Vaccine Against Different Forms Of Leishmaniasis Biology Essay

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Among various diseases that plague the tropical and sub tropical regions of the world, is Leishmaniasis which is caused by the protozoan parasite of the Leishmania genus. A vaccine against different forms of Leishmaniasis is feasible as well as essential and hence, this research project deals with the expression of gp63, a protein present on the Leishmania surface which is also a potential vaccine candidate, in mammalian cells.

Leishmaniasis is a major, vector-borne, tropical disease rampant in large areas of the tropics, subtropics and the Mediterranean basin which, incapacitates and kills thousands. This parasitic infection caused by the obligate, intracellular protozoan of the genus Leishmania (family Trypanosomatidae), has become an important global health problem as there are about 2 million new cases reported annually and 350 million people are at risk and yet there is no vaccine and few drugs that are effectual.

Humans are infected by at least 20 Leishmania species which are transmitted by various species of sandflies and cause a gamut of diseases which can be broadly divided into four classes: (i) visceral leishmaniasis (VL; also known as kala-azar); (ii) cutaneous leishmaniasis (CL); (iii) muco-cutaneous leishmaniasis (also known as espundia) and (iv) post-kala-azar dermal leishmaniasis (PKDL).

In case of CL, the macrophages present in the dermis of the infected individual are attacked by different species of Leishmania which result in ulceration and formation of nodules in the skin of the patient amongst other clinical presentations and prognoses. The lesions formed heal slowly in immmunocompetent individuals but leave disfiguring scars. Most cases of CL are caused by L. major, L. mexicana and L. vianna and there are three and four organisms in the L. mexicana and L. vianna subgenera, respectively that are known to infect human beings1.

The Leishmania parasite life cycle comprises of a promastigote and amastigote form. During transmission by female phlebotomine sandflies, the promastigote form of the parasite is internalized by the macrophages and dendritic cells present in the dermis of humans and it loses its flagella to transform into the amastigote form. The amastigotes then multiply within and destroy the host cell to further infect other phagocytic cells and they are transported to various parts of the body via the vascular and lymphatic systems to finally infect the liver, spleen and bone marrow.

Acute T-cell unresponsiveness to Leishmania antigens2 and the production of interleukin-10 (IL-10)3 contribute to the inability to control Leishmania infection. Thus, the host specific cell mediated immune (CMI) response is important in controlling the infection which is vindicated by the observation that malnutrition or co-infection with immunosuppressive diseases like HIV increases the risk of developing VL4,5,6. The control of Leishmanial infections has been observed to be mediated by Th1 type immune response7 and an acute VL leads to a decrease in levels of interferon (IFN)-γ and IL-12, the signature cytokines for Th1 immune response8. Thus, leishmanial antigens that primarily stimulate Th1 responses are considered as 'potential protective antigens' and those that principally stimulate a Th2 response to be associated with pathology.

Treatment of Leishmaniasis using drugs requires long-term medication, which is expensive and maybe toxic. Hence, developing a vaccine for leishmaniasis has been a goal for a century, but there are still no effective vaccines9. Amongst the various approaches, DNA vaccines are easily prepared, cheap and able to raise a range of T helper immune responses and hence, seem to be a viable option.

No effective vaccine has been developed successfully for this disease10 though the simple nature of the parasite lifecycle and the fact that strong immunity is acquired by cured individuals; indicate that it should be possible to develop a vaccine against Leishmaniasis. Vaccines based on live attenuated parasites has been tested on animal models with variable success but the major challenge remains to identify mechanisms to prevent undefined random genetic mutations and potential reversion to virulence. Thus, use of DNA vaccines is a feasible alternative, especially as they are able to induce type 1 and CD8+ cytotoxic T lymphocyte responses11,12. Previously, studies to examine the protective efficacy of DNA vaccines comprising of A2 and nucleoside hydrolase (NH) antigens have been done in mice infected with L. amazonensis and L. chagasi13. NH/A2 DNA immunized mice produced higher levels of IFN- in response to both specific recombinant proteins (rNH or rA2), but exhibited increased edema and parasite loads after L. amazonensis infection, as compared to A2 DNA immunized animals13. DNA vaccine encoding the LACK (Leishmania homolog of receptors for activated C kinase) antigen was found to elicit protective immunity in mice having L. major infection14 but although the LACK DNA vaccine stimulated a vigorous parasite-specific Th1 immune response (IFN- but not IL-4 production); it did not induce protection against cutaneous or systemic L. donovani challenge15. Thus, identification of an appropriate antigen and investigating its ability to elicit a protective immune response against VL is still to be achieved.

A plasmid harbouring the gp63 gene was the first DNA vaccine against Leishmaniasis16. In this study the vaccinated mice were partially protected from L. major infections and levels of IFN-γ but not IL-4 were increased. In another study employing vervet monkeys17, vaccination with recombinant gp63 provided partial protection against challenge with virulent L. major promastigotes. So, the success of these vaccines using the gp63 molecule needs to be further elucidated and to accomplish that the present study has been undertaken in which the gp63 gene is being cloned into the TOPO vector for further cloning into a mammalian expression vector leading to expression of gp63 in mammalian cells.


1) Bulking of VR1012-gp63 and PCR-II TOPO vector

2) Extraction of gp63 insert from VR1012-gp63 plasmid

3) Cloning of gp63 into PCR-II TOPO vector

Plan of Work

1) Bulking of VR1012-gp63 and PCR-II TOPO vector

The VR1012 vector containing the gp63 gene as insert and PCR-II TOPO vector will be bulked by transforming into bacterial cells (XL1Blue). will then be screened and DNA isolated from the positive clones by employing plasmid DNA extraction kits according to standard protocol. The purity of the DNA obtained will be ascertained by agarose gel electrophoresis and spectrophotometry.

2) Extraction of gp63 insert from VR1012-gp63 plasmid

Using the VR1012-gp63 plasmid DNA isolated above, polymerase chain reaction (PCR) would be set up using appropriate forward and reverse primers and PCR conditions to amplify the gp63 insert. This PCR product will be run on a low melting point (LMP) agarose gel and the insert band extracted using a kit for extraction of DNA from gel bands.

3) Cloning of gp63 into PCR-II TOPO vector

After ascertaining the purity and quantity of the DNA obtained from the PCR product, as stated above, a ligation will be set up of the gp63 insert with the PCR-II TOPO vector backbone. The ligated DNA will then be transformed into XL1B cells. Bacterial colonies obtained after blue-white selection, will confirm the presence of positive clones harbouring the gp63 gene.