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

Abstract

BackgroundConsiderable evidence has accumulated that multiple viruses, bacteria, and protozoa manipulate interleukin-10 (IL-10)-mediated signaling through the IL-10 receptor (IL-10R) in ways that could enable establishment of a persistent microbial infection. This suggests that inhibition of pathogen targeting of IL-10/IL-10R signaling could prevent microbial persistence. Human cytomegalovirus (HCMV) and rhesus cytomegalovirus (RhCMV) express a viral interleukin-10 (cmvIL-10 and rhcmvIL-10, respectively) with comparable immune modulating properties in vitro to that of their host's cellular IL-10 (cIL-10). A prior study noted that rhcmvIL-10 alters innate and adaptive immunity to RhCMV in vivo, 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.Methods and FindingsAs 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.ConclusionThis 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.

Vaccination against a Virus-Encoded Cytokine Significantly Restricts Viral Challenge

“…Using a DNA prime-protein boost immunization strategy (23), four RhCMV-uninfected macaques were primed with plasmid vectors for rhcmvIL-10M1 and rhcmvIL-10M2 and boosted three times with insect cell-expressed rhcmvIL-10M1 and -M2 proteins (20). Vaccinees developed rhcmvIL-10-specific binding antibodies and antibodies that neutralized wild-type (WT) rhcmvIL-10 function (19) (Fig. 1A and B).…”

“…The M1 and M2 variants of rhcmvIL-10 each contain two site-directed amino acid changes that prevent the recombinant proteins from (i) binding IL-10R and (ii) suppressing proinflammatory cytokine production by LPS-activated PBMC. Four RhCMV-uninfected animals were immunized with rhcmvIL-10M1 and -M2 plasmids at week 0, as previously described (23), and subsequently boosted with rhcmvIL-10M1/M2 protein at weeks 6, 12, and 26, as described previously (19,20). In brief, DNA recombinant expression constructs, pND/rhcmvIL-10M1 and pND/rhcmvIL-10M2 (20), were purified using an endotoxin-free plasmid purification kit (Qiagen), and the DNA concentration was determined spectrophotometrically.…”

“…Enzyme-linked immunosorbent assays (ELISAs) for rhcmvIL-10 binding, RhCMV binding antibodies, and RhCMV neutralizing (fibroblast) antibodies (NAb) were performed as described previously (19,20,23,27). The avidity of rhcmvIL-10-specific antibodies was quantified, as described previously (19).…”

“…Neutralizing antibodies against rhcmvIL-10 function were characterized by a bioassay measuring the ability of rhesus plasma to neutralize wild-type rhcmvIL-10-mediated suppression of IL-12 production in LPS-stimulated PBMC (19,20).…”

“…In support of this, rhesus macaques inoculated with an RhCMV variant lacking rhcmvIL-10 function exhibit increased inflammatory responses and greater RhCMV-specific antibody and T cell responses than animals inoculated with the parental virus expressing rhcmvIL-10 (18). A mechanistic basis for these observations is suggested by the fact that rhcmvIL-10 suppresses the production of IL-12 in lipopolysaccharide (LPS)-activated peripheral blood mononuclear cells (PBMC) (19,20). Together, these studies highlight HCMV modulation of host immunity and lead to the hypothesis that strategies that prevent HCMV-mediated activation of IL-10R signaling during the earliest stage of primary infection should significantly alter the virus-host relationship to augment host antiviral immunity.…”