Leishmania donovani, which is responsible for visceral leishmaniasis (VL), poses severe public health challenges, necessitating novel strategies to decrease its incidence. The epitopes predicted with immune informatics tools provide the opportunity for the development of a good vaccine candidate. The immunogenicity of sand fly salivary proteins SP02, SP12, SP13, SP14, SP15, SP19, and SP56 and known Leishmania MHC class I and class II epitopes was evaluated. Among the sand fly salivary proteins the most prominent ten B cell epitopes based on their immunogenicity, stability and safety were derived from these proteins. Two remarkable vaccination candidates that were considered safe, SP14 and SP15, were "LPTILAHSPAGASVNQ" (1.244) and "RLLIKDYVVTRKVVKD" (1.178), respectively. These T cell epitopes included thirteen verified MHC I and six MHC class II epitopes. All known immunodominant epitopes were used in the vaccine build together with possible linkers and an adjuvant at the N-terminus to improve immunogenicity. The proposed vaccine was extensively tested to establish its stability, non-allergic reaction, and nontoxicity, all of which were confirmed.The vaccine construct's stable interaction with TLR4 highlighted important hydrogen bonds necessary for stable receptor engagement. Indicative of a robust Th1 response, immune simulations showed an elivated IgG and IgM titers, substantial helper and cytotoxic T-cell responses, and elevated levels of IFN-γ, IL-2, and IL-12, further supporting the vaccine construct's potential as a promising candidate for leishmaniasis control. However, experimental validations will determine its potential as an effective vaccine candidate.

Neglected tropical disease, visceral leishmaniasis is a significant health issue globally, caused by Leishmania donovani, a protozoan parasite, transmitted by an insect vector sandfly Phlebotomus (P). argentipes. Understanding the disease transmission dynamics is crucial, with P. argentipes being the primary vector in the Indian subcontinent. The study utilized citation network analysis (CNA) to investigate the scientific literature on the sandfly species P. argentipes , focusing on the distribution of publications over the past 80 years. A total of 2601 publications were initially retrieved, refined through manual screening to 400 relevant articles published by 1209 authors in 150 journals in the last eight decades. Data included publication details, such as association of authors, citations, journals, and affiliations. CNA involved the use of VOSviewer software for creating bibliographic networks, visualizing and analyzing citation networks. Core publications highlighted key research areas, including vector control, taxonomy, genetics, and epidemiology. Collaboration across countries and disciplines was evident, emphasizing the global effort to combat visceral leishmaniasis. By analyzing extensive publication data, CNA revealed key research clusters and the most cited works, showing that research into visceral leishmaniasis transmission and vector control has intensified since the 2000s, with significant contributions from India which is highly endemic for visceral leishmaniasis followed by USA, and the UK.

Background Hepatitis C virus (HCV) is a common cause of liver cirrhosis and hepatocellular carcinoma. Globally, nearly 71 million people have chronic HCV infection, and approximately 399,000 dies annually. In patients without cirrhosis, HCV infection is treated with 12 weeks of sofosbuvir/velpatasvir combination. Results from available small, single-centre observational studies suggest that the sofosbuvir/velpatasvir combination given for 8 weeks may be as effective as the standard 12 weeks of treatment. We propose to compare the treatment response of 12 weeks versus 8 weeks of sofosbuvir/velpatasvir in non-cirrhotic people with chronic HCV infection. Methods This multicentric, randomized, open-label, non-inferiority trial will include 880 (2 arms x 440) treatment naïve, viraemic (HCV RNA >10,000 IU/mL), non-cirrhotic adults (age >18 years) with chronic hepatitis C. People who are at high-risk for HCV reinfection such as haemophiliacs, people who inject drugs, those on maintenance hemodialysis or having HIV will be excluded. The presence or absence of cirrhosis will be determined with a combination of history, examination, ultrasound, liver stiffness measured with transient elastography, APRI, FIB-4, and esophagogastroduodenoscopy. Participants will be randomized to receive either 8- or 12-week sofosbuvir/velpatasvir treatment. A blood specimen will be collected before starting the treatment (to determine the HCV genotype), after 4 weeks of treatment (for early virological response), and at 12 weeks after treatment discontinuation for SVR12. Discussion The study will provide data on the efficacy of 8 weeks of treatment as compared to the standard of care (12 weeks) in non-cirrhotic patients with chronic HCV infection. Treatment for a shorter duration may improve treatment compliance, reduce the cost of treatment, and ease the treatment implementation from a public health perspective. Trial registration Registered with Clinical Trial Registry of India ( http://ctri.nic.in ) Registration No. CTRI/2022/03/041368 [Registered on: 24/03/2022]—Trial Registered Prospectively.

The magnitude and duration of immune response to COVID-19 vaccination in older adults are known to be adversely affected due to immunosenescence and inflammaging. The threat of emerging variants warrants studies on immune response in older adults to primary vaccination and booster doses so as to understand the effectiveness of vaccines in countering the threat of emerging variants. Non-human primates (NHPs) are ideal translational models, as the immunological responses in NHPs are similar to those in humans, so it enables us to understand host immune responses to the vaccine. We initially studied humoral immune responses in aged rhesus macaques employing a three-dose regimen of BBV152, an inactivated SARS-CoV-2 vaccine. Initially, the study investigated whether the third dose enhances the neutralizing antibody (Nab) titer against the homologous virus strain (B.1) and variants of concern (Beta and Delta variants) in aged rhesus macaques immunized with BBV152, adjuvanted with Algel/Algel-IMDG (imidazoquinoline). Later, we also attempted to understand cellular immunity in terms of lymphoproliferation against γ-inactivated SARS-CoV-2 B.1 and delta in naïve and vaccinated rhesus macaques after a year of the third dose. Following the three-dose regimen with 6 µg of BBV152 with Algel-IMDG, animals had increased Nab responses across all SARS-CoV-2 variants studied, which suggested the importance of booster dose for the enhanced immune response against SARS-CoV-2-circulating variants. The study also revealed the pronounced cellular immunity against B.1 and delta variants of SARS-CoV-2 in the aged rhesus macaques even after a year of vaccination.

Microbial pattern recognition critically contributes to innate response, both at extracellular and intracellular cytosolic surveillance pathway (CSP) interface. However, the role of pattern recognition by host innate receptors in CSP is poorly understood in Leishmania donovani infection. Here, we have demonstrated that cytosolic targeting of L.donovani DNA (Ld-DNA) inhibits macrophage responsiveness to IFNɣ, through decreased MHC-II expression and lowered pSTAT1 (Y701) levels, involving host three-prime repair exonuclease-1 (TREX-1). The Ld-DNA potently induced type-1 IFNs, i.e. significant over-production of IFNβ through activation of the IRF pathway. Interestingly, knockdown of TRIF or MyD88 expression in macrophages had no effect on cytosolic Ld-DNA transfection-mediated IFN-β production, indicating involvement of a TLR independent pathway. Contrastingly, Ld-DNA failed to induce IFNβ in both TBK-1 and IRF3KO knockout macrophages. Although IFNβ was not induced by Ld-DNA in STING- knockout macrophages, STING alone was not enough for the induction. Evidently, besides STING, Ld-DNA recognition for induction of IFNβ critically required cytosolic cyclic GMP-AMP synthase (cGAS). Furthermore, the cGAS dependent targeting of Ld-DNA induced IFNβ over-production that contributed to antimony resistance in L.donovani infection. We provide the first evidence that enhanced cytosolic sensing of Ld-DNA in infection by antimony resistant (SBR-LD), but not antimony sensitive L.donovani strains (SBS-LD), was critically regulated by host MDRs, multi drug resistant associated protein 1 (MRP 1) and permeability glycoprotein (P-gp) in macrophages. Collectively, our results disclose Ld-DNA as a vital pathogen associated molecular pattern (PAMP) driving host Type-I IFN responses and antimony resistance. The findings may help in future development of policies for novel anti-leishmanial therapeutics.

Background Leishmania contains a concatenated mitochondrial DNA, kDNA. Universal minicircle sequence binding protein (UMSBP), a mitochondrial protein, initiates kDNA replication by binding with a conserved universal minicircle sequence (UMS) of kDNA. Here, we describe first time in L. donovani the regulation of DNA binding activity of UMSBP and the role of UMSBP in virulence. Methods Insilco and EMSA study were performed to show UMS-binding activity of UMSBP. Tryparedoxin(TXN)-tryparedoxin peroxidase(TXNPx) assay as well as co-overexpression of cytochrome-b5 reductase-like protein (CBRL) and tryparedoxin in L. donovani were done to know the regulation of DNA binding activity of UMSBP. Knockout and episomal-expression constructs of UMSBP were transfected in L. donovani . The cell viability assay and immunofluorescence study to know the status of kDNA were performed. Macrophages were infected with transfected parasites. mRNA level of cytochrome b, activity of complex-III, intracellular ATP level of both transfected promastigotes and amastigotes as well as ROS concentration and the level of apoptosis of transfected promastigotes were measured. Level of oxidative phosphorylation of both transfected and un-transfected amastigotes were compared. Burden of transfected amastigotes in both macrophages and BALB/c mice were measured. Results L. donovani UMSBP is capable of binding with UMS, regulated by redox through mitochondrial enzymes, TXN, TXNPx and CBRL. Depletion of UMSBP (LdU −/− ) caused kDNA loss, which decreased cytochrome-b expression [component of complex-III of electron transport chain (ETC)] and leads to the disruption of complex-III activity, decreased ATP generation, increased ROS level and promastigotes exhibited apoptosis like death. Interestingly, single knockout of UMSBP (LdU −/+ ) has no effect on promastigotes survival. However, single knockout in intracellular amastigotes demonstrate loss of mRNA level of cytochrome-b, disruption in the activity of complex-III and reduced production of ATP in amastigotes than wild type. This process interfere with the oxidative-phosphorylation and thereby completely inhibit the intracellular proliferation of LdU −/+ amastigotes in human macrophages and in BALB/c mice. Amastigotes proliferation was restored as wild type after episomal expression of LdUMSBP in LdU −/+ parasites (LdU −/+ AB). Conclusion The LdUMSBP regulates leishmanial mitochondrial respiration and pathogenesis. So, LdUMSBP may be an attractive target for rational drug designing and LdU −/+ parasites could be considered as a live attenuated vaccine candidate against visceral leishmaniasis.

aDivision of Molecular Biology, Rajendra Memorial Research Institute of Medical Sciences, Agamkuan, Patna, Bihar, India bDepartment of Microbiology, All India Institute of Medical Sciences, Phulwarisharif, Patna, Bihar, India cNational Institute of Pharmaceutical Education and Research, Export Promotion Industrial Park, Hajipur, Vaishali, Bihar, India dDepartment of Bioinformatics, Rajendra Memorial Research Institute of Medical Sciences, Agamkuan, Patna, Bihar, India

Adenosine 3',5'-cyclic monophosphate (cAMP) is a stress sensor molecule that transduces the cellular signal when Leishmania donovani moves from insect vector to mammalian host. At this stage, the parasite membrane-bound receptor adenylate cyclase predominantly produces cAMP to cope with the oxidative assault imposed by host macrophages. However, the role of soluble adenylate cyclase of L. donovani (LdHemAC) has not been investigated fully. In the present investigation, we monitored an alternative pool of cAMP, maintained by LdHemAC. The elevated cAMP effectively transmits signals by binding to Protein Kinase A (PKA) present in the cytosol and regulates antioxidant gene expression and phosphorylates several unknown PKA substrate proteins. Menadione-catalyzed production of reactive oxygen species (ROS) mimics host oxidative condition in vitro in parasites where cAMP production and PKA activity were found increased by ~1.54 ± 0.35, and ~1.78 ± 0.47-fold, respectively while expression of LdHemAC gene elevated by ~2.18 ± 0.17-fold. The LdHemAC sense these oxidants and became activated to cyclize ATP to enhance the cAMP basal level that regulates antioxidant gene expression to rescue parasites from oxidative stress. In knockdown parasites (LdHemAC-KD), the downregulated antioxidant genes expression, namely, Sod (2.30 ± 0.46), Pxn (2.73 ± 0.15), Tdr (2.7 ± 0.12), and Gss (1.57 ± 0.15) results in decreased parasite viability while in overexpressed parasites (LdHemAC-OE), the expression was upregulated by ~5.7 ± 0.35, ~2.57 ± 0.56, ~4.7 ± 0.36, and ~2.4 ± 0.83, respectively, which possibly overcomes ROS accumulation and enhances viability. Furthermore, LdHemAC-OE higher PKA activity regulates phosphorylation of substrate proteins (~56 kDs in membrane fraction and ~25 kDs in the soluble fraction). It reduced significantly when treated with inhibitors like DDA, Rp-cAMP, and H-89 and increased by ~2.1 ± 0.28-fold, respectively under oxidative conditions. The LdHemAC-KD was found less infective to RAW 264.7 macrophages and more prone to oxidative damage as compared to LdHemAC-OE and control parasites. Together, this study demonstrates mechanistic links among LdHemAC, cAMP, and PKA in parasite survival and invasion under host oxidative condition.

Summary Statement Leishmania secretes over 151 proteins during in vitro cultivation. Cellular functions of one such novel protein: mevalonate kinase is discussed here; signifying its importance in Leishmania infection. Visceral Leishmaniasis is a persistent infection, caused by Leishmania donovani in Indian subcontinent. This persistence is partly due to phagocytosis and evasion of host immune response. The underlying mechanism involves secretory proteins of Leishmania parasite; however, related studies are meagre. We have identified a novel secretory Leishmania donovani glycoprotein, Mevalonate kinase (MVK), and shown its importance in parasite internalization and immuno-modulation. In our studies, MVK was found to be secreted maximum after 1 h temperature stress at 37°C. Its secretion was increased by 6.5-fold in phagolysosome-like condition (pH ~5.5, 37°C) than at pH ~7.4 and 25°C. Treatment with MVK modulated host immune system by inducing interleukin-10 and interleukin-4 secretion, suppressing host’s ability to kill the parasite. Peripheral blood mononuclear cell (PBMC)-derived macrophages infected with mevalonate kinase-overexpressing parasites showed an increase in intracellular parasite burden in comparison to infection with vector control parasites. Mechanism behind the increase in phagocytosis and immunosuppression was found to be phosphorylation of mitogen-activated protein (MAP) kinase pathway protein, Extracellular signal-regulated kinases-1/2, and actin scaffold protein, cortactin. Thus, we conclude that Leishmania donovani Mevalonate kinase aids in parasite engulfment and subvert the immune system by interfering with signal transduction pathways in host cells, which causes suppression of the protective response and facilitates their persistence in the host. Our work elucidates the involvement of Leishmania in the process of phagocytosis which is thought to be dependent largely on macrophages and contributes towards better understanding of host pathogen interactions.

In host-pathogen interactions, exosomal secretions are crucial for cell to cell communication and have an established role in immunomodulation. Protozoans, including Leishmania, modulates their host vesicular secretions for better survival; although the role of exosomal secretions in unresponsive against sodium antimony gluconate (SAG) has never been documented. In this study, the exosomal proteome of RAW macrophages infected with either SAG responsive (SAGS) or SAG unresponsive (SAGR) L. donovani parasites has been compared with uninfected RAW macrophages. Proteins isolated from exosomes were labelled with iTRAQ reagents; followed by subsequent LC-TOF/-MS analysis. In total, 394 proteins (p < 0.05) were identified which were shared common among all sets. Highly differentially expressed proteins were sorted by log2 value -1 and +1 as down regulated and up regulated respectively which yielded 58 proteins in SAGR and 41 proteins during SAGS infection. Out of the 58 proteins identified during SAGR infection, 17 proteins were of immune modulatory function. Network visualization model and pathway analysis revealed the interactions among these proteins via different immunological pathways with reported involvement of some proteins in SAG resistance and host immune modulation. Hence, the differential abundance of immune pathway related proteins in exosomes of infected host during SAGR infection supports the immune modulatory strategy adopted by SAG resistant parasites for enhanced survival .

Leishmaniasis comprises of a wide variety of diseases, caused by protozoan parasite belonging to the genus Leishmania. Leishmania parasites undergo different types of stress during their lifetime and have developed strategies to overcome this damage. Identifying the mechanistic approach used by the parasite in dealing with the stress is of immense importance for unfolding the survival strategy adopted by the parasite. Mevalonate kinase (MVK) is an important regulatory factor in the mevalonate pathway in both bacteria and eukaryotes. In this study, we explored the role of Leishmania donovani mevalonate kinase (LdMVK) in parasite survival under stress condition. Hydrogen peroxide (H2O2) and menadione, the two known oxidants were used to carry out the experiments. The MVK expression was found to be up regulated ∼2.1 fold and ∼2.3 fold under oxidative stress condition and under the effect of anti-Leishmania drug, AmBisome respectively. The cell viability declined under the effect of MVK inhibitor viz: vanadyl sulfate (VS). The level of intracellular ROS was also found to be increased under the effect of MVK inhibitor. To confirm the findings, LdMVK over expression (LdMVK OE) and LdMVK knockdown (LdMVK KD) parasites were generated. The level of ergosterol, an important component of plasma membrane in L. donovani, was observed and found to be reduced by nearly 60 % in LdMVK KD parasite and increased by nearly 30 % in LdMVK OE parasites as compared to wild type. However, the ergosterol content was found to be elevated under oxidative stress. Furthermore, LdMVK was also found to be associated with maintaining the plasma membrane integrity and also in preventing the peroxidation of cellular lipids when exposed to oxidative stress. The above data clearly suggests that MVK has a vital role in protecting the parasite from oxidative stress. These findings may also explore the contribution of LdMVK in drug unresponsiveness which may help in future rational drug designing for leishmaniasis.

Visceral leishmaniasis is characterized by mixed production of Th1/2 cytokines and the disease is established by an enhanced level of Th2 cytokine. CD4+ T cells are main cell type which produces Th1/2 cytokine in the host upon Leishmania infection. However, the regulatory mechanism for Th1/2 production is not well understood. In this study, we co-cultured mice CD4+ T cells with Leishmania donovani infected and uninfected macrophage for the identification of dysregulated miRNAs in CD4+ T cells by next-generation sequencing. Here, we identified 604 and 613 known miRNAs in CD4+ T cells in control and infected samples respectively and a total of only 503 miRNAs were common in both groups. The expression analysis revealed that 112 miRNAs were up and 96 were down-regulated in infected groups, compared to uninfected control. Nineteen up-regulated and 17 down-regulated miRNAs were statistically significant (p < 0.05), which were validated by qPCR. Further, using insilco approach, we identified the gene targets of significant miRNAs on the basis of CD4+ T cell biology. Eleven up-regulated miRNAs and 9 down-regulated miRNAs were associated with the cellular immune responses and Th1/2 dichotomy upon Leishmania donovani infection. The up-regulated miRNAs targeted transcription factors that promote differentiation of CD4+ T cells towards Th1 phenotype. While down-regulated miRNAs targeted the transcription factors that facilitate differentiation of CD4+ T cells towards Th2 populations. The GO and pathway enrichment analysis also showed that the identified miRNAs target the pathway and genes related to CD4+ T cell biology which plays important role in Leishmania donovani infection.

Visceral leishmaniasis (VL) is a disease caused by protozoan species of the genus Leishmania and is transmitted through bites from the Phlebotomus sand fly; it is associated with considerable morbidity and mortality in many parts of world, including India. Reports on the protective role played by saliva proteins of Lutozomyia longipalpis, Phlebotomus papatasi and Phlebotomus duboscqi. are available. However, no studies have explored the salivary proteins of P. argentipes, which is the known proven vector for the transmission of VL in the Indian sub-continent. Herein we revealed the presence of two proteins of 14.2 and one protein of 13.6 kDa in Indian strain P. argentipes which is absolute identical to previously reported protein of SP15 family (PagSP01, PagSP02 and PagSP07) of P. argentipes of NIH colony, USA. In an experimental study on P. argentipes from Bihar, India, we demonstrated that a strong humoral and cellular immune response was triggered to reduce the concomitant Leishmania load in groups of immunized mice. The immunized group produced a considerable amount of IgG antibodies, and their splenocytes generated TH1 cytokines (IL-12, IFN-γ) with the support of delayed-type hypersensitivity (DTH) reactivity in such mice at the challenged site. We summarize from our data that some identical proteins to previous from SP15 family protein of 14.2 and 13.6 kDa molecular size, derived from Indian P. argentipes and reported its first time, can also be significant in resolution of VL infection after modulation of host protective T cell response in VL.

Leishmania donovani, the causative agent of Indian visceral leishmaniasis has to face several barriers of the immune system inside the mammalian host for its survival. The complement system is one of the first barriers and consists of a well-balanced network of proteases including S1A family serine proteases (SPs). Inhibitor of serine peptidases (ISPs) is considered as inhibitor of S1A family serine peptidases and is reported to be present in trypanosomes, including Leishmania. In our previous study, we have deciphered the role of ISPs [LdISP1 and L. donovani inhibitor of serine peptidases 2 (LdISP2)] in the survival of L. donovani inside the sandfly midgut. However, the role of theses ISPs in the survival of L. donovani inside mammalian host still remains elusive. In the present study, we have deciphered the inhibitory effect of LdISPs on the host complement S1A serine peptidases, such as C1r/C1s and MASP1/MASP2. Our study suggested that although both rLdISP1 and rLdISP2 inferred strong interaction with C1complex and MBL-associated serine proteases (MASPs) but rLdISP2 showed the stronger inhibitory effect on MASP2 than rLdISP1. Moreover, we found that rLdISP2 significantly reduces the formation of C3, C5 convertase, and membrane attacking complex (MAC) by lectin pathway (LP) resulting in significant reduction in serum mediated lysis of the parasites. The role of LdISP2 on neutrophil elastase-mediated C5aR signaling was also evaluated. Notably, our results showed that infection of macrophages with ISP2-overexpressed Leishmania parasites significantly induces the expression of C5aR both at the transcript and translational level. Simultaneously, infection with ISP2KD parasites results in downregulation of host PI3K/AKT phosphorylation and increased in IL-12 production. Taken together, our findings clearly suggest that LdISP2 promotes parasite survival inside host by inhibiting MAC formation and complement-mediated lysis via LP and by upregulation of C5aR signaling.

Currently, there is no approved vaccine for visceral leishmaniasis (VL) caused by L. donovani. The ability to manipulate Leishmania genome by eliminating or introducing genes necessary for parasites' survival considered as the powerful strategy to generate the live attenuated vaccine. In the present study fructose-1,6-bisphosphatase (LdFBPase) gene deleted L. donovani (Δfbpase) was generated using homologous gene replacement strategy. Though LdFBPase gene deletion (Δfbpase) does not affect the growth of parasite in the promastigote form but axenic amastigotes display a marked reduction in their capacity to multiply in vitro inside macrophages and in vivo in Balb/c mice. Though Δfbpase L. donovani parasite persisted in BALB/c mice up to 12 weeks but was unable to cause infection, we tested its ability to protect against a virulent L. donovani challenge. Notably, intraperitoneal immunisation with live Δfbpase parasites displayed the reduction of parasites load in mice spleen and liver post challenge. Moreover, immunised BALB/c mice showed a reversal of T cell anergy and high levels of NO production that result in the killing of the parasite. A significant, correlation was found between parasite clearance and elevated IFNγ, IL12, and IFNγ/IL10 ratio compared to IL10 and TGFβ in immunised and challenged mice. Results suggested the generation of protective Th1 type immune response which induced significant parasite clearance at 12-week, as well as 16 weeks post, challenged immunised mice, signifying sustained immunity. Therefore, we propose that Δfbpase L. donovani parasites can be a live attenuated vaccine candidate for VL and a good model to understand the correlatives of protection in visceral leishmaniasis.