Inclusion of the Guinea Pig Cytomegalovirus Pentameric Complex in a Live Virus Vaccine Aids Efficacy against Congenital Infection but Is Not Essential for Improving Maternal and Neonatal Outcomes.

The development of a vaccine against congenital human cytomegalovirus (HCMV) infection is a major priority. The pentameric complex (PC) of virion envelope proteins gH, gL, UL128, UL130, and UL131A is a key vaccine target. To determine the importance of immunity to the homologous PC encoded by guinea pig cytomegalovirus (GPCMV) in preventing congenital CMV, PC-intact and PC-deficient live-attenuated vaccines were generated and directly compared for immunogenicity and efficacy against vertical transmission in a vertical transmission model. A virulent PC-intact GPCMV (PC/intact) was modified by galK mutagenesis either to abrogate PC expression (PC/null; containing a frame-shift mutation in GP129, homolog of UL128) or to delete genes encoding three MHC Class I homologs and a protein kinase R (PKR) evasin while retaining the PC (3DX/Δ145). Attenuated vaccines were compared to sham immunization in a two-dose preconception subcutaneous inoculation regimen in GPCMV seronegative Hartley guinea pigs. Vaccines induced transient, low-grade viremia in 5/12 PC/intact-, 2/12 PC/null-, and 1/11 3DX/Δ145-vaccinated animals. Upon completion of the two-dose vaccine series, ELISA titers for the PC/intact group (geometic mean titer (GMT) 13,669) were not significantly different from PC/null (GMT 8127) but were significantly higher than for the 3DX/Δ145 group (GMT 6185; p < 0.01). Dams were challenged with salivary gland-adapted GPCMV in the second trimester. All vaccines conferred protection against maternal viremia. Newborn weights were significantly lower in sham-immunized controls (84.5 ± 2.4 g) compared to PC/intact (96 ± 2.3 g), PC/null (97.6 ± 1.9 g), or 3DX/Δ145 (93 ± 1.7) pups (p < 0.01). Pup mortality in sham-immunized controls was 29/40 (73%) and decreased to 1/44 (2.3%), 2/46 (4.3%), or 4/40 (10%) in PC/intact, PC/null, or 3DX/Δ145 groups, respectively (all p < 0.001 compared to control). Congenital GPCMV transmission occurred in 5/44 (11%), 16/46 (35%), or 29/38 (76%) of pups in PC/intact, PC/null, or 3DX/Δ145 groups, versus 36/40 (90%) in controls. For infected pups, viral loads were lower in pups born to vaccinated dams compared to controls. Sequence analysis demonstrated that infected pups in the vaccine groups had salivary gland-adapted GPCMV and not vaccine strain-specific sequences, indicating that congenital transmission was due to the challenge virus and not vaccine virus. We conclude that inclusion of the PC in a live, attenuated preconception vaccine improves immunogenicity and reduces vertical transmission, but PC-null vaccines are equal to PC-intact vaccines in reducing maternal viremia and protecting against GPCMV-related pup mortality.

A Fully Protective Congenital CMV Vaccine Requires Neutralizing Antibodies to Viral Pentamer and gB Glycoprotein Complexes but a pp65 T-Cell Response Is Not Necessary.

A vaccine against congenital cytomegalovirus infection is a high priority. Guinea pig cytomegalovirus (GPCMV) is the only congenital CMV small animal model. GPCMV encodes essential glycoprotein complexes for virus entry (gB, gH/gL/gO, gM/gN) including a pentamer complex (gH/gL/GP129/GP131/GP133 or PC) for endocytic cell entry. The cohorts for protection against congenital CMV are poorly defined. Neutralizing antibodies to the viral glycoprotein complexes are potentially more important than an immunodominant T-cell response to the pp65 protein. In GPCMV, GP83 (pp65 homolog) is an evasion factor, and the GP83 mutant GPCMV has increased sensitivity to type I interferon. Although GP83 induces a cell-mediated response, a GP83-only-based vaccine strategy has limited efficacy. GPCMV attenuation via GP83 null deletion mutant in glycoprotein PC positive or negative virus was evaluated as live-attenuated vaccine strains (GP83dPC+/PC-). Vaccinated animals induced antibodies to viral glycoprotein complexes, and PC+ vaccinated animals had sterilizing immunity against wtGPCMV challenge. In a pre-conception vaccine (GP83dPC+) study, dams challenged mid-2nd trimester with wtGPCMV had complete protection against congenital CMV infection without detectable virus in pups. An unvaccinated control group had 80% pup transmission rate. Overall, gB and PC antibodies are key for protection against congenital CMV infection, but a response to pp65 is not strictly necessary.

Comparative Analysis of Roseoloviruses in Humans, Pigs, Mice, and Other Species.

Viruses of the genus Roseolovirus belong to the subfamily Betaherpesvirinae, family Herpesviridae. Roseoloviruses have been studied in humans, mice and pigs, but they are likely also present in other species. This is the first comparative analysis of roseoloviruses in humans and animals. The human roseoloviruses human herpesvirus 6A (HHV-6A), 6B (HHV-6B), and 7 (HHV-7) are relatively well characterized. In contrast, little is known about the murine roseolovirus (MRV), also known as murine thymic virus (MTV) or murine thymic lymphotrophic virus (MTLV), and the porcine roseolovirus (PRV), initially incorrectly named porcine cytomegalovirus (PCMV). Human roseoloviruses have gained attention because they can cause severe diseases including encephalitis in immunocompromised transplant and AIDS patients and febrile seizures in infants. They have been linked to a number of neurological diseases in the immunocompetent including multiple sclerosis (MS) and Alzheimer's. However, to prove the causality in the latter disease associations is challenging due to the high prevalence of these viruses in the human population. PCMV/PRV has attracted attention because it may be transmitted and pose a risk in xenotransplantation, e.g., the transplantation of pig organs into humans. Most importantly, all roseoloviruses are immunosuppressive, the humoral and cellular immune responses against these viruses are not well studied and vaccines as well as effective antivirals are not available.

CD8+ T Cells Prevent Lethality from Neonatal Murine Roseolovirus Infection.

A recently described mouse homolog of the human roseoloviruses, murine roseolovirus (MRV), causes loss of peripheral and thymic CD4+ cells during neonatal infection of BALB/c mice. Despite significant disruptions to the normal adaptive immune response, infected BALB/c mice reproducibly recover from infection, consistent with prior studies on a related virus, mouse thymic virus. In this article, we show that, in contrast to published studies on mouse thymic virus, MRV appears to robustly infect neonatal C57BL/6 (B6) mice, causing severe depletion of thymocytes and peripheral T cells. Moreover, B6 mice recovered from infection. We investigated the mechanism of thymocyte and T cell loss, determining that the major thymocyte subsets were infected with MRV; however, CD4+ and CD4+CD8- T cells showed increased apoptosis during infection. We found that CD8+ T cells populated MRV-infected thymi. These CD8+ T cells expressed markers of activation, had restricted TCR repertoire, and accumulated intracellular effector proteins, consistent with a cytotoxic lymphocyte phenotype and suggesting their involvement in viral clearance. Indeed, absence of CD8+ T cells prevented recovery from MRV infection and led to lethality in infected animals, whereas B cell-deficient mice showed CD4+ T cell loss but recovered from infection without lethality. Thus, these results demonstrate that CD8+ T cells are required for protective immunity against a naturally occurring murine pathogen that infects the thymus and establish a novel infection model for MRV in B6 mice, providing the foundation for detailed future studies on MRV with the availability of innumerable mutant mice on the B6 background.

The essential role of guinea pig cytomegalovirus (GPCMV) IE1 and IE2 homologs in viral replication and IE1-mediated ND10 targeting.

Guinea pig cytomegalovirus (GPCMV) immediate early proteins, IE1 and IE2, demonstrated structural and functional homologies with human cytomegalovirus (HCMV). GPCMV IE1 and IE2 co-localized in the nucleus with each other, the viral polymerase and guinea pig ND10 components (gpPML, gpDaxx, gpSp100, gpATRX). IE1 showed direct interaction with ND10 components by immunoprecipitation unlike IE2. Additionally, IE1 protein disrupted ND10 bodies. IE1 mutagenesis mapped the nuclear localization signal to the C-terminus and identified the core domain for gpPML interaction. Individual knockout of GPCMV GP122 or GP123 (IE2 and IE1 unique exons respectively) was lethal to the virus. However, an IE1 mutant (codons 234-474 deleted), was viable with attenuated viral growth kinetics and increased susceptibility to type I interferon (IFN-I). In HCMV, the IE proteins are important T cell target antigens. Consequently, characterization of the homologs in GPCMV provides a basis for their evaluation in candidate vaccines against congenital infection.

Comparison of monovalent glycoprotein B with bivalent gB/pp65 (GP83) vaccine for congenital cytomegalovirus infection in a guinea pig model: Inclusion of GP83 reduces gB antibody response but both vaccine approaches provide equivalent protection against pup mortality.

Cytomegalovirus (CMV) subunit vaccine candidates include glycoprotein B (gB), and phosphoprotein ppUL83 (pp65). Using a guinea pig cytomegalovirus (GPCMV) model, this study compared immunogenicity, pregnancy outcome, and congenital viral infection following pre-pregnancy immunization with a three-dose series of modified vaccinia virus Ankara (MVA)-vectored vaccines consisting either of gB administered alone, or simultaneously with a pp65 homolog (GP83)-expressing vaccine. Vaccinated and control dams were challenged at midgestation with salivary gland-adapted GPCMV. Comparisons included ELISA and neutralizing antibody responses, maternal viral load, pup mortality, and congenital infection rates. Strikingly, ELISA and neutralization titers were significantly lower in the gB/GP83 combined vaccine group than in the gB group. However, both vaccines protected against pup mortality (63.2% in controls vs. 11.4% and 13.9% in gB and gB/GP83 combination groups, respectively; p<0.0001). Reductions in pup viral load were noted for both vaccine groups compared to control, but preconception vaccination resulted in a significant reduction in GPCMV transmission only in the monovalent gB group (26/44, 59% v. 27/34, 79% in controls; p<0.05). We conclude that, using the MVA platform, the addition of GP83 to a gB subunit vaccine interferes with antibody responses and diminishes protection against congenital GPCMV infection, but does not decrease protection against pup mortality.

Development of a novel, guinea pig-specific IFN-γ ELISPOT assay and characterization of guinea pig cytomegalovirus GP83-specific cellular immune responses following immunization with a modified vaccinia virus Ankara (MVA)-vectored GP83 vaccine.

The guinea pig (Cavia porcellus) provides a useful animal model for studying the pathogenesis of many infectious diseases, and for preclinical evaluation of vaccines. However, guinea pig models are limited by the lack of immunological reagents required for characterization and quantification of antigen-specific T cell responses. To address this deficiency, an enzyme-linked immunospot (ELISPOT) assay for guinea pig interferon (IFN)-γ was developed to measure antigen/epitope-specific T cell responses to guinea pig cytomegalovirus (GPCMV) vaccines. Using splenocytes harvested from animals vaccinated with a modified vaccinia virus Ankara (MVA) vector encoding the GPCMV GP83 (homolog of human CMV pp65 [gpUL83]) protein, we were able to enumerate and map antigen-specific responses, both in vaccinated as well as GPCMV-infected animals, using a panel of GP83-specific peptides. Several potential immunodominant GP83-specific peptides were identified, including one epitope, LGIVHFFDN, that was noted in all guinea pigs that had a detectable CD8+ response to GP83. Development of a guinea pig IFN-γ ELISPOT should be useful in characterization of additional T cell-specific responses to GPCMV, as well as other pathogens. This information in turn can help focus future experimental evaluation of immunization strategies, both for GPCMV as well as for other vaccine-preventable illnesses studied in the guinea pig model.

A live guinea pig cytomegalovirus vaccine deleted of three putative immune evasion genes is highly attenuated but remains immunogenic in a vaccine/challenge model of congenital cytomegalovirus infection.

Live attenuated vaccines for prevention of congenital cytomegalovirus infections encode numerous immune evasion genes. Their removal could potentially improve vaccine safety and efficacy. To test this hypothesis, three genes encoding MHC class I homologs (presumed NK evasins) were deleted from the guinea pig cytomegalovirus genome and the resulting virus, 3DX, was evaluated as a live attenuated vaccine in the guinea pig congenital infection model. 3DX was attenuated in vivo but not in vitro. Vaccination with 3DX produced elevated cytokine levels and higher antibody titers than wild type (WT) virus while avidity and neutralizing titers were similar. Protection, assessed by maternal viral loads and pup mortality following pathogenic viral challenge during pregnancy, was comparable between 3DX and WT and significant compared to naïve animals. These results suggest that the safety and perhaps efficacy of live attenuated human cytomegalovirus vaccines could be enhanced by deletion of viral immunomodulatory genes.

Macrophage inflammatory proteins in cytomegalovirus-related inner ear injury.

OBJECTIVE: Inner ear inflammation triggered by CMV infection may play a role in CMV-related auditory pathogenesis. The purpose of the study was to determine if a virally encoded macrophage inflammatory protein played a role in CMV-related hearing loss. DESIGN: Mutagenesis was performed with deletion of a guinea pig CMV macrophage inflammatory protein. Intracochlear inoculations were performed on three groups of animals (n = 18). Group 1 received sterile viral media, Group 2 received wild-type CMV virus, and Group 3 received "knockout" (KO) virus with a deleted immunomodulation gene. Baseline and postinoculation ABRs were obtained. ELISA and PCR were performed and temporal bones examined. SUBJECTS: Eighteen guinea pigs. RESULTS: The KO group had significantly better hearing than the WT group. There were no significant differences between the KO and sham groups. The WT group had significant hearing loss at all frequencies. Inflammation and fibrosis were noted in the WT temporal bones only. CONCLUSIONS: Virally encoded macrophage inflammatory proteins appear to play a significant role in CMV-related hearing loss.

Protection against congenital cytomegalovirus (CMV) disease, conferred by a replication-disabled, bacterial artificial chromosome (BAC)-based DNA vaccine.

It is unclear if protective immunity can be conferred by a cytomegalovirus (CMV) vaccine encoding a single protein subunit, or if multiple viral genes need to be targeted. Using the guinea pig model of congenital CMV infection, these studies examined the immunogenicity and efficacy of a DNA vaccine based on the guinea pig cytomegalovirus (GPCMV) genome cloned as a non-infectious BAC plasmid, modified by transposon insertion into the homolog of the HCMV tegument protein, UL48. Following vaccination of female Hartley guinea pigs with BAC DNA, adverse GPCMV-related pregnancy outcome were assessed after establishment of pregnancy, followed by GPCMV third-trimester challenge. Animals immunized with recombinant BACmid engendered anti-GPCMV antibodies by ELISA assay. Immunogenicity of BAC plasmid DNA was augmented by inclusion of the lipid adjuvant, DOTMA/DOPE, in the vaccine regimen. Among pups born to 12 control (sham-immunized) dams challenged with GPCMV in the third trimester, mortality was 23/35 (66%). In contrast, among evaluable pregnancy outcomes in pups born to 10 BAC-immunized pregnant dams, preconception immunization resulted in reduced pup mortality, to 10/34 pups (29%; p<0.005 versus control, Fisher's exact test). In addition, vaccinated dams had reduced viral load, compared to controls, as assessed by quantitative, real-time PCR.

A macrophage inflammatory protein homolog encoded by guinea pig cytomegalovirus signals via CC chemokine receptor 1.

Cytomegaloviruses encode homologs of cellular immune effector proteins, including chemokines (CKs) and CK receptor-like G protein-coupled receptors (GPCRs). Sequence of the guinea pig cytomegalovirus (GPCMV) genome identified an open reading frame (ORF) which predicted a 101 amino acid (aa) protein with homology to the macrophage inflammatory protein (MIP) subfamily of CC (beta) CKs, designated GPCMV-MIP. To assess functionality of this CK, recombinant GPCMV-MIP was expressed in HEK293 cells and assayed for its ability to bind to and functionally interact with a variety of GPCRs. Specific signaling was observed with the hCCR1 receptor, which could be blocked with hMIP -1alpha in competition experiments. Migration assays revealed that GPCMV-MIP was able to induce chemotaxis in hCCR1-L1.2 cells. Antisera raised against a GST-MIP fusion protein immunoprecipitated species of approximately 12 and 10 kDa from GPCMV-inoculated tissue culture lysates, and convalescent antiserum from GPCMV-infected animals was immunoreactive with GST-MIP by ELISA assay. These results represent the first substantive in vitro characterization of a functional CC CK encoded by a cytomegalovirus.