Development and use of a 293 cell line expressing lac repressor for the rescue of recombinant adenoviruses expressing high levels of rabies virus glycoprotein.

142

Herpesvirus glycoprotein complex gH/gL provides a core entry function through interactions with the fusion protein gB and can also influence tropism through receptor interactions. The Epstein-Barr virus gH/gL and gH/gL/gp42 serve both functions for entry into epithelial and B cells, respectively. Human cytomegalovirus (HCMV) gH/gL can be bound by the UL128-131 proteins or gO. The phenotypes of gO and UL128-131 mutants suggest that gO-gH/gL interactions are necessary for the core entry function on all cell types, whereas the binding of UL128-131 to gH/gL likely relates to a distinct receptor-binding function for entry into some specific cell types (e.g., epithelial) but not others (e.g., fibroblasts and neurons). There are at least eight isoforms of gO that differ by 10 to 30% of amino acids, and previous analysis of two HCMV strains suggested that some isoforms of gO function like chaperones, disassociating during assembly to leave unbound gH/gL in the virion envelope, while others remain bound to gH/gL. For the current report, we analyzed the gH/gL complexes present in the virion envelope of several HCMV strains, each of which encodes a distinct gO isoform. Results indicate that all strains of HCMV contain stable gH/gL/gO trimers and gH/gL/UL128-131 pentamers and little, if any, unbound gH/gL. TR, TB40/e, AD169, and PH virions contained vastly more gH/gL/gO than gH/gL/UL128-131, whereas Merlin virions contained mostly gH/gL/UL128-131, despite abundant unbound gO remaining in the infected cells. Suppression of UL128-131 expression during Merlin replication dramatically shifted the ratio toward gH/gL/gO. These data suggest that Merlin gO is less efficient than other gO isoforms at competing with UL128-131 for binding to gH/gL. Thus, gO diversity may influence the pathogenesis of HCMV through effects on the assembly of the core versus tropism gH/gL complexes.

Section: Methodsmentioning

confidence: 99%

Comparative Analysis of gO Isoforms Reveals that Strains of Human Cytomegalovirus Differ in the Ratio of gH/gL/gO and gH/gL/UL128-131 in the Virion Envelope

Zhou

Wechsler

et al. 2013

142

“…We could rescue the latter only after introducing TetO sites into the HCMV promoter and growth of the virus in the Flp-In T-REx-293 cells. This observation demonstrates the value of inducible systems to regulate vaccine transgene expression (33,40), enabling the largescale production of vectors encoding any antigen of interest. Both Ad6 vectors induced T cell responses in the experimental animals, and as expected, a broader response targeting all encoded antigens (E1, E2, and p7) was induced by Ad6E1E2p7, suggesting that this vector is a superior candidate vaccine (41,42).…”

Section: Discussionmentioning

confidence: 99%

Combined Adenovirus Vector and Hepatitis C Virus Envelope Protein Prime-Boost Regimen Elicits T Cell and Neutralizing Antibody Immune Responses

Chmielewska

Naddeo

Capone

et al. 2014

Despite the recent progress in the development of new antiviral agents, hepatitis C virus (HCV) infection remains a major global health problem, and there is a need for a preventive vaccine. We previously reported that adenoviral vectors expressing HCV nonstructural proteins elicit protective T cell responses in chimpanzees and were immunogenic in healthy volunteers. Furthermore, recombinant HCV E1E2 protein formulated with adjuvant MF59 induced protective antibody responses in chimpanzees and was immunogenic in humans. To develop an HCV vaccine capable of inducing both T cell and antibody responses, we constructed adenoviral vectors expressing full-length and truncated E1E2 envelope glycoproteins from HCV genotype 1b. Heterologous prime-boost immunization regimens with adenovirus and recombinant E1E2 glycoprotein (genotype 1a) plus MF59 were evaluated in mice and guinea pigs. Adenovirus prime and protein boost induced broad HCV-specific CD8 Hepatitis C virus (HCV) infection is a major global health problem affecting an estimated 170 million people worldwide, occurring among persons of all ages, genders, races, and regions of the world. Chronically infected subjects are at risk of developing progressive liver disease, including cirrhosis, and primary hepatocellular carcinoma (1). Although the recent introduction of directly acting antiviral drugs (DAAs) has improved therapy for chronic HCV and interferon (IFN)-free regimens are on the horizon (2), treatment success may be limited by a range of factors, including awareness of infection status, access to and cost of therapy, relative efficacy of different regimens for specific HCV genotypes, adverse effects, comorbidities (e.g., cirrhosis or HIV coinfection), and host factors. For these reasons, the development of a safe, effective, and affordable preventive vaccine against HCV is the optimal long-term goal to control the global epidemic (3).Approximately 20% of infected individuals clear the virus spontaneously, and resolution is associated with HLA type and with potent, multispecific, and long-lasting T cell responses (4). T cell depletion experiments with chimpanzees confirmed the essential role of cellular immunity in controlling HCV infection (5). Moreover, antibodies targeting the HCV envelope glycoproteins have been shown to neutralize infection in vitro (6, 7) and to protect against virus challenge in the human liver-Alb-uPA/SCID murine model (8)(9)(10). A recent report demonstrated that spontaneous clearance of HCV is associated with the early appearance of a broadly neutralizing antibody response (11). Recombinant E1E2 glycoproteins have been shown to induce cross-neutralizing antibody responses against het-

“…Nonreplicating (E1 Ϫ ) Ad vectors that express HCMV TR gH and gL have been described (42). Ad vectors expressing TR gO, AdTRgO and AdTRgO(co), were generating using a commercial (Microbix, Toronto, Canada) modification of the method of Matthews et al (32). Briefly, the gO gene (UL74) was PCR amplified from the TR genome (in the case of AdTRgO) or synthesized by GeneArt (Regensburg, Germany) as a codon-optimized gene [in the case of AdTRgO(co)], and then these gO genes were ligated into shuttle plasmid pDC316(io) (Microbix).…”

Section: Methodsmentioning

confidence: 99%

“…However, this vector did not stably express gO. Thus, a second Ad vector was constructed in which the TR gO gene was coupled to a promoter element containing a lac operator sequence that is repressed in 293 cells expressing the lac repressor (32). AdTRgO stably expressed gO through numerous passages.…”

Section: Fig 3 Pulse-chase Analysis Of the Maturation Of N-linked Omentioning

confidence: 99%

Human Cytomegalovirus TR Strain Glycoprotein O Acts as a Chaperone Promoting gH/gL Incorporation into Virions but Is Not Present in Virions

Ryckman

Chase

Johnson

2010

Human cytomegalovirus (HCMV) produces the following two gH/gL complexes: gH/gL/gO and gH/gL/ UL128-131. Entry into epithelial and endothelial cells requires gH/gL/UL128-131, and we have provided evidence that gH/gL/UL128-131 binds saturable epithelial cell receptors to mediate entry. HCMV does not require gH/gL/UL128-131 to enter fibroblasts, and laboratory adaptation to fibroblasts results in mutations in the UL128-131 genes, abolishing infection of epithelial and endothelial cells. HCMV gO-null mutants produce very small plaques on fibroblasts yet can spread on endothelial cells. Thus, one prevailing model suggests that gH/gL/gO mediates infection of fibroblasts, while gH/gL/UL128-131 mediates entry into epithelial/endothelial cells. Most biochemical studies of gO have involved the HCMV lab strain AD169, which does not assemble gH/gL/UL128-131 complexes. We examined gO produced by the low-passage clinical HCMV strain TR. Surprisingly, TR gO was not detected in purified extracellular virus particles. In TR-infected cells, gO remained sensitive to endoglycosidase H, suggesting that the protein was not exported from the endoplasmic reticulum (ER). However, TR gO interacted with gH/gL in the ER and promoted export of gH/gL from the ER to the Golgi apparatus. Pulse-chase experiments showed that a fraction of gO remained bound to gH/gL for relatively long periods, but gO eventually dissociated or was degraded and was not found in extracellular virions or secreted from cells. The accompanying report by P. T. Wille et al. (J. Virol., 84:2585-2596, 2010) showed that a TR gO-null mutant failed to incorporate gH/gL into virions and that the mutant was unable to enter fibroblasts and epithelial and endothelial cells. We concluded that gO acts as a molecular chaperone, increasing gH/gL ER export and incorporation into virions. It appears that gO competes with UL128-131 for binding onto gH/gL but is released from gH/gL, so that gH/gL (lacking UL128-131) is incorporated into virions. Thus, our revised model suggests that both gH/gL and gH/gL/UL128-131 are required for entry into epithelial and endothelial cells.