Cytomegalovirus pp65 limits dissemination but is dispensable for persistence
Daniel Malouli, Scott G. Hansen, Ernesto S. Nakayasu, Emily E. Marshall, Colette M. Hughes, Abigail B. Ventura, Roxanne M. Gilbride, Matthew S. Lewis, Guangwu Xu, Craig Kreklywich, Nathan Whizin, Miranda Fischer, Alfred W. Legasse, Kasinath Viswanathan, Don Siess, David G. Camp II, Michael K. Axthelm, Christoph Kahl, Victor R. DeFilippis, Richard D. Smith, Daniel N. Streblow, Louis J. Picker, Klaus Früh
Daniel Malouli, Scott G. Hansen, Ernesto S. Nakayasu, Emily E. Marshall, Colette M. Hughes, Abigail B. Ventura, Roxanne M. Gilbride, Matthew S. Lewis, Guangwu Xu, Craig Kreklywich, Nathan Whizin, Miranda Fischer, Alfred W. Legasse, Kasinath Viswanathan, Don Siess, David G. Camp II, Michael K. Axthelm, Christoph Kahl, Victor R. DeFilippis, Richard D. Smith, Daniel N. Streblow, Louis J. Picker, Klaus Früh
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Research Article Virology

Cytomegalovirus pp65 limits dissemination but is dispensable for persistence

Abstract

The most abundantly produced virion protein in human cytomegalovirus (HCMV) is the immunodominant phosphoprotein 65 (pp65), which is frequently included in CMV vaccines. Although it is nonessential for in vitro CMV growth, pp65 displays immunomodulatory functions that support a potential role in primary and/or persistent infection. To determine the contribution of pp65 to CMV infection and immunity, we generated a rhesus CMV lacking both pp65 orthologs (RhCMVΔpp65ab). While deletion of pp65ab slightly reduced growth in vitro and increased defective particle formation, the protein composition of secreted virions was largely unchanged. Interestingly, pp65 was not required for primary and persistent infection in animals. Immune responses induced by RhCMVΔpp65ab did not prevent reinfection with rhesus CMV; however, reinfection with RhCMVΔUS2-11, which lacks viral-encoded MHC-I antigen presentation inhibitors, was prevented. Unexpectedly, induction of pp65b-specific T cells alone did not protect against RhCMVΔUS2-11 challenge, suggesting that T cells targeting multiple CMV antigens are required for protection. However, pp65-specific immunity was crucial for controlling viral dissemination during primary infection, as indicated by the marked increase of RhCMVΔpp65ab genome copies in CMV-naive, but not CMV-immune, animals. Our data provide rationale for inclusion of pp65 into CMV vaccines but also demonstrate that pp65-induced T cell responses alone do not recapitulate the protective effect of natural infection.

Authors

Daniel Malouli, Scott G. Hansen, Ernesto S. Nakayasu, Emily E. Marshall, Colette M. Hughes, Abigail B. Ventura, Roxanne M. Gilbride, Matthew S. Lewis, Guangwu Xu, Craig Kreklywich, Nathan Whizin, Miranda Fischer, Alfred W. Legasse, Kasinath Viswanathan, Don Siess, David G. Camp II, Michael K. Axthelm, Christoph Kahl, Victor R. DeFilippis, Richard D. Smith, Daniel N. Streblow, Louis J. Picker, Klaus Früh

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Figure 5

T cells induced by heterologous prime-boost vaccination with pp65b do not protect against superinfection with ΔUS2-11.

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T cells induced by heterologous prime-boost vaccination with pp65b do no...
(A) Three CMV-negative RMs were vaccinated with 1 mg pND/pp65b and boosted with 5 × 108 PFUs MVApp65b at 6 and 12 weeks after the initial vaccination (black). As controls, 3 CMV-negative RMs were vaccinated with the parental pND plasmid not expressing any antigen and boosted with WT MVA at 6 and 12 weeks after the initial vaccination (green). At 18 weeks after the initial DNA vaccination, both groups of animals were challenged with 107 PFUs ΔUS2-11gag. The top row shows the specific T cell responses to pp65, whereas the bottom row shows specific T cell responses to SIVgag. T cells were isolated from peripheral blood (PBMCs). The corresponding T cell responses obtained from BAL fluid are shown in Supplemental Figure 3. The production of anti-RhCMV antibodies in pp65-vaccinated animals (Rm23557, Rm27814, Rm27838) was compared to that in control-vaccinated animals (Rm23672, Rm25052, Rm27821) prior to and upon challenge with RhCMV-ΔUS2-11. At the indicated time points, RhCMV-specific end point antibody (IgG, IgA, IgM) titers were measured in plasma from each animal by ELISA using lysates from fibroblasts infected with either (B) RhCMV-Δpp65 or (C) WT-RhCMV as the capture antigen. (D) Viral proteins recognized by the antibodies were detected by Western blotting. Lysates of cells infected with WT-RhCMV or RhCMV-Δpp65 were separated by SDS-PAGE and immunoblotted with antisera from the pp65-vaccinated animal (Rm27838) or a control-vaccinated animal (Rm23672). Asterisks denote the pp65 proteins. The results from these 2 animals are representative of the responses observed in the other animals of each group.