Limits...
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.

Show MeSH

Related in: MedlinePlus

L1 binding and neutralizing antibodies in sera of mice immunized with pL1op and psL1op. (A) Mice (n = 5) received empty vector, pL1op or psL1op by gene gun on day 0 and after 3 and 6 weeks. Mice were bled at 3, 6 and 8 weeks after the first immunization. Antibody binding to L1 was determined by ELISA. Arrows point to days of immunization. (B) Neutralizing antibodies (IC50) were measured at 8 weeks using a flow cytometry assay.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2654435&req=5

Figure 3: L1 binding and neutralizing antibodies in sera of mice immunized with pL1op and psL1op. (A) Mice (n = 5) received empty vector, pL1op or psL1op by gene gun on day 0 and after 3 and 6 weeks. Mice were bled at 3, 6 and 8 weeks after the first immunization. Antibody binding to L1 was determined by ELISA. Arrows point to days of immunization. (B) Neutralizing antibodies (IC50) were measured at 8 weeks using a flow cytometry assay.

Mentions: Mice were inoculated with plasmids expressing L1op and sL1op to determine whether the different levels of expression translated into higher antibody responses. Three DNA immunizations were administered by gene gun at three-week intervals. L1 binding antibodies were detected in the sera at 3 weeks after the first immunization with L1op or sL1op, however the latter had a 16-fold higher titer (Figure 3A). In both groups of immunized mice, the titers rose after each successive immunization but the difference narrowed so that it was about 4-fold after the second and third immunizations (Figure 3A).


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)

L1 binding and neutralizing antibodies in sera of mice immunized with pL1op and psL1op. (A) Mice (n = 5) received empty vector, pL1op or psL1op by gene gun on day 0 and after 3 and 6 weeks. Mice were bled at 3, 6 and 8 weeks after the first immunization. Antibody binding to L1 was determined by ELISA. Arrows point to days of immunization. (B) Neutralizing antibodies (IC50) were measured at 8 weeks using a flow cytometry assay.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: L1 binding and neutralizing antibodies in sera of mice immunized with pL1op and psL1op. (A) Mice (n = 5) received empty vector, pL1op or psL1op by gene gun on day 0 and after 3 and 6 weeks. Mice were bled at 3, 6 and 8 weeks after the first immunization. Antibody binding to L1 was determined by ELISA. Arrows point to days of immunization. (B) Neutralizing antibodies (IC50) were measured at 8 weeks using a flow cytometry assay.
Mentions: Mice were inoculated with plasmids expressing L1op and sL1op to determine whether the different levels of expression translated into higher antibody responses. Three DNA immunizations were administered by gene gun at three-week intervals. L1 binding antibodies were detected in the sera at 3 weeks after the first immunization with L1op or sL1op, however the latter had a 16-fold higher titer (Figure 3A). In both groups of immunized mice, the titers rose after each successive immunization but the difference narrowed so that it was about 4-fold after the second and third immunizations (Figure 3A).

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