Equine herpesvirus 1 glycoprotein D expressed in Pichia pastoris is hyperglycosylated and elicits a protective immune response in the mouse model of EHV-1 disease

Ruitenberg, K. M.; Gilkerson, James R.; Wellington, J. E.; Love, Daria N.; Whalley, J. M.

doi:10.1016/s0168-1702(01)00337-9

Cited by 20 publications

(9 citation statements)

References 48 publications

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“…The second band at *40 kD could be indicative of extra glycosylation, which has been observed before for recombinant glycoproteins expressed in Pichia Pastoris [ (Ruitenberg et al 2001)]. Interestingly, also when p/RDS is expressed in COS cells, it migrates as a doublet on SDS-PAGE, which was ascribed to the presence of both glycosylated and nonglycosylated forms (Muller-Weeks et al 2002), suggesting that also in the present study at least part of the protein expressed in Pichia is glycosylated.…”

Section: Expression Of P/rds In Pichia Pastorissupporting

confidence: 84%

Expression and structural characterization of peripherin/RDS, a membrane protein implicated in photoreceptor outer segment morphology

et al. 2009

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Peripherin/RDS is a member of the tetraspanin family of integral membrane proteins and plays a major role in the morphology of photoreceptor outer segments. Peripherin/RDS has a long extracellular loop (hereafter referred to as the LEL domain), which is vital for its function. Point mutations in the LEL domain often lead to impaired photoreceptor formation and function, making peripherin/RDS an important drug target. Being a eukaryotic membrane protein, acquiring sufficient peripherin/ RDS for biophysical characterisation represents a significant challenge. Here, we describe the expression and characterisation of peripherin/RDS in Drosophila melangolaster Schneider (S2) insect cells and in the methylotrophic yeast Pichia pastoris. The wild-type peripherin/RDS and the retinitis pigmentosa causing P216L mutant from S2 cells are characterised using circular dichroism (CD) spectroscopy. The structure of peripherin/RDS and of a pathogenic mutant is assessed spectroscopically for the first time. These findings are evaluated in relation to a three-dimensional model of the functionally important LEL domain obtained by protein threading.

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Section: Expression Of P/rds In Pichia Pastorissupporting

confidence: 84%

Expression and structural characterization of peripherin/RDS, a membrane protein implicated in photoreceptor outer segment morphology

et al. 2009

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“…EHV-1 gD, a 55-58-kDa glycoprotein, is a component of the virion envelope, is inserted into the membrane of EHV-1-infected cells, and is required for virus entry and cell-tocell spread (14,16,53). Glycoprotein D of alphaherpesviruses, including EHV-1, elicits virus-specific B-and T-cell responses that are often associated with protective immunity in vivo (5,27,40,42,49,50,52,56). Recently, Ruitenberg et al (41) showed that immunization with EHV-1 gD DNA followed by immunization with recombinant gD protein in a prime/boost inoculation schedule induced neutralizing antibodies and afforded protection from challenge in Balb/c mice.…”

Section: Discussionmentioning

confidence: 99%

Cytokine Profiles and Long-Term Virus-Specific Antibodies Following Immunization of CBA Mice with Equine Herpesvirus 1 and Viral Glycoprotein D

Zhang

Frampton²,

Osterrieder³

et al. 2003

Viral Immunology

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Equine herpesvirus 1 (EHV-1)-specific antibody-secreting cells (ASC) isolated from the lung and spleen of mice at 12 months after immunization with attenuated EHV-1 KyA, heat-killed KyA, or recombinant viral glycoprotein D (rgD) assessed by ELISPOT showed a three- to fivefold increase in three immunoglobulin isotypes at 3 days post-challenge with pathogenic EHV-1 RacL11 as compared to control mice. ELISPOT assays demonstrated a high frequency of cells secreting proinflammatory tumor necrosis factor-alpha (TNF-alpha), interferon gamma (IFN-gamma), and interleukin 4 (IL-4) in the lungs in response to infection with KyA or RacL11 or immunization with rgD. Cytokine production elicited by EHV-1 KyA or RacL11 infection revealed similar frequencies of EHV-1-specific IFN-gamma and IL-4 spot forming cells in the mediastinal lymph nodes and spleen. However, KyA induced significantly greater amounts of IFN-gamma producing cells in the lungs than did RacL11. Intranasal immunization with KyA or rgD induced long-term immunity that provided protection against pathogenic EHV-1 challenge infection at 12 months post-immunization. Overall, the data indicate that immunization with infectious KyA or rgD induces significant levels of cytokines, virus-specific ASC in the lungs and spleen, and long-term virus specific B-cell responses.

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“…VC2-EHV-1-gD stimulated strong virus-neutralizing (VN) activity in comparison to that seen in both unvaccinated and VC2-vaccinated animals, and this VN activity was similar to that generated by the commercial EHV-1 killed virus vaccine. It has been reported that intranasal or intramuscular immunization with EHV-1 gD produces antigen-specific IgG responses (3,4,8,10,11,(22)(23)(24)(25). In addition, the presence of VN antibody prior to EHV-1 infection was shown to be associated with a reduction in the amount and duration of nasopharyngeal virus shedding (26,27).…”

Section: Figmentioning

confidence: 99%

“…Thus, these proteins are major antigenic determinants for both humoral and cellular immune responses and have been utilized as subunit vaccines (2)(3)(4). Specifically, immunization with EHV-1 glycoprotein D (gD) has been shown to generate protective immune responses in mice and horses (3,(5)(6)(7)(8)(9)(10)(11). EHV-1 and herpes simplex virus 1 (HSV-1) glycoproteins D are required for entry into cells and cell-to-cell fusion, a function which is conserved in most but not all alphaherpesviruses (12).…”

mentioning

confidence: 99%

Intramuscular Immunization of Mice with the Live-Attenuated Herpes Simplex Virus 1 Vaccine Strain VC2 Expressing Equine Herpesvirus 1 (EHV-1) Glycoprotein D Generates Anti-EHV-1 Immune Responses in Mice

Liu

Stanfield

Chouljenko

et al. 2017

J Virol

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Vaccination remains the best option to combat equine herpesvirus 1 (EHV-1) infection, and several different strategies of vaccination have been investigated and developed over the past few decades. Herein, we report that the liveattenuated herpes simplex virus 1 (HSV-1) VC2 vaccine strain, which has been shown to be unable to enter into neurons and establish latency in mice, can be utilized as a vector for the heterologous expression of EHV-1 glycoprotein D (gD) and that the intramuscular immunization of mice results in strong antiviral humoral and cellular immune responses. The VC2-EHV-1-gD recombinant virus was constructed by inserting an EHV-1 gD expression cassette under the control of the cytomegalovirus immediate early promoter into the VC2 vector in place of the HSV-1 thymidine kinase (UL23) gene. The vaccines were introduced into mice through intramuscular injection. Vaccination with both the VC2-EHV-1-gD vaccine and the commercially available vaccine Vetera EHV XP 1/4 (Vetera; Boehringer Ingelheim Vetmedica) resulted in the production of neutralizing antibodies, the levels of which were significantly higher in comparison to those in VC2-and mock-vaccinated animals (P Ͻ 0.01 or P Ͻ 0.001). Analysis of EHV-1-reactive IgG subtypes demonstrated that vaccination with the VC2-EHV-1-gD vaccine stimulated robust IgG1 and IgG2a antibodies after three vaccinations (P Ͻ 0.001). Interestingly, Vetera-vaccinated mice produced significantly higher levels of IgM than mice in the other groups before and after challenge (P Ͻ 0.01 or P Ͻ 0.05). Vaccination with VC2-EHV-1-gD stimulated strong cellular immune responses, characterized by the upregulation of both interferonand tumor necrosis factor-positive CD4 ϩ T cells and CD8 ϩ T cells. Overall, the data suggest that the HSV-1 VC2 vaccine strain may be used as a viral vector for the vaccination of horses as well as, potentially, for the vaccination of other economically important animals.IMPORTANCE A novel virus-vectored VC2-EHV-1-gD vaccine was constructed using the live-attenuated HSV-1 VC2 vaccine strain. This vaccine stimulated strong humoral and cellular immune responses in mice, suggesting that it could protect horses against EHV-1 infection.KEYWORDS EHV-1, HSV-1, equine herpesvirus, live vector vaccines, glycoprotein D, immune response, mouse model

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Equine herpesvirus 1 glycoprotein D expressed in Pichia pastoris is hyperglycosylated and elicits a protective immune response in the mouse model of EHV-1 disease

Cited by 20 publications

References 48 publications

Expression and structural characterization of peripherin/RDS, a membrane protein implicated in photoreceptor outer segment morphology

Expression and structural characterization of peripherin/RDS, a membrane protein implicated in photoreceptor outer segment morphology

Cytokine Profiles and Long-Term Virus-Specific Antibodies Following Immunization of CBA Mice with Equine Herpesvirus 1 and Viral Glycoprotein D

Intramuscular Immunization of Mice with the Live-Attenuated Herpes Simplex Virus 1 Vaccine Strain VC2 Expressing Equine Herpesvirus 1 (EHV-1) Glycoprotein D Generates Anti-EHV-1 Immune Responses in Mice

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