Christopher O. Anjili

Vervet monkeys vaccinated with killed Leishmania major parasites and interleukin-12 develop a type 1 immune response but are not protected against challenge infection

ABSTRACT Leishmania major is a protozoan parasite that causes chronic cutaneous lesions that often leave disfiguring scars. Infections in mice have demonstrated that leishmanial vaccines that include interleukin-12 (IL-12) as an adjuvant are able to induce protective immunity. In this study, we assessed the safety, immunopotency, and adjuvant potential of two doses of IL-12 when used with a killed L. major vaccine in vervet monkeys. The induction of cell-mediated immunity following vaccination was determined by measuring delayed-type hypersensitivity, in vitro lymphocyte proliferation, and gamma interferon (IFN-γ) production. Protection was assessed by challenging the animals with L. major parasites and monitoring the course of infection. At low doses of IL-12 (10 μg), a small increase in the parameters of cell-mediated immunity was observed, relative to those in animals that received antigen without IL-12. However, none of these animals were protected against a challenge infection. At higher doses of IL-12 (30 μg), a substantial increase in Leishmania-specific immune responses was observed, and monkeys immunized with antigen and IL-12 exhibited an IFN-γ response that was as great as that in animals that had resolved a primary infection and were immune. Nevertheless, despite the presence of correlates of protection, the disease course was only slightly altered, and protection was low compared to that in self-cured monkeys. These data suggest that protection against leishmaniasis may require more than the activation of Leishmania-specific IFN-γ-producing T cells, which has important implications for designing a vaccine against leishmaniasis.

Vaccination of vervet monkeys against cutaneous leishmaniosis using recombinant Leishmania 'major surface glycoprotein' (gp63).

Vervet monkeys (Cercopithicus aethiops) were shown to give a positive delayed-type hypersensitivity (DTH) reaction to gp63, a major surface glycoprotein of Leishmania parasites, and also produce antibodies to the molecule following a triple vaccination with a total dose of 150 micrograms of recombinant gp63 mixed with Bacille Calmette Guerin (BCG). However, peripheral blood leucocytes (PBL) from these animals neither proliferated nor produced any interferon-gamma (IFN-gamma) following in vitro stimulation with the antigen. Analysis of lymphocyte subsets following vaccination did not reveal any striking phenotypic alteration of cellular sub-populations in PBL. When vaccinated animals were rechallenged, via the needle, with virulent Leishmania major promastigotes containing salivary gland extracts from vector sandflies, only partial protection was achieved. We concluded from these studies that rgp63 produced in Escherichia coli is a safe vaccine molecule which gives only partial protection following vaccination in the vervet monkey host. The molecule requires further improvement for vaccine and/or immunodiagnosis application.

The chemotactic effect of Phlebotomus duboscqi (diptera: psychodidae) salivary gland lysates to murine monocytes

The possibility that salivary gland lysates of Phlebotomus duboscqi are able to attract vertebrate monocytes was investigated. In vitro studies showed that salivary gland lysates of P. duboscqi, the vector of Leishmania major in Kenya, are chemotactic to mouse peritoneal monocytes. This attraction of monocytes by vector salivary gland lysates may form part of the mechanisms through which sandfly saliva ensures successful parasitization of macrophages in a susceptible host by Leishmania parasites.

IFN-gamma and delayed-type hypersensitivity are associated with cutaneous leishmaniasis in vervet monkeys following secondary rechallenge with Leishmania major.

IFN‐γ levels and delayed‐type hypersensitivity (DIM) responses were evaluated in vervet monkeys, following secondary infection with leishmania major (L. major). The animals had previously been vaccinated with leishmanial antigen, exposed to a primary infection and allowed to self‐cure. Supernatants of peripheral blood mononuclear cell (PBMC) cultures, stimulated with either L. major antigen or Concanavalin A (Con A), were examined for the presence of IFN‐γ in a double sandwich enzyme‐linked immunosorbent ASSAY (ELISA). Significant levels of IFN‐γ were detected during active disease and following Self‐cure in both antigen and Con A supernatants Higher levels of IFN‐y were, however, present during active disease as compared with after self‐cure. Positive and strong DTH responses were elicited in all experimental animals, following intradermal injection of fixed promastigotes (5×107/animal) before rechallenge, during active infection and following self‐cure. Again, strongest DTH responses were obtained during active infection as compared with the other sampling point

Experimental infection of domestic goats with Leishmania major through bites of infected Phlebotomus duboscqi and needle inoculation of culture-derived promastigotes.

Anjili, C.O., Olobo, J.O., Mbati, P.A., Robert, L. and Githure, J.I., 1994. Experimental infection of domestic goats with Leishmania major through bites of infected Phlebotomus duboscqi and needle inoculation of culture-derived promasfigotes. Veterinary Research Communications, 18 (4), 301-305

EXPERIMENTAL INFECTION OF DOMESTIC SHEEP WITH CULTURE-DERIVED LEISHMANIA DONOVANI PROMASTIGOTES

Domestic sheep were intradermally inoculated with culture-derived stationary phase Leishmania donovani promastigotes. Sampling of site of inoculation, liver and spleen for 244 days showed that this parasite can stay alive in the skin for up to 28 days post-inoculation. Apart from pyrexia that was evident in all the animals for 42 days, no other symptoms of kala-azar were seen. No parasites were recovered from the visceral organs throughout the sampling period, suggesting that sheep are not susceptible to infection with L. donovani. It is therefore unlikely that sheep can be synanthropic reservoirs for this parasite.

The effect of immune sera from hamsters immunized with sandfly gut and whole body extract antigens on the fecundity and mortality of Phlebotomus duboscqi (Diptera: Psychodidae)

Phlebotomus duboscqi were fed on hamsters previously immunized with different concentrations of homogenized crude sandfly gut antigen and supernatant obtained from whole body extract. The humoral response in the rodents was quantified at different times post-immunization by the enzyme-linked immunosorbent assay. Sandflies were fed on either immunized or saline control hamsters and the effect of the blood meals on sandfly feeding, survival and fecundity was investigated. The humoral response in immunized hamsters as measured by the presence of P. duboscqi-specific IgG antibodies was significantly greater (P < 0.05) as compared to the controls. This difference was noted in sera collected on 15 and 25 days post-immunization. Sandflies fed on immunized hamsters had a significantly higher mortality (P < 0.05) and decreased egg production (P < 0.05) than those fed on unimmunized control hamsters.

A simple method for maintaining, detecting and recovering virulent Leishmania donovani in hamsters

The ability of hamsters (Mesocricetus auratus) to retain amastigotes of Leishmania donovani at cutaneous sites was examined. Following intradermal inoculation of L. donovani stationary phase culture promastigotes in fore and hind footpads, nasal area and belly skin, cultures of aspirates taken fortnightly from these sites showed that amastigotes can survive in the skin for up to 10 months without visceralizing. Hairless cutaneous sites were better at retaining L. donovani amastigotes than the hairy belly skin. L. donovani promastigotes cultivated from aspirates of sites of inoculation were highly virulent. The skin is suggested as one of the sites where viscerotropic L. donovani can remain cryptic for a long time before the infection either visceralizes or is aborted. Skins of hamsters when inoculated intradermally can serve as an easy site for maintaining, detecting and recovering virulent L. donovani without killing the hamster.

F1 Cross-Breed Between Susceptible BALB/c and Resistant Swiss mice Infected with Leishmania major Exhibit an Intermediate Phenotype for Lesion Sizes and Type 1 Cytokines but Show Low Level of Total IgG Antibodies

Our current understanding of the host immune response during leishmaniases largely derives from studies performed in mice due to the intrusive techniques required to study infected human patients. Swiss mice are highly resistant to Leishmania infections in concordance with observed response in humans, while BALB/c mice indicate a high‐susceptibility phenotype. Developing a cross‐breed between BALB/c and Swiss mice may have important consequences on disease development, immune responses and parasite killing, as yet, response of the cross‐breed to Leishmania infection is superficial. The aim of the present study was to determine disease course and immune responses in F1 cross‐breed between BALB/c and Swiss albino mice infected with L. major. Three mice groups were infected intradermally with stationary‐phase L. major parasites with parental strains (BALB/c and Swiss albino) as controls. Lesion development was monitored weekly for 8 weeks and monocyte chemotactic protein (MCP‐1), macrophage inflammatory protein (MIP‐1α), interferon‐gamma (IFN‐γ) and IgG antibody quantified by enzyme‐linked immunosorbent assay. The data were analysed using one‐way analysis of variance and Tukey–Kramer test. Results indicated F1 mice having intermediate lesion sizes, type 1 cytokine levels and footpad parasite loads as compared to the parental strains. However, the F1 mice had low levels of IgG antibodies and parasite burden in the spleen. (P < 0.05). This study concludes that the F1 cross‐breed between resistant and susceptible mice may be used as a requisite model to study the role of genetics in leishmaniases and perhaps other intracellular parasites.