DEVELOPMENT OF DNA VACCINE ENCODING MODIFIED GLYCOPROTEIN GENE AGAINST RABIES
DEVELOPMENT OF DNA VACCINE ENCODING MODIFIED GLYCOPROTEIN GENE AGAINST RABIES
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Date
2008-06
Authors
OSINUBI, Modupe Olubusola Vivian
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Abstract
A study aimed at the identification and antigenic characterization of existing rabies virus
strains in Nigeria with subsequent classification into their phylogenetic groups, and the
development of an immunogenic DNA vaccine that will protect against such major
circulating rabies variants in Nigeria was conducted. Plasmids encoding
glycoprotein/modified glycoprotein genes of bat and dog rabies variants, respectively,
were developed and tested for potency in mice using multiple administrations, single
administration and various routes. From the source of sample collection, Seller‟s staining
method was used to confirm rabies infection in the one hundred and sixty-five (165)
samples collected. However, only fifty-one (51) samples, which accounted for 30.9% of
the total samples, were found positive using direct fluorescent antibody (DFA) technique.
Also, fourteen (14) of the positive samples which were prepared according to mouse
inoculation test (MIT) technique and inoculated into pools of suckling mice did not kill
them and no signs suggestive of rabies were observed. It was inferred that due to the
deficiencies in preservation and transportation of the samples, the viability of the viruses
could not be sustained. Ribonucleic acid (RNA) was extracted from 13 (25.5%) of the
fifty-one (51) samples that were positive to DFA, using TRIZOL reagent. The integrity of
the extracted RNA remained intact (quantitation range of 1.5 to 2.4 at A260/A280).
Analysis of the phylogenetic relationships performed on the complete Nucleoprotein (N)
coding gene established them to belong to the genotype 1, comprising of typical dog rabies
virus originating from West Africa (Africa 2 group). Two samples appeared very closely
situated to Africa 3 group, which are classified to be related to African mongoose and
represents a wild life rabies variant. These two isolates could probably be suggested to be
related to the wild life rabies variants. In the multiple intramuscular (i.m) vaccination
schedules using the CDAG3, neutralizing antibody was detected in 60% of the vaccinated
mice as early as day 21 which gradually increased after booster shots were administered.
On challenge with street rabies variant belonging to the African 2, (West African dog
rabies virus), all the mice in this group survived. In the subcutaneous (s.c) multiple
administration group, neutralizing antibody was first noticed by day 62 after a booster had
been given though only 20% of these had neutralizing antibody level greater than the
protective level of 0.5 IU/ml recommended by WHO. Out Out of the 60% that showed traces of
antibody production, only 20% survived the challenge. While in the oral (p.o) route,
neutralizing antibody was detected in just 20% and all the mice died after challenge.
Multiple administration using s.c and p.o routes were found not protective in this research.
CDAG3 single dose i.m route showed neutralizing antibody by Day 30 with 80% of the
mice surviving the challenge. Similar observation was recorded for the single intra-dermal
route (i.d) that received only one tenth of the DNA vaccine. Results obtained from
WCBVG and controls using different administration regimens and routes did not show
neutralizing antibodies and did not protect the mice from challenge. It was concluded that
WCBVG does not protect against the street rabies strain. Statistical analysis using Fisher
exact test – two tailed probability, showed that the i.m and the i.d routes were statistically
significant compared with the control (P < 0.01). Also there was statistical significance
when comparing i.m route to the s.c routes in multiple administration (P < 0.05). All
mice that did not survive the challenge were tested for rabies antigen in their brains using the DFA and Direct Rapid Immunohistochemistry Technique (DRIT) and all were positive
to the tests conducted.
Description
A PhD DISSERTATION SUBMITTED TO THE POSTGRADUATE SCHOOL
AHMADU BELLO UNIVERSITY
IN PARTIAL FULFILLMENTOF THE REQUIRMENTS FOR THE AWARD OF
DOCTOR OF PHILOSOPHY IN VETERINARY MEDICINE
DEPARTMENT OF VETERINARY SURGERY AND MEDICINE,
FACULTY OF VETERINARY MEDICINE,
AHMADU BELLO UNIVERSITY,
ZARIA, NIGERIA
Keywords
DEVELOPMENT,, DNA VACCINE ENCODING MODIFIED GLYCOPROTEIN GENE,, RABIES.