Clin Exp Vaccine Res.  2014 Jul;3(2):202-211. 10.7774/cevr.2014.3.2.202.

Immunogenicity and efficacy of a plasmid DNA rabies vaccine incorporating Myd88 as a genetic adjuvant

• Ullas PT
• Desai A
• Madhusudana SN

• KMID: 1730625
• DOI: http://doi.org/10.7774/cevr.2014.3.2.202

Abstract

PURPOSE
Myeloid differentiation factor 88 (Myd88), a ubiquitous Toll-like receptor adaptor molecule, has been reported to play important roles in B cell responses to infections and vaccination. The present study evaluated the effects of genetic adjuvanting with Myd88 on the immune responses to a plasmid DNA rabies vaccine.
MATERIALS AND METHODS
Plasmids encoding rabies glycoprotein alone (pIRES-Rgp) or a fragment of Myd88 gene in addition (pIRES-Rgp-Myd) were constructed and administered intramuscularly or intrademally in Swiss albino mice (on days 0, 7, and 21). Rabies virus neutralizing antibody (RVNA) titres were estimated in the mice sera on days 14 and 28 by rapid fluorescent focus inhibition test. The protective efficacy of the constructs was evaluated by an intracerebral challenge with challenge virus standard virus on day 35.
RESULTS
Co-expression of Myd88 increased RVNA responses to pIRES-Rgp by 3- and 2-folds, following intramuscular and intradermal immunization, respectively. pIRES-Rgp protected 80% of the mice following intramuscular and intradermal immunizations, while pIRES-Rgp-Myd afforded 100% protection following similar administrations.
CONCLUSION
Genetic adjuvanting with Myd88 enhanced the RVNA responses and protective efficacy of a plasmid DNA rabies vaccine. This strategy might be useful for rabies vaccination of canines in the field, and needs further evaluation.

Keyword

Rabies; DNA vaccines; Adjuvants; Myeloid differentiation factor 88

MeSH Terms

Animals
Antibodies, Neutralizing
DNA*
Glycoproteins
Immunization
Mice
Myeloid Differentiation Factor 88
Plasmids*
Rabies
Rabies Vaccines*
Rabies virus
Toll-Like Receptors
Vaccination
Vaccines, DNA
Antibodies, Neutralizing
DNA
Glycoproteins
Myeloid Differentiation Factor 88
Rabies Vaccines
Toll-Like Receptors
Vaccines, DNA

Figure

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  Fig. 1 Scheme of plasmid constructs (linearized). (A) pIRES-Rgp. (B) pIRES-Rgp-Myd.

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  Fig. 2 Gel image showing polymerase chain reaction amplification of rabies virus glycoprotein gene. Lane 1, lambda DNA/EcoRI+HindIII marker; lane 2, ~1.6 kb band indicative of full-length glycoprotein gene.

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  Fig. 3 Gel image showing polymerase chain reaction amplification of Myd88 gene fragment. Lane 1, GeneRuler 100 bp DNA ladder; lane 2, 891 bp band indicative of Myd88 gene fragment.

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  Fig. 4 Images of BHK-21 cells fluorescently stained for expression of G and MyD88. Cells were grown in 24-well plates and mock-transfected (A, B) or transfected with a liposomal complex of pIRES-Rgp-Myd (C, D). Forty-eight hours later, the cells were stained sequentially with a murine anti-G antibody and a rabbit polyclonal anti-Myd88 antibody, followed by species-specific secondary IgG conjugated with FITC or TRITC, respectively. Note the lack of specific fluorescence in mock-transfected cells, and the apple-green fluorescence (indicative of expression of G) and orange-red fluorescence (indicative of MyD88 expression) in pIRES-Rgp-Myd transfected cells, following staining (images at a total magnification of ×400).

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  Fig. 5 Graph showing the survival rates of immunized mice following an intracerebral challenge with CVS-11 strain of rabies virus. Results show data compiled from challenge study in the intramuscular and intradermal immunization groups. NS, normal saline.

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