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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

Cell surface expression of modified L1 proteins determined by confocal microscopy and flow cytometry. (A) BS-C-1 cells were transfected with empty vector, pL1op, or psL1op and stained with anti-L1 mAb (7D11) followed by anti-mouse IgG FITC and viewed by confocal microscopy. Upper panel shows confocal fluorescent images and the lower panel shows a merge of confocal fluorescent and differential interference contrast images. (B) BS-C-1 cells were transfected as in panel A. After 24 h, non-permeabilized cells were incubated with MAb 7D11 followed by anti-mouse IgG antibody conjugated to fluorescein isothiocyanate, fixed with paraformaldehyde and analyzed by flow cytometry with gating on L1 positive cells.
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Figure 2: Cell surface expression of modified L1 proteins determined by confocal microscopy and flow cytometry. (A) BS-C-1 cells were transfected with empty vector, pL1op, or psL1op and stained with anti-L1 mAb (7D11) followed by anti-mouse IgG FITC and viewed by confocal microscopy. Upper panel shows confocal fluorescent images and the lower panel shows a merge of confocal fluorescent and differential interference contrast images. (B) BS-C-1 cells were transfected as in panel A. After 24 h, non-permeabilized cells were incubated with MAb 7D11 followed by anti-mouse IgG antibody conjugated to fluorescein isothiocyanate, fixed with paraformaldehyde and analyzed by flow cytometry with gating on L1 positive cells.

Mentions: Cell surface expression of L1op and sL1op were analyzed by confocal microscopy and flow cytometry of unpermeabilized cells using MAb 7D11, which recognizes correctly folded and disulfide bonded L1 [21,34]. Confocal microscopic analysis indicated that cells expressing sL1op were more frequent and stained more brightly than those expressing L1op (Figure 2A). This impression was supported by more quantitative flow cytometry experiments (Figure 2B). In three separate experiments the mean fluorescence intensity of cell surface L1 expression by sL1op was 2.3, 2.9 and 3.4 times higher than by L1op.


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)

Cell surface expression of modified L1 proteins determined by confocal microscopy and flow cytometry. (A) BS-C-1 cells were transfected with empty vector, pL1op, or psL1op and stained with anti-L1 mAb (7D11) followed by anti-mouse IgG FITC and viewed by confocal microscopy. Upper panel shows confocal fluorescent images and the lower panel shows a merge of confocal fluorescent and differential interference contrast images. (B) BS-C-1 cells were transfected as in panel A. After 24 h, non-permeabilized cells were incubated with MAb 7D11 followed by anti-mouse IgG antibody conjugated to fluorescein isothiocyanate, fixed with paraformaldehyde and analyzed by flow cytometry with gating on L1 positive cells.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Cell surface expression of modified L1 proteins determined by confocal microscopy and flow cytometry. (A) BS-C-1 cells were transfected with empty vector, pL1op, or psL1op and stained with anti-L1 mAb (7D11) followed by anti-mouse IgG FITC and viewed by confocal microscopy. Upper panel shows confocal fluorescent images and the lower panel shows a merge of confocal fluorescent and differential interference contrast images. (B) BS-C-1 cells were transfected as in panel A. After 24 h, non-permeabilized cells were incubated with MAb 7D11 followed by anti-mouse IgG antibody conjugated to fluorescein isothiocyanate, fixed with paraformaldehyde and analyzed by flow cytometry with gating on L1 positive cells.
Mentions: Cell surface expression of L1op and sL1op were analyzed by confocal microscopy and flow cytometry of unpermeabilized cells using MAb 7D11, which recognizes correctly folded and disulfide bonded L1 [21,34]. Confocal microscopic analysis indicated that cells expressing sL1op were more frequent and stained more brightly than those expressing L1op (Figure 2A). This impression was supported by more quantitative flow cytometry experiments (Figure 2B). In three separate experiments the mean fluorescence intensity of cell surface L1 expression by sL1op was 2.3, 2.9 and 3.4 times higher than by L1op.

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