Protection against Congenital CMV Infection Conferred by MVA-Vectored Subunit Vaccines Extends to a Second Pregnancy after Maternal Challenge with a Heterologous, Novel Strain Variant

Maternal reinfection of immune women with novel human cytomegalovirus (HCMV) strains acquired during pregnancy can result in symptomatic congenital CMV (cCMV) infection. Novel animal model strategies are needed to explore vaccine-mediated protections against maternal reinfection. To investigate this in the guinea pig cytomegalovirus (GPCMV) model, a strictly in vivo-passaged workpool of a novel strain, the CIDMTR strain (dose, 1 × 107 pfu) was used to infect dams that had been challenged in a previous pregnancy with the 22122 strain, following either sham-immunization (vector only) or vaccination with MVA-vectored gB, gH/gL, or pentameric complex (PC) vaccines. Maternal DNAemia cleared by day 21 in the glycoprotein-vaccinated dams, but not in the sham-immunized dams. Mean pup birth weights were 72.85 ± 10.2, 80.0 ± 6.9, 81.4 ± 14.1, and 89.38 ± 8.4 g in sham-immunized, gB, gH/gL, and PC groups, respectively (p < 0.01 for control v. PC). Pup mortality in the sham-immunized group was 6/12 (50%), but reduced to 3/35 (8.6%) in combined vaccine groups (p = 0.0048). Vertical CIDMTR transmission occurred in 6/12 pups (50%) in the sham-vaccinated group, compared to 2/34 pups (6%) in the vaccine groups (p = 0.002). We conclude that guinea pigs immunized with vectored vaccines expressing 22122 strain-specific glycoproteins are protected after a reinfection with a novel, heterologous clinical isolate (CIDMTR) in a second pregnancy.

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.

The Placental Response to Guinea Pig Cytomegalovirus Depends Upon the Timing of Maternal Infection

Human cytomegalovirus (HCMV) infects the placenta, and these placental infections can cause fetal injury and/or demise. The timing of maternal HCMV infection during pregnancy is a determinant of fetal outcomes, but how development affects the placenta’s susceptibility to infection, the likelihood of placental injury post-infection, and the frequency of transplacental HCMV transmission remains unclear. In this study, guinea pig cytomegalovirus (GPCMV) was used to model primary maternal infection and compare the effects of infection at two different times on the placenta. When guinea pigs were infected with GPCMV at either 21- or 35-days gestation (dGA), maternal and placental viral loads, as determined by droplet digital PCR, were not significantly affected by the timing of maternal infection. However, when the transcriptomes of gestational age-matched GPCMV-infected and control placentas were compared, significant infection-associated changes in gene expression were only observed after maternal infection at 35 dGA. Notably, transcripts associated with immune activation (e.g. Cxcl10, Ido1, Tgtp1, and Tlr8) were upregulated in the infected placenta. A GPCMV-specific in situ hybridization assay detected rare infected cells in the main placenta after maternal infection at either time, and maternal infection at 35 dGA also caused large areas of GPCMV-infected cells in the junctional zone. As GPCMV infection after mid-gestation is known to cause high rates of stillbirth and/or fetal growth restriction, our results suggest that the placenta becomes sensitized to infection-associated injury late in gestation, conferring an increased risk of adverse pregnancy outcomes after cytomegalovirus infection.

Guinea Pig Cytomegalovirus Protective T Cell Antigen GP83 Is a Functional pp65 Homolog for Innate Immune Evasion and Pentamer-Dependent Virus Tropism

ABSTRACT The guinea pig is the only small animal model for congenital cytomegalovirus (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 CMVs 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 nonfibroblast infection, had normal growth kinetics on fibroblasts but was highly impaired on epithelial and trophoblast cells. GP83dPC+ virus was highly sensitive to type I interferon (IFN-I), suggesting that 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 the fibroblast cell receptor platelet-derived growth factor receptor alpha (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 various levels of protection against wild-type virus, with a protective response against the 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. IMPORTANCE Congenital 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 nonfibroblast 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 adenovirus (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.

A trimeric capable gB CMV vaccine provides limited protection against a highly cell associated and epithelial tropic strain of cytomegalovirus in guinea pigs

Multiple strains of human cytomegalovirus (HCMV) can cause congenital cytomegalovirus (cCMV) by primary or secondary infection. The viral gB glycoprotein is a leading vaccine candidate, essential for infection of all cell-types, and immunodominant antibody target. Guinea pig cytomegalovirus (GPCMV) is the only small animal model for cCMV. Various gB vaccines have shown efficacy but studies have utilized truncated gB and protection against prototype strain 22122 with preferential tropism to fibroblasts despite encoding a gH-based pentamer complex for non-fibroblast infection. A highly cell-associated novel strain of GPCMV (TAMYC) with 99 % identity in gB sequence to 22122 exhibited preferred tropism to epithelial cells. An adenovirus vaccine encoding full-length gB (AdgB) was highly immunogenic and partially protected against 22122 strain challenge in vaccinated animals but not when challenged with TAMYC strain. GPCMV studies with AdgB vaccine sera on numerous cell-types demonstrated impaired neutralization (NA50) compared to fibroblasts. GPCMV-convalescent sera including pentamer complex antibodies increased virus neutralization on non-fibroblasts and anti-gB depletion from GPCMV-convalescent sera had minimal impact on epithelial cell neutralization. GPCMV(PC+) 22122-convalescent animals challenged with TAMYC exhibited higher protection compared to AdgB vaccine. Overall, results suggest that antibody response to both gB and PC are important components of a GPCMV vaccine.

Identification and functional analyses of a cell-death inhibitor encoded by guinea pig cytomegalovirus gp38.1 in cell culture and in animals

Cytomegaloviruses (CMVs) employ an array of strategies designed to interfere with host defence responses against pathogens. Studies on such evasion mechanisms are important for understanding the pathogenesis of CMV diseases. Although guinea pig CMV (GPCMV) provides a useful animal model for congenital CMV infection, its evasion strategies are not fully elucidated. Here, we analysed a genome locus that may encode gene products for the GPCMV evasion mechanisms and found the following. (1) RACE analyses identified five transcripts in the GP38-gp38.4 locus, one of which was a spliced product encoding gp38.1. Similarities in the splicing pattern and gene position of gp38.1 to human CMV UL37 and its exon 1 encoding vMIA (viral mitochondria-localized inhibitor of apoptosis) suggest that the gp38.1 gene encodes an apoptosis inhibitor. (2) In a transient transfection assay, gp38.1 localized in the mitochondria and relocated BAX from the cytoplasm to the mitochondria, although its co-localization with BAK was not evident. Further, the expression of gp38.1 partially reduced staurosporine-induced apoptosis. (3) GPCMV defective in the gp38.1 ORF (Δ38.1) and the virus that rescues the defect (r38.1) were generated. Guinea pig fibroblast cells infected with Δ38.1 died earlier than r38.1-infected cells, which resulted in the lower yields of Δ38.1. (4) In animals, viral loads in the spleens of r38.1-infected guinea pigs were higher than those in the spleens of Δ38.1-infected animals. In conclusion, although GPCMV gp38.1 exerts a vMIA-like function, its inhibitory effect was not robust, suggesting the presence of additional inhibitory molecule(s), such as a BAK-specific inhibitor.

Convalescent Immunity to Guinea Pig Cytomegalovirus Induces Limited Cross Strain Protection against Re-Infection but High-Level Protection against Congenital Disease

The guinea pig is the only small animal model for congenital cytomegalovirus (cCMV) but requires guinea pig cytomegalovirus (GPCMV). Current GPCMV research utilizes prototype strain 22122, which limits the translational impact of GPCMV as numerous human CMV strains exist and cCMV is possible in the setting of re-infection. A novel strain of GPCMV (TAMYC) exhibited differences to 22122 in various glycoproteins with GP74 (gO homolog) the most variable (25% difference). Antibody ELISAs for TAMYC-convalescent animals evoked similar immune response to viral glycoprotein complexes (gB, gH/gL, gM/gN, pentamer) and cell-mediated response to pp65 homolog (GP83). Convalescent sera from TAMYC-infected animals neutralized GPCMV infection on fibroblasts but was less effective on epithelial cells. TAMYC-convalescent animals were not protected from dissemination of heterogenous virus challenge (22122). However, in a cCMV protection study, TAMYC-convalescent animals challenged mid-pregnancy (22122) exhibited high-level protection against cCMV compared to seronegative animals with pup transmission reduced from 80% (control) to 12%. Overall, pre-existing immunity in guinea pigs provides limited ability to prevent GPCMV re-infection by a different viral strain but provides a high level of protection against cCMV in heterogenous strain challenge. This level of cross protection against cCMV should be a prerequisite of any CMV vaccine.