Vaccines for visceral leishmaniasis: A review.

JIM-11997; No of Pages 12 Journal of Immunological Methods xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

Journal of Immunological Methods journal homepage: www.elsevier.com/locate/jim

Review

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Vaccines for visceral leishmaniasis: A review

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Keerti Jain, N.K. Jain ⁎

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Pharmaceutical Nanotechnology Research Laboratory, ISF College of Pharmacy, Moga, Punjab 142001, India

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a b s t r a c t

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Article history: Received 24 August 2014 Received in revised form 21 February 2015 Accepted 28 March 2015 Available online xxxx

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Keywords: Visceral leishmaniasis Vaccine Immune response Antigen Protein

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Contents

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Visceral leishmaniasis, which is also known as Kala-Azar, is one of the most severely neglected tropical diseases recognized by the World Health Organization (WHO). The threat of this debilitating disease continues due to unavailability of promising drug therapy or human vaccine. An extensive research is undergoing to develop a promising vaccine to prevent this devastating disease. In this review we compiled the findings of recent research with a view to facilitate knowledge on experimental vaccinology for visceral leishmaniasis. Various killed or attenuated parasite based first generation vaccines, second generation vaccines based on antigenic protein or recombinant protein, and third generation vaccines derived from antigen-encoding DNA plasmids including heterologous prime-boost Leishmania vaccine have been examined for control and prevention of visceral leishmaniasis. Vaccines based on recombinant protein and antigenencoding DNA plasmids have given promising results and few vaccines including Leishmune®, Leishtec, and CaniLeish® have been licensed for canine visceral leishmaniasis. A systematic investigation of these vaccine candidates can lead to development of promising vaccine for human visceral leishmaniasis, most probably in the near future. © 2015 Published by Elsevier B.V.

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Introduction . . . . . . . . . . . . . . . . . . Immune responses in leishmaniasis . . . . . . . Antileishmanial vaccines . . . . . . . . . . . . 3.1. First generation antileishmanial vaccines . 3.2. Second generation antileishmanial vaccines 3.3. Third generation antileishmanial vaccines . 4. Novel drug delivery systems for vaccine delivery . 5. Conclusions . . . . . . . . . . . . . . . . . . Conflict of interest . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . .

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⁎ Corresponding author at: Pharmaceutical Nanotechnology Research Laboratory, ISF College of Pharmacy, Moga, Punjab 142001, India. Tel./fax: +91 7582 264712. E-mail addresses: [email protected] (K. Jain), [email protected] (N.K. Jain).

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1. Introduction

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Infectious diseases like malaria, tuberculosis, filariasis, visceral leishmaniasis, leprosy, and HIV infection pose enormous burden on world health. Hence it is necessary to control these diseases and their outbreaks. Leishmaniasis has been elected by the World Health Organization (WHO) among the category-1 diseases described as emerging and uncontrolled diseases and

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http://dx.doi.org/10.1016/j.jim.2015.03.017 0022-1759/© 2015 Published by Elsevier B.V.

Please cite this article as: Jain, K., Jain, N.K., Vaccines for visceral leishmaniasis: A review, J. Immunol. Methods (2015), http:// dx.doi.org/10.1016/j.jim.2015.03.017

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Leishmania parasites are transmitted by the bite of infected 122 sandfly, reside in host macrophages and cause infection due to 123 its ability to evade and attenuate microbicidal function of 124

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and African continents including India, Nepal, Bangladesh and East Africa (Van Griensven and Diro, 2012). Around 350 million populations are at risk of contracting this parasitic infection and about 1.6 million new cases are likely to occur per annum. Visceral leishmaniasis is fatal in 85–90% untreated patients (Stockdale and Newton, 2013). Visceral leishmaniasis is one of the fatal diseases in the Indian subcontinent due to increasing resistance to conventional drugs, inadequate treatment and HIV–leishmania co-infection; hence it is necessary to develop novel drugs/drug delivery systems/vaccine targets to fulfill the needs of visceral leishmaniasis therapy. Additionally cost of treatment as well as drug identification and development, restricts commercial production of antileishmanial agents. Further toxicity, long treatment course and limited efficacy are other confinement of antileishmanial therapy. Hence scientists are focusing on the immunotherapy and immunochemotherapy for control and treatment of visceral leishmaniasis (Roatt et al., 2014; Kumar et al., 2015; Islamuddin et al., 2015; Jain et al., 2015a, 2015b). The development of various vaccine candidates including live or killed parasites, defined leishmanial antigenic proteins as well as antigenic salivary proteins of sandfly vector with successful results obtained in animal experiments strongly supports the opportunity for developing vaccine against visceral leishmaniasis as well as other forms of leishmaniasis (Lee et al., 2012; Alvar et al., 2013). In the following sections we will discuss various vaccine candidates, which have shown some promising results against experimental visceral leishmaniasis, along with the role of immune responses in the treatment and control of visceral leishmaniasis.

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its prevention is majorly based on three parameters including control of vector, animal reservoir control and research and development of potential vaccine candidates. Instead of extensive research and execution of various programs by health system, the prevalence of visceral leishmaniasis has increased particularly in Indian subcontinent (Abdian et al., 2011; John et al., 2011; Matlashewski et al., 2011; Stockdale and Newton, 2013). Inefficient antileishmanial drugs, unavailability of human leishmania vaccine, diversity and complexity of leishmania parasite, varied response with geographical distribution, complexity and severity of diseases, emergence of resistance due to improper use of medicines and unawareness of general public resulted in this prevalence. Current treatment strategies for visceral leishmaniasis are greatly hampered by cost of available drugs and emergence of resistance to antileishmanial drugs. Hence it is necessary to understand immunology of visceral leishmaniasis to develop an effective vaccine against this severe ailment (Jain and Jain, 2013; Kaye and Aebischer, 2011; Nagill and Kaur, 2011). Leishmaniases comprises a group of diseases caused by protozoan parasite belonging to genus Leishmania. Based on the main clinical symptoms, these complex diseases may be classified into three groups, namely; visceral leishmaniasis, cutaneous leishmaniasis and mucocutaneous leishmaniasis. Visceral leishmaniasis is the most severe form of leishmaniasis, which may be fatal if not treated and is caused by parasite Leishmania donovani and Leishmania infantum (also known as Leishmania chagasi). Transmission of visceral leishmaniasis occurs by the bite of a sand fly belonging to genus Phlebotomus and Lutzomiya (De Oliveira et al., 2009; Van Griensven and Diro, 2012). Life cycle of Leishmania parasite along with strategy to control infection is presented in Fig. 1. Visceral leishmaniasis exists in two forms, zoonotic and anthroponotic, caused by L. infantum and L. donovani, respectively. The zoonotic form of disease exists in the Mediterranean region and American continent whereas the anthroponotic form is prevalent in Asian

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K. Jain, N.K. Jain / Journal of Immunological Methods xxx (2015) xxx–xxx

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Fig. 1. Life cycle of visceral leishmaniasis parasite and potential strategies for control of infection. 1) Transfer of flagellate promastigotes form of Leishmania parasite by the bite of sandfly vector to human; 2) invasion of macrophages by promastigote; 3) transformation of promastigotes into amastigotes form; 4) multiplication of amastigotes within macrophages which infects new cells; 5) transfer of amastigotes into vector during human bite; 6) release of amastigotes in the midgut of sandfly; 7) transformation of amastigotes into promastigotes; 8) multiplication of promastigotes; and 9) migration of promastigotes to pharyngeal valve.



146 147 148 149 150 151 152 153 154 155 156 157 158 159 160 161 162 163 164 165 166 Q8 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182 183 184 185

229

Although initial studies suffered with objectionable side effects resulting into clinical complications, later various leishmanial antigens have been explored as promising vaccine candidate for leishmaniasis. These antigens included killed or live attenuated Leishmania parasite (first generation), recombinant Leishmania proteins (second generation), DNA encoding Leishmania proteins (third generation), and immunomodulators (Abdian et al., 2011; Nagill and Kaur, 2011; Araújo et al., 2011; Singh and Sundar, 2012). Admittedly at present there is no vaccine available for human being against leishmaniasis yet many scientists are investigating the possibility of vaccination against leishmaniasis (Araújo et al., 2011; Singh et al., 2012). The major challenges in the development of vaccine against leishmaniasis are the complexity associated with the antigenicity, uneven response by the host,

230 231 Q9

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3 . Antileishmanial vaccines

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186 187

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been elucidated that CD4+ T (helper T lymphocytes) as well as CD8+ T (cytotoxic T lymphocytes) cells play important role in conferring defense against and cure of visceral leishmaniasis (Basu et al., 2005; Basu et al., 2007). Polarized immune response is very important in the treatment of leishmaniasis, because the Th1 response protects animals from infection while Th2 immune response supports the persistence of infection. In various experimental and clinical studies, it has been observed that Th1 type immune responses (IL-12, IFN-γ, IL-2 and TNF-α) confer protection against leishmaniasis while Th2 [IL-4, IL-5, IL-10, IL-13 and transforming growth factor-β (TGF-β)] responses are related to vulnerability to disease (Jain and Jain, 2013; Choudhury et al., 2013; Ponte et al., 2012). These facts have been explored by many scientists to develop possible vaccine candidates for prophylactic cure against visceral leishmaniasis (Abdian et al., 2011; Choudhury et al., 2013; Araújo et al., 2011). Protection from visceral leishmaniasis strongly depends on host immune response since it involves different levels of tissue-specific immunity and immune-mediated tissue damage. Further it is possible to activate antiparasitic cell-mediated immune responses and inhibition of negative immune regulators in visceral leishmaniasis infected patients (Faleiro et al., 2014). Chowdhury et al. (2015) reported that a TLR2-ligand, arabinosylated lipoarabinomannan, isolated from Mycobacterium smegmatis, act as a strong immunomodulator by modulating TLR2 and MAPK signaling as well as IFN-γ signaling and can confer protection against L. donovani infection. In this study scientists investigated that apart from TLR2 and MAPK signaling, IFN-γ signaling is also involved in its activity, since it is an important immune response, which mediates nitric oxide generation and activation of macrophages and T-cells, suppressed by Leishmania parasite (Chowdhury et al., 2015). This is an important finding since activation of Th1 immunity and IFN-γ signaling is very critical for host immunity to restrict visceral leishmaniasis infection. For successful control of leishmaniasis it is necessary to take into account the elicited immune response, particularly in case of vaccine development. Effective immune response can play important role in the alleviation of this severe disease as achieved by the production of toxic nitrogen and oxygen species, which can destroy the amastigotes residing intracellularly within the macrophages.

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host by modulation of innate and adaptive immunological responses. Now the inactivated phagocytic macrophages serve as safe shelter for the growth of Leishmania parasite. Since Leishmania infection is opportunistic to immune-suppression hence instigation of strong immune response is important for treatment and prevention of visceral leishmaniasis. Clinical application of currently available antileishmanial drugs is limited by serious adverse effect, narrow therapeutic indices and emerging resistance. To inhibit growth of parasites triggering of Th1 immune response is essential for activation of oxidative burst including nitric oxide burst which is subverted by Leishmania parasite in susceptible individuals. In order to improve antileishmanial therapy by reducing the cost of treatment, toxicity and increasing efficacy of various strategies like novel chemotherapeutic molecules, drug delivery systems, herbal drugs, targeted delivery, drug targets and vaccines are being investigated by scientists. Development of therapeutics based on leishmanial antigens, cytokines and antibodies which can stimulate preferential Th1 immune responses is one of the major challenges in the development of vaccine against leishmaniasis (Ali et al., 2014). Islamuddin et al. (2015) reported that n-hexane fractions of Artemisia annua leaves and seeds showed in vitro apoptotic antileishmanial activity. Further they also observed that oral administration of A. annua leaves and seeds to ten-week post infected Balb/c mice resulted in approximately 90 and 95% reduction in the hepatic and splenic parasite burden, respectively, with preferential stimulation of Th1 immune responses elicited by increase in the level of IFN-γ and suppression of Th2 immune responses with decreased level of IL-4 and IL-10. After in vitro antigen recall, a significant increase in lyphoproliferative responses (with increased level of IFN-γ producing CD4+ and CD8+ T lymphocytes and nitrite) was observed. Finally researchers concluded that A. annua leaves and seeds can provide two-pronged attack on Leishmanial parasite by leishmanicidal action and preferential stimulation of Th1-immunity (Islamuddin et al., 2015). The severity of leishmanial infection is determined by two factors; one is the causative species and second is the immune response (Choudhury et al., 2013; Saha et al., 2011). Immune responses play important role in the establishment of infection or protection from any infection. The Toll Like Receptors (TLRs) participate in the control of infections by regulation of innate immune response mediated by pathogen-associated molecular patterns (PAMPs) like glycolipids, peptidoglycans and lipopeptides, common in a large group of microorganisms. TLR mediates immune responses by production of tumor necrosis factor-α (TNF-α), IL-12 and nitric oxide. Out of 11 members of the TLR family, TLR4 and TLR9 primarily contribute in immune response against leishmanial infection. It has been also suggested that these two TLRs play possible role in the antileishmanial mechanism of miltefosine. Thus these receptors could also be important factor in designing the suitable vaccine candidate against leishmanial infection as well as targeted drug delivery system for treatment of leishmaniasis (Saha et al., 2011; Mukherjee et al., 2012). Immune responses play important role in the control and cure of infection as well as resistance to re-infection. One of the important aspects in the development of vaccine against leishmaniasis is the awareness about the most effective machinery of the immune system against leishmaniasis. It has

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t1:1 t1:2

Table 1 First generation vaccine candidates for visceral leishmaniasis.

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3.1. First generation antileishmanial vaccines

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The killed and attenuated Leishmania parasites constituted the first generation antileishmanial vaccine (Table 1). Leishvaccine [antigenic preparation of L. amazonensis (strain IFLA/BR/1967/PH8) with BCG as adjuvant] is an example of first generation vaccine explored for prophylactic treatment from canine visceral leishmaniasis. In its immunological profile it is observed to elicit initial alterations in the innate immunity mediated by neutrophils and eosinophils followed by delayed

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2011). Various killed or attenuated Leishmania parasites, leishmanial proteins and DNA encoding leishmanial proteins have been found successful in providing prophylactic protection against visceral leishmaniasis in in vitro and in vivo studies (Nagill and Kaur, 2011; Araújo et al., 2011; Singh and Sundar, 2012).

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variability in the different species of the Leishmania, and cost associated with the development (Singh and Sundar, 2012). The success of Leishmune® out of the two licensed canine vaccine; Leishmune® and Leishtec in Brazil (Leishmune®: FML-saponin formulation; Leishtec: L. donovani A2 proteinadenovirus) has encouraged the scientists for the development of human leishmaniasis vaccine. CaniLeish® (LiESP/ QA-21) vaccine is the next in this list which has also showed Th1 dominated immune response which persisted for a full year (Bongiorno et al., 2013; Moreno et al., 2014; Gradoni, 2015; Martin et al., 2014) (Fig. 2). First generation vaccine suffered the problem of provoking variable immune responses in human beings (Choudhury et al., 2013). Subsequently various Leishmania proteins and DNA vaccines have been investigated for conferring protection against leishmaniasis, which lead to some success in terms of Leishmune®, Leish-111f (Abdian et al., 2011; Araújo et al.,

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Fig. 2. Antileishmanial vaccine candidates under clinical trials.

Live attenuated Leishmania parasite

t1:4

Antigen

t1:5 t1:6 t1:7 t1:8 t1:9 t1:10 t1:11 t1:12 t1:13

L. donovani L. infantum L. donovani L. infantum L. donovani L. tarentolae L. amazonensis

t1:14

Antigen

Adjuvant

References

t1:15 t1:16 t1:17

Alum precipitated autoclaved L. major Autoclaved or heat killed L. donovani Autoclaved L. major

BCG (Bacillus Calmette Guerin) – BCG

Khalil et al. (2006) Nagill et al. (2009) Satti et al. (2001)

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t1:3

Description of antigen

Adjuvant

Reference

Radio-attenuated L. donovani SIR2 (silent information regulatory 2) deficient L. infantum amastigotes Long term culture of LiESP promastigotes

– – Gentamycin

Datta et al. (2015) Silvestre et al. (2007) Daneshvar et al. (2003)

BT1 (biopterin transporter) knock out promastigotes Live attenuated nonpathogenic L. tarentolae promastigotes Photodynamic vaccination with suicidal mutants of L. amazonensis

– – –

Papadopoulou et al. (2002) Breton et al. (2005) Kumari et al. (2009)

Killed Leishmania parasite


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270 271 272 273 274 275 276


modifications on monocytes, activation of CD4+ T, CD8+ T cells and B cells with induction of mixed cytokine profile 279 including interferon-γ and IL-4 (Araujo et al., 2008, 2011). 280 Jamshidi et al. (2011) investigated the effect of immunotherapy 281 Q10 with autoclaved Leishmania major and Mycobacterium 282 vaccae antigens with antileishmanial chemotherapeutic agent, 283 glucantime against experimental canine visceral leishmaniasis 284 induced in Mongrel dogs. In this investigation, chemotherapy 285 with glucantime showed rapid therapeutic effect with relapse in 286 one member whereas immunotherapy with M. vaccae resulted 287 in slow therapeutic benefit. Groups treated with L. major and 288 M. vaccae immunotherapy and combination of immunotherapy 289 with chemotherapy showed inhibition of parasite with recur290 rence of disease in one member of each group. From this study, 291 scientists concluded that immunotherapy could assist as adju292 vant in treatment of visceral leishmaniasis provided a thorough 293 investigation is performed to develop successful vaccine 294 candidate against visceral leishmaniasis (Jamshidi et al., 2011). 295 Datta et al. (2012) investigated the efficacy of γ-irradiated 296 (radio-attenuated) L. donovani parasite in elucidation of immu297 nity. The immunoprophylactic efficacy of this radio-attenuated 298 parasite was investigated against experimental murine visceral 299 leishmaniasis induced in Balb/c mice. Radio-attenuated 300 Leishmania parasite was found to be significantly effective in 301 protecting mice against L. donovani infection after two doses at a 302 time interval of 15 days, which was further confirmed by 303 increased Th1 immune response with reduced Th2 immunity 304 (Datta et al., 2012). Later Datta et al. (Datta et al., 2015) observed 305 that the Balb/c mice immunized via intramuscular route 306 with radio-attenuated L. donovani parasite cleared the parasite 307 by T-cell restoration which resulted in killing of L. donovani 308 parasite by PDK1, PI3K and p38MAPK signaling pathways 309 leading to increased release of nitric oxide (Datta et al., 2015). 310 Although first generation vaccine provided some level of 311 Q11 protection against leishmaniasis, it suffered from the limitation 312 of variable immune response as well as intolerable toxicity. To 313 combat these limitations various Leishmania proteins have 314 been examined by scientists as antigens to elucidate immune 315 response.

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3.2. Second generation antileishmanial vaccines

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Second generation vaccines include purified Leishmania parasite proteins as well as recombinant Leishmania proteins (Table 2). Leishmune® is a second generation vaccine, which has shown promising results in canine visceral leishmaniasis. It is a veterinary vaccine, which has been licensed in Brazil to protect dogs and to block the transmission of disease from dogs to humans via bite of sandfly vector in Americas and Mediterranean endemic region where leishmaniasis is an immunosuppressive zoonotic disease. Constitutively Leishmune® is the Fucose Mannose Ligand (FML)-saponin vaccine containing FML purified fraction from L. donovani promastigotes and saponins as adjuvants including QS21 and two deacylated saponins. The adjuvanticity and protection ability of these saponins could be enhanced by remodeling them in such a way like addition of one sugar unit in the glycosidic moiety attached to C-28, which leads to increase in the length and hydrophilicity of the sugar chain. Selective immunological responses were observed in dogs immunized with Leishmune® including early phenotypic changes in neutrophils and monocytes, selective stimulation of

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CD8+ T-cells with induction of specific proinflammatory response mediated by interferon-γ and nitric oxide (Araújo et al., 2011; Nico et al., 2012). A recombinant L. donovani protein p45 (rLDp45), which is protein belonging to the family of methionin aminopeptidase, was also found capable to evoke substantial protective Th1 immune response, confirmed by increased level of expression of mRNA for IFN-γ, IL-12, TNF-α and IL-12 with simultaneous decrease in IL-4 and TGF-β, to protect hamsters from experimentally induced visceral leishmaniasis (Gupta et al., 2012). An antigen specific for amastigote form of Leishmania parasite A2, when formulated in recombinant form (with saponin, alum and IL-12 or expressed by attenuated adenovirus) was found to be effective as vaccine in animals for protection against leishmaniasis. This antigen has restrained epitopes for CD4+ T and CD8+ T cells (Fernandes et al., 2012). Agallou et al. (2012) examined the efficacy of bone-marrow derived dendritic cells pulsed with histone H1 of L. infantum in the prophylactic protection against leishmaniasis and found it to be a potential vaccine candidate as it was capable of elucidating the polarized Th1 immune response against leishmaniasis, which is the primary goal in the development of Leishmania vaccine (Singh and Sundar, 2012; Agallou et al., 2012). Chakravarty et al. (2011) found LEISH-F1 (recombinant Leishmania polyprotein LEISH-F1 antigen) + monophosphoryl lipid A with squalene or MPL-SE (adjuvant) vaccine (three injections administered subcutaneously on days 0, 28 and 56) to be safe, well tolerated and immunogenic in an open label, dose-escalating, uncontrolled clinical trial continued for 168 days. Study was carried out with healthy Indian adult volunteers (three cohorts of 6 DAT-negative and 6 DATpositive volunteers) and the vaccine showed immunogenic potential evidenced by the production of cytokines including IFN-γ from induced T-cell (Chakravarty et al., 2011). Various excreted/secreted proteins of Leishmania parasites have been identified and explored for immunogenic potential. These proteins play important role in the virulence of Leishmania parasite owing to their active participation in the infection and suppression of host immune system. Since these proteins are important in the pathogenesis of leishmaniasis hence it is expected that they could be exploited for inducing long-lasting immunity against leishmaniasis (DebRoy et al., 2010; Gour et al., 2012). Gour et al. (2012) examined a series of 17 excreted/ secreted proteins obtained from in vitro culture of L. donovani promastigotes as antigen for immunostimulant activity. These proteins were divided into five groups having proteins with different molecular weights i.e. F1 (11, 13, 16 kDa), F2 (18, 21, 23 kDa), F3 (26, 29, 33 kDa), F4 (35, 42, 51 kDa) and F5 (54, 58, 64, 70, 80 kDa). The antigenicity of leishmanial excreted/ secreted antigens was determined by cytokine production, macrophage effector functions and lymphoproliferation; wherein fractions F1 and F2 showed the highest immunogenic potential suggesting that leishmanial excreted/secreted antigenic proteins is a probable candidate in the vista of antileishmanial vaccines (Gour et al., 2012). Immunization of Balb/c mice by secretory L. donovani serine protease (pSP) with IL-12 as adjuvant protected the mice from the load of parasite and infection of visceral leishmaniasis (Choudhury et al., 2013). Saliva of sand fly vector plays an important role in visceral leishmaniasis infection after infective bite to the host. MartinMartin et al. (2013) identified the antigens present in saliva of

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6 t2:1 t2:2 t2:3


Table 2 Second generation or purified antigen (purified Leishmania antigens or live recombinant bacteria expressing Leishmania antigens) Leishmania vaccine for visceral leishmaniasis. Antigen


Adjuvant

References

t2:5 t2:6 t2:7

P0 CPI and CPII GP63

Acidic ribosomal protein P0 of Leishmania infantum L. infantum cysteine proteinases A and B Surface expressed glycoprotein GP63, or leishmanolysin

Pereira et al. (2015) Saljoughian et al. (2013a) Elfaki et al. (2012)

t2:8

FML

Fucose mannose ligand

t2:9 t2:10

GP36 LiESAp

t2:11 t2:12

rLdccys1 SLA

Purified antigen from FML complex Naturally excreted/secreted antigens purified from culture supernatant of L. infantum promastigotes Recombinant cysteine proteinase Soluble leishmanial antigen

– Cationic solid lipid nanoparticles Salmonella typhimurium or BCG or cationic liposomes or monophosphoryl lipid A-trehalose dicorynomycolate (MPL-TDM) Saponins (Riedel De Haen, QuilA, QS21), IL12, BCG Saponin MDP (Muramyl dipeptide)

t2:13

rLdp45

t2:14 t2:15

rLelF-2 rLdPDI

t2:16 t2:17 t2:18 t2:19

rA2 KMP-11 rLeish-111f (Polyprotein vaccine)

t2:21 t2:22

pSP rSMT

t2:23

Histone H1

t2:24 t2:25

HSP70 and HSP83 LBSap

t2:26

GP63 and HSP70

t2:27

rLiHyp1

402 403 404 405 406 407 408 409 410 411 412 413 414

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Cocktail vaccine (GP63 + HSP70 + MPL-A and GP63 + HSP70 + ALD) hypothetical Leishmania amastigote-specific protein (LiHyp1)

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Phlebotomus argentipes, one of the female sandfly vectors of visceral leishmaniasis, using western blot, two-dimensional electrophoresis, MALDI-TOF, MALDI-TOF/TOF and de novo sequencing technique. Based on the outcomes of the investigation the authors suggested that in designing of vector-borne vaccine for visceral leishmaniasis, specific antigens for different sand fly vectors are needed to be chosen (Martin-Martin et al., 2013). Apart from identification of suitable antigen, appropriate antigen delivery and induction of strong Leishmania specific Th1 type immune response are the other two important parameters in the design of antileishmanial vaccine. Kumar et al. (2015) performed sub-proteome analysis of the 95 spots of membrane-enriched protein (MEP) fractions of L. donovani using matrix asserted laser desorption ionization-time of flight/ mass spectrometry (MALDI-TOF/MS) and classified 72 spots on the basis of their biological functions. Such exploration of various functional proteins of L. donovani parasite could be

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400 401

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Ldp27

398 399

D

P. acnes CpG-ODN MPL-SE (monophosphoryl lipid A plus squalene) and/or Glucantime

Oliveira-Freitas et al. (2006) Paraguai de Souza et al. (2001) Lemesre et al. (2007)

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P

– –

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Q1 t2:20

397

Recombinant L. donovani p45 (rLdp45). Ldp45 is a member of the methionine aminopeptidase family L. donovani elongation factor-2 protein (LelF-2) Protein disulfide isomerase (PDI) is a chaperone involved in virulence and survival of Leishmania parasite. (rLdPDI: recombinant L. donovani protein disulfide isomerase) Recombinant protein specific to L. donovani amastigotes Kinetoplastid membrane protein-11 (KMP-11) Leish-111f is a polyprotein derived by fusion of three antigens namely L. major homologue of eukaryotic thiol-specific antioxidant (TSA), L. major stress inducible protein 1 (LmSTI1) & L. braziliensis elongation and initiation factor (LeIF) L. donovani amastigote-specific protein p27 (Ldp27), which is a component of an active cytochrome C oxidase complex Secretory L. donovani serine protease Sterol 24-c-methyltransferase (SMT) is one of the identified Ags. SMT is an enzyme involved in biosynthesis of ergosterol, which is a target molecule of leishmanicidal and fungicidal drug amphotericin B Bone marrow-derived dendritic cells (BM-DCs) pulsed with the L. infantum histone H1 Heat shock protein L. braziliensis antigens plus saponin (LBSap) vaccine

BCG/Propionibacterium acnes Monophosphoryl lipid-trehalose dicorynomycolate (MPL-TDM) –

F

t2:4

Ferreira et al. (2008) Ravindran et al. (2012)

Gupta et al. (2012) Kushawaha et al. (2011) Kushawaha et al. (2012)

Fernandes et al. (2012) Agallou et al. (2011) Trigo et al. (2010)

–

Dey et al. (2013)

IL-12 MPL-SE

Choudhury et al. (2013) Goto et al. (2007)

CpG ODNs

Agallou et al. (2012)

MPLA, ALD Saponins MPL-A, ALD

Chakravarty et al. (2011) Resende et al. (2013), Vitoriano-Souza et al. (2013) Kaur et al. (2013)

Saponin

Martins et al. (2013)

helpful in investigating new therapeutic targets as well as vaccine targets for treatment and prophylactic prevention of visceral leishmaniasis and may be helpful in elucidating host– parasite relationships which could be vital in management of this severe debilitating disease (Kumar et al., 2015). Second generation vaccines showed improved response over first generation vaccines. To further increase the specificity and potency of vaccination, third generation vaccines including DNA encoding Leishmania proteins and prime boost vaccine are being developed, which are discussed in the following section.

415

3.3. Third generation antileishmanial vaccines

425

Vaccine based on the Kinetoplastid membrane protein-11 (KMP-11) DNA construct of L. donovani parasite conferred immunity against both antimony responsive and antimony resistant Leishmania parasites in golden hamsters. In the event of exploring the epitopes for CD8+ T cells by scanning the

426 427


416 417 418 419 420 421 422 423 424

428 429 430


450 451 452 453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468

F

R O O

448 449

P

446 447

4. Novel drug delivery systems for vaccine delivery

E

444 445

R

442 443

R

440 441

t3:3

Antigen

t3:4 t3:5 t3:6 t3:7 t3:8 t3:9

Antigen-encoding DNA plasmid vaccines ORFF Open reading frame fragment A2 Antigen specific to L. donovani amastigotes LACK Leishmania homologue of receptors for activated C kinase KMP-11 Kinetoplastid membrane protein-11 NH36 Nucleoside hydrolase (NH36) is a vital enzyme of L. donovani used in the synthesis of parasite DNA.

t3:10 t3:11 t3:12 t3:13 t3:14 t3:15 t3:16 t3:17 t3:18 t3:19

Development of an appropriate vaccine delivery system is an important part of vaccination strategy for any infectious diseases. Till date various novel strategies based on liposomes, electroporation, polymeric nanoparticles, bioadhesive nanoparticles, solid lipid nanoparticles, dendrimers, carbon nanotubes etc. have been evaluated for vaccine delivery with promising results to some extent. Most of these strategies are based on nanomaterials which have their own advantages attributed to their nanoscale dimensions (Jain et al., 2012, 2013, 2014; Bagre et al., 2013; Kesharwani et al., 2014; Mehra et al., 2015; Saljoughian et al., 2013a).

O

438 439

N C

436 437

D

Table 3 Third generation antileishmanial vaccine for visceral leishmaniasis.

435

E

t3:1 t3:2

433 434

Freund's adjuvant) vaccine, pVAX-P1 plasmid DNA vaccine/ peptide vaccine (single dose or double dose including prime dose and boost dose) was evaluated in hamster against experimentally induced visceral leishmaniasis. From the results, the authors found that DNA vaccine in double dose (prime + boost dose) protected the hamsters with decrease in mortality rate as determined by enhanced Th1 immune response with reduced parasite burden (up to 75.68%) in spleen, suggesting L. donovani ribosomal P1 gene as potential DNA vaccine candidate (Masih et al., 2011). Heat shock proteins are one of the most explored surface proteins for immunization against leishmaniasis, which are the chaperons, also known as natural adjuvants. Lipophosphoglycan 3 (LPG3) is one of the members of HSP90 proteins, which is the Leishmania homologous with GRP94 (glucose regulated protein 94). This protein has been found very efficient in stimulating the immune response against Leishmania parasite in experimental leishmaniasis model. The prime-boost (DNA/Protein) vaccine of LPG3 was found more efficient in stimulating immunogenic response in Balb/c mice compared to DNA/DNA vaccine (Abdian et al., 2011). Details of potential third generation vaccines for visceral leishmaniasis including antigen-encoding DNA plasmid vaccines and heterologous prime-boost vaccines are presented in Table 3.

C

469

complete sequence of KMP-11 with overlapping nonapeptides it was observed that 30 peptides of this protein was able to trigger CD8+ T cells followed by secretion of interferon-γ. This study further confirmed that KMP-11 could serve as promising candidate for vaccination against leishmaniasis by processing and presenting antigen via the MHC-1 pathway (Basu et al., 2005, 2007). In Latin America the causative agent for leishmaniasis is L. chagasi for which Lutzomyia longipalpis sandfly is the vector. A DNA plasmid coding a salivary protein of L. longipalpis (LJM19) has also been found effective partially in protecting hamsters from visceral leishmaniasis and its lethal conclusions, even after two to five months with reduced parasitic load and normal hematological profile (Gomes et al., 2008). Later it was also observed that when both DNA plasmid encoding KMP11 and LJM19 were used in combination, the protection conferred by them was almost equivalent to the protection rendered by both plasmids, unaccompanied (da Silva et al., 2011). Pereira et al. (2015) investigated acidic ribosomal protein P0 and nucleosomal histones of L. infantum to control visceral leishmaniasis in hamsters. Vaccination was done with plasmid DNA only (homologous) or plasmid DNA plus recombinant protein and adjuvant (heterologous immunization) which showed production of high level of antibodies and predominant cellular immune response, respectively. Both L. infantum LiP0 and HIS antigens were found immunogenic in hamsters and only LiP0 antigen was also found to be able to confer a significant degree of protection against L. infantum. Further the research work also showed that immunization strategy is also critical in development of a potential vaccine candidate since that homologous strategy i.e. L. infantum LiP0 antigen administered in a DNA formulation was found to be a potential vaccine candidate than heterologous strategy comprised of pcDNA-LiP0 followed by rLiP0 boost (Pereira et al., 2015). Masih et al. (2011) developed a DNA vaccine for visceral leishmaniasis by molecular cloning of an acidic ribosomal protein P1 gene of L. donovani (LdP1) by PCR amplified complete open reading frame (ORF) in pQE or pVAX vector. The prophylactic efficacy of recombinant protein rLdP1 (with

T

431 432

7

U


Heterologous prime-boost vaccines LACK Leishmania homologue of receptors for activated C kinase prime boost vaccine CPI + CPII L. infantum cysteine proteinase type I (CPI) and II (CPII) prime boost vaccine CPIII L. infantum cysteine proteinase type III (CPIII) prime boost vaccine KM P-11 Kinetoplastid membrane protein-11 A2 + CPI + CPII A2 antigen and cysteine proteinases (CPI and CPII) of L. donovani rLdP1 Recombinant L. donovani ribosomal P1 gene (rLdP1) TRYP Tryparedoxin peroxidase (TRYP) Ldccys1 Cysteine proteinase of 30 kDa from L. chagasi

Expression vector

References

pcDNA3.1 A2-ex pressing Lactococcus lactis pCl-neo pCMV-LIC VR1012

Sukumaran et al. (2003) Yam et al. (2011) Sinha et al. (2013) Santos et al. (2012) Gamboa-Leon et al. (2006)

Recombinant vaccinia virus (rVV)/ modified vaccinia virus Ankara (MVA) PCB6

Ramos et al. (2008)

pcDNA

Khoshgoo et al. (2008)

rVV pcDNA pVAX Modified vaccinia virus Ankara (MVA) pcDNA3

Guha et al. (2013) Saljoughian et al. (2013b) (Masih et al., 2011) Ferreira et al. (2008) Carson et al. (2009)

Rafati et al. (2006)


470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493

494 495 496 497 498 499 500 501 502 503 504 505

8

Table 4 Clinical trials on vaccine for visceral leishmaniasis.

U

t4:3

Vaccine details

Trial

Disease

Volunteers

Results

References

t4:4

Leishmune® or Leish-Tec®

Field trial

Zoonotic canine visceral leishmaniasis

Dogs

Fernandes et al. (2014)

t4:5 t4:6

Gentamicin-attenuated Leishmania infantum

Field trial

t4:7

LEISH-F1 + MPL-SE vaccine

t4:8

Leish-111f + MPL-SE

Open-label, dose-escalating, uncontrolled clinical trial Open Trial and blinded Trial

t4:9

LiESAp-MDP

Field trial

t4:10 t4:11

Field trials

Canine visceral leishmaniasis

t4:12 t4:13 t4:14 t4:15 t4:16 t4:17Q2 t4:18

DNA/modified vaccinia virus Ankara (MVA) prime/boost vaccine Alum-precipitated autoclaved Leishmania major (Alum/ALM) vaccine + Bacille Calmette– Guérin (BCG) and sodium stibogluconate (SSG) or vaccine diluents and SSG LiESAp-MDP vaccine

Randomized double-blind trial

Post-kala-azar dermal (PKDL) leishmaniasis

Leishmune® or Leish-Tec® were found equivalent in terms of aspects, parasitism, IgG seropositivity, or dog infectiousness. Leishmune®The stimulated higher levels of IgG, IgG1, and IgG2 whereas LeishTec(®) exhibited adverse reactions with greater frequency and severity. Gentamicin-attenuated L. infantum induced a significant and strong protective effect against canine visceral leishmaniasis in the endemic area. Vaccine was found safe and immunogenic in healthy human volunteers. A promising tool for control of visceral leishmaniasis in dogs and humans. Strong and long-lasting cell-mediated immune response which is important for protection against infection of visceral leishmaniasis. CLA ELISA will provide sensitive estimates of L. infantum infection incidence in DNA/MVA vaccinated dogs, to improve overall specificity. SSG + Alum/ALM + BCG were found safe and immunogenic with significant healing potentials in persistent PKDL lesions which was probably attributed to immunochemotherapy augmented IFN-gamma production.

t4:19 t4:20

Alum precipitated Leishmania major with BCG

An extended phase II study in children Double-blind randomized efficacy field trial

Visceral leishmaniasis Canine visceral leishmaniasis

Naturally exposed dogs [205 (Vaccinated animals) +209 (Placebotreated animals)] 544 healthy, leishmanin non-reactive children with age less than 15 years Three hundred and forty-seven healthy dogs

Study of safety and immunogenicity Field trial in an endemic area of Brazil Trial in healthy Sudanese volunteers Phase III trial of efficacy in an endemic area of Brazil Randomized, double-blind, BCG-controlled trial in Sudan

Visceral leishmaniasis

Twenty-four healthy adult volunteers

Canine visceral leishmaniasis Visceral leishmaniasis

Naturally exposed dogs

Canine visceral leishmaniasis Visceral leishmaniasis

Naturally exposed dogs

t4:21

FML-QuilA saponin vaccine

t4:22 t4:23

Autoclaved L. major (ALM) with BCG FML-vaccine

t4:24 t4:25

Autoclaved L. major (ALM) with or without BCG

N

C

O

Canine visceral leishmaniasis

R

Visceral leishmaniasis

Dogs

R

Healthy human volunteers

Canine visceral leishmaniasis Canine visceral leishmaniasis

Field trial in the endemic areas Canine visceral of the South of France leishmaniasis

Dogs Dogs

E

C

Dogs

Human

T

E

D

Human

Human

Daneshvar et al. (2014)

Chakravarty et al. (2011) Trigo et al. (2010) Bourdoiseau et al. (2009) Carson et al. (2009)

Musa et al. (2008)

A significant, long-lasting and strong protective effect against canine visceral leishmaniasis.

Lemesre et al. (2007)

Vaccine was found safe and immunogenic.

Khalil et al. (2006)

Vaccine was safe and well tolerated after 16 months follow-up and vaccine was found to be 69.3% effective Vaccine was found safe and induced a strong delayed hypersensitivity reaction Significant, long-lasting and strong protective effect was observed 76.9% volunteers produced significant levels of interferon-gamma in response to L. major antigen. Vaccine showed a significant, long-lasting and strong protective effect Two doses of ALM plus BCG did not offer significant protective immunity against visceral leishmaniasis compared with BCG alone

Mohebali et al. (2004)

P

R O

O

F

Kamil et al. (2003) Borja-Cabrera et al. (2002) Satti et al. (2001) Da Silva et al. (2000) Khalil et al. (2000)



t4:1 t4:2


506 507

535

5. Conclusions

536 537

U

N C

O

R

R

E

The results of various researches strongly support the possibility for immunoprophylaxis of visceral leishmaniasis. 538 Further, it seems that current vaccine strategy for visceral 539 leishmaniasis majorly relies on identification of appropriate 540 surface antigens of Leishmania parasite. One of the major 541 constraints in the development of vaccine for visceral leish542 maniasis is the requirement of combining two or more antigens 543 to conserve antigenic properties for various Leishmania species 544 as well amastigote and promastigote phases of parasite. As 545 observed, combination of L. donovani A2 antigen with cystein 546 proteinases CPI and CPII with cationic solid lipid nanoparticles 547 as adjuvant was found very effective in reducing the parasite 548 burden and increasing the production of nitric oxide. Recently, 549 Q15 cocktail vaccine containing LD31, LD51 polypeptides derived 550 from amastigote form of L. donovani with saponin adjuvant 551 showed high immunogenicity with maximum protection in 552 L. donovani infected mice (Kaur et al., 2015). 553 Although identification of appropriate antigen is the major 554 stumbling block to the road map of vaccine for leishmaniasis 555 medical and epidemiologically relevant viewpoint strongly 556 supports the possibility of vaccine for prevention of visceral 557 leishmaniasis. Currently scientists are investigating single as 558 well as combination of two or more Leishmania antigens with 559 or without suitable adjuvant and results with animal experi560 ments are showing promising results and therefore future 561 clinical trials may result into a successful antileishmanial 562 vaccine. The veterinary antileishmanial vaccine, Leishmune®, 563 being effective, could pave the way for development of a 564 promising vaccine for prophylactic protection against visceral

565 566 567 568 569 570 571 Q16 572 573 574 575 576 577 578 579 580 581 582 583 584 585 586

D

P

R O O

F

leishmaniasis in human. Despite few controversial reports, there is strong possibility that the systematic investigation of vaccine candidates through well planned experimental models followed by clinical trials may result in the development of a promising antileishmanial vaccine for visceral leishmaniasis. A list of clinical trial on vaccine for visceral leishmaniasis is summarized in Table 4. From the critical examination of a large number of vaccine candidates explored for visceral leishmaniasis, it could be concluded that protein- or DNA-based second and third generation vaccines may be capable to confer longterm protection from visceral leishmaniasis. Similarly, results on humoral and cellular immune responses of various researches strongly support the possibility of antileishmanial vaccine. Cumulative reports reveal that the molecular mechanism of host defense for visceral leishmaniasis may facilitate the development of effective antileishmanial vaccine. Use of novel drug delivery systems, suitable adjuvant or combination of two or more leishmanial antigens may fascinate the development of a promising vaccine. Further, more systematic and well planned research is needed to investigate potential antigenic candidate for development of safe and effective vaccine for human visceral leishmaniasis.

587

Acknowledgments

589

Conflict of interest

T

E

The authors report no conflict of interest.

C

Various novel delivery systems have been also examined for delivery of antileishmanial vaccines. Liposomes and nanopar508 ticle based formulations have been evaluated for delivery of 509 antileishmanial vaccine candidates for their adjuvant property 510 to stimulate continued immune response and also resulted in 511 significant protection and strong stimulation of Th1 immune 512 response against visceral leishmaniasis (Saljoughian et al., 513 2013a, 2013b; Joshi et al., 2014). Saljoughian et al. (2013a) 514 investigated two vaccine delivery strategies based on electro515 poration and cationic solid lipid nanoparticles to deliver 516 antileishmanial vaccine comprised of a DNA vaccine harboring 517 the L. donovani A2 antigen along with L. infantum cysteine 518 proteinases (CPA and CPB). In the result of this study the 519 efficiency of cationic solid lipid nanoparticles based vaccine 520 delivery system was found to bee equivalent with electropo521 ration method of vaccine delivery (Saljoughian et al., 2013a). In 522 another study, Saljoughian and co-workers investigated the 523 efficacy of Live/Live and DNA/Live vaccine as prime-boost 524 Q14 regimens. Leishmania tarentolae expressing the L. donovani A2 525 antigen along with cysteine proteinases [CPA and CPB without 526 its unusual C-terminal extension {CPB(-CTE)}] as a tri-fusion 527 gene was used as a live vaccine and pcDNA encoding tri-fusion 528 gene was used as DNA primer and formulated with cationic 529 solid lipid nanoparticles. Developed formulation of prime-boost 530 A2-CPA-CPB(-CTE)-recombinant L. tarentolae was found able 531 to protect Balb/c mice from infection of L. infantum due to 532 stimulated Th1-type immune responses indicated by increased 533 production of IFN-γ with reduction in level of IL-10 (Saljoughian 534 et al., 2013b).

9

588

Author Keerti Jain is grateful to the Council of Scientific and 590 Industrial Research (CSIR), New Delhi, India for providing 591 Q17 financial support in the form of Senior Research Fellowship. 592 References

593

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Therapeutic vaccines for leishmaniasis.

Therapeutic options for visceral leishmaniasis.

Epidemiology of visceral leishmaniasis.

Visceral leishmaniasis in Libya--review of 21 cases.

Research priorities for elimination of visceral leishmaniasis.

Immunity to visceral leishmaniasis: implications for immunotherapy.

Amphotericin B for visceral leishmaniasis in hemodialysis.

Development of Leishmania vaccines in the era of visceral leishmaniasis elimination.

Mimotope-based vaccines of Leishmania infantum antigens and their protective efficacy against visceral leishmaniasis.

Visceral leishmaniasis in a Scottish child.

Visceral leishmaniasis mimicking Richter transformation.

Visceral leishmaniasis with Roth spots.

Cryoglobulinemic purpura in visceral leishmaniasis.

Granulocyte function in visceral leishmaniasis.

Biomarkers of safety and immune protection for genetically modified live attenuated leishmania vaccines against visceral leishmaniasis - discovery and implications.

Visceral leishmaniasis after kidney transplantation: report of a new case and a review of the literature.

Molecular tools for diagnosis of visceral leishmaniasis: systematic review and meta-analysis of diagnostic test accuracy.

Phlebotomine sand flies (Diptera: Psychodidae) transmitting visceral leishmaniasis and their geographical distribution in China: a review.

Transmission Dynamics of Visceral Leishmaniasis in the Indian Subcontinent - A Systematic Literature Review.

Decentralized control of human visceral leishmaniasis in endemic urban areas of Brazil: a literature review.

Accounting for False Positive HIV Tests: Is Visceral Leishmaniasis Responsible?

Combined Immune Therapy for the Treatment of Visceral Leishmaniasis.

Nanoliposomal artemisinin for the treatment of murine visceral leishmaniasis.