Biology subjects

McGregor, A.

Publications and source records attributed to McGregor, A..

3 recordsLinked to original sources

bioRxiv2026

Cytomegalovirus pentamer dependent endocytic cellular infection utilizes redundant entry receptors in the guinea pig model

2.The guinea pig with guinea pig cytomegalovirus (GPCMV) is the only small animal model for congenital CMV (cCMV). GPCMV cell entry is dictated by specific viral gH/gL-based complexes: gH/gL/gO trimer (direct entry); pentamer complex, PC (endocytic entry). GPCMV gB as the fusogenic protein is also essential for all entry pathways. PDGFRA and NRP2 are receptors for direct and endocytic virus entry respectively based on strain 13 animal fibroblast ATCC cell line studies. All non-fibroblast guinea pig cell lines are derived from Dunkin-Hartley animals, the focus of cCMV studies. GPCMV infection of Dunkin-Hartley embryo fibroblasts (GEFh) and epithelial cells were compared. Knockout of PDGFRA on GEFh cells prevented GPCMV(PC-) direct entry but not endocytic GPCMV(PC+) infection, demonstrating both pathways of infection. Fibroblast generated virus poorly infected epithelial cells compared to epithelial virus stock, which exhibited full tropism to all cell types. Guinea pig epithelial cell lines are NRP2-positive and PDGFRA-negative requiring PC for GPCMV infection. Epithelial and GEFh cells, but not strain 13 fibroblasts, additionally expressed ThBD. In immunoprecipitation assays, PC and ThBD interacted unlike CD46 receptor candidate targeting gH/gL. Double-knockout of NRP2/ThBD in epithelial cells impaired infection unlike single knockouts. Individual ectopic species-specific receptor expression restored infection on double-knockout epithelial (NRP2/ThBD) and fibroblast (PDGFRA/NRP2) cell lines. Knockout of NRP2/ThBD receptors did not enhance GPCMV neutralization by gB antibodies on PDGFRA-negative cells demonstrating a limitation of a gB vaccine strategy. Overall, GPCMV and HCMV similarity for receptors and cell tropism maintains the translational importance of this model. 3. Impact StatementA CMV vaccine is a high priority as congenital cytomegalovirus (cCMV) is a leading cause of hearing loss and cognitive impairment in newborns. CMV species-specificity requires animal studies to utilize species-specific virus. The guinea pig is the only small animal model for cCMV and guinea pig cytomegalovirus (GPCMV) encodes functional HCMV homolog glycoprotein complexes for cell entry including a gH/gL-based PC for endocytic cell entry. The viral fusogenic gB glycoprotein is required for GPCMV infection of all cell types but antibody based gB vaccines fail to fully protect against endocytic infection. The PC is potentially an important vaccine antibody target, but PC-based cell entry is only partially understood and poorly characterized for GPCMV. Identifying GPCMV cell entry receptors is critical to the understanding of virus tropism and disease especially since the PC is necessary for cCMV. Correlation with HCMV improves translational impact of guinea pig based cCMV intervention strategies.

microbiology↗

bioRxiv2025

Selective knockout of key CMV receptors in fetal cells blocks direct and endocytic pathways of entry in the guinea pig

Cytomegalovirus is a leading cause of congenital disease and a vaccine is a high priority. Guinea pig with guinea pig cytomegalovirus (GPCMV) is the only small animal model for congenital CMV (cCMV). GPCMV encodes functionally essential viral entry glycoprotein complexes similar to HCMV, which are neutralizing antibody targets. As with HCMV, GPCMV has two pathways of cell entry (direct and endocytic). Common to both pathways and essential for infection is the fusogenic viral glycoprotein gB. Additional gH/gL-based complexes are necessary for receptor interaction and cell entry: gH/gL/gO trimer (direct); pentamer complex, PC (endocytic). Direct cell entry requires host PDGFRA receptor and viral trimer. An endocytic PC receptor has not been identified for GPCMV or any animal CMV. GPCMV endocytic entry was blocked by acidic flux inhibition but not direct cell entry, requiring knockout of PDGFRA. We hypothesized that cellular knockout of GPCMV direct and endocytic receptors would completely block infection. Two PC receptor candidates, guinea pig NRP2 and CD147, present on all established guinea pig cell lines were evaluated. Results demonstrated that NRP2 interacted with PC unlike CD147 in immunoprecipitation assays. Double knockout of PDGFRA and NRP2 completely blocked GPCMV but had no impact on control HSV-1 infection. In contrast, CD147/PDGFRA double knockout had limited inhibition of GPCMV and no impact on HSV-1. Ectopic expression of cell receptors restored infection to normal levels on knockout cell lines. Overall, results demonstrate GPCMV conservation with HCMV for key receptors and cell entry pathways enhancing the translational importance of this model. ImportanceCongenital CMV is a leading cause of hearing loss and cognitive impairment in newborns and a vaccine is a high priority. Species-specificity of HCMV requires animal model studies to utilize species-specific virus. The guinea is the only small animal model for cCMV and GPCMV encodes functional HCMV homolog glycoprotein complexes for cell entry via direct or endocytic pathways. The gB glycoprotein is required for infection of all cell types but a gB vaccine fails to fully protect against cCMV. GPCMV encodes a functional PC required for infection of different cell types via endocytic pathway. The PC has emerged as an important vaccine antibody target but PC-based cell entry is only partially understood and poorly characterized for GPCMV. Identifying GPCMV cell entry receptors is critical to the understanding of virus tropism and disease in this model. Correlation with HCMV improves translational impact of a GPCMV vaccine and antiviral cCMV intervention.

microbiology↗

bioRxiv2025

Complete cross strain protection against congenital cytomegalovirus infection requires a vaccine encoding key antibody (gB) and T-cell (immediate early 1 protein) viral antigens

AbstractO_ST_ABSBackgroundC_ST_ABSCytomegalovirus is a leading cause of congenital disease and multiple strains enable congenital CMV (cCMV) from both primary and non-primary infection. A cross-strain protective cCMV vaccine is a high priority. The guinea pig is the only small animal model for cCMV and guinea pig cytomegalovirus (GPCMV) encodes functional homolog proteins including cell entry gB glycoprotein and non-structural immediate early 1 protein (IE1), essential for lytic infection. A gB vaccine antibody response fails to provide horizontal protection against highly cell-associated clinical GPCMV strain TAMYC compared to prototype strain 22122. Previously, a recombinant defective adenovirus (Ad) vaccine encoding IE1, a T-cell antigen, provided high-level cCMV protection. In this study, we hypothesized that a combined Ad-based strategy encoding trimeric gB complex and IE1 (AdgB+AdIE1) could improve cross-strain protection against cCMV compared to a gB vaccine (AdgB). MethodsA preconception vaccine study evaluated the immune response and ability of vaccines to provide cross-strain protection against cCMV. Seronegative female animals were assigned into three vaccine groups: Group 1 (AdgB); Group 2 (AdgB+AdIE1); Group 3 (no vaccine). Animals were vaccinated following a previously defined protocol and antibody ELISAs were used to evaluate gB immune response (AD1, prefusion gB and wild type gB). Additionally, an IFN{gamma}-ELISPOT assay evaluated IE1 T-cell response. During second trimester dams were challenged with GPCMV (22122 and TAMYC) and pregnancy went to term where viral loads in pup target organs and placentas were evaluated. ResultsVaccinated dams elicited a higher neutralizing antibody response to gB than natural convalescent immunity and antibodies recognized homolog AD1 gB domain as well as prefusion gB with response surpassing natural immunity. Group 2 animals additionally elicited a T-cell response to IE1. Evaluation of viral load in pups demonstrated that AdgB+AdIE1 vaccine reduced GPCMV transmission to below detectable limits compared to 91.7% in unvaccinated group. In contrast, AdgB reduced cCMV transmission to 12% in pups. ConclusionComplete cross-strain cCMV protection is a significant milestone in this model and achieved by inclusion of an antibody response to trimeric gB and T-cell response to IE1. Importantly, gB and IE1 responses can synergize and increase protection against cCMV unlike prior approaches.

immunology↗