Elsevier

Parasitology International

Volume 63, Issue 6, December 2014, Pages 826-834
Parasitology International

Short-term protection conferred by Leishvacin® against experimental Leishmania amazonensis infection in C57BL/6 mice

https://doi.org/10.1016/j.parint.2014.07.010Get rights and content

Highlights

  • We protected mice against Leishmania amazonensis infection with Leishvacin® and adjuvant.

  • Protection was dependent on IFN-γ and nitric oxide.

  • Protection was lost at later time points after challenge.

  • Loss of protection was characterized by increased lesion size and parasitism.

  • Loss of protection correlated with decreased levels of IFN-γ and IL-2.

Abstract

To date, there is no vaccine available against human leishmaniasis. Although some vaccination protocols can induce immunity in murine models, they fail to induce protection in humans. The reasons for that remain unclear. The aim of the present study was to characterize the changes in the pattern of the immune response during subcutaneous vaccination with Leishvacin® in mice. We also investigated whether IFN-γ and nitric oxide synthase are indispensable for the protection elicited by the vaccine. C57BL/6 WT vaccinated mice showed smaller lesions and fewer numbers of parasites in footpads until 8 weeks post-infection. Up to this time, they produced higher levels of IFN-γ, IL-2, IL-4, IL-17A and IL-10 and higher specific antibody response than control non-vaccinated mice. Moreover, we showed that IFN-γ, most likely by induction of iNOS expression, is essential for immunity. However, after 12 weeks of infection, we observed loss of difference in lesion size and parasite burden between the groups. Loss of resistance was associated with the disappearance of differences in cytokine patterns between vaccinated and control mice, but not of antibody response, which remained different until a later time of infection. The reversal of resistance to L. amazonensis could not be explained by upregulation of regulatory cytokines. Our data point to a subversion of the host immune response by L. amazonensis even when a protective response was previously induced.

Introduction

Leishmaniasis is a disease with a spectrum of clinical manifestations, depending on the species of Leishmania and the state of the host's immunity. Clinical manifestations include localized cutaneous leishmaniasis, mucocutaneous leishmaniasis, diffuse cutaneous leishmaniasis and the systemic form, visceral leishmaniasis. This disease is a relevant public health problem, affecting about 90 countries in the world. Statistical studies have estimated that there are 0.9–1.6 million new cases per year of cutaneous and visceral leishmaniasis [1], [2]. To this day, there is no vaccine available against leishmaniasis and the traditional treatment is based on pentavalent antimonials, which have been associated with antimony-resistant strains of Leishmania and toxicity [3], [4]. Also, prophylactic measures for cutaneous leishmaniasis are ineffective. Therefore, the development of a vaccine would be the most effective measure to eliminate this disease worldwide [5]. Mouse models, albeit useful, are, however, not a completely accurate reproduction of the human disease [6]. Nevertheless, experimental models, especially mouse models, have been the startpoint of choice to test for the efficacy of vaccines.

Resistance to Leishmania major, which causes cutaneous leishmaniasis in the Old World, is mediated by Th1 immune responses. IFN-γ and TNF-α are important mediators that induce NO production by macrophages, which are consequently able to kill the parasite, as evidenced in experimental models [7]. Therefore, protocols of vaccination against some species of Leishmania aim at inducing polarization to Th1 responses. Some studies have found a correlation between higher production of IFN-γ and protection induced by vaccination [8], [9]. However, other studies have demonstrated that increased production of IFN-γ was not enough to induce immunity [10] or was not confirmed to be related to protection [11], [12]. On the other hand, immune response to Leishmania amazonensis infection, which is responsible for different clinical manifestations such as localized cutaneous and diffuse cutaneous leishmaniasis in the New World [13], [14], is different from that induced by L. major. Differently from L. major infection, susceptibility to L. amazonensis is more associated with a weak Th1 immune response than with a polarization to a Th2 immune response [15]. IFN-γ is, surprisingly, able to induce the proliferation of amastigote forms of the parasite in vitro [16]. Despite these differences, immunization protocols against L. amazonensis have the same aim: to induce the production of high levels of IFN-γ and polarization to a Th1 immune response. However, a few studies have shown that this kind of response in L. amazonensis infection does not lead to healing [17], [18].

Leishvacin® is a vaccine composed of killed promastigote forms of L. amazonensis strain PH8 (IFLA/BR/67/PH8). This vaccine has been shown to induce the production of IFN-γ by murine splenic cells and the production of anti-Leishmania IgG and IgM antibodies, as well as to promote proliferation of murine lymphocytes and to confer protection to C57BL/10 and C57BL/6 mice [19], [20], [21], [22]. Although this vaccine induces protection in mice, it fails to induce protective immunity against cutaneous leishmaniasis in humans [23], even with the administration of BCG as adjuvant [24].

The aim of this work was to characterize the immune responses induced by Leishvacin® during L. amazonensis infection in mice, through the identification of components of cellular and humoral immunity, and its persistence for several weeks after infection. We chose to use Leishvacin ® as part of an effort to elucidate the reasons for the contrast between its experimental success and the lack of protection in human trials. The use of adjuvant (Corynebacterium parvum) was necessary, since parasite antigens by themselves do not lead to protection in mice [25]. Furthermore, we analyzed the role of IFN-γ in this immunization protocol.

Section snippets

Animals

Four- to 6-week-old male and female C57BL/6 mice were obtained from the Bioterism Center (CEBIO), Instituto de Ciências Biológicas, Universidade Federal de Minas Gerais. IFN-γ −/− (B6.129S7-Ifngtm1Ts/J) and iNOS −/− (B6.129P2-Nos2tm1Lau/J) were purchased from The Jackson Laboratory (Glesnsville, NJ, USA). Mice were kept in conventional conditions with barriers, controlled light cycle and controlled temperature. Animals were fed a commercial diet for rodents (Labina-Purina, SP, Brazil) ad libitum.

Leishvacin® induces short-term protection against L. amazonensis

We followed the course of infection of vaccinated and control C57BL/6 mice challenged with 1 × 105 stationary phase promastigote forms of L. amazonensis in the hind footpad. Immunized mice showed smaller lesions when compared to control mice at 5 weeks of infection. Lesions continued to be smaller in immunized mice until 8 weeks post-infection, when the experiment was terminated (Fig. 1A). In addition to smaller lesions, vaccinated mice presented fewer parasites than the control group at the site

Discussion

Protection induced by Leishvacin® in mice has been previously shown. Immunization induced increased production of IFN-γ, although a positive correlation between protection and this increase in C57BL/10 mice was not found [12]. Although our group has shown, in C57BL/6 mice, that Leishvacin® was able to confer protection against challenge with L. amazonensis for up to 10 weeks post-infection, the mechanisms of protection were not clear [20], [21]. Immunization did not require CD8+ T cells and

Acknowledgments

The authors are indebted to Antonio Mesquita Vaz for expert animal care and to Biomm S.A. for providing Leishvacin®. This work was supported by FAPEMIG (grant numbers REC 32011/99 and EDT-468/07) and CNPq (grant numbers 304776/2009-2 and 571093/2008-6). MBHC, LMAS, LMS, RG, WLT, LCCA and LQV are CNPq fellows.

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