Animal models for vaccine studies for visceral leishmaniasis

Visceral leishmaniases (VL) or kala-azar is the most dreaded and devastating amongst the various forms of leishmaniases. The disease, though localized in certain areas only, has gained immense importance because of high mortality rate, mainly in children. The parasite is responsible for a spectrum of clinical syndromes, which can, in most extreme cases, go from an asymptomatic infection to a fatal form of VL. Chemotherapeutic measures, alone are not sufficient to control and contain the disease. As an alternate strategy, vaccination is also under experimental and clinical trails. The situation unquestionably demands the use of proper screening system, rationale chemical synthesis, vaccine development and targeted vaccine delivery. Thus, development of an acceptable vaccine is not an easy task. While the factors, which determine clinical outcomes, are in part, a feature of the parasite, it is the nature of the host and its genetic make up and immune status that play crucial role. The prerequisite of reliable animal model is that it should have a considerably good correlation with the clinical situation and is expected to mimic the pathological features and immunological responses observed in humans when exposed to a variety of Leishmania spp. with different pathogenic characteristics. Many experimental animal models like rodents, dogs and monkeys have been developed, each with specific features, but none accurately reproduces what happens in humans. In addition to the nature of the host, the major difference between natural and experimental infections is the parasite inoculum; in natural conditions, the infected sand fly vector deposits a few hundred metacyclic promastigotes into the dermis of the host, whereas experimental infections are induced by the injection (subcutaneous or intravenous) of millions of promastigotes grown in axenic cultures in vitro or amastigotes recovered from infected spleens.In public health terms, VL is the disease of humans and dogs (which may be considered secondary or ‘accidental’ hosts in the leishmanial life cycle) who often exhibit severe clinical signs and symptoms when infected, whereas reservoir hosts generally show a few, minor or no signs. This situation makes the definition of a suitable laboratory model a difficult one since the various experimental hosts may behave either like a reservoir or an accidental host. This review discusses the concept of animal models for VL and provides a critical evaluation of the most common experimental models and their respective advantages and disadvantages. Particular emphasis is given to the value of using mouse, hamster, dog and primate models, especially in the context of testing potential antileishmanial vaccines

Assessment of soil physical health and productivity of Kharkhoda and Gohana blocks of Sonipat district (Haryana), India

In order to assess soil health of Kharkhoda and Gohana blocks of Sonipat district (a part of western Yamuna canal irrigated region), important parameters namely pH, electrical conductivity (EC), texture, bulk density (BD), saturated hydraulic conductivity (HC), soil organic carbon (OC), available water retension capacity (AWRC) and non capillary pores (NCP) were measured by collecting undisturbed soil samples in nearly 66 villages. Soil physical rating index (PI) method was used to compute PI which was an indicator of soil physical health of thatregion. Results revealed that in Gohana and Kharkhoda blocks, nearly 90% area had pH <8.0 and EC>4 dS m-1, which indicated that soils were saline. Prediction maps of soil BD showed that 75% of the total area in 15-30 cm soil layer had BD above >1.6 mg m-3, which indicated the presence of hard pan in subsurface. HC data of subsurface layer also showed that 60% of the area had values<0.5 cm hr-1 which reconfirmed the presence of hard pan. For both surface as well as subsurface soil layers, mostly AWC was >10% which indicated adequate water retention capacity of these soils. However 85% of subsurface had poor soil aeration capacity as indicated NCP range < 10 %. Prediction map of PI for subsurface layer showed that majority of area had PI<0.4 which indicated that expected yield of the crop cannot be more than 70% of the potential yield even under normal or higher levels of fertilizer and water inputs

Leishmania infantum Amastigotes Enhance HIV-1 Production in Cocultures of Human Dendritic Cells and CD4+ T Cells by Inducing Secretion of IL-6 and TNF-α

Visceral leishmaniasis (VL) is a potentially deadly parasitic disease afflicting millions worldwide. Although itself an important infectious illness, VL has also emerged as an opportunistic disease among patients infected with HIV-1. This is partly due to the increasing overlap between urban regions of high HIV-1 transmission and areas where Leishmania is endemic. Furthermore, VL increases the development and clinical progression of AIDS-related diseases. Conversely, HIV-1-infected individuals are at greater risk of developing VL or suffering relapse. Finally, HIV-1 and Leishmania can both productively infect cells of the macrophage-dendritic cell lineage, resulting in a cumulative deficiency of the immune response. We therefore studied the effect of Leishmania infantum on HIV-1 production when dendritic cells (DCs) are cocultured with autologous CD4+ T cells. We show that amastigotes promote virus replication in both DCs and lymphocytes, due to a parasite-mediated production of soluble factors by DCs. Micro-beads array analyses indicate that Leishmania infantum amastigotes infection induces a higher secretion of several cytokines in these cells, and use of specific neutralizing antibodies revealed that the Leishmania-induced increase in HIV-1 replication is due to IL-6 and TNF-α. These findings suggest that Leishmania's presence within DC/T-cell conjugates leads to an enhanced HIV-1 production

Intake of nutrient supplements affects multiplication of Leishmania donovani in hamsters

The role of the essential nutrients, vitamins A,B (complex), C and E and iron, as prophylactic as well as supportive therapy in experimental visceral leishmaniasis (VL), was studied in hamsters. Prophylactic administration of vitamin C (50, 100 and 250 mg/kg) from day15 to day 0 (15 doses) significantly reduced the intake of Leishmania donovani in hamsters but had no therapeutic effect. In contrast, vitamins A,B complex and E and iron, whether used prophylactically or therapeutically, promoted parasite multiplication. The efficacy of sodium stibogluconate, a reference antileishmanial drug, was appreciably improved in animals administered prophylactically with vitamin C. However, supplementation of vitamin C during established infections resulted in reduced drug action. The results show that the prophylactic use of vitamin C may prevent the onset of leishmania infection and cautions against the indiscriminate use of nutrient supplements such as vitamin A, B complex, and E and iron in VL endemic areas

Efficacy of human β-casein fragment (54–59) and its synthetic analogue compound 89/215 against Leishmania donovani in hamsters

The characteristic feature of visceral leishmaniasis (VL) is the profound impairment of immune system of the infected host, which contributes significantly to the partial success of antileishmanial chemotherapy. Since in VL, cure is the combinatorial effect of drug and immune status of the host, the rationale approach towards antileishmanial chemotherapy would be to potentiate the immune functioning of the host to extract desired results. Towards this direction several rationally designed analogues of human β-casein fragment (54–59) were evaluated for their ability to stimulate the non-specific resistance in hamsters against Leishmania donovani infection. By virtue of being derived from the food protein casein derivatives may be devoid of unwanted side effects associated with the substances of microbial origin, e.g. muramyl dipeptide (MDP). Out of this one peptide Val-Glu-Gly-Ile-Pro-Tyr (compound 89/215) had been reported to have such activity. In this communication, the prophylactic and therapeutic efficacy of the peptide along with its natural sequence has been evaluated in detail against experimental VL in hamsters. Their use as an adjunct to chemotherapy was also explored. Human β-casein fragment, compound 89/215 and MDP were tested in vivo at various dose levels wherein compound 89/215 showed superiority over MDP at 3 mg/kg × 2 given intraperitoneally (i.p.). Compound 89/215 sensitized peritoneal macrophages acquired considerable resistance and only 24% of the cells were found infected in comparison to control peritoneal macrophages where 76.4% of the cells were found infected. Similarly, the efficacy of sodium antimony gluconate (SAG) in hamsters pretreated with compound 89/215 enhanced significantly (P < 0.001). This peptide also exhibited considerably good therapeutic efficacy when evaluated either alone or in combination with SAG in established infection of L. donovani

Selection of adjuvants for vaccines targeting specific pathogens

Introduction: Adjuvants form an integral component in most of the inactivated and subunit vaccine formulations. Careful and proper selection of adjuvants helps in promoting appropriate immune responses against target pathogens at both innate and adaptive levels such that protective immunity can be elicited. Areas covered: Herein, we describe the recent progress in our understanding of the mode of action of adjuvants that are licensed for use in human vaccines or in clinical or pre-clinical stages at both innate and adaptive levels. Different pathogens have distinct characteristics, which require the host to mount an appropriate immune response against them. Adjuvants can be selected to elicit a tailor-made immune response to specific pathogens based on their unique properties. Identification of biomarkers of adjuvanticity for several candidate vaccines using omics-based technologies can unravel the mechanism of action of modern and experimental adjuvants. Expert opinion: Adjuvant technology has been revolutionized over the last two decades. In-depth understanding of the role of adjuvants in activating the innate immune system, combined with systems vaccinology approaches, have led to the development of next-generation, novel adjuvants that can be used in vaccines against challenging pathogens and in specific target populations

Isolation of integral membrane proteins of Leishmania promastigotes and evaluation of their prophylactic potential in hamsters against experimental visceral leishmaniasis

The integral membrane proteins (IMP's) of promastigotes of two virulent strain of Leishmania (L.) donovani Dd8 and Leishmania (L.) infantum LV9 and one avirulent viscerotropic strain Leishmania tropica UR6 were extracted by phase separation technique using a non-ionic detergent “Triton X-114”. This detergent is homogeneous at 0 °C but divides in an aqueous phase and a detergent phase at above 20 °C. The phase partition resulted in solubilisation of hydrophilic proteins in aqueous phase, and IMP's with an amphiphilic nature were recovered in the detergent phase. The strain wise quantitative recovery rates of IMP's were estimated. These proteins were purified by chill methanol centrifugation and used as vaccinogen, alone or in combination with adjuvant against L. donovani challenge in hamster model. Among all the combinations, hamsters immunised with IMP of L. donovani (Dd8) in combination with CFA resulted in 75% parasite inhibition in spleen (P < 0.001), however, 61.14% (P < 0.001) and 77.60% (P < 0.001) parasitic inhibition was achieved in liver and bone marrow respectively as compared to their unvaccinated counter part. Similar combinations with UR6 and LV9 strain inhibited the parasite establishment up to 65.12% (P < 0.001) and 66.87% (P < 0.001), respectively on splenic site. The specific IgG level against (Dd8 strain) soluble leishmania promastigote antigen was monitored at different stages by enzyme linked immunosorbent assay (ELISA) corresponds to the level of parasitic establishment. Similar observations were made in cases of LV9 and UR6 strains. However, significant lymphoproliferative response to IMPs of Dd8 strain (SI 3.5–4.9, P < 0.001) was noticed in all IMP + CFA vaccinated animals. Thus, this study will provide a lead for more manipulative trials to develop a subunit vaccine against the fatal disease

Immunostimulatory potential and proteome profiling of Leishmania donovanisoluble exogenous antigens

Summary: Isolation of the soluble exogenous antigens (SEAgs), its immune response study and proteome profiling is an essential prerequisite for understanding the molecular pathogenesis of Leishmania donovani. The immunostimulatory potential of L. donovani SEAgs, purified from culture of L. donovani clinical isolate, was evaluated for their ability to induce cellular responses in treated/cured hamsters. SEAgs induced significant proliferative responses in lymphocytes (SI 5·6 ± 2·3; P < 0·01) isolated from cured hamster. In addition, significant NO production in response to SEAgs was also noticed in macrophages of hamsters, mouse and human cell lines (J774A-1 and THP1). Western blot analyses with antibodies against proteophosphoglycan (PPG; surface-expressed and secreted molecule) of L. donovani revealed that PPG molecules are also present in L. donovani SEAgs. Mass spectrometry (MS)-based proteome analysis of 12 protein bands of SEAgs through MALDI-TOF/TOF endorsed the identification of some Th1-stimulatory immunogenic proteins. These immunogenic proteins may offer increased hope for the discovery of new promising vaccine candidates against visceral leishmaniasis (VL). The overall results suggest that immunostimulatory molecules are present in the SEAgs, which may be further exploited, for developing a subunit vaccine against VL a fatal human disease