A TB40/E-Derived Human Cytomegalovirus Genome with an Intact US-Gene Region and a Self-Excisable BAC Cassette for Immunological Research

Sampaio, Kerstin Laib; Weyell, Anja; Subramanian, Narmadha; Wu, Zhourui; Sinzger, Christian

doi:10.2144/000114606

Cited by 32 publications

(31 citation statements)

References 33 publications

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“…Validation of the putative receptor interaction sites was performed on the background of AD169-derived BAC clone pHB5 (62) and a novel, previously unpublished BAC construct of the highly endotheliotropic strain VHL/E (63). The recombinant VHL/E-BAC19 construct was generated by homologous recombination in HFFs using the vector plasmid pEB1097 as described previously (64). A single clone was chosen according to the prototype orientation of the genome and was transferred into bacterial strain GS1783 (65) for further mutagenesis.…”

Section: Cells and Virusesmentioning

confidence: 99%

The N Terminus of Human Cytomegalovirus Glycoprotein O Is Important for Binding to the Cellular Receptor PDGFRα

Stegmann¹,

Rothemund²,

Sampaio³

et al. 2019

J Virol

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The human cytomegalovirus (HCMV) glycoprotein complex gH/gL/gO is required for the infection of cells by cell-free virions. It was recently shown that entry into fibroblasts depends on the interaction of gO with the platelet-derived growth factor receptor alpha (PDGFR␣). This interaction can be blocked with soluble PDGFR␣-Fc, which binds to HCMV virions and inhibits entry. The aim of this study was to identify parts of gO that contribute to PDGFR␣ binding. In a systematic mutational approach, we targeted potential interaction sites by exchanging conserved clusters of charged amino acids of gO with alanines. To screen for impaired interaction with PDGFR␣, virus mutants were tested for sensitivity to inhibition by soluble PDGFR␣-Fc. Two mutants with mutations within the N terminus of gO (amino acids 56 to 61 and 117 to 121) were partially resistant to neutralization. To validate whether these mutations impair interaction with PDGFR␣-Fc, we compared binding of PDGFR␣-Fc to mutant and wild-type virions via quantitative immunofluorescence analysis. PDGFR␣-Fc staining intensities were reduced by 30% to 60% with mutant virus particles compared to wild-type particles. In concordance with the reduced binding to the soluble receptor, virus penetration into fibroblasts, which relies on binding to the cellular PDGFR␣, was also reduced. In contrast, PDGFR␣-independent penetration into endothelial cells was unaltered, demonstrating that the phenotypes of the gO mutant viruses were specific for the interaction with PDGFR␣. In conclusion, the mutational screening of gO revealed that the N terminus of gO contributes to efficient spread in fibroblasts by promoting the interaction of virions with its cellular receptor. IMPORTANCE The human cytomegalovirus is a highly prevalent pathogen that can cause severe disease in immunocompromised hosts. Currently used drugs successfully target the viral replication within the host cell, but their use is restricted due to side effects and the development of resistance. An alternative approach is the inhibition of virus entry, for which understanding the details of the initial virus-cell interaction is desirable. As binding of the viral gH/gL/gO complex to the cellular PDGFR␣ drives infection of fibroblasts, this is a potential target for inhibition of infection. Our mutational mapping approach suggests the N terminus as the receptor binding portion of the protein. The respective mutants were partially resistant to inhibition by PDGFR␣-Fc but also attenuated for infection of fibroblasts, indicating that such mutations have little if any benefit for the virus. These findings highlight the potential of targeting the interaction of gH/gL/gO with PDGFR␣ for therapeutic inhibition of HCMV.

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Section: Cells and Virusesmentioning

confidence: 99%

The N Terminus of Human Cytomegalovirus Glycoprotein O Is Important for Binding to the Cellular Receptor PDGFRα

Stegmann¹,

Rothemund²,

Sampaio³

et al. 2019

J Virol

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“…For this purpose, we used RV-TB40-BAC KL7 -SE-EGFP. This EGFP-expressing virus is derived from low-passage HCMV strain TB40/E and contains an intact US-gene region encoding all immunoevasins (US2, US3, US6, and US11) that downregulate MHC class I presentation (44). We infected the GBM cell lines LN18, U343, and U251 with RV-TB40-BAC KL7 -SE-EGFP at a multiplicity of infection (MOI) of 0.3.…”

Section: Resultsmentioning

confidence: 99%

“…HCMV strain TB40/E and the corresponding bacterial artificial chromosome TB40/E-BAC (clone 4) as well as RV-TB40-BAC KL7 -SE-EGFP, an enhanced green fluorescent protein (EGFP)-expressing virus derived from TB40/E (44), were kindly provided by Christian Sinzger, University of Ulm, Ulm, Germany. The advantages of TB40/E are high titer growth in cell culture similar to lab strains and cell tropism resembling recent clinical isolates (45).…”

Section: Methodsmentioning

confidence: 99%

Development of a Human Cytomegalovirus (HCMV)-Based Therapeutic Cancer Vaccine Uncovers a Previously Unsuspected Viral Block of MHC Class I Antigen Presentation

et al. 2019

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Human cytomegalovirus (HCMV) induces a uniquely high frequency of virus-specific effector/memory CD8+ T-cells, a phenomenon termed “memory inflation”. Thus, HCMV-based vaccines are particularly interesting in order to stimulate a sustained and strong cellular immune response against cancer. Glioblastoma multiforme (GBM) is the most aggressive primary brain tumor with high lethality and inevitable relapse. The current standard treatment does not significantly improve the desperate situation underlining the urgent need to develop novel approaches. Although HCMV is highly fastidious with regard to species and cell type, GBM cell lines are susceptible to HCMV. In order to generate HCMV-based therapeutic vaccine candidates, we deleted all HCMV-encoded proteins (immunoevasins) that interfere with MHC class I presentation. The aim being to use the viral vector as an adjuvant for presentation of endogenous tumor antigens, the presentation of high levels of vector-encoded neoantigens and finally the repurposing of bystander HCMV-specific CD8+ T cells to fight the tumor. As neoantigen, we exemplarily used the E6 and E7 proteins of human papillomavirus type 16 (HPV-16) as a non-transforming fusion protein (E6/E7) that covers all relevant antigenic peptides. Surprisingly, GBM cells infected with E6/E7-expressing HCMV-vectors failed to stimulate E6-specific T cells despite high level expression of E6/E7 protein. Further experiments revealed that MHC class I presentation of E6/E7 is impaired by the HCMV-vector although it lacks all known immunoevasins. We also generated HCMV-based vectors that express E6-derived peptide fused to HCMV proteins. GBM cells infected with these vectors efficiently stimulated E6-specific T cells. Thus, fusion of antigenic sequences to HCMV proteins is required for efficient presentation via MHC class I molecules during infection. Taken together, these results provide the preclinical basis for development of HCMV-based vaccines and also reveal a novel HCMV-encoded block of MHC class I presentation.

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“…HB5, a derivative of the laboratory strain AD169, was obtained from Borst et al (60). TB40/E-BAC KL7 (here denoted TB40/E) is a derivative of the endotheliotropic strain TB40/E (61) that carries an intact UL US-gene region (62). TB40-BAC4-IE-GLuc is a derivative of TB40/E expressing the Gaussia luciferase under the HCMV IE promoter at an ectopic position (41).…”

Section: Methodsmentioning

confidence: 99%

Dense Bodies of a gH/gL/UL128/UL130/UL131 Pentamer-Repaired Towne Strain of Human Cytomegalovirus Induce an Enhanced Neutralizing Antibody Response

Lehmann

Falk²,

Büscher

et al. 2019

J Virol

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The development of a vaccine against human cytomegalovirus infection (HCMV) is a high-priority medical goal. The viral pentameric protein complex consisting of glycoprotein H (gH)/gL/UL128-131A (PC) is considered to be an important vaccine component. Its relevance to the induction of a protective antibody response is, however, still a matter of debate. We addressed this issue by using subviral dense bodies (DBs) of HCMV. DBs are exceptionally immunogenic. Laboratory HCMV strain DBs harbor important neutralizing antibody targets, like the glycoproteins B, H, L, M, and N, but they are devoid of the PC. To be able to directly compare the impact of the PC on the levels of neutralizing antibody (NT-abs) responses, a PC-positive variant of the HCMV laboratory strain Towne was established by bacterial artificial chromosome (BAC) mutagenesis (Towne-UL130rep). This strain synthesized PC-positive DBs upon infection of fibroblasts. These DBs were used in side-by-side immunizations with PC-negative Towne DBs. Mouse and rabbit sera were tested to address the impact of the PC on DB immunogenicity. The neutralizing antibody response to PC-positive DBs was superior to that of PC-negative DBs, as tested on fibroblasts, epithelial cells, and endothelial cells and for both animal species used. The experiments revealed the potential of the PC to enhance the antibody response against HCMV. Of particular interest was the finding that PC-positive DBs induced an antibody response that blocked the infection of fibroblasts by a PC-positive viral strain more efficiently than sera following immunizations with PC-negative particles. IMPORTANCE Infections with the human cytomegalovirus (HCMV) may cause severe and even life-threatening disease manifestations in newborns and immunosuppressed individuals. Several strategies for the development of a vaccine against this virus are currently pursued. A critical question in this respect refers to the antigenic composition of a successful vaccine. Using a subviral particle vaccine candidate, we show here that one protein complex of HCMV, termed the pentameric complex (PC), enhances the neutralizing antibody response against viral infection of different cell types. We further show for the first time that this not only relates to the infection of epithelial or endothelial cells; the presence of the PC in the particles also enhanced the neutralizing antibody response against the infection of fibroblasts by HCMV. Together, these findings argue in favor of including the PC in strategies for HCMV vaccine development.

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A TB40/E-Derived Human Cytomegalovirus Genome with an Intact US-Gene Region and a Self-Excisable BAC Cassette for Immunological Research

Cited by 32 publications

References 33 publications

The N Terminus of Human Cytomegalovirus Glycoprotein O Is Important for Binding to the Cellular Receptor PDGFRα

The N Terminus of Human Cytomegalovirus Glycoprotein O Is Important for Binding to the Cellular Receptor PDGFRα

Development of a Human Cytomegalovirus (HCMV)-Based Therapeutic Cancer Vaccine Uncovers a Previously Unsuspected Viral Block of MHC Class I Antigen Presentation

Dense Bodies of a gH/gL/UL128/UL130/UL131 Pentamer-Repaired Towne Strain of Human Cytomegalovirus Induce an Enhanced Neutralizing Antibody Response

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