Successful vaccination against Leishmania donovani infection in Indian langur using alum-precipitated autoclaved Leishmania major with BCG

Abstract

Autoclaved Leishmania major (ALM) along with BCG, presently undergoing phase II clinical trial by WHO for its vaccine potential against cutaneous leishmaniasis, has been successfully evaluated in single and triple dose schedules against L. donovani in Indian langurs (Presbytis entellus). Encouraged with the results, another formulation alum-precipitated ALM (provided by WHO) along with BCG has been evaluated in this system. Eight monkeys were vaccinated with alum-precipitated ALM+BCG (1 mg of each per animal) while four were kept as unvaccinated controls. All were challenged with 100×10⁶ amastigotes i.v. on day 60 post vaccination. Parasitic assessment in splenic tissue was performed on day 45, 90 and 180 p.c. Initially, seven of the eight vaccinated monkeys developed infection (two to six amastigotes per 1000 cell nuclei), which resolved by day 180 p.c., while the eighth monkey had a parasite burden of 14 amastigotes per 1000 cell nuclei on day 45 p.c. and died on day 130 p.c. On the other hand, there was progressive infection in unvaccinated control animals and three out of four died between days 110 and 120 p.c., and one monkey, which had low parasite burden, died on day 178 p.c. Prior to challenge, there was an initial rise in antileishmanaial antibodies in the vaccinated group compared to the unvaccinated control group, which later came down to normal level, while it remained higher in the unvaccinated control group. An increasing pattern of antigen-specific proliferative responses and interferon-γ level to the two antigens — autoclaved L. donovani (ALD) and ALM — was observed in vaccinated monkeys throughout the experiment. There was a good correlation between parasite burden and IFN-γ level on days 90 and 180 p.c., indicating IFN-γ response as a sensitive parameter of immune status. The findings suggest alum-precipitated ALM+BCG as a potential vaccine against visceral leishmaniasis and warrants clinical trials.

Introduction

Kala-azar (visceral leishmaniasis) is endemic in parts of Eastern India. The available antileishmanial drugs are toxic, have serious side-effects and are associated with numerous relapses and there is an increasing incidence of drug resistance [1], [2]. Alternate strategies, such as immunoprophylaxis and vaccination, need to be developed for the control of this disease.

Infection with Leishmania donovani is associated with defective impaired cellular responses and a conserved humoral response [3], [4], [5], [6], [7], [8], [9], [10]. These altered immune responses are restored after successful chemotherapy [4], [9], [11], [12]. Individuals who recover from leishmaniasis are usually refractory to further infection, an observation which suggests that vaccination is a feasible prospect.

Considerable efforts have been made towards vaccine development for cutaneous leishmaniasis [13], [14], [15], [16], [17], [18] and killed promastigotes with or without BCG as an adjuvant has been evaluated in human trials [19], [20], [21], [22], [23], [24], [25]. The vaccines developed for cutaneous disease were found to be protective against visceral leishmaniasis (VL) caused by L. donovani in a monkey model [26]. However, multiple doses were required for complete protection.

For the widespread acceptance of a vaccine, it is desirable that maximum protection should be conferred with single dose schedule. There has been considerable effort to improve vaccine adjuvants in order to improve immunogenicity. Aluminium hydroxide gel [27] or water in oil emulsions [28] in combination with murine rIL-12 was found to enhance the protective immune response to HIV antigens in mice [27]. This was characterized by both increased antibody production and TH1 cytokine response. In a primate model of cutaneous leishmaniasis, killed promastigotes gave effective protection when used in conjunction with alum and rhIL-12 [29]. These animals showed a distinct Th1 response. Alum has been conventionally used as an adjuvant for a wide range of antibody-inducing vaccines. However, it retains antigen at the site of injection and the slow release of antigen may have beneficial effects on diseases needing a TH1 boost. Following the successful use of alum in primates, an alum-precipitated L. major vaccine was prepared by Dr R. Hashemi-Fesharki, Razi Institute of Iran, Tehran and was provided by TDR/WHO for its prophylactic assessment against VL. In this study, this vaccine has been evaluated in conjunction with BCG in a single dose schedule against L. donovani in the langur model.