Exploratory algorithm to devise multi-epitope subunit vaccine by investigating Leishmania donovani membrane proteins

Visceral leishmaniasis (VL) is a deadly parasitic infection which affects poorest to poor population living in the endemic countries. Increasing resistant to existing drugs, disease burden and a significant number of deaths, necessitates the need for an effective vaccine to prevent the VL infection. This study employed a combinatorial approach to develop a multi-epitope subunit vaccine by exploiting Leishmania donovani membrane proteins. Cytotoxic T- and helper T-lymphocyte binding epitopes along with suitable adjuvant and linkers were joined together in a sequential manner to design the subunit vaccine. The occurrence of B-cell and IFN-γ inducing epitopes approves the ability of subunit vaccine to develop humoral and cell-mediated immune response. Physiochemical parameters of vaccine protein were also assessed followed by homology modeling, model refinement and validation. Moreover, disulfide engineering was performed for the increasing stability of the designed vaccine and molecular dynamics simulation was performed for the comparative stability purposes and to conform the geometric conformations. Further, molecular docking and molecular dynamics simulation study of a mutated and non-mutated subunit vaccine against TLR-4 immune receptor were performed and respective complex stability was determined. In silico cloning ensures the expression of designed vaccine in pET28a(+) expression vector. This study offers a cost-effective and time-saving way to design a novel immunogenic vaccine that could be used to prevent VL infection.

Communicated by Ramaswamy H. Sarma     

Designing of a Novel Fusion Protein Vaccine Candidate Against Human Visceral Leishmaniasis (VL) Using Immunoinformatics and Structural Approaches

Leishmaniasis is caused by an obligate intracellular protozoan parasite. The clinical forms of leishmaniasis differ from cutaneous leishmaniasis, mucocutaneous leishmaniasis and visceral leishmaniasis (VL) which depend on the parasite species and the host’s immune responses. There are significant challenges to the available anti-leishmanial drug therapy, particularly in severe forms of disease, and the rise of drug resistance has made it more difficult. Currently, no licensed vaccines have been introduced to the market for the control and elimination of VL. A potential target for use in candidate vaccines against leishmaniasis has been shown to be leishmania Kinetoplastid membrane protein-11 (KMP-11) antigen. In this study, we chose KMP-11 antigen as target antigen in our vaccine construct. In addition, B-type flagellin (fliC) was used as an adjuvant for enhancing vaccine immunogenicity. The GSGSGSGSGSG linker was applied to link the KMP-11 antigen and fliC (KMP-11-fliC) to construct our fusion protein. Bioinformatics approaches such as; 3D homology modeling, CTL, B-cell, MHC class I and II epitopes prediction, allergenicity, antigenicity evaluations, molecular docking, fast simulations of flexibility of docked complex and in silico cloning were employed to analysis and evaluation of various properties of the designed fusion construct. Computational results showed that our engineered structure has the potential for proper stimulation of cellular and humoral immune responses against VL. Consequently, it could be proposed as a candidate vaccine against VL according to these data and after verifying the efficacy of the candidate vaccine through in vivo and in vitro immunological tests.

     

Designing B‐ and T‐cell multi‐epitope based subunit vaccine using immunoinformatics approach to control Zika virus infection

The Zika virus is a rapidly spreading Aedes mosquito‐borne sickness, which creates an unanticipated linkage birth deformity and neurological turmoil. This study represents the use of the combinatorial immunoinformatics approach to develop a multiepitope subunit vaccine using the structural and nonstructural proteins of the Zika virus. The designed subunit vaccine consists of cytotoxic T‐lymphocyte and helper T‐lymphocyte epitopes accompanied by suitable adjuvant and linkers. The presence of humoral immune response specific B‐cell epitopes was also confirmed by B‐cell epitope mapping among vaccine protein. Further, the vaccine protein was characterized for its allergenicity, antigenicity, and physiochemical parameters and found to be safe and immunogenic. Molecular docking and molecular dynamics studies of the vaccine protein with the toll‐like receptor‐3 were performed to ensure the binding affinity and stability of their complex. Finally, in silico cloning was performed for the effective expression of vaccine construct in the microbial system (Escherichia coli K12 strain). Aforementioned approaches result in the multiepitope subunit vaccine which may have the ability to induce cellular as well as humoral immune response. Moreover, this study needs the experimental validation to prove the immunogenic and protective behavior of the developed subunit vaccine.     

Genome-based approaches to develop epitope-driven subunit vaccines against pathogens of infective endocarditis

Infective endocarditis (IE) has emerged as a public health problem due to changes in the etiologic spectrum and due to involvement of resistant bacterial strains with increased virulence. Developing potent vaccine is an important strategy to tackle IE. Complete genome sequences of eight selected pathogens of IE paved the way to design common T-cell driven subunit vaccines. Comparative genomics and subtractive genomic analysis were applied to identify adinosine tri phosphate (ATP)-binding cassette (ABC) transporter ATP-binding protein from Streptococcus mitis (reference organism) as common vaccine target. Reverse vaccinology technique was implemented using computational tools such as ProPred, SYFPEITHI, and Immune epitope database. Twenty-one T-cell epitopes were predicted from ABC transporter ATP-binding protein. Multiple sequence alignment of ABC transporter ATP-binding protein from eight selected IE pathogens was performed to identify six conserved T-cell epitopes. The six selected T-cell epitopes were further evaluated at structure level for HLA-DRB binding through homology modeling and molecular docking analysis using Maestro v9.2. The proposed six T-cell epitopes showed better binding affinity with the selected HLA-DRB alleles. Subsequently, the docking complexes of T-cell epitope and HLA-DRBs were ranked based on XP Gscore. The T-cell epitope (208-LNYITPDVV-216)–HLA-DRB1^?0101 (1T5?W) complex having the best XP Gscore (?13.25?kcal/mol) was assessed for conformational stability and interaction stability through molecular dynamic simulation for 10?ns using Desmond v3.2. The simulation results revealed that the HLA-DRB–epitope complex was stable throughout the simulation time. Thus, the epitope would be ideal candidate for T-cell driven subunit vaccine design against infective endocarditis.     

Efficient generation of monoclonal antibodies from single rhesus macaque antibody secreting cells

Nonhuman primates (NHPs) are used as a preclinical model for vaccine development, and the antibody profiles to experimental vaccines in NHPs can provide critical information for both vaccine design and translation to clinical efficacy. However, an efficient protocol for generating monoclonal antibodies from single antibody secreting cells of NHPs is currently lacking. In this study we established a robust protocol for cloning immunoglobulin (IG) variable domain genes from single rhesus macaque (Macaca mulatta) antibody secreting cells. A sorting strategy was developed using a panel of molecular markers (CD3, CD19, CD20, surface IgG, intracellular IgG, CD27, Ki67 and CD38) to identify the kinetics of B cell response after vaccination. Specific primers for the rhesus macaque IG genes were designed and validated using cDNA isolated from macaque peripheral blood mononuclear cells. Cloning efficiency was averaged at 90% for variable heavy (VH) and light (VL) domains, and 78.5% of the clones (n = 335) were matched VH and VL pairs. Sequence analysis revealed that diverse IGHV subgroups (for VH) and IGKV and IGLV subgroups (for VL) were represented in the cloned antibodies. The protocol was tested in a study using an experimental dengue vaccine candidate. About 26.6% of the monoclonal antibodies cloned from the vaccinated rhesus macaques react with the dengue vaccine antigens. These results validate the protocol for cloning monoclonal antibodies in response to vaccination from single macaque antibody secreting cells, which have general applicability for determining monoclonal antibody profiles in response to other immunogens or vaccine studies of interest in NHPs.     

A novel recombinant Leishmania donovani p45, a partial coding region of methionine aminopeptidase, generates protective immunity by inducing a Th1 stimulatory response against experimental visceral leishmaniasis

The development of a vaccine against visceral leishmaniasis (VL) conferring long-lasting immunity remains a challenge. Identification and proteomic characterization of parasite proteins led to the detection of p45, a member of the methionine aminopeptidase family. To our knowledge the present study is the first known report that describes the molecular and immunological characterization of p45. Recombinant Leishmania donovani p45 (rLdp45) induced cellular responses in cured hamsters and generated Th1-type cytokines from peripheral blood mononuclear cells of cured/endemic VL patients. Immunization with rLdp45 exerted considerable prophylactic efficacy (～85%) supported by an increase in mRNA expression of iNOS, IFN-γ, TNF-α and IL-12 and decrease in TGF-β and IL-4, indicating its potential as a vaccine candidate against VL.     

Structure-based virtual screening,molecular docking,ADMET and molecular simulations to develop benzoxaborole analogs as potential inhibitor against Leishmania donovani trypanothione reductase

Visceral leishmaniasis (VL) is the most fatal form of leishmaniasis and it affects 70 countries worldwide. Increasing drug resistant for antileishmanial drugs such as miltefosine, sodium stibogluconate and pentamidine has been reported in the VL endemic region. Amphotericin B has shown potential antileishmanial activity in different formulations but its cost of treatment and associated nephrotoxicity have limited its use by affected people living in the endemic zone. To control the VL infection in the affected countries, it is necessary to develop new antileishmanial compounds with high efficacy and negligible toxicity. Computer aided programs such as binding free energy estimation; ADMET prediction and molecular dynamics simulation can be used to investigate novel antileishmanial molecules in shorter duration. To develop antileishmanial lead molecule, we performed standard precision (SP) docking for 1160 benzoxaborole analogs along with reference inhibitors against trypanothione reductase of Leishmania parasite. Furthermore, extra precision (XP) docking, ADMET prediction, prime MM-GBSA was conducted over 115 ligands, showing better docking score than reference inhibitors to get potential antileishmanial compounds. Simultaneously, area under the curve (AUC) was estimated using ROC plot to validate the SP and XP docking protocol. Later on, two benzoxaborole analogs with best MM-GBSA ΔG-bind were subjected to molecular simulation and docking confirmation to ensure the ligand interaction with TR. The presented drug discovery based on computational study confirms that BOB27 can be used as a potential drug candidate and warrants further experimental investigation to fight against VL in endemic areas.     

Protection of red snapper (Lutjanus sanguineus) against Vibrio alginolyticus with a DNA vaccine containing flagellin flaA gene

Aims: The main aims of this study were to construct a DNA vaccine containing flagellin flaA gene from Vibrio alginolyticus strain HY9901 and to explore the potential application of pcDNA‐flaA as a DNA vaccine candidate for red snapper (Lutjanus sanguineus). Methods and Results: Plasmid DNA encoding flagellin flaA gene (designated as pcDNA‐flaA) was used as a DNA vaccine to immunize red snapper. The distribution, expression and immunoprotection of the DNA vaccine were analysed in tissues of the red snapper by PCR, RT‐PCR and challenge test. PCR results indicated that pcDNA‐flaA distributed in liver, spleen, kidney, gill and injection site muscle at 7–28 days after vaccination. RT‐PCR results indicated that the flaA gene was expressed in all above tissues of vaccinated fish at 7–28 days after vaccination. In addition, fish receiving the DNA vaccine developed a protective response to live V. alginolyticus challenge 28 days post inoculation, the relative per cent survival (RPS) was 88%. Conclusions: This study showed that injection of pcDNA‐flaA induced an efficient, systemic and antigen‐specific immune response in red snapper, which makes it an effective vaccine candidate against V. alginolyticus infection. Significance and Impact of the Study: The finding that red snapper does adequately respond to pcDNA‐flaA intramuscular injection makes pcDNA‐flaA a promising candidate for DNA vaccine treatment. Furthermore, the availability of red snapper for foreign gene expression represents a useful model to develop effective prophylactic strategies and opens new perspectives for the treatment of bacterial pathogens of marine cultured fish.     

Proteome-wide screening for designing a multi-epitope vaccine against emerging pathogen Elizabethkingia anophelis using immunoinformatic approaches

Abstract

Elizabethkingia anophelis is an emerging human pathogen causing neonatal meningitis, catheter-associated infections and nosocomial outbreaks with high mortality rates. Besides, they are resistant to most antibiotics used in empirical therapy. In this study, therefore, we used immunoinformatic approaches to design a prophylactic peptide vaccine against E. anophelis as an alternative preventive measure. Initially, cytotoxic T-lymphocyte (CTL), helper T-lymphocyte (HTL), and linear B-lymphocyte (LBL) epitopes were predicted from the highest antigenic protein. The CTL and HTL epitopes together had a population coverage of 99.97% around the world. Eventually, six CTL, seven HTL, and two LBL epitopes were selected and used to construct a multi-epitope vaccine. The vaccine protein was found to be highly immunogenic, non-allergenic, and non-toxic. Codon adaptation and in silico cloning were performed to ensure better expression within E. coli K12 host system. The stability of the vaccine structure was also improved by disulphide bridging. In addition, molecular docking and dynamics simulation revealed strong and stable binding affinity between the vaccine and toll-like receptor 4 (TLR4) molecule. The immune simulation showed higher levels of T-cell and B-cell activities which was in coherence with actual immune response. Repeated exposure simulation resulted in higher clonal selection and faster antigen clearance. Nevertheless, experimental validation is required to ensure the immunogenic potency and safety of this vaccine to control E. anophelis infection in the future.

Communicated by Ramaswamy H. Sarma     

Contriving multiepitope subunit vaccine by exploiting structural and nonstructural viral proteins to prevent Epstein–Barr virus-associated malignancy

Cancer is one of the common lifestyle diseases and is considered to be the leading cause of death worldwide. Epstein–Barr virus (EBV)-infected individuals remain asymptomatic; but under certain stress conditions, EBV may lead to the development of cancers such as Burkitt’s and Hodgkin’s lymphoma and nasopharyngeal carcinoma. EBV-associated cancers result in a large number of deaths in Asian and African population, and no effective cure has still been developed. We, therefore, tried to devise a subunit vaccine with the help of immunoinformatic approaches that can be used for the prevention of EBV-associated malignancies. The epitopes were predicted through B-cell, cytotoxic T lymphocytes (CTL), and helper T lymphocytes (HTL) from the different oncogenic proteins of EBV. A vaccine was designed by combining the B-cell and T-cell (HTL and CTL) epitopes through linkers, and for the enhancement of immunogenicity, an adjuvant was added at the N-terminal. Further, homology modeling was performed to generate the 3D structure of the designed vaccine. Moreover, molecular docking was performed between the designed vaccine and immune receptor (TLR-3) to determine the interaction between the final vaccine construct and the immune receptor complex. In addition, molecular dynamics was performed to analyze the stable interactions between the ligand final vaccine model and receptor TLR-3 molecule. Lastly, to check the expression of our vaccine construct, we performed in silico cloning. This study needed experimental validation to ensure its effectiveness and potency to control malignancy.     

Potential use of chitosan nanoparticles for oral delivery of DNA vaccine in Asian sea bass (Lates calcarifer) to protect from Vibrio (Listonella) anguillarum

In recent years, attention has been focused on the possibility of utilizing DNA vaccines in fish aquaculture. A successful regime for intramuscular injection of naked DNA into fish has been developed and novel methods to deliver this DNA to fish are under investigation. The potential of chitosan as a polycationic gene carrier for oral administration has been explored since 1990s. The present study examines the potential efficacy of DNA vaccine against Vibrio anguillarum through oral route using chitosan nanoparticles encapsulation. The porin gene of V. anguillarum was used to construct DNA vaccine using pcDNA 3.1, a eukaryotic expression vector and the construct was named as pVAOMP38. The chitosan nanoparticles were used to deliver the constructed plasmid. In vitro and in vivo expression of porin gene was observed in sea bass kidney cell line (SISK) and in fish, respectively by fluorescent microscopy. The cytotoxicity of chitosan encapsulated DNA vaccine construct was analyzed by MTT assay and it was found that the cytotoxicity of pVAOMP38/chitosan was quite low. Distribution of gene in different tissues was studied in fish fed with the DNA (pVAOMP38) encapsulated in chitosan by using immunohistochemistry. The results indicate that DNA vaccine can be easily delivered into fish by feeding with chitosan nanoparticles. After oral vaccination Asian sea bass were challenged with Vibrio anguillarum by intramuscular injection. A relative percent survival (RPS) rate of 46% was recorded. The results indicate that Sea bass (Lates calcarifer) orally vaccinated with chitosan-DNA (pVAOMP38) complex showed moderate protection against experimental V. anguillarum infection.     

Leishmania donovani Triose Phosphate Isomerase: A Potential Vaccine Target against Visceral Leishmaniasis

Visceral leishmaniasis (VL) is one of the most important parasitic diseases with approximately 350 million people at risk. Due to the non availability of an ideal drug, development of a safe, effective, and affordable vaccine could be a solution for control and prevention of this disease. In this study, a potential Th1 stimulatory protein- Triose phosphate isomerase (TPI), a glycolytic enzyme, identified through proteomics from a fraction of Leishmania donovani soluble antigen ranging from 89.9–97.1 kDa, was assessed for its potential as a suitable vaccine candidate. The protein- L. donovani TPI (LdTPI) was cloned, expressed and purified which exhibited the homology of 99% with L. infantum TPI. The rLdTPI was further evaluated for its immunogenicity by lymphoproliferative response (LTT), nitric oxide (NO) production and estimation of cytokines in cured Leishmania patients/hamster. It elicited strong LTT response in cured patients as well as NO production in cured hamsters and stimulated remarkable Th1-type cellular responses including IFN-ã and IL-12 with extremely lower level of IL-10 in Leishmania-infected cured/exposed patients PBMCs in vitro. Vaccination with LdTPI-DNA construct protected naive golden hamsters from virulent L. donovani challenge unambiguously (∼90%). The vaccinated hamsters demonstrated a surge in IFN-ã, TNF-á and IL-12 levels but extreme down-regulation of IL-10 and IL-4 along with profound delayed type hypersensitivity and increased levels of Leishmania-specific IgG2 antibody. Thus, the results are suggestive of the protein having the potential of a strong candidate vaccine.     

Studies of the conformational stability of invasion plasmid antigen B from Shigella

Shigella spp. are the causative agent of shigellosis, the second leading cause of diarrhea in children of ages 2–5. Despite many years of research, a protective vaccine has been elusive. We recently demonstrated that invasion plasmid antigens B and D (IpaB and IpaD) provide protection against S. flexneri and S. sonnei. These proteins, however, have very different properties which must be recognized and then managed during vaccine formulation. Herein, we employ spectroscopy to assess the stability of IpaB as well as IpgC (invasion protein gene), IpaB's cognate chaperone, and the IpaB/IpgC complex. The resulting data are mathematically summarized into a visual map illustrating the stability of the proteins and their complex as a function of pH and temperature. The IpaB/IpgC complex exhibits thermal stability at higher pH values but, though initially stable, quickly unfolds with increasing temperature when maintained at lower pH. In contrast, IpaB is a much more complex protein exhibiting increased stability at higher pH, but shows initial instability at lower pH values with pH 5 showing a distinct transition. IpgC precipitates at and below pH 5 and is stable above pH 7. Most strikingly, it is clear that complex formation results in stabilization of the two components. This work serves as a basis for the further development of IpaB as a vaccine candidate as well as extends our understanding of the structural stability of the Shigella type III secretion system.     

Strategies to overexpress enterotoxigenic <Emphasis Type="Italic">Escherichia coli</Emphasis> (ETEC) colonization factors for the construction of oral whole-cell inactivated ETEC vaccine candidates

Enterotoxigenic Escherichia coli (ETEC) is an important cause of diarrheal disease and deaths among children in developing countries and the major cause of traveler's diarrhea (TD). Since surface protein colonization factors (CFs) of ETEC are important for pathogenicity and immune protection is mainly mediated by locally produced IgA antibodies in the gut, much effort has focused on the development of an oral CF-based vaccine. The most extensively studied ETEC candidate vaccine is the rCTB-CF ETEC vaccine, containing recombinantly produced cholera B subunit and the most commonly encountered ETEC CFs on the surface of whole inactivated bacteria. Initial clinical trials with this vaccine showed significant immune responses against the key antigens in different age groups in Bangladesh and Egypt and protection against more severe TD in Western travelers. However, when tested in a phase-III trial in Egyptian infants, the protective efficacy of the vaccine was found to be low, indicating the need to improve the immunogenicity of the vaccine, e.g., by increasing the levels of the protective antigens. This review describes different strategies for the construction of recombinant nontoxigenic E. coli and Vibrio cholerae candidate vaccine strains over-expressing higher amounts of ETEC CFs than clinical ETEC isolates selected to produce high levels of the respective CF, e.g., those ETEC strains which have been used in the rCTB-CF ETEC vaccine. Several different expression vectors containing the genes responsible for the expression and assembly of the examined CFs, all downstream of the powerful tac promoter, which could be maintained either with or without antibiotic selection, were constructed. Expression from the tac promoter was under the control of the lacI ^q repressor present on the plasmids. Following induction with isopropyl-β-d-thiogalactopyranoside, candidate vaccine strains over-expressing single CFs, unnatural combinations of two CFs, and also hybrid forms of ETEC CFs were produced. Specific monoclonal antibodies against the major subunits of the examined CF were used to quantify the amount of the surface-expressed CF by a dot-blot assay and inhibition ELISA. Oral immunization with formalin- or phenol-inactivated recombinant bacteria over-expressing the CFs was found to induce significantly higher antibody responses compared to immunization with the previously used vaccine strains. We therefore conclude that our constructs may be useful as candidate strains in an oral whole-cell inactivated CF ETEC vaccine.     

Recombinant NAD-dependent SIR-2 Protein of Leishmania donovani: Immunobiochemical Characterization as a Potential Vaccine against Visceral Leishmaniasis

BackgroundThe development of a vaccine conferring long-lasting immunity remains a challenge against visceral leishmaniasis (VL). Immunoproteomic characterization of Leishmania donovani proteins led to the identification of a novel protein NAD+-dependent Silent Information regulatory-2 (SIR2 family or sirtuin) protein (LdSir2RP) as one of the potent immunostimulatory proteins. Proteins of the SIR2 family are characterized by a conserved catalytic domain that exerts unique NAD-dependent deacetylase activity. In the present study, an immunobiochemical characterization of LdSir2RP and further evaluation of its immunogenicity and prophylactic potential was done to assess for its possible involvement as a vaccine candidate against leishmaniasis.Conclusion/SignificanceThe immunobiochemical characterization strongly suggest the potential of rLdSir2RP as vaccine candidate against VL and supports the concept of its being effective T-cell stimulatory antigen.     

Construction of a trivalent candidate Shigella vaccine strain with host-vector balanced-lethal system

A trivalent liveShigella vaccine candidate FSD01 against S.flexneri 2a, S.sonnei and S.dysenteriae I was constructed. This candidate strain was based on the S.flexneri 2a vaccine T32. By homologous recombination exchange, the chromosomalasd gene of T32 was site-specifically inactivated, resulting in the strain unable to grow normally in LB broth, while anotherasd gene of S.mutans was employed to construct an Asd⁺ complementary vector. This combination ofasd ^- host/Asd⁺ vector formed a balanced-lethal expression system in T32 strain. By use of this system, two important protective antigen genes coding for S.sonnei Form I antigen and Shiga toxin B subunit were cloned and expressed in T32, which led to the construction of trivalent candidate vaccine FSD01. Experimental results showed that this strain was genetically stable, but its recombinant plasmid was non-resistant. Moreover, it was able to effectively express trivalent antigens in one host and induce protective responses in mice against the challenges of the above threeShigella strains.     

Clinical and Parasitological Protection in a Leishmania infantum-Macaque Model Vaccinated with Adenovirus and the Recombinant A2 Antigen

Background

Visceral leishmaniasis (VL) is a severe vector-born disease of humans and dogs caused by Leishmania donovani complex parasites. Approximately 0.2 to 0.4 million new human VL cases occur annually worldwide. In the new world, these alarming numbers are primarily due to the impracticality of current control methods based on vector reduction and dog euthanasia. Thus, a prophylactic vaccine appears to be essential for VL control. The current efforts to develop an efficacious vaccine include the use of animal models that are as close to human VL. We have previously reported a L. infantum-macaque infection model that is reliable to determine which vaccine candidates are most worthy for further development. Among the few amastigote antigens tested so far, one of specific interest is the recombinant A2 (rA2) protein that protects against experimental L. infantum infections in mice and dogs.

Methodology/Principal Findings

Primates were vaccinated using three rA2-based prime-boost immunization regimes: three doses of rA2 plus recombinant human interleukin-12 (rhIL-12) adsorbed in alum (rA2/rhIL-12/alum); two doses of non-replicative adenovirus recombinant vector encoding A2 (Ad5-A2) followed by two boosts with rA2/rhIL-12/alum (Ad5-A2+rA2/rhIL12/alum); and plasmid DNA encoding A2 gene (DNA-A2) boosted with two doses of Ad5-A2 (DNA-A2+Ad5-A2). Primates received a subsequent infectious challenge with L. infantum. Vaccines, apart from being safe, were immunogenic as animals responded with increased pre-challenge production of anti-A2-specific IgG antibodies, though with some variability in the response, depending on the vaccine formulation/protocol. The relative parasite load in the liver was significantly lower in immunized macaques as compared to controls. Protection correlated with hepatic granuloma resolution, and reduction of clinical symptoms, particularly when primates were vaccinated with the Ad5-A2+rA2/rhIL12/alum protocol.

Conclusions/Significance

The remarkable clinical protection induced by A2 in an animal model that is evolutionary close to humans qualifies this antigen as a suitable vaccine candidate against human VL.     

Efficient expression of Human papillomavirus 16 E7 oncoprotein fused to C-terminus of Tobacco mosaic virus (TMV) coat protein using molecular chaperones in Escherichia coli

The Human papillomavirus 16 (HPV16) E7 oncoprotein is a promising candidate for development of anti-cancer therapeutic vaccine. We have prepared the expression construct carrying mutagenized E7 oncoprotein fused to the C-terminus of Tobacco mosaic virus (TMV) coat protein via 15 amino acids β-sheet linker. The fusion protein was expressed in Escherichia coli MC 1061 cells. We have obtained high level expression, but most of the protein remained in insoluble inclusion bodies. To increase the ratio of soluble protein various molecular chaperones (TF, DnaK-DnaJ-GrpE, GroEL-GroES) were used. The immunological reactivity of expressed recombinant protein was evaluated with anti-E7 and anti-TMV antibodies. The distribution of expressed product during ultracentrifugation on sucrose gradient was studied.