Leishmanization revisited: Immunization with a naturally attenuated cutaneous Leishmania donovani isolate from Sri Lanka protects against visceral leishmaniasis

Leishmaniasis is a neglected tropical disease caused by Leishmania protozoa and associated with three main clinical presentations: cutaneous, mucocutaneous and visceral leishmaniasis. Visceral leishmaniasis is the second most lethal parasitic disease after malaria and there is so far no human vaccine. Leishmania donovani is a causative agent of visceral leishmaniasis in South East Asia and Eastern Africa. However, in Sri Lanka, L. donovani causes mainly cutaneous leishmaniasis, while visceral leishmaniasis is rare. We investigate here the possibility that the cutaneous form of L. donovani can provide immunological protection against the visceral form of the disease, as a potential explanation for why visceral leishmaniasis is rare in Sri Lanka. Subcutaneous immunization with a cutaneous clinical isolate from Sri Lanka was significantly protective against visceral leishmaniasis in BALB/c mice. Protection was associated with a mixed Th1/Th2 response. These results provide a possible rationale for the scarcity of visceral leishmaniasis in Sri Lanka and could guide leishmaniasis vaccine development efforts.     

Leishmania genome analysis and high-throughput immunological screening identifies tuzin as a novel vaccine candidate against visceral leishmaniasis

Leishmaniasis is a neglected tropical disease caused by Leishmania species. It is a major health concern affecting 88 countries and threatening 350 million people globally. Unfortunately, there are no vaccines and there are limitations associated with the current therapeutic regimens for leishmaniasis. The emerging cases of drug-resistance further aggravate the situation, demanding rapid drug and vaccine development. The genome sequence of Leishmania, provides access to novel genes that hold potential as chemotherapeutic targets or vaccine candidates. In this study, we selected 19 antigenic genes from about 8000 common Leishmania genes based on the Leishmania major and Leishmania infantum genome information available in the pathogen databases. Potential vaccine candidates thus identified were screened using an in vitro high throughput immunological platform developed in the laboratory. Four candidate genes coding for tuzin, flagellar glycoprotein-like protein (FGP), phospholipase A1-like protein (PLA1) and potassium voltage-gated channel protein (K VOLT) showed a predominant protective Th1 response over disease exacerbating Th2. We report the immunogenic properties and protective efficacy of one of the four antigens, tuzin, as a DNA vaccine against Leishmania donovani challenge. Our results show that administration of tuzin DNA protected BALB/c mice against L. donovani challenge and that protective immunity was associated with higher levels of IFN-γ and IL-12 production in comparison to IL-4 and IL-10. Our study presents a simple approach to rapidly identify potential vaccine candidates using the exhaustive information stored in the genome and an in vitro high-throughput immunological platform.     

Characterization and immunogenicity in mice of recombinant influenza haemagglutinins produced in Leishmania tarentolae

The membrane displayed antigen haemagglutinin (HA) from several influenza strains were expressed in the Leishmania tarentolae system. This non-conventional expression system based on a parasite of lizards, can be readily propagated to high cell density (>10⁸ cells/mL) in a simple incubator at 26 °C. The genes encoding HA proteins were cloned from six influenza strains, among these being a 2009 A/H1N1 pandemic strain from swine origin, namely A/California/07/09(H1N1). Soluble HA proteins were secreted into the cell culture medium and were easily and successfully purified via a His-Tag domain fused to the proteins. The overall process could be conducted in less than 3 months and resulted in a yield of approximately 1.5–5 mg of HA per liter of biofermenter culture after purification. The recombinant HA proteins expressed by L. tarentolae were characterized by dynamic light scattering and were observed to be mostly monomeric. The L. tarentolae recombinant HA proteins were immunogenic in mice at a dose of 10 μg when administered twice with an oil-in-water emulsion-based adjuvant. These results suggest that the L. tarentolae expression system may be an alternative to the current egg-based vaccine production.     

Alum-precipitated autoclaved Leishmania major plus bacille Calmette-Guérrin, a candidate vaccine for visceral leishmaniasis: safety, skin-delayed type hypersensitivity response and dose finding in healthy volunteers

In a previous efficacy study, autoclaved Leishmania major (ALM) + bacille Calmette—Guérrin (BCG) vaccine was shown to be safe, but not superior to BCG alone, in protecting against visceral leishmaniasis. From June 1999 to June 2000, we studied the safety and immunogenicity of different doses of alum-precipitated ALM + BCG vaccine mixture administered intradermally to evaluate whether the addition of alum improved the immunogenicity of ALM. Twenty-four healthy adult volunteers were recruited and sequentially allocated to receive either 10 μg, 100 μg, 200 μg, or 400 μg of leishmanial protein in the alum-precipitated ALM + BCG vaccine mixture. Side effects were minimal for all doses and confined to the site of injection. All volunteers in the 10 μg, 100 μg, and 400 μg groups had a leishmanin skin test (LST) reaction of ? 5 mm by day 42 and this response was maintained when tested after 90 d. Only 1 volunteer out of 5 in the 200 μg group had a LST reaction of ? 5mm by day 42 and the reasons for the different LST responses in this group are unclear. This is the first time that an alum adjuvant with ALM has been in used in humans and the vaccine mixture was safe and induced a strong delayed type hypersensitivity (DTH) reaction in the study volunteers. On the basis of this study we suggest that 100 μg of leishmanial protein in the vaccine mixture is a suitable dose for future efficacy studies, as it induced the strongest DTH reaction following vaccination.     

A chimeric vaccine combined with adjuvant system induces immunogenicity and protection against visceral leishmaniasis in BALB/c mice

In Brazil, canine visceral leishmaniasis is an important public health problem due to its alarming growth. The high prevalence of infected dogs reinforces the need for a vaccine for use in prophylactic vaccination campaigns. In the present study, we evaluate the immunogenicity and protection of the best dose of Chimera A selected through the screening of cytokines production important in disease. BALB/c mice were vaccinated subcutaneously with three doses and challenged intravenously with 1 × 10⁷ L. infantum promastigotes. Spleen samples were collected to assess the intracellular cytokine profile production, T cell proliferation and parasite load. At first, three different doses of Chimera A (5 μg, 10 μg and 20 μg) were evaluated through the production of IFN-γ and IL-10 cytokines. Since the dose of 20 μg showed the best results, it was chosen to continue the study. Secondarily, Chimera A at dose of 20 μg was formulated with Saponin plus Monophosphoryl lipid A. Vaccination with Chimera A alone and formulated with SM adjuvant system was able to increase the percentage of the proliferation of specific T lymphocytes and stimulated a Th1 response with increased levels of IFN-γ, TNF-α and IL-2, and decreased of IL-4 and IL-10. The vaccine efficacy through real-time PCR demonstrated a reduction in the splenic parasite load in animals that received Chimera A formulated with the SM adjuvant system (92%). Additionally, we observed increased levels of nitric oxide in stimulated-culture supernatants. The Chimera A formulated with the SM adjuvant system was potentially immunogenic, being able to induce immunoprotective mechanisms and reduce parasite load. Therefore, the use of T-cell multi-epitope vaccine is promising against visceral leishmaniasis.     

Treatment of canine visceral leishmaniasis by the vaccine Leish-111f + MPL-SE

Immunotherapy of canine visceral leishmaniasis (CVL) may provide an alternative to both marginally effective chemotherapy and undesired euthanasia of infected dogs and could have a great impact not only on animal welfare, but also on control of human disease. Therefore, we examined the potential immunotherapeutic efficacy of the subunit vaccine Leish-111f + MPL-SE, which has undergone rigorous preclinical testing and been demonstrated safe in human clinical trials. Two separate trials were performed in Salvador, Brazil, to evaluate the vaccine for therapeutic efficacy against CVL caused by natural infection: an Open Trial and a Blinded Trial. In the Open Trial 59 dogs with clinically active CVL were sequentially allocated to four groups: group 1 received Leish-111f + MPL-SE; group 2 was treated with Glucantime; group 3 received a combination of the vaccine and Glucantime; and group 4 was given no treatment. At the 6-month assessment, the 13 non-treated dogs had either died or showed no clinical improvement. In contrast, most dogs in groups 1–3 showed initial improvement (100%, 80%, and 92%, respectively). Upon evaluation for a mean of 36 months after therapy, the following cure rates were observed: 75% for group 1 dogs (exact 95% confidence interval [CI] 43–95%), 64% for group 2 dogs (exact 95% CI 31–89%), and 50% for group 3 dogs (exact 95% CI 19–81%). Therapeutic efficacy of the Leish-111f + MPL-SE vaccine was reconfirmed in a subsequent Blinded Trial. The vaccine was effective for mild cases of CVL and was compromised in dogs with severe disease. Although further studies are required to understand mechanisms of action, the Leish-111f + MPL-SE vaccine is a promising tool to control VL in both dogs and humans.     

Antibiotic resistance free plasmid DNA expressing LACK protein leads towards a protective Th1 response against Leishmania infantum infection

Canine visceral leishmaniasis is a serious public health concern in the Mediterranean basin since dogs are the main Leishmania infantum reservoir. However, there is not a vaccination method in veterinary use in this area, and therefore the development of a vaccine against this parasite is essential for the possible control of the disease. Previous reports have shown the efficacy of heterologous prime-boost vaccination with the pCIneo plasmid and the poxvirus VV (both Western Reserve and MVA strains) expressing L. infantum LACK antigen against canine leishmaniasis. As pCIneo-LACK plasmid contains antibiotic resistance genes, its use as a profilactic method is not recommended. Hence, the antibiotic resistance gene free pORT-LACK plasmid is a more suitable tool for its use as a vaccine. Here we report the protective and immunostimulatory effect of the prime-boost pORT-LACK/MVA-LACK vaccination tested in a canine experimental model. Vaccination induced a reduction in clinical signs and in parasite burden in the liver, an induction of the Leishmania-specific T cell activation, as well as an increase of the expression of Th1 type cytokines in PBMC and target organs.     

Immunogenicity of candidate chimeric DNA vaccine against tuberculosis and leishmaniasis

Mycobacterium tuberculosis and Leishmania donovani are important intracellular pathogens, especially in Indian context. In India and other South East Asian countries, both these infections are highly endemic and in about 20% cases co-infection of these pathogens is reported. For both these pathogens cell mediated immunity plays most important role. The available treatment of these infections is either prolonged or cumbersome or it is ineffective in controlling the outbreaks and spread. Therefore, potentiation of a common host defense mechanism can be used to prevent both the infections simultaneously. In this study we have developed a novel chimeric DNA vaccine candidate comprising the esat-6 gene of M. tuberculosis and kinesin motor domain gene of L. donovani. After developing this novel chimera, its immunogenicity was studied in mouse model. The immune response was compared with individual constructs of esat-6 and kinesin motor domain. The results showed that immunization with chimeric DNA vaccine construct resulted in stronger IFN-γ and IL-2 response against kinesin (3012 ± 102 and 367.5 ± 8.92 pg/ml) and ESAT-6 (1334 ± 46.5 and 245.1 ± 7.72 pg/ml) in comparison to the individual vaccine constructs. The reciprocal immune response (IFN-γ and IL-2) against individual construct was lower (kinesin motor domain: 1788 ± 36.48 and 341.8 ± 9.801 pg/ml and ESAT-6: 867.0 ± 47.23 and 170.8 ± 4.578 pg/ml, respectively). The results also suggest that using the chimeric construct both proteins yielded a reciprocal adjuvant affect over each other as the IFN-γ production against chimera vaccination is statistically significant (p < 0.0001) than individual construct vaccination. From this pilot study we could envisage that the chimeric DNA vaccine construct may offer an attractive strategy in controlling co-infection of leishmaniasis and tuberculosis and have important implication in future vaccine design.     

Clostridium perfringens epsilon toxin mutant Y30A-Y196A as a recombinant vaccine candidate against enterotoxemia

Epsilon toxin (Etx) is a β-pore-forming toxin produced by Clostridium perfringens toxinotypes B and D and plays a key role in the pathogenesis of enterotoxemia, a severe, often fatal disease of ruminants that causes significant economic losses to the farming industry worldwide. This study aimed to determine the potential of a site-directed mutant of Etx (Y30A-Y196A) to be exploited as a recombinant vaccine against enterotoxemia. Replacement of Y30 and Y196 with alanine generated a stable variant of Etx with significantly reduced cell binding and cytotoxic activities in MDCK.2 cells relative to wild type toxin (>430-fold increase in CT₅₀) and Y30A-Y196A was inactive in mice after intraperitoneal administration of trypsin activated toxin at 1000× the expected LD₅₀ dose of trypsin activated wild type toxin. Moreover, polyclonal antibody raised in rabbits against Y30A-Y196A provided protection against wild type toxin in an in vitro neutralisation assay. These data suggest that Y30A-Y196A mutant could form the basis of an improved recombinant vaccine against enterotoxemia.     

Leishmania infantum-induced primary and challenge infections in rhesus monkeys (Macaca mulatta): a primate model for visceral leishmaniasis

Visceral leishmaniasis (VL) was experimentally induced in rhesus macaques (Macaca mulatta) by intravenously inoculating 2 x 10(7)amastigotes/kg of body weight of Leishmania infantum. The macaques developed a systemic disease showing characteristic features of human VL such as fever, diarrhoea, body weight loss, anaemia, hypergammaglobulinaemia and transient lymphocytosis, as well as lymph node, liver and/or spleen enlargement. Nine weeks after infection, one primate showed pronounced weight loss, became moribund and was euthanized. The necropsy findings included granulomas composed of parasite-containing macrophages, lymphocytes and plasma cells in the liver, spleen and lymph nodes. The remaining macaques had a sustained course of infection but developed a mild-to-moderate illness that subsequently showed evidence of self-cure. Of note, pathological findings included a typical cell-mediated immunity-induced granulomatous reaction that had an effect on the control of parasite replication. All infected monkeys responded with increased production of anti-Leishmania-specific IgG antibodies. Despite the fact that clinical resistance to L. infantum was not consistently associated with a parasite-specific cell-mediated immune response, drug-cured macaques from the primary infection acquired immunity to homologous re-infection. These findings point to the feasibility of using the L. infantum macaque model for pre-clinical evaluation of novel chemotherapeutics or vaccine candidates for human VL.     

Mucosal and systemic immunization with a novel attenuated pneumococcal vaccine candidate confer serotype independent protection against Streptococcus pneumoniae in mice

Despite the availability of effective vaccines, Streptococcus pneumoniae is still one of the major infectious diseases causing substantial morbidity and mortality in children under 5 years old. In this study, we demonstrate the protective efficacy of S. pneumoniae SPY1, a novel live attenuated vaccine strain against pneumococcal infection in murine models. This strain was characterized by defects in three important pneumococcal virulence factors including capsule, teichoic acids and pneumolysin. The lactate dehydrogenase assays and in vivo animal experiments demonstrated a significantly attenuated virulence and a reduced nasopharyngeal colonization for the SPY1 strain. We also show that mucosal and systemic immunization with the live SPY1 strain induced protective immune responses against pneumococci. Mucosal immunization with SPY1 offered better protection against colonization challenge with strains TIGR4 and serotype 19F than systemic SPY1 immunization. In invasive infection models, mucosal vaccination with the SPY1 strain conferred complete protection against D39 and clinical serotype 6B and 3 strains. Notably, intranasal vaccination with the SPY1 strain conferred superior protection against pneumococcal invasive disease compared with the commercial available vaccines. SPY1 strain was shown to elicit high levels of serotype-independent antibodies and a mixed cellular immune response. Besides, the SPY1 serum was able to passively protect mice against invasive challenge with D39 strain, indicating the protective effect of the antibody-mediated responses. Together, the SPY1 strain may be a promising live vaccine strain to protect pneumococcal infection.     

The emergence of concurrent HIV-1/AIDS and visceral leishmaniasis in Northeast Brazil

HIV has become increasingly prevalent in the Northeast region of Brazil where Leishmania infantum chagasi is endemic, and concurrent AIDS and visceral leishmaniasis (VL) has emerged. In this study, persons with HIV/AIDS and VL (n = 17) had a mean age of 37.3 years (range 29-53 years) compared with 12.5 years (1-80 years) for persons with VL alone (n = 2836). Males accounted for 88% of cases with concurrent VL and AIDS and 65% of those with VL alone. The mean CD4 count and antileishmanial antibody titre were lower and recurrence of VL and death were more likely with co-infection. Considering the prevalences of L.i. chagasi and HIV in the region, this may herald the emergence of an important public health problem.     

Recombinant attenuated Listeria monocytogenes vaccine expressing Francisella tularensis IglC induces protection in mice against aerosolized Type A F. tularensis

Fransicella tularensis, the causative agent of tularemia, is in the top category (Category A) of potential agents of bioterrorism. To develop a safer vaccine against aerosolized F. tularensis, we have employed an attenuated Listeria monocytogenes, which shares with F. tularensis an intracellular and extraphagosomal lifestyle, as a delivery vehicle for F. tularensis antigens. We constructed recombinant L. monocytogenes (rLm) vaccines stably expressing seven F. tularensis proteins including IglC (rLm/iglC), and tested their immunogenicity and protective efficacy against lethal F. tularensis challenge in mice. Mice immunized intradermally with rLm/iglC developed significant cellular immune responses to F. tularensis IglC as evidenced by lymphocyte proliferation and CD4+ and CD8+ T-cell intracellular expression of interferon gamma. Moreover, mice immunized with rLm/iglC were protected against lethal challenge with F. tularensis LVS administered by the intranasal route, a route chosen to mimic airborne infection, and, most importantly, against aerosol challenge with the highly virulent Type A F. tularensis SchuS4 strain.     

Generation of growth arrested Leishmania amastigotes: A tool to develop live attenuated vaccine candidates against visceral leishmaniasis

Visceral leishmaniasis (VL) is fatal if not treated and is prevalent widely in the tropical and sub-tropical regions of world. VL is caused by the protozoan parasite Leishmania donovani or Leishmania infantum. Although several second generation vaccines have been licensed to protect dogs against VL, there are no effective vaccines against human VL [1]. Since people cured of leishmaniasis develop lifelong protection, development of live attenuated Leishmania parasites as vaccines, which can have controlled infection, may be a close surrogate to leishmanization. This can be achieved by deletion of genes involved in the regulation of growth and/or virulence of the parasite. Such mutant parasites generally do not revert to virulence in animal models even under conditions of induced immune suppression due to complete deletion of the essential gene(s). In the Leishmania life cycle, the intracellular amastigote form is the virulent form and causes disease in the mammalian hosts. We developed centrin gene deleted L. donovani parasites that displayed attenuated growth only in the amastigote stage and were found safe and efficacious against virulent challenge in the experimental animal models. Thus, targeting genes differentially expressed in the amastigote stage would potentially attenuate only the amastigote stage and hence controlled infectivity may be effective in developing immunity. This review lays out the strategies for attenuation of the growth of the amastigote form of Leishmania for use as live vaccine against leishmaniasis, with a focus on visceral leishmaniasis.     

Display of Eimeria tenella EtMic2 protein on the surface of Saccharomyces cerevisiae as a potential oral vaccine against chicken coccidiosis

S. cerevisiae is generally regarded as safe and benign organism and its surface display system may be used as a unique eukaryotic expression system that is suitable for expressing eukaryotic antigen. In addition to the convenience of vaccine delivery, the yeast cell wall has been shown to enhance the innate immunity when immunized with the yeast live oral vaccine. In the present study, we expressed the chicken coccidian E. tenella EtMic2, a microneme protein, on the surface of the S. cerevisiae and evaluated it as a potential oral vaccine for chicken against E. tenella challenge. The protective efficacy against a homologous challenge was evaluated by body weight gains, lesion scores and fecal oocyst shedding. The results showed that the live oral vaccine can improve weight gains, reduced cecal pathology and lower oocyst fecal shedding compared with non immunized controls. In addition, the yeast oral vaccine could stimulate humoral as well as cell mediate immune responses. These results suggested that EtMic2 displayed on the cell surface of S. cerevisiae could be used as potential live vaccine against chicken coccidiosis.     

Saccharomyces cerevisiae as a vaccine against coccidioidomycosis

Disseminated coccidioidomycosis is a life-threatening infection. In these studies, we examined protection against systemic murine coccidioidomycosis by vaccination with heat-killed Saccharomyces cerevisiae (HKY). CD-1 mice received HKY subcutaneously or by oral gavage with or without adjuvants once weekly beginning 3 or 4 weeks prior to infection; oral live Saccharomyces was also studied. All HKY sc regimens were equivalent, prolonging survival (P ≤ 0.005) and reducing fungal burden versus controls. Oral live Saccharomyces, but not HKY, prolonged survival (P = 0.03), but did not reduce fungal burden. Survival of mice given HKY was equivalent to vaccination with formalin-killed spherules, but inferior in reduction of fungal burden. HKY was superior to a successful recombinant vaccine, PRA plus adjuvant. This novel heterologous protection afforded by HKY vaccination offers a new approach to a vaccine against coccidioidomycosis.     

Systematic analysis of attenuated Coxsackievirus expressing a foreign gene as a viral vaccine vector

Recombinant viruses expressing foreign antigens may provide a convenient vaccine vector capable of inducing preventative immunity. In this study, we explored the capacity of highly attenuated Coxsackievirus B3 (CVB3) to act as a recombinant vector to deliver foreign genes into experimental animals for the purpose of vaccination. The infectious cDNA of highly attenuated CVB3, YYFF, which has been reported previously (Vaccine 27:1974), was used to construct a recombinant YYFF cDNA (YYFF-HCV) by inserting a truncated form of hepatitis C virus (HCV) envelope protein E2 as an antigenic marker immediately upstream from the gene encoding the VP4 capsid protein. In YYFF-HCV-infected HeLa cells, HCV E2 expression was confirmed by immunoblotting and fluorescence microscopy. YYFF-HCV induced the production of antibodies and the cytotoxic T-lymphocyte (CTL) response to HCV E2 in the inoculated mice. Moreover, YYFF-HCV induced no inflammation in the virus-immunized mice. These results demonstrate that recombinant CVB3 expressing a foreign gene can act as a live vaccine vector capable of inducing humoral and cell-mediated immune responses directed against a foreign protein.     

Atypical American visceral leishmaniasis caused by disseminated Leishmania amazonensis infection presenting with hepatitis and adenopathy

Leishmania amazonensis is widely recognised as a cause of cutaneous leishmaniasis in Latin America, but it can also disseminate to produce atypical visceral leishmaniasis with hepatitis and lymphadenopathy. The patient, an 8-year-old Brazilian boy, presented with a febrile illness and hepatosplenomegaly, elevated liver enzymes and generalised adenopathy. Serological tests using antigens of L. chagasi, the typical cause of visceral leishmaniasis in Latin America, were inconclusive. Leishmania amazonensis was eventually isolated in a culture of a lymph node. The patient recovered fully after treatment with meglumine antimoniate. As this case illustrates, L. amazonensis produces a spectrum of disease that includes atypical American visceral leishmaniasis with evidence of hepatocellular injury and generalised lymphadenopathy.