To investigate the therapeutic potential of immunochemotherapy with cisplatin + 78 kDa + MPL‐A against Leishmania donovani in BALB/c mice

Leishmaniasis has recently garnered attention as one of the diseases ‘most neglected’ by drug research and development, as the current therapeutic modalities available for the patients are ridden with unacceptable toxicity due to high dosage of the drug, prolonged treatment schedules, resistance and prohibitive costs. A successful chemotherapy requires a restoration of immune response; therefore, we combined Leishmania‐specific 78 kDa antigen (with or without adjuvant MPL‐A) along with a novel drug cisplatin in infected BALB/c mice and did its comparative analysis with chemotherapy and immunotherapy alone. Animals that were treated with immunochemotherapy showed maximum curative potential as demonstrated by a marked reduction in parasite load. Delayed‐type hypersensitivity response to leishmanial antigens has been widely used to assess the level of host protection to the disease. An increased delayed‐type hypersensitivity (DTH) response was observed in animals given immunotherapy or chemotherapy or immunochemotherapy; however, maximum DTH response was observed in animals treated with cisplatin + 78 kDa + MPL‐A. These animals were also found to exhibit higher IgG2a levels greater cytokine (IFN‐γ and IL‐2) concentrations suggesting the generation of a strong Th1 type of immune response which is responsible for resolution of the disease.     

Cross‐protective efficacy from a immunogen firstly identified in Leishmania infantum against tegumentary leishmaniasis

Experimental vaccine candidates have been evaluated to prevent leishmaniasis, but no commercial vaccine has been proved to be effective against more than one parasite species. LiHyT is a Leishmania‐specific protein that was firstly identified as protective against Leishmania infantum. In this study, LiHyT was evaluated as a vaccine to against two Leishmania species causing tegumentary leishmaniasis (TL): Leishmania major and Leishmania braziliensis. BALB/c mice were immunized with rLiHyT plus saponin and lately challenged with promastigotes of the two parasite species. The immune response generated was evaluated before and 10 weeks after infection, as well as the parasite burden at this time after infection. The vaccination induced a Th1 response, which was characterized by the production of IFN‐γ, IL‐12 and GM‐CSF, as well as by high levels of IgG2a antibodies, after in vitro stimulation using both the protein and parasite extracts. After challenge, vaccinated mice showed significant reductions in their infected footpads, as well as in the parasite burden in the tissue and organs evaluated, when compared to the control groups. The anti‐Leishmania Th1 response was maintained after infection, being the IFN‐γ production based mainly on CD4⁺ T cells. We described one conserved Leishmania‐specific protein that could compose a pan‐Leishmania vaccine.     

Cocktail of gp63 and Hsp70 induces protection against Leishmania donovani in BALB/c mice

The 63-kDa antigen of Leishmania donovani is a membrane-anchored matrix metalloprotease that has been shown to be involved in the infection process. We have shown that this antigen alone generates a Th1 type of protective response that is partial but when the animals are primed with the antigen along with the Hsp70, the level of protection is raised significantly, which is demonstrated by a considerable reduction in parasite load of immunized animals when compared to the infected controls. Delayed-type hypersensitivity responses to leishmanin were measured as an index of cell-mediated immune response and were found to be higher in immunized animals when compared to the infected controls, the maximum being in the animals immunized with cocktail of both the antigens. Maximum IgG2a and minimum IgG1 levels were observed in this group of animals. These animals also generated maximum levels of IFN-γ and IL-2 and minimum levels of IL-4 and IL-10 pointing towards the generation of a protective Th1 response and the suppression of the Th2 type of immune response.     

Cysteine proteinase type I, encapsulated in solid lipid nanoparticles induces substantial protection against Leishmania major infection in C57BL/6 mice

Appropriate adjuvant, proper antigen(s) and a suitable formulation are required to develop stable, safe and immunogenic vaccines. Leishmanial cysteine proteinase type I (CPB) is a promising vaccine candidate; nevertheless, it requires a delivery system to induce a potent immune response. Herein, solid lipid nanoparticles (SLN) have been applied for CPB [with and without C‐terminal extension (CTE)] formulation to utilize as a vaccine against Leishmania major infection in C57BL/6 mice. Therefore, SLN‐CPB and SLN‐CPB^?CTE formulations were prepared from cetyl palmitate and cholesterol, using melt emulsification method. After intraperitoneal vaccination and subsequent L. major challenge, a strong antigen‐specific T‐helper type 1 (Th1) immune response was induced compared to control groups. Lymph node cells from immunized mice displayed lower parasite burden, higher IFN‐γ, IgG2a and lower IL‐4 production, indicating that robust Th1 immune response had been induced. Our results revealed that CTE is not necessary for inducing protective responses against L. major infection as the IFN‐γ/IL‐4 ratio was significantly higher, whereas IgG1 responses were lower in the SLN‐CPB^?CTE vaccinated group, post‐challenge. Thus, SLN‐CPB^?CTE was shown to induce specific Th1 immune responses to control L. major infection, through effective antigen delivery to the peritoneal antigen presenting cells.     

Th1 immune response to Plasmodium falciparum recombinant thrombospondin‐related adhesive protein (TRAP) antigen is enhanced by TLR3‐specific adjuvant,poly(I:C) in BALB/c mice

Sporozoite‐based malaria vaccines have provided a gold standard for malaria vaccine development, and thrombospondin‐related adhesive protein (TRAP) serves as the main vaccine candidate antigen on sporozoites. As recombinant malaria vaccine candidate antigens are poorly immunogenic, additional appropriate immunostimulants, such as an efficient adjuvant, are highly essential to modulate Th1‐cell predominance and also to induce a protective and long‐lived immune response. In this study, polyinosinic:polycytidylic acid [poly(I:C)], the ligand of TLR3, was considered as the potential adjuvant for vaccines targeting stronger Th1‐based immune responses. For this purpose, BALB/c mice were immunized with rPfTRAP delivered in putative poly(I:C) adjuvant, and humoural and cellular immune responses were determined in different immunized mouse groups. Delivery of rPfTRAP with poly(I:C) induced high levels and titres of persisted and also high‐avidity anti‐rPfTRAP IgG antibodies comparable to complete Freund's adjuvant (CFA)/incomplete Freund’s adjuvant (IFA) adjuvant after the second boost. In addition, rPfTRAP formulated with poly(I:C) elicited a higher ratio of IFN‐γ/IL‐5, IgG2a/IgG1, and IgG2b/IgG1 than with CFA/IFA, indicating that poly(I:C) supports the induction of a stronger Th1‐based immune response. This is a first time study which reveals the potential of rPfTRAP delivery in poly(I:C) to increase the level, avidity and durability of both anti‐PfTRAP cytophilic antibodies and Th1 cytokines.     

PEGylation of cationic liposomes encapsulating soluble Leishmania antigens reduces the adjuvant efficacy of liposomes in murine model

Although there have been several attempts to develop a vaccine against leishmaniasis, no vaccine in human has been developed yet. Liposomes consisting of 1, 2‐dioleoyl‐3‐trimethylammonium‐propane (DOTAP) encapsulating soluble Leishmania antigens (SLA) enhance protective immunity of SLA against Leishmania major infection in mice. However, they immobilized at the injection site because of their positive charge. To overcome the problem, shielding the surface charge with polyethylene glycol (PEGylation) was chosen in this study. Liposomal SLA consisting different concentrations of PEG (1.9%‐15% mol) were prepared. BALB/c mice were immunized three times in 3 weeks intervals with different formulations. Lesion development and parasite burden in footpad and spleen were evaluated to specify the type of generated immune response and extent of protection. Th1/Th2 cytokine profiles and IgG isotypes were also analysed. The maximum protection was observed in mice immunized with Lip‐SLA or pLip‐SLA (1.9%) due to smaller footpad swelling, reduction in parasite load, an increase in IgG2a and IFN‐γ production. Our results showed that immunization of mice with a high level of PEG (>7.5%) did not improve protective immunity of liposomal SLA. The presence of PEG, particularly more than 3.75%, is not recommended for protection against leishmaniasis.     

Cationic solid–lipid nanoparticles are as efficient as electroporation in DNA vaccination against visceral leishmaniasis in mice

The use of an appropriate delivery system has recently emerged as a promising approach for the development of effective vaccination against visceral leishmaniasis (VL). Here, we compare two vaccine delivery systems, namely electroporation and cationic solid–lipid nanoparticle (cSLN) formulation, to administer a DNA vaccine harbouring the L. donovani A2 antigen along with L. infantum cysteine proteinases [CPA and CPB without its unusual C‐terminal extension (CPB^?CTE)] and evaluate their potential against L. infantum challenge. Prime‐boost administration of the pcDNA‐A2‐CPA‐CPB^?CTE delivered by either electroporation or cSLN formulation protects BALB/c mice against L. infantum challenge and that protective immunity is associated with high levels of IFN‐γ and lower levels of IL‐10 production, leading to a strong Th1 immune response. At all time points, the ratio of IFN‐γ: IL‐10 induced upon restimulation with rA2‐rCPA‐rCPB and F/T antigens was significantly higher in vaccinated animals. Moreover, Th2‐efficient protection was elicited through a high humoral immune response. Nitric oxide production, parasite burden and histopathological analysis were also in concordance with other findings. Overall, these data indicate that similar to the electroporation delivery system, cSLNs as a nanoscale vehicle of Leishmania antigens could improve immune response, hence indicating the promise of these strategies against visceral leishmaniasis.     

DNA vaccine against visceral leishmaniasis: a promising approach for prevention and control

The visceral leishmaniasis (VL) caused by Leishmania donovani parasite severely affects large populations in tropical and subtropical regions of the world. The arsenal of drugs available is limited, and resistance is common in clinical field isolates. Therefore, vaccines could be an important alternative for prevention against VL. Recently, some investigators advocated the protective efficacy of DNA vaccines, which induces the T cell‐based immunity against VL. The vaccine antigens are selected as conserved in various Leishmania species and provide a viable strategy for DNA vaccine development. Our understanding for DNA vaccine development against VL is not enough and much technological advancement is required. Improved formulations and methods of delivery are required, which increase the uptake of DNA vaccine by cells; optimization of vaccine vectors/encoded antigens to augment and direct the host immune response in VL. Despite the many genes identified as vaccine candidates, the disappointing potency of the DNA vaccines in VL underscores the challenges encountered in the efforts to translate efficacy in preclinical models into clinical realities. This review will provide a brief background of DNA vaccines including the insights gained about the design, strategy, safety issues, varied candidates, progress and challenges that play a role in their ability against VL.     

Prophylactic properties of a Leishmania‐specific hypothetical protein in a murine model of visceral leishmaniasis

In this work, the effect of vaccination of a newly described Leishmania infantum antigenic protein has been studied in BALB/c mice infected with this parasite species. The LiHyD protein was characterized after a proteomic screening performed with the sera from dogs suffering visceral leishmaniasis (VL). Its recombinant version was expressed, purified and administered to BALB/c mice in combination with saponin. As a result of vaccination and 10 weeks after challenge using an infective dose of L. infantum stationary promastigotes, vaccinated mice showed lower parasite burdens in different organs (liver, spleen, bone marrow and footpads’ draining lymph nodes) than mice inoculated with the adjuvant alone or the vaccine diluent. Protected mice showed anti‐Leishmania IgG2a antibodies and a predominant IL‐12‐driven IFN‐γ production (mainly produced by CD4⁺ T cells) against parasite proteins, whereas unprotected controls showed anti‐Leishmania IgG1 antibodies and parasite‐mediated IL‐4 and IL‐10 responses. Vaccinated mice showed an anti‐LiHyD IgG2a humoral response, and their spleen cells were able to secrete LiHyD‐specific IFN‐γ, IL‐12 and GM‐CSF cytokines before and after infection. The protection was correlated with the Leishmania‐specific production on nitric oxide. Altogether, the results indicate that the new LiHyD protein could be considered in vaccine formulations against VL.     

Total Leishmania antigens with Poly(I:C) induce Th1 protective response

Our proposal was to develop a vaccine based on total Leishmania antigens (TLA) adjuvanted with polyinosinic‐polycytidylic acid [Poly(I:C)] able to induce a Th1 response which can provide protection against Leishmania infection. Mice were vaccinated with two doses of TLA‐Poly(I:C) administered by subcutaneous route at 3‐week interval. Humoral and cellular immune responses induced by the immunization were measured. The protective efficacy of the vaccine was evaluated by challenging mice with infective promastigotes of Leishmania (Leishmania) amazonensis into the footpad. Mice vaccinated with TLA‐Poly(I:C) showed a high anti‐Leishmania IgG titre, as well as increased IgG1 and IgG2a subclass titres compared with mice vaccinated with the TLA alone. The high IgG2a indicated a Th1 bias response induced by the TLA‐Poly(I:C) immunization. Accordingly, the cellular immune response elicited by the formulation was characterized by an increased production of IFN‐γ and no significant production of IL‐4. The TLA‐Poly(I:C) immunization elicited good protection, which was associated with decreased footpad swelling, a lower parasite load and a reduced histopathological alteration in the footpad. Our findings demonstrate a promising vaccine against cutaneous leishmaniasis that is relatively economic and easy to develop and which should be taken into account for preventing leishmaniasis in developing countries.     

Immune effector mechanisms in malaria: An update focusing on human immunity

The past decade has witnessed dramatic decreases in malaria‐associated mortality and morbidity around the world. This progress has largely been due to intensified malaria control measures, implementation of rapid diagnostics and establishing a network to anticipate and mitigate antimalarial drug resistance. However, the ultimate tool for malaria prevention is the development and implementation of an effective vaccine. To date, malaria vaccine efforts have focused on determining which of the thousands of antigens expressed by Plasmodium falciparum are instrumental targets of protective immunity. The antigenic variation and antigenic polymorphisms arising in parasite genes under immune selection present a daunting challenge for target antigen selection and prioritization, and is a given caveat when interpreting immune recall responses or results from monovalent vaccine trials. Other immune evasion strategies executed by the parasite highlight the myriad of ways in which it can become a recurrent infection. This review provides an update on immune effector mechanisms in malaria and focuses on our improved ability to interrogate the complexity of human immune system, accelerated by recent methodological advances. Appreciating how the human immune landscape influences the effectiveness and longevity of antimalarial immunity will help explain which conditions are necessary for immune effector mechanisms to prevail.     

Vaccination of dogs against Babesia canis infection using parasite antigens from in vitro culture

Groups of five dogs were vaccinated with different Babesia canis vaccine formulations. It appeared that partial protection against challenge infection was obtained when using parasite antigens from in vitro culture in combination with saponin. Protection was evident as a decrease in parasitaemia after challenge and was associated with the presence of serum antibodies against Babesia parasites. In addition, parasite antigen derived from in vitro culture supernatant exhibited more protective activity than somatic parasite antigen, in that a less marked fall of haematocrit values was found after challenge infection. The fall of haematocrit value observed in the animals immunized with somatic parasite antigen was not different from that observed in the adjuvant control group.     

Immune response in susceptible BALB/c mice immunized with DNA encoding Lipophosphoglycan 3 of Leishmania infantum

Visceral leishmaniasis is a serious parasitic infection that the development of an effective vaccine is necessary to control the disease. Lipophosphoglycan 3 (LPG3) is essential for the synthesis of glycoconjugates as parasite virulence factors. In this study, we evaluated the immunogenicity of Leishmania infantum LPG3 gene as a DNA vaccine against murine visceral leishmaniasis. For this purpose, BALB/c mice were immunized subcutaneously with the DNA encoding LPG3 either alone or in combination with recombinant heat shock protein 70 (rHSP70). Next, its immunogenicity and protective efficacy were evaluated in the immunized mice. The results showed a mixed Th1/Th2 response following immunization, which was associated with the production of both IFN‐γ and IL‐10 by splenocytes compared with control groups but did not lead to reduction in the splenic parasite burden. Serum levels of IgG antibody isotypes indicated no significant difference between the LPG3 DNA and the empty vector. In addition, the co‐administration of rHSP70 with the DNA vaccine offered no additive protective advantage on experimental infectious challenge. Thus, we propose to strengthen the immunogenic potential of L. infantum LPG3 in prime‐boost approach with a powerful adjuvant to elicit a robust parasite‐specific protective Th1 response.     

Intradermal immunization of rats with plasmid DNA encoding Schistosoma mansoni 28 kDa glutathione S-transferase

Direct administration of plasmid DNA encoding an antigen represents an attractive approach to vaccination against infectious diseases, particularly in developing countries where easy-to-handle and cost-effective vaccines are needed. We have investigated the potential of DNA immunization to induce a specific antibody response against Schistosoma mansoni , using plasmid-DNA encoding the protective antigen, S. mansoni 28 kDa glutathione S-transferase (Sm28GST). Since S. mansoni parasite penetrates into its host through the skin, this tissue was chosen for plasmid DNA delivery. Following plasmid DNA administration into the skin of rats, the parasite antigen was detected in skin cells by immunohistochemistry. Three administrations of 200 μg plasmid at 14 day intervals led to the induction of a long-lasting specific IgG antibody response in the sera of immunized rats, with a predominance of IgG2a and IgG2b subclasses. Sera of immunized animals were able to mediate antibody-dependent cellular cytotoxicity in vitro , leading to the specific killing of parasite larvae. A parasite challenge performed on plasmid DNA-immunized animals induced a strong and rapid boosting effect on the specific IgG antibody response. These results demonstrate the potential of genetic immunization via the skin with plasmid DNA encoding Sm28GST for inducing immune responses with protective patterns against an S. mansoni infection .     

Comparative efficacy of the Schistosoma mansoni nucleic acid vaccine,Sm23, following microseeding or gene gun delivery

Sm23 is an integral membrane protein expressed widely in the human parasitic worm Schistosoma mansoni. Sm23 has already been shown to elicit protective immune responses following immunization with peptides or DNA constructs. In this study, we evaluated the immunogenicity and the protective efficacy of the Sm23 DNA vaccine using two different intradermal DNA delivery methods: microseeding and gene gun. Using both techniques, all mice immunized with the Sm23-pcDNA construct generated Sm23-specific immunoglobulin (Ig)G antibody, while mice immunized with the control plasmid, pcDNA, did not. Antibody isotypes analysis revealed that microseeding elicited mainly IgG2a and IgG2b antibodies, with relatively low levels of IgG1 and IgG3. The relative IgG1/IgG2a ratio was 0.03, indicative of a Th1 type immune response. In contrast, gene gun immunization resulted in significantly higher levels of IgG1 and IgG3. The relative IgG1/IgG2a ratio in this case was 11, indicative of a Th2 type immune response. No significant difference in the levels of IgG2b was observed. Coimmunization with plasmid DNA encoding either interleukin (IL)-12 or IL-4 by microseeding did not affect the levels of IgG1, while the levels of IgG2a and IgG2b were reduced. On the other hand, the levels of IgG3 were significantly increased by IL-4, but unchanged by IL-12. Importantly, in all experiments, the Sm23-pcDNA vaccine provided statistically significant levels of protection against challenge infection. Microseeding immunizations resulted in higher levels of protection (31-34% protection) than gene gun immunization (18% protection). This suggests that the Th1 type immune response elicited by microseeding immunization was responsible for the higher protection levels. However, the protective effect of the vaccine was not affected by coadministering plasmids encoding either IL-12 or IL-4 using the microseeding technique.     

Administration of naloxone in combination with recombinant Plasmodium vivax AMA‐1 in BALB/c mice induces mixed Th1/Th2 immune responses

Naloxone (NLX) has the ability to shift the immune response to a Th1 profile. Therefore, the adjuvant efficacy of NLX with recombinant P. vivax apical membrane antigen‐1(rPvAMA‐1) in BALB/c mice was evaluated. Mice were immunized subcutaneously with purified rPvAMA‐1 formulated with NLX (doses of 5 mg/kg body weight) alone or in combination with IFA. A significant increase in anti‐PvAMA‐1 IgG antibody after the second boost (mean OD₄₉₀ = 2·08 and 2·17, in groups received, rPvAMA‐1/NLX and rPvAMA‐1/NLX/IFA, respectively) was detected. IgG1 and IgG2b were the predominant isotypes in all immunized mouse groups. In immunized mice with rPvAMA‐1/NLX (mean: 1036 pg/mL) and with rPvAMA‐1/NLX/IFA (mean: 1024 pg/mL), IFN‐γ was elicited in response to rPvAMA‐1 after the second boost. No detectable IL‐4 secretion was determined in all tested groups. In conclusion, the administration of NLX alone or NLX/IFA with rPvAMA‐1 in BALB/c mice, which induced mixed Th1/Th2 immune responses, was comparable with that of the same recombinant antigen with CFA/IFA adjuvant. The results indicate that NLX alone may possibly not be considered as a potent Th1 adjuvant in PvAMA‐1‐based vaccine. However, in order to modulate immune responses from mixed Th1/Th2 to strong and protective Th1 response, further study is warranted on combination of NLX with other adjuvants such as CpG motifs or MPL in proper vaccine formulation. Additionally, dose–response study is necessary to determine the effect of different doses of antigen combined with NLX (at various doses) in Balb/c mice.     

TLR4 agonist protects against Trypanosoma cruzi acute lethal infection by decreasing cardiac parasite burdens

E6020 is a synthetic agonist of Toll-like receptor-4 (TLR4). The purpose of this study was to evaluate the effect of different doses of E6020-SE on Trypanosoma cruzi-specific immune responses and its ability to confer protection against acute lethal infection in mice. Forty female BALB/c were infected with 500 trypomastigotes of T cruzi H1 strain, divided into four groups (n = 10) and treated at 7- and 14-day post-infection (dpi) with different doses of E6020-SE or PBS (control). Survival was followed for 51 days, mice were euthanized and hearts were collected to evaluate parasite burden, inflammation and fibrosis. We found significantly higher survival and lower parasite burdens in mice injected with E6020-SE at all doses compared to the control group. However, E6020-SE treatment did not significantly reduce cardiac inflammation or fibrosis. On the other hand, E6020-SE modulated Th1 and Th2 cytokines, decreasing IFN-γ and IL-4 in a dose-dependent manner after stimulation with parasite antigens. We conclude that E6020-SE alone increased survival by decreasing cardiac parasite burdens in BALB/c mice acutely infected with T cruzi but failed to prevent cardiac damage. Our results suggest that for optimal protection, a vaccine antigen is necessary to balance and orient a protective immune response.     

T‐cell profiles elicited by Toxoplasma gondii in acutely/chronically infected humans

Toxoplasma gondii (T. gondii) is an obligate intracellular protozoan parasite that can infect almost all warm‐blooded species and induce a chronic infection in human hosts. The aim of this work was to investigate Th1, Th2, Th17 and Treg polarization, induced by four important T. gondii antigens (SAG1, ROP1, GRA8 and MAG1) in acutely and chronically infected patients. For this purpose, SAG1, ROP1, GRA8 and MAG1 were expressed as recombinant proteins, purified, and used to evaluate the proinflammatory and regulatory immune response profiles in seropositive and seronegative individuals. Our results show that SAG1 and ROP1 elicited a proinflammatory profile (INF‐γ, IL‐12 and IL‐17) in individuals in the acute phase, whereas MAG1 and GRA8 induced a regulatory pattern (Treg and TGF‐β) in chronically infected patients. These results reveal fundamental differences in T‐cell polarization induced by T. gondii antigens, which could have important implications in the immunopathogenesis of the disease and in future proposals of therapeutic strategies.