Development of a multivalent DNA vaccine against pathogenic leptospiral infection in laboratory animal model
Leptospirosis is neglected emerging zoonoses, occurring both in urban environments as well as rural regions worldwide. During occupational and recreational activities, humans that come in direct contact with infected animals or environments contaminated by the urine of reservoir animals are at a...
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| Format: | Thesis |
| Language: | English |
| Published: |
2017
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| Subjects: | |
| Online Access: | http://psasir.upm.edu.my/id/eprint/68599/1/FPV%202018%205%20IR.pdf |
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| Summary: | Leptospirosis is neglected emerging zoonoses, occurring both in urban
environments as well as rural regions worldwide. During occupational and
recreational activities, humans that come in direct contact with infected
animals or environments contaminated by the urine of reservoir animals are
at a higher risk of infection. Prevention is basically through improved
hygienic measures and rodent control which is very difficult to achieve
particularly in developing countries. Development of an effective vaccine
against leptospirosis remains a challenge. The heat-killed whole-cell vaccine
has been seen to produce some undesirable side effects which include pain,
nausea and fever, short term immunity and serovar restricted protection.
Multi-epitope peptide DNA vaccines are effective against some viruses and
they have recently been shown to have potential efficacy against some
bacterial diseases including leptospires. They are also known mimic antigen
processing and presentation during natural infection and can induce more
potent immunoreaction than the whole protein vaccine. The aim of this
study is to develop a multivalent DNA vaccine that can stimulate significant
antibody production that will aid the control and prevention of leptospirosis
using hamster model. Antigenic B cell epitopes from highly conserved
leptospiral genes LipL32, LipL41, OmpL1, Loa22 and LigA were predicted
using bioinformatics tools, assembled and linked using Gly-Ser spacer and
chemically synthesized. The vaccine constructs were composed of the
lipopolyscharide genes (LipL32, LipL41), the outer membrane porin and
outer membrane-like protein (OmpL1, Loa22), the immunoglobulin-like protein (LigA) and the final construct that is a combination of all the other
constructs. The synthesized DNA was cloned in a pBudCE4.1 mammalian
expression vector. The multivalent DNA(s) were expressed (invitro) and
confirmed by indirect immunofluorescence antibody test. The indirect
immunofluorescence test showed that the recombinant protein was
expressed in CHO-K1 cells and it reacted with antibodies against the V5 and
Myc epitope tags fused to the cloned expression plasmid vector at the 5’ end
of the constructs. To evaluate the efficacy of the DNA vaccines, 3-4 weeks old
golden Syrian hamsters were immunized with 150μg of the vaccine in equal
volume of incomplete Freund’s adjuvant. Subsequently, all the hamster
groups were challenged with L. interrogans Copenhageni Fiocruz strain
except the control group. Analysis of humoral immune response by
microscopic agglutination tests showed agglutinating antibodies production
(p<0.05) by the immunized hamsters and the antibodies were
immunologically cross-reactive with a range of reference pathogenic
leptospira strains including L. interrogans, L. borgpetersenii, L. weilii, L.
santorasai belonging to different serogroups. Similarly, all the vaccines were
able to stimulate secretion of neutralizing antibodies that prevented the
growth of a number of pathogenic leptospira species as indicated by the
invitro growth inhibition test using a panel of 19 serovars. Histopathological
evaluation of kidneys of challenged hamsters showed that the vaccine
significantly reduced kidney colonization by the leptospires as indicated by
the moderate to severe pathological lesions observed in the kidney tissues. In
conclusion, the multi-epitope chimeric DNA vaccine proves to be a
promising antigen for the prevention of renal colonization and urinary
shedding of the bacteria, which is the major contributing factor for the
persistence of the bacteria in susceptible animals as well as as the
environment. It also showed great potential for stimulation of cross-reactive
immunity against a broad range of pathogenic leptospira serovars. |
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