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Inclusion of the Viral Pentamer Complex in a Vaccine Design Greatly Improves Protection against Congenital Cytomegalovirus in the Guinea Pig Model.

ABSTRACT: A vaccine against congenital cytomegalovirus (cCMV) is a high priority. The guinea pig is a small-animal model for cCMV. A disabled infectious single-cycle (DISC) viral vaccine strain based on a guinea pig cytomegalovirus (GPCMV) capsid mutant was evaluated. A previous version of this vaccine did not express the gH/gL-based pentamer complex (PC) and failed to fully protect against cCMV. The PC is necessary for GPCMV epithelial cell/trophoblast tropism and congenital infection and is a potentially important neutralizing antigen. Here, we show that a second-generation PC-positive (PC+) DISC (DISCII) vaccine induces neutralizing antibodies to the PC and other glycoproteins and a cell-mediated response to pp65 (GP83). Additionally, a CRISPR/Cas9 strategy identified guinea pig platelet-derived growth factor receptor alpha (PDGFRA) to be the receptor for PC-independent infection of fibroblast cells. Importantly, PDGFRA was absent in epithelial and trophoblast cells, which were dependent upon the viral PC for infection. Virus neutralization by DISCII antibodies on epithelial and trophoblast cells was similar to that in sera from wild-type virus-infected animals and dependent in part on PC-specific antibodies. In contrast, sera from PC-negative virus-infected animals poorly neutralized virus on non-fibroblast cells. DISCII-vaccinated animals were protected against congenital infection, in contrast to a nonvaccinated group. The target organs of pups in the vaccine group were negative for wild-type virus, unlike those of pups in the control group, with GPCMV transmission being approximately 80%. Overall, the DISCII vaccine had 97% efficacy against cCMV. The complete protection provided by this PC+ DISC vaccine makes the possibility of the use of this approach against human cCMV attractive.IMPORTANCE Cytomegalovirus (CMV) is a leading cause of congenital disease in newborns, and an effective vaccine remains an elusive goal. The guinea pig is the only small-animal model for cCMV. Guinea pig cytomegalovirus (GPCMV) encodes a glycoprotein pentamer complex (PC) for entry into non-fibroblast cells, including placental trophoblasts, to enable cCMV. As with human cytomegalovirus (HCMV), GPCMV uses a specific cell receptor (PDGFRA) for fibroblast entry, but other receptors are required for non-fibroblast cells. A disabled infectious single-cycle (DISC) GPCMV vaccine strain induced an antibody immune response to the viral pentamer to enhance virus neutralization on non-fibroblast cells, and vaccinated animals were fully protected against cCMV. Inclusion of the PC as part of a vaccine design dramatically improved vaccine efficacy, and this finding underlines the importance of the immune response to the PC in contributing toward protection against cCMV. This vaccine represents an important milestone in the development of a vaccine against cCMV.

SUBMITTER: Choi KY

PROVIDER: S-EPMC6819920 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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Inclusion of the Viral Pentamer Complex in a Vaccine Design Greatly Improves Protection against Congenital Cytomegalovirus in the Guinea Pig Model.

Choi K Yeon KY El-Hamdi Nadia S NS McGregor Alistair A

Journal of virology 20191029 22

A vaccine against congenital cytomegalovirus (cCMV) is a high priority. The guinea pig is a small-animal model for cCMV. A disabled infectious single-cycle (DISC) viral vaccine strain based on a guinea pig cytomegalovirus (GPCMV) capsid mutant was evaluated. A previous version of this vaccine did not express the gH/gL-based pentamer complex (PC) and failed to fully protect against cCMV. The PC is necessary for GPCMV epithelial cell/trophoblast tropism and congenital i ...[more]

PMID: 31484753

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Project description:The guinea pig is the only small animal model for congenital CMV but requires species-specific guinea pig cytomegalovirus (GPCMV). Tegument protein GP83 is the presumed homolog of HCMV pp65 but gene duplication in the UL82-UL84 homolog locus in various animal CMV made it unclear if GP83 was a functional homolog. A GP83 null deletion mutant GPCMV (GP83dPC+) generated in the backdrop of glycoprotein pentamer complex (PC) positive virus, required for non-fibroblast infection, had normal growth kinetics on fibroblasts but was highly impaired on epithelial and trophoblast cells. GP83dPC+ virus was highly sensitive to IFN-I suggesting GP83 had an innate immune evasion function. GP83 interacted with cellular DNA sensors guinea pig IFI16 and cGAS indicating a role in the cGAS/STING pathway. Ectopically expressed GP83 in trophoblast cells restored GP83dPC+ virus growth. Additionally, mutant virus growth was restored in epithelial cells by expression of bovine viral diarrhea virus (BVDV) NPRO protein targeting IRF3 as part of the cGAS/STING pathway or alternatively by expression of fibroblast cell receptor PDGFRA. HCMV pp65 is a T cell target antigen and a recombinant adenovirus encoding GP83 was evaluated as a vaccine. In GPCMV challenge studies, vaccinated animals had varying levels of protection against wild type virus with a protective response against 22122 prototype strain but little protection against a novel clinical strain of GPCMV (TAMYC), despite 100% identity in GP83 protein sequences. Overall, GP83 is a functional pp65 homolog with novel importance for epithelial cell infection but a GP83 T cell response provides limited vaccine efficacy.ImportanceCongenital CMV (cCMV) is a leading cause of cognitive impairment and deafness in newborns and a vaccine is a high priority. The guinea pig is the only small animal model for cCMV but requires guinea pig cytomegalovirus (GPCMV). The translational impact of GPCMV research is potentially reduced if the virus does not encode functional HCMV homolog proteins. This study demonstrates that tegument protein GP83 (pp65 homolog) is involved in innate immune evasion and highly important for infection of non-fibroblast cells via the viral glycoprotein pentamer complex (PC)-dependent endocytic entry pathway. The PC pathway is highly significant for virus dissemination and disease in the host, including cCMV. A GP83 candidate Ad-vaccine strategy in animals induced a cell-mediated response but failed to provide cross strain protection against a novel clinical strain of GPCMV. Results suggest that the pp65 antigen provides very limited efficacy as a stand-alone vaccine, especially in cross strain protection.

Project description:Development of a cytomegalovirus (CMV) vaccine is a major public health priority due to the risk of congenital infection. A key component of a vaccine is thought to be an effective neutralizing antibody response against the viral glycoproteins necessary for cell entry. Species specificity of human CMV (HCMV) precludes direct studies in an animal model. The guinea pig is the only small animal model for congenital cytomegalovirus infection. Analysis of the guinea pig CMV (GPCMV) genome indicates that it potentially encodes homologs to the HCMV glycoproteins (including gB, gH, gL, gM, gN and gO) that form various cell entry complexes on the outside of the virus: gCI (gB); gCII (gH/gL/gO); gCIII (gM/gN). The gB homolog (GP55) has been investigated as a candidate subunit vaccine but little is known about the other homolog proteins. GPCMV glycoproteins were investigated by transient expression studies which indicated that homolog glycoproteins to gN and gM, or gH, gL and gO were able to co-localize in cells and generate respective homolog complexes which could be verified by immunoprecipitation assays. ELISA studies demonstrated that the individual complexes were highly immunogenic in guinea pigs. The gO (GP74) homolog protein has 13 conserved N-glycosylation sites found in HCMV gO. In transient expression studies, only the glycosylated protein is detected but in virus infected cells both N-glycosylated and non-glycosylated gO protein were detected. In protein interaction studies, a mutant gO that lacked N-glycosylation sites had no impact on the ability of the protein to interact with gH/gL which indicated a potential alternative function associated with these sites. Knockout GPCMV BAC mutagenesis of the respective glycoprotein genes (GP55 for gB, GP75 for gH, GP115 for gL, GP100 for gM, GP73 for gN and GP74 for gO) in separate reactions was lethal for virus regeneration on fibroblast cells which demonstrated the essential nature of the GPCMV glycoproteins. The gene knockout results were similar to HCMV, except in the case of the gO homolog, which was non-essential in epithelial tropic virus but essential in lab adapted GPCMV. Overall, the findings demonstrate the similarity between HCMV and GPCMV glycoproteins and strengthen the relevance of this model for development of CMV intervention strategies.

Project description: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.

Project description:BackgroundCytomegalovirus (CMV) is a leading infectious cause of neurologic deficits, both in the settings of congenital and perinatal infection, but few animal models exist to study neurodevelopmental outcomes. This study examined the impact of neonatal guinea pig CMV (GPCMV) infection on spatial learning and memory in a Morris water maze (MWM) model.MethodsNewborn pups were challenged intraperitoneally (i.p.) with a pathogenic red fluorescent protein-tagged GPCMV, or sham inoculated. On days 15-19 post infection (p.i.), pups were tested in the MWM. Viral loads were measured in blood and tissue by quantitative PCR (qPCR), and brain samples collected at necropsy were examined by histology and immunohistochemistry.ResultsViremia (DNAemia) was detected at day 3 p.i. in 7/8 challenged animals. End-organ dissemination was observed, by qPCR, in the lung, liver, and spleen. CD4-positive (CD4+) and CD8-positive (CD8+) T cell infiltrates were present in brains of challenged animals, particularly in periventricular and hippocampal regions. Reactive gliosis and microglial nodules were observed. Statistically significant spatial learning and memory deficits were observed by MWM, particularly for total maze distance traveled (p < 0.0001).ConclusionNeonatal GPCMV infection in guinea pigs results in cognitive defects demonstrable by the MWM. This neonatal guinea pig challenge model can be exploited for studying antiviral interventions.ImpactCMV impairs neonatal neurocognition and memory in the setting of postnatal infection. The MWM can be used to examine memory and learning in a guinea pig model of neonatal CMV infection. CD4+ and CD8+ T cells infiltrate the brain following neonatal CMV challenge. This article demonstrates that the MWM can be used to evaluate memory and learning after neonatal GPCMV challenge. The guinea pig can be used to examine central nervous system pathology caused by neonatal CMV infection and this attribute may facilitate the study of vaccines and antivirals.

Dataset Information

Inclusion of the Viral Pentamer Complex in a Vaccine Design Greatly Improves Protection against Congenital Cytomegalovirus in the Guinea Pig Model.

Publications

Inclusion of the Viral Pentamer Complex in a Vaccine Design Greatly Improves Protection against Congenital Cytomegalovirus in the Guinea Pig Model.

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