Blaise Dondji

Heterologous Prime-Boost Vaccination with the LACK Antigen Protects against Murine Visceral Leishmaniasis

ABSTRACT This study reports the efficacy of a heterologous prime-boost vaccination using DNA and vaccinia viruses (Western Reserve [WR] virus and modified [attenuated] vaccinia virus Ankara [MVA]) expressing the LACK antigen (Leishmania homologue of receptors for activated C kinase) and an intradermal murine infection model employing Leishmania infantum. At 1 month postinfection, vaccinated mice showed high levels of protection in the draining lymph node (240-fold reduction in parasite burden) coupled with significant levels of gamma interferon (20 to 200 ng/ml) and tumor necrosis factor alpha/lymphotoxin (8 to 134 pg/ml). Significant but lower levels of protection (6- to 30-fold) were observed in the spleen and liver. Comparable levels of protection were found for mice boosted with either LACK-WR or LACK-MVA, supporting the use of an attenuated vaccinia virus-based vaccine against human visceral leishmaniasis.

Murine visceral leishmaniasis: IgM and polyclonal B‐cell activation lead to disease exacerbation

In visceral leishmaniasis, the draining LN (DLN) is the initial site for colonization and establishment of infection after intradermal transmission by the sand fly vector; however, little is known about the developing immune response within this site. Using an intradermal infection model, which allows for parasite visceralization, we have examined the ongoing immune responses in the DLN of BALB/c mice infected with Leishmania infantum. Although not unexpected, at early times post‐infection there is a marked B‐cell expansion in the DLN, which persists throughout infection. However, the characteristics of this response were of interest; as early as day 7 post‐infection, polyclonal antibodies (TNP, OVA, chromatin) were observed and the levels appeared comparable to the specific anti‐leishmania response. Although B‐cell‐deficient JhD BALB/c mice are relatively resistant to infection, neither B‐cell‐derived IL‐10 nor B‐cell antigen presentation appear to be primarily responsible for the elevated parasitemia. However, passive transfer and reconstitution of JhD BALB/c with secretory immunoglobulins, (IgM or IgG; specific or non‐specific immune complexes) results in increased susceptibility to L. infantum infection. Further, JhD BALB/c mice transgenetically reconstituted to secrete IgM demonstrated exacerbated disease in comparison to WT BALB/c mice as early as 2 days post‐infection. Evidence suggests that complement activation (generation of C5a) and signaling via the C5a receptor (CD88) is related to the disease exacerbation caused by IgM rather than cytokine levels (IL‐10 or IFN‐γ). Overall these studies indicate that polyclonal B‐cell activation, which is known to be associated with human visceral leishmaniasis, is an early and intrinsic characteristic of disease and may represent a target for therapeutic intervention.

Characterization of the immune response to Leishmania infantum recombinant antigens

Leishmaniases have a high prevalence in tropical countries. In order to improve existing diagnostic systems based on total Leishmania proteins, and to identify antigen candidates for vaccine development, an intensive search for the identification of antigens was performed using molecular biology techniques. In this study, the immune response to three L. infantum recombinant antigens was evaluated. Upon stimulation with KMP11, mononuclear cells from leishmaniasis patients produced high levels of IL-10, while a predominant IFN-gamma production could be observed in cultures stimulated with H2A and soluble Leishmania antigen. All the recombinant antigens induced very little IL-5. KMP11 decreased IFN-gamma production by 48% in cultures of peripheral blood mononuclear cells from cutaneous leishmaniasis patients who had been stimulated with soluble Leishmania antigen. Furthermore, antibodies to KMP11 were detected in the sera from all patients with visceral leishmaniasis and in the majority of the sera from patients with cutaneous leishmaniasis or individuals with asymptomatic L. chagasi infection. Thus, KMP11 is recognized by cells and sera of patients with different clinical forms of leishmaniasis, and KMP11, through IL-10 production, proved to be a potent antigen in modulating type 1 immune response.

Intradermal NKT cell activation during DNA priming in heterologous prime‐boost vaccination enhances T cell responses and protection against Leishmania

Heterologous prime‐boost vaccination employing DNA‐vaccinia virus (VACV) modality using the Leishmania homologue of receptors for activated C kinase (LACK) (p36) antigen has been shown to elicit protective immunity against both murine cutaneous and visceral leishmaniasis. However, DNA priming is known to have limited efficacy; therefore in the current study the effect of NKT cell activation using α‐galactosyl‐ceramide (αGalCer) during intradermal DNAp36 priming was examined. Vaccinated mice receiving αGalCer + DNAp36 followed by a boost with VVp36 appeared to be resolving their lesions and had at ten‐ to 20‐fold higher reductions in parasite burdens. NKT cell activation during αGalCer + DNAp36 priming resulted in higher numbers of antigen‐reactive effector CD4+ and CD8+ T cells producing granzyme and IFN‐γ, with lower levels of IL‐10. Although immunodepletion studies indicate that both CD4 and CD8 T cells provide protection in the vaccinated mice, the contribution of CD4+ T cells was significantly increased in mice primed with DNAp36 together with αGalCer. Notably 5 months after boosting, mice vaccinated with DNAp36 + αGalCer continued to show sustained and heightened T cell immune responses. Thus, heterologous prime‐boost vaccination using αGalCer during priming is highly protective against murine cutaneous leishmaniasis, resulting in the heightened activation and development of CD4 and CD8 T cells (effector and memory T cells).

Role for Nitric Oxide in Hookworm-Associated Immune Suppression

ABSTRACT Hookworm infection is a major cause of anemia and malnutrition in resource-poor countries. Human and animal studies suggest that infection with these intestinal nematodes is associated with impaired cellular immunity, characterized by reduced lymphocyte proliferation in response to both parasite and heterologous antigens. We report here data from studies aimed at defining mechanisms through which hookworms modulate the host cellular immune response. Splenocytes and mesenteric lymph node (MLN) cells from hamsters infected with Ancylostoma ceylanicum showed minimal proliferation in response to mitogen at days 20 and 30 postinfection (p.i.), with partial recovery noted at day 70 p.i. The proliferative capacity of enriched splenocyte T-cell preparations from infected animals following stimulation with hookworm antigens was partially restored in the presence of antigen-presenting cells from uninfected hamsters. Analysis by fluorescence-activated cell sorting revealed that hookworm infection is associated with reduced percentages of both CD4+ and surface immunoglobulin G-positive lymphocytes in the spleen and MLN cells. Splenocytes from infected hamsters also secreted more nitric oxide (NO) in culture than did those from naïve animals. Inhibition of NO secretion was associated with partial restoration of the proliferative capacity of splenocytes from infected animals in response to concanavalin A, suggesting a role for NO in mediating this effect. Together, these data demonstrate that hookworm infection is associated with impaired function of antigen-presenting cells and depletion of important lymphocyte subpopulations and also suggests a role for NO in parasite-induced immunosuppression.

Assessment of Laboratory and Field Assays of Sunlight-Induced Killing of Mosquito Larvae by Photosensitizers

Abstract The effectiveness of light-induced killing of mosquito larvae in the presence of photosensitizers was studied with larvae of Aedes aegypti (L.), Anopheles stephensi (Liston), and Culex quinquefasciatus Say grown in the laboratory and of Cx. quinquefasciatus grown under field conditions. Tested photosensitizers included xanthene, chlorin, and porphyrin derivatives. All the larvae were treated at the fourth instar. Preliminary laboratory experiments showed a light-induced lethal effect of Rose Bengal (RB) on three species of mosquito larvae. Compared with other photosensitizers, RB seemed to be more efficient at even lower concentration than chlorin (e6) and chlorophyllin on Ae. aegypti larvae. Among the four porphyrin derivatives, i.e., chloroquinoline tetraphenyl propioamidoporphine, tetraphenyl porphine tetrasulfonate, hematoporphyrin (HP), and tetraphenylporphine-propionic acid porphine, HP was the only effective photosensitizer on Ae. aegypti larvae. The best conditions for field tests using RB were conducted on Cx. quinquefasciatus in Bobo-Dioulasso, Burkina Faso. The mortality induced by RB varied from 80 to 96% obtained with unfiltered cesspit water to 0.4 to 6.7% in cesspits with a heavy load of organic materials, thus providing the basis for further developments of this technique under field conditions.

What determines the success or failure of intracellular cutaneous parasites? Lessons learned from leishmaniasis

Most parasitic skin infections are averted by very efficient strategies of preventing pathogen invasion. Innate immune cells such as mast cells, macrophages and dendritic cells are responsible for detecting parasites and for recruiting proinflammatory cells that help to contain and control the pathogen at sites of infection. This induces efficient adaptive immunity, which is crucially important for parasite control. Using the example of cutaneous leishmaniasis, we highlight how the skin utilizes different strategies to prevent skin infection and how containment of the infection to the skin site may reduce the harm that otherwise may result for the entire organism.

CD4+ T cells mediate mucosal and systemic immune responses to experimental hookworm infection.

Hookworm infection is associated with anaemia and malnutrition in many resource‐limited countries. Ancylostoma hookworms have previously been shown to modulate host cellular immune responses through multiple mechanisms, including reduced mitogen‐mediated lymphocyte proliferation, impaired antigen presentation/processing, and relative reductions in CD4+ T cells in the spleen and mesenteric lymph nodes. Syrian hamsters were depleted of CD4+ for up to 9 days following intraperitoneal injection (200 μg) of a murine anti‐mouse CD4 monoclonal IgG (clone GK1·5). CD4+ T‐cell‐depleted hamsters infected with the hookworm Ancylostoma ceylanicum exhibited a threefold higher mean intestinal worm burden and more severe anaemia than animals that received isotype control IgG. In addition, depletion of CD4+ T cells was associated with impaired cellular and humoral (serum and mucosal) immune responses to hookworm antigens. These data demonstrate an effector role for CD4+ T cells in hookworm immunity and disease pathogenesis. Ultimately, these studies may yield important insights into the relationship between intestinal nematode infections and diseases that are associated with CD4+ T‐cell depletion, including HIV.

Clinical features and epidemiology of cutaneous leishmaniasis and Leishmania major/HIV co-infection in Cameroon: results of a large cross-sectional study

Cutaneous leishmaniasis (CL) is endemic in Central Africa, including Cameroon. However, data on its prevalence and co-infection with HIV are scarce. Here we present the results of a large cross-sectional study reporting the prevalence, clinical features and species identification of CL and HIV co-infection in northern Cameroon. A total of 32 466 subjects were clinically screened for CL during a door-to-door survey, followed by parasitological diagnosis in the field laboratory. Amongst the subjects surveyed, 146 (0.4%) were diagnosed with active CL. Seven (4.8%) of these 146 CL patients tested positive for HIV-1 and/or HIV-2. The number of lesions per CL patient ranged from 1 to 20. Three of the five subjects with >10 active lesions were co-infected with HIV. In both CL and HIV co-infected subjects, three successful parasite isolates were identified as Leishmania major by PCR. This first report of L. major/HIV co-infection in Cameroon and Central Africa confirms the endemicity of CL in the region and highlights a worsened CL pathology in HIV co-infected individuals. These findings provide important data necessary for the development and implementation of successful control programmes against CL and HIV in this geographical area.

Previous exposure to a low infectious dose of Leishmania major exacerbates infection with Leishmania infantum in the susceptible BALB/c mouse

The geographic distribution of Leishmania major overlaps with several other species of Leishmania. This study seeks to examine what effect previous exposure to L. major has on the outcome of infection with Leishmania infantum, the agent of virulent visceral leishmaniasis. The L. major immune response is well characterized by a strong Th1 response leading to resolution and protection against subsequent re-infection. A contrasting Th2 immune response leads to disseminated disease, while the role Th17 cytokines may play in Leishmania infection is still being explored. The cytokine profile, antibody titer, and parasite burden were evaluated in the susceptible BALB/c mouse after L. infantum infection in either naïve mice or those previously infected with a low/self-healing dose of L. major. Only IL-4 expression in mice previously exposed to L. major was found to be significantly increased over controls, a cytokine with an ambiguous role in L. infantum infection. However, disease exacerbation, with a notably higher parasite burden, was observed in the L. major exposed mice compared to the L. infantum only. Cross-reactive antibodies were seen in both groups of infected mice regardless of their immune history. Studies have shown a role for opsonizing antibodies leading to increased disease in visceral leishmaniasis. We speculate that cross-reactive antibodies may be playing a role in augmenting visceral disease in mice with immunological memory to L. major.