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Engineering the vaccinia virus L1 protein for increased neutralizing antibody response after DNA immunization.

Shinoda K, Wyatt LS, Irvine KR, Moss B - Virol. J. (2009)

Bottom Line: Therefore, safer DNA and protein vaccines are being investigated.Addition of a signal sequence to the N-terminus of L1 increased cell surface expression as shown by confocal microscopy and flow cytometry of transfected cells.Removal of the transmembrane domain led to secretion of L1 into the medium.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892-3210, USA. shinodak@niaid.nih.gov

ABSTRACT

Background: The licensed smallpox vaccine, comprised of infectious vaccinia virus, has associated adverse effects, particularly for immunocompromised individuals. Therefore, safer DNA and protein vaccines are being investigated. The L1 protein, a component of the mature virion membrane that is conserved in all sequenced poxviruses, is required for vaccinia virus entry into host cells and is a target for neutralizing antibody. When expressed by vaccinia virus, the unglycosylated, myristoylated L1 protein attaches to the viral membrane via a C-terminal transmembrane anchor without traversing the secretory pathway. The purpose of the present study was to investigate modifications of the gene expressing the L1 protein that would increase immunogenicity in mice when delivered by a gene gun.

Results: The L1 gene was codon modified for optimal expression in mammalian cells and potential N-glycosylation sites removed. Addition of a signal sequence to the N-terminus of L1 increased cell surface expression as shown by confocal microscopy and flow cytometry of transfected cells. Removal of the transmembrane domain led to secretion of L1 into the medium. Induction of binding and neutralizing antibodies in mice was enhanced by gene gun delivery of L1 containing the signal sequence with or without the transmembrane domain. Each L1 construct partially protected mice against weight loss caused by intranasal administration of vaccinia virus.

Conclusion: Modifications of the vaccinia virus L1 gene including codon optimization and addition of a signal sequence with or without deletion of the transmembrane domain can enhance the neutralizing antibody response of a DNA vaccine.

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Related in: MedlinePlus

Expression of truncated L1 proteins detected by Western blotting. BS-C-1 cells were transfected with plasmids and cells (A) and media (B) were harvested separately and analyzed by SDS-PAGE followed by Western blotting with polyclonal L1 antibody. In panel C, the Western blot of the cell lysate was probed with antibody to glyceraldehyde 3-phosphate dehydrogenase as a loading control. Proteins were detected by chemiluminescence. Lanes: 1, pL1op; 2, psL1op; 3, pL1optr; 4, psL1optr; 5, empty vector; 6, lysate from VACV-infected cells. The positions and masses in kDa of marker proteins are shown on the left.
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Figure 4: Expression of truncated L1 proteins detected by Western blotting. BS-C-1 cells were transfected with plasmids and cells (A) and media (B) were harvested separately and analyzed by SDS-PAGE followed by Western blotting with polyclonal L1 antibody. In panel C, the Western blot of the cell lysate was probed with antibody to glyceraldehyde 3-phosphate dehydrogenase as a loading control. Proteins were detected by chemiluminescence. Lanes: 1, pL1op; 2, psL1op; 3, pL1optr; 4, psL1optr; 5, empty vector; 6, lysate from VACV-infected cells. The positions and masses in kDa of marker proteins are shown on the left.

Mentions: Additional constructs were made by truncating the genes encoding L1op and sL1op at amino acid 185 in order to remove the C-terminal transmembrane domain. Analysis of cell extracts by SDS-PAGE and Western blotting indicated that the truncated L1op (L1optr) migrated as a single band, more rapidly that the authentic L1 made by VACV (Figure 4A). The truncated form with a signal peptide (sL1optr) migrated as two bands corresponding to uncleaved and cleaved signal peptide forms (Figure 4A). The cleaved form of sL1optr was also present in the medium with only a small amount of the uncleaved form, whereas neither the truncated form without a signal peptide nor the full-length form with a signal peptide were secreted into the medium (Figure 4B). In Fig. 4C, the Western blot of the cell lysate was probed with antibody to glyceraldehyde 3-phosphate dehydrogenase as a loading control.


Engineering the vaccinia virus L1 protein for increased neutralizing antibody response after DNA immunization.

Shinoda K, Wyatt LS, Irvine KR, Moss B - Virol. J. (2009)

Expression of truncated L1 proteins detected by Western blotting. BS-C-1 cells were transfected with plasmids and cells (A) and media (B) were harvested separately and analyzed by SDS-PAGE followed by Western blotting with polyclonal L1 antibody. In panel C, the Western blot of the cell lysate was probed with antibody to glyceraldehyde 3-phosphate dehydrogenase as a loading control. Proteins were detected by chemiluminescence. Lanes: 1, pL1op; 2, psL1op; 3, pL1optr; 4, psL1optr; 5, empty vector; 6, lysate from VACV-infected cells. The positions and masses in kDa of marker proteins are shown on the left.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2654435&req=5

Figure 4: Expression of truncated L1 proteins detected by Western blotting. BS-C-1 cells were transfected with plasmids and cells (A) and media (B) were harvested separately and analyzed by SDS-PAGE followed by Western blotting with polyclonal L1 antibody. In panel C, the Western blot of the cell lysate was probed with antibody to glyceraldehyde 3-phosphate dehydrogenase as a loading control. Proteins were detected by chemiluminescence. Lanes: 1, pL1op; 2, psL1op; 3, pL1optr; 4, psL1optr; 5, empty vector; 6, lysate from VACV-infected cells. The positions and masses in kDa of marker proteins are shown on the left.
Mentions: Additional constructs were made by truncating the genes encoding L1op and sL1op at amino acid 185 in order to remove the C-terminal transmembrane domain. Analysis of cell extracts by SDS-PAGE and Western blotting indicated that the truncated L1op (L1optr) migrated as a single band, more rapidly that the authentic L1 made by VACV (Figure 4A). The truncated form with a signal peptide (sL1optr) migrated as two bands corresponding to uncleaved and cleaved signal peptide forms (Figure 4A). The cleaved form of sL1optr was also present in the medium with only a small amount of the uncleaved form, whereas neither the truncated form without a signal peptide nor the full-length form with a signal peptide were secreted into the medium (Figure 4B). In Fig. 4C, the Western blot of the cell lysate was probed with antibody to glyceraldehyde 3-phosphate dehydrogenase as a loading control.

Bottom Line: Therefore, safer DNA and protein vaccines are being investigated.Addition of a signal sequence to the N-terminus of L1 increased cell surface expression as shown by confocal microscopy and flow cytometry of transfected cells.Removal of the transmembrane domain led to secretion of L1 into the medium.

View Article: PubMed Central - HTML - PubMed

Affiliation: Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Maryland 20892-3210, USA. shinodak@niaid.nih.gov

ABSTRACT

Background: The licensed smallpox vaccine, comprised of infectious vaccinia virus, has associated adverse effects, particularly for immunocompromised individuals. Therefore, safer DNA and protein vaccines are being investigated. The L1 protein, a component of the mature virion membrane that is conserved in all sequenced poxviruses, is required for vaccinia virus entry into host cells and is a target for neutralizing antibody. When expressed by vaccinia virus, the unglycosylated, myristoylated L1 protein attaches to the viral membrane via a C-terminal transmembrane anchor without traversing the secretory pathway. The purpose of the present study was to investigate modifications of the gene expressing the L1 protein that would increase immunogenicity in mice when delivered by a gene gun.

Results: The L1 gene was codon modified for optimal expression in mammalian cells and potential N-glycosylation sites removed. Addition of a signal sequence to the N-terminus of L1 increased cell surface expression as shown by confocal microscopy and flow cytometry of transfected cells. Removal of the transmembrane domain led to secretion of L1 into the medium. Induction of binding and neutralizing antibodies in mice was enhanced by gene gun delivery of L1 containing the signal sequence with or without the transmembrane domain. Each L1 construct partially protected mice against weight loss caused by intranasal administration of vaccinia virus.

Conclusion: Modifications of the vaccinia virus L1 gene including codon optimization and addition of a signal sequence with or without deletion of the transmembrane domain can enhance the neutralizing antibody response of a DNA vaccine.

Show MeSH
Related in: MedlinePlus