class II antigen processing through the study of viral antigens. Curr Opin Virol 22:71-76
Frey, Tiffany R; Lehmann, Michael H; Ryan, Colton M et al. (2017) Ectromelia virus accumulates less double-stranded RNA compared to vaccinia virus in BS-C-1 cells. Virology 509:98-111
Forsyth, Katherine S; Eisenlohr, Laurence C (2016) Giving CD4+ T cells the slip: viral interference with MHC class II-restricted antigen processing and presentation. Curr Opin Immunol 40:123-9