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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

Effects of signal peptide and presence or absence of transmembrane domain on VACV neutralizing antibodies. Mice (n = 5) were immunized at 0 time and weeks 2, 4 and 6 with plasmids using a gene gun. Mice were bled at 2 weeks after each of the first three immunizations and 3 weeks after the last. Neutralizing activity was determined by flow cytometry. Arrows point to days of immunization
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Figure 5: Effects of signal peptide and presence or absence of transmembrane domain on VACV neutralizing antibodies. Mice (n = 5) were immunized at 0 time and weeks 2, 4 and 6 with plasmids using a gene gun. Mice were bled at 2 weeks after each of the first three immunizations and 3 weeks after the last. Neutralizing activity was determined by flow cytometry. Arrows point to days of immunization

Mentions: Mice were inoculated four times by gene gun with plasmids expressing L1optr and sL1optr as well as plasmids expressing full-length versions of L1 in order to compare their immunogenicities. Sera were collected at two weeks after each of the first three immunizations and three weeks after the fourth. Very low neutralizing titers were detected after the first immunization which were boosted after the second and third (Figure 5). The highest neutralizing titers were measured after the third immunization with psL1optr and psL1op, the plasmids containing L1 with signal peptide sequences. The drop in titers after the fourth immunization could be due to the absence of boosting and an additional week before assay.


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)

Effects of signal peptide and presence or absence of transmembrane domain on VACV neutralizing antibodies. Mice (n = 5) were immunized at 0 time and weeks 2, 4 and 6 with plasmids using a gene gun. Mice were bled at 2 weeks after each of the first three immunizations and 3 weeks after the last. Neutralizing activity was determined by flow cytometry. Arrows point to days of immunization
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Effects of signal peptide and presence or absence of transmembrane domain on VACV neutralizing antibodies. Mice (n = 5) were immunized at 0 time and weeks 2, 4 and 6 with plasmids using a gene gun. Mice were bled at 2 weeks after each of the first three immunizations and 3 weeks after the last. Neutralizing activity was determined by flow cytometry. Arrows point to days of immunization
Mentions: Mice were inoculated four times by gene gun with plasmids expressing L1optr and sL1optr as well as plasmids expressing full-length versions of L1 in order to compare their immunogenicities. Sera were collected at two weeks after each of the first three immunizations and three weeks after the fourth. Very low neutralizing titers were detected after the first immunization which were boosted after the second and third (Figure 5). The highest neutralizing titers were measured after the third immunization with psL1optr and psL1op, the plasmids containing L1 with signal peptide sequences. The drop in titers after the fourth immunization could be due to the absence of boosting and an additional week before assay.

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