Host Immune Responses to a Viral Immune Modulating Protein: Immunogenicity of Viral Interleukin-10 in Rhesus Cytomegalovirus-Infected Rhesus Macaques

, consistent with a central role for rhcmvIL-10 during acute virus-host interactions. Since cmvIL-10 and rhcmvIL-10 are extremely divergent from the cIL-10 of their respective hosts, vaccine-mediated neutralization of their function could inhibit establishment of viral persistence without inhibition of cIL-10.As a prelude to evaluating cmvIL-10-based vaccines in humans, the rhesus macaque model of HCMV was used to interrogate peripheral and mucosal immune responses to rhcmvIL-10 in RhCMV-infected animals. ELISA were used to detect rhcmvIL-10-binding antibodies in plasma and saliva, and an IL-12-based bioassay was used to quantify plasma antibodies that neutralized rhcmvIL-10 function. rhcmvIL-10 is highly immunogenic during RhCMV infection, stimulating high avidity rhcmvIL-10-binding antibodies in the plasma of all infected animals. Most infected animals also exhibited plasma antibodies that partially neutralized rhcmvIL-10 function but did not cross-neutralize the function of rhesus cIL-10. Notably, minimally detectable rhcmvIL-10-binding antibodies were detected in saliva.This study demonstrates that rhcmvIL-10, as a surrogate for cmvIL-10, is a viable vaccine candidate because (1) it is highly immunogenic during natural RhCMV infection, and (2) neutralizing antibodies to rhcmvIL-10 do not cross-react with rhesus cIL-10. Exceedingly low rhcmvIL-10 antibodies in saliva further suggest that the oral mucosa, which is critical in RhCMV natural history, is associated with suboptimal anti-rhcmvIL-10 antibody responses

Comparison of the pathogenesis of the highly passaged MCMV Smith strain with that of the low passaged MCMV HaNa1 isolate in BALB/c mice upon oronasal inoculation

Murine cytomegalovirus (MCMV) Smith strain is widely used in mouse models to study HCMV infections. Due to high serial passages, MCMV Smith has acquired genetic and biological changes. Therefore, a low passaged strain would be more relevant to develop mouse models. Here, the pathogenesis of an infection with MCMV Smith was compared with that of an infection with a low passaged Belgian MCMV isolate HaNa1 in BALB/c adult mice following oronasal inoculation with either a low (10(4) TCID50/mouse) or high (10(6) TCID50/mouse) inoculation dose. Both strains were mainly replicating in nasal mucosa and submandibular glands for one to two months. In nasal mucosa, MCMV was detected earlier and longer (1-49 days post inoculation (dpi)) and reached higher titers with the high inoculation dose compared to the low inoculation dose (14-35 dpi). In submandibular glands, a similar finding was observed (high dose: 7-49 dpi; low dose: 14-42 dpi). In lungs, both strains showed a restricted replication. In spleen, liver and kidneys, only the Smith strain established a productive infection. The infected cells were identified as olfactory neurons and sustentacular cells in olfactory epithelium, macrophages and dendritic cells in NALT, acinar cells in submandibular glands, and macrophages and epithelial cells in lungs for both strains. Antibody analysis demonstrated for both strains that IgG(2a) was the main detectable antibody subclass. Overall, our results show that significant phenotypic differences exist between the two strains. MCMV HaNa1 has been shown to be interesting for use in mouse models in order to get better insights for HCMV infections in immunocompetent humans

Design and Analysis of Rhesus Cytomegalovirus IL-10 Mutants as a Model for Novel Vaccines against Human Cytomegalovirus

Human cytomegalovirus (HCMV) expresses a viral ortholog (CMVIL-10) of human cellular interleukin-10 (cIL-10). Despite only ∼26% amino acid sequence identity, CMVIL-10 exhibits comparable immunosuppressive activity with cIL-10, attenuates HCMV antiviral immune responses, and contributes to lifelong persistence within infected hosts. The low sequence identity between CMVIL-10 and cIL-10 suggests vaccination with CMVIL-10 may generate antibodies that specifically neutralize CMVIL-10 biological activity, but not the cellular cytokine, cIL-10. However, immunization with functional CMVIL-10 might be detrimental to the host because of its immunosuppressive properties.Structural biology was used to engineer biologically inactive mutants of CMVIL-10 that would, upon vaccination, elicit a potent immune response to the wild-type viral cytokine. To test the designed proteins, the mutations were incorporated into the rhesus cytomegalovirus (RhCMV) ortholog of CMVIL-10 (RhCMVIL-10) and used to vaccinate RhCMV-infected rhesus macaques. Immunization with the inactive RhCMVIL-10 mutants stimulated antibodies against wild-type RhCMVIL-10 that neutralized its biological activity, but did not cross-react with rhesus cellular IL-10.This study demonstrates an immunization strategy to neutralize RhCMVIL-10 biological activity using non-functional RhCMVIL-10 antigens. The results provide the methodology for targeting CMVIL-10 in vaccine, and therapeutic strategies, to nullify HCMV's ability to (1) skew innate and adaptive immunity, (2) disseminate from the site of primary mucosal infection, and (3) establish a lifelong persistent infection