Good Candidates For Vaccine Development Research Articles

Bioinformatic Approach of B and T Cell Epitopes of PLD and CP40 Proteins of Corynebacterium pseudotuberculosis ovis Mexican Isolate 2J-L towards a Peptide-Based Vaccine.

Mapping B and T cell epitopes constitutes an important action for peptide vaccine design. PLD and CP40 virulence factors of Corynebacterium pseudotuberculosis biovar ovis, a causal agent of Caseous Lymphadenitis, have been evaluated in a murine model as good candidates for vaccine development. Therefore, the goal of this work was to in silico analyze B and T cell epitopes of the PLD and CP40 proteins of a Mexican isolate of Corynebacterium pseudotuberculosis ovis. The Immune Epitope Data Base and Resource website was employed to predict the linear and conformational B-cell, T CD4+, and T CD8+ epitopes of PLD and CP40 proteins of Corynebacterium pseudotuberculosis ovis Mexican strain 2J-L. Fifty B cell epitopes for PLD 2J-L and forty-seven for CP40 2J-L were estimated. In addition, T CD4+ and CD8+ cell epitopes were predicted for PLD 2J-L (MHC I:16 epitopes, MHC II:10 epitopes) and CP40 2J-L (MHC I: 15 epitopes, MHC II: 13 epitopes). This study provides epitopes, paying particular attention to sequences selected by different predictor programs and overlap sequences as B and T cell epitopes. PLD 2J-L and CP40 2J-L protein epitopes may aid in the design of a promising peptide-based vaccine against Caseous Lymphadenitis in Mexico.

Production of Zika Virus Virus-Like Particles.

Zika virus (ZIKV) is a mosquito-transmitted virus that has caused major outbreaks of disease around the world over the last few years. The infectious ZIKV consists of a structural protein outer shell surrounding a nucleocapsid. Virus-like particles (VLP) consist of the outer structural protein shell, but without the nucleocapsid, and are hence noninfectious. VLP, however, are structurally equivalent to the native virus and thus present a similar antigenic profile. These properties make them good candidates for vaccine development. ZIKV VLP can be generated on a laboratory scale by cloning the relevant structural proteins into a eukaryotic expression vector and transfecting the construct into mammalian cells. The secreted VLP can be harvested from the culture medium and purified by sucrose cushion ultracentrifugation. Validation of the VLP is achieved through western blotting and electron microscopy.

Effective Platform for the Production of Recombinant Outer Membrane Vesicles in Gram-Negative Bacteria.

Bacterial outer membrane vesicles (OMVs) typically contain multiple immunogenic molecules that include antigenic proteins, making them good candidates for vaccine development. In animal models, vaccination with OMVs has been shown to confer protective immune responses against many bacterial diseases. It is possible to genetically introduce heterologous protein antigens to the bacterial host that can then be produced and relocated to reside within the OMVs by means of the host secretion mechanisms. Accordingly, in this study we sought to develop a novel platform for recombinant OMV (rOMV) production in the widely used bacterial expression host species, Escherichia coli. Three different lipoprotein signal peptides including their Lol signals and tether sequences-from Neisseria meningitidis fHbp, Leptospira interrogans LipL32, and Campylobactor jejuni JlpA-were combined upstream to the GFPmut2 model protein, resulting in three recombinant plasmids. Pilot expression studies showed that the fusion between fHbp and GFPmut2 was the only promising construct; therefore, we used this construct for large-scale expression. After inducing recombinant protein expression, the nanovesicles were harvested from cell-free culture media by ultrafiltration and ultracentrifugation. Transmission electron microscopy demonstrated that the obtained rOMVs were closed, circular single-membrane particles, 20-200 nm in size. Western blotting confirmed the presence of GFPmut2 in the isolated vesicles. Collectively, although this is a non-optimized, proof-of-concept study, it demonstrates the feasibility of this platform in directing target proteins into the vesicles for OMV-based vaccine development.

Immuno-informatics Analysis to Identify Novel Vaccine Candidates and Design of a Multi-Epitope Based Vaccine Candidate Against Theileria parasites.

Theileriosis poses a serious threat to ruminants in tropical and subtropical countries. It is a tick-borne disease, caused by an apicomplexan parasite, Theileria. The high disease burden in animals causes huge economic losses to marginal farmers. Further, with increasing cases of resistance to commonly used drugs, it is highly desirable to develop better and cost-effective vaccines against theileriosis. The only available vaccine, live attenuated parasite vaccine, has many drawbacks and hence is unsuitable for controlling this disease. Immuno-informatics has emerged as a useful tool in down selection of potential molecules for vaccine development. In this study, we have used an immuno-informatics driven genome-wide screening strategy to identify potential vaccine targets containing important and effective dominant immunogens against Theileria. The proteome of Theileria annulata was screened for proteins with probability of plasma membrane localization or GPI anchor. The proteins non-homologous to the host (bovine) were selected and their antigenicity was analyzed. The B-cell epitopes were identified in the selected proteins and mapped in the modeled structure of the proteins. A total of 19 linear epitopes in 12 proteins, exposed in the extracellular space and having the potential to induce protective antibodies were obtained. Additionally, CTL epitopes which are peptides with 9-mer core sequence, were also identified, modeled and docked with bovine MHC-I structures. The CTL epitopes showing high binding energy with the bovine MHC-I were further engineered in silico to design a putative multi-epitope vaccine candidate against Theileria parasites. The docking studies and molecular dynamics studies with the predicted multi-epitope vaccine candidate and modeled bovine TLR4 exhibited strong binding energy, suggesting that the complex is stable and the putative multi-epitope vaccine candidate can be a potentially good candidate for vaccine development.

Molecular docking of immunogenic peptide of Toxoplasma gondii and encapsulation with polymer as vaccine candidate

Toxoplasma gondii is one of the most widely spread parasitic organisms in the world. T. gondii causes primary, chronic infection and mortality. Major surface antigen 1 is the most abundant tachyzoite surface protein and highly conserved between species and causes strong humoural response. Some studies showed that the peptide sequence of surface antigen has immunity. Therefore, tachyzoite surface antigenic peptide sequence is one of the good candidates for vaccine development. However, conformational information and delivery systems are very important parameters for vaccine development. Computational chemistry which is used as an effective method to perform drug or vaccine design provides important information on structure–activity relationship, biological effects of functional groups, molecular geometry, design of enzyme inhibitors and antagonists. The interaction of immunological peptides with protein systems was carried out by means of computing the free energy of binding using the molecular docking technique. Due to the major histocompatibility complex (MHC), proteins play a substantial role for adaptive immunity, the crystal structure of a MHC class I, which plays a pivotal role in the adaptive branch of the immune system, was preferred for docking calculations. A delivery system based on poly(lactic-co-glycolic acid) (PLGA) nanoparticles and peptide loaded PLGA nanoparticles was prepared in this study to improve the bioavailability of tachyzoite surface antigenic peptide sequence. Double emulsion method (water-in-oil-in-water or w/o/w) was used for synthesis of PLGA and peptide loaded PLGA nanoparticles. The average particle size, polydispersity index and zeta potential values of PLGA and peptide loaded PLGA nanoparticles were measured with zeta-sizer by using dynamic light scattering (DLS) technique. The scanning electron microscope (SEM) (Zeiss Supra 50 V) was used for imagining the peptide loaded PLGA nanoparticles. Cell toxicity of nanoparticles was assayed on AGS (gastric adenocarcinoma) cell line. To evaluate mitochondrial activity of cells and toxicity studies, XTT methods were carried out. In this study, we aimed to obtain specific immunological peptide loaded PLGA nanoparticles and characterize the formation with FTIR, zeta sizer and SEM imaging, and evaluate cytotoxicity and carry out molecular docking calculations of peptide–MHC protein in order to enlight in vivo events as vaccine candidate against T. gondii.

Transient Expression of an Immunogenic Envelope Attachment Glycoprotein of Nipah Virus in Nicotiana benthamiana

The Nipah virus is highly virulent to swine and humans. The envelope attachment glycoprotein (G) of Nipah virus plays a key role in viral entry and induction of neutralizing antibody in mammalian hosts, thus is considered a good candidate for vaccine development. Plant transient expression systems are gaining recognition as a viable alternative for the production of vaccine antigens. In this study, we expressed the Nipah virus G protein heterologously in Nicotiana benthamiana using an agroinfiltration approach. The highest expression of recombinant G protein in N. benthamiana at RNA and protein levels was detected on day 9 post-infiltration. Western blot analysis demonstrated that the purified G protein reacted specifically with rabbit anti-Nipah Virus serum, indicating its potential for vaccine use.

Recombinant TgHSP70 Immunization Protects against Toxoplasma gondii Brain Cyst Formation by Enhancing Inducible Nitric Oxide Expression.

Toxoplasma gondii is known to cause congenital infection in humans and animals and severe disease in immunocompromised individuals; consequently development of vaccines against the parasite is highly necessary. Under stress conditions, T. gondii expresses the highly immunogenic heat shock protein 70 (TgHSP70). Here, we assessed the protective efficacy of rTgHSP70 immunization combined with Alum in oral ME-49 T. gondii infection and the mechanisms involved on it. It was observed that immunized mice with rTgHSP70 or rTgHSP70 adsorbed in Alum presented a significantly reduced number of cysts in the brain that was associated with increased iNOS+ cell numbers in the organ, irrespective the use of the adjuvant. Indeed, ex vivo experiments showed that peritoneal macrophages pre-stimulated with rTgHSP70 presented increased NO production and enhanced parasite killing, and the protein was able to directly stimulate B cells toward antibody producing profile. In addition, rTgHSP70 immunization leads to high specific antibody titters systemically and a mixed IgG1/IgG2a response, with predominance of IgG1 production. Nonetheless, it was observed that the pretreatment of the parasite with rTgHSP70 immune sera was not able to control T. gondii internalization and replication by NIH fibroblast neither peritoneal murine macrophages, nor anti-rTgHSP70 antibodies were able to kill T. gondii by complement-mediated lysis, suggesting that these mechanisms are not crucial to resistance. Interestingly, when in combination with Alum, rTgHSP70 immunization was able to reduce inflammation in the brain of infected mice and in parallel anti-rTgHSP70 immune complexes in the serum. In conclusion, immunization with rTgHSP70 induces massive amounts of iNOS expression and reduced brain parasitism, suggesting that iNOS expression and consequently NO production in the brain is a protective mechanism induced by TgHSP70 immunization, therefore rTgHSP70 can be a good candidate for vaccine development against toxoplasmosis.

Antigenicity Analysis and Molecular Characterization of Two Outer Membrane Proteins of Vibrio alginolyticus Strain VA2 as Vaccine Candidates in Tiger Grouper Culture

Vibriosis is one of the most prevalent fish diseases caused by bacteria from the genus Vibrio. An effective method to counter Vibriosis is vaccination which increases the resistance of the immune system to subsequent pathogen infection. In this study, Vibrio alginolyticus strain VA2 isolated from infected tiger groupers was selected for determination of the antigenicity and molecular characterization of its Outer Membrane Proteins (OMPs). Protein profiling of OmpK and OmpW were evaluated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) while their antigenicity were tested by Western Blot. Results of the Western Blot assay revealed three antigenic OMP bands about 23, 31 and 34 kDa. Sequencing results showed that the full length of the target genes OmpK and OmpW of V. alginolyticus strain VA2 were 846 and 642 bp, respectively. Multiple alignment analysis showed that both regions of OmpK and OmpW were highly conserved among Vibrio species, thus are good candidates for vaccine development. Phylogenetic analysis of OmpK and OmpW showed that most branches in which they were grouped with had bootstrap values higher than 50%, indicating high confidence level of the branches. The antigenic sites of OmpK and OmpW proteins were predicted which were 34 and 27, respectively, thus are good candidates for vaccine development.

Immunogenicity of Novel Mumps Vaccine Candidates Generated by Genetic Modification

Mumps is a highly contagious human disease, characterized by lateral or bilateral nonsuppurative swelling of the parotid glands and neurological complications that can result in aseptic meningitis or encephalitis. A mumps vaccination program implemented since the 1960s reduced mumps incidence by more than 99% and kept the mumps case numbers as low as hundreds of cases per year in the United States before 2006. However, a large mumps outbreak occurred in vaccinated populations in 2006 and again in 2009 in the United States, raising concerns about the efficacy of the vaccination program. Previously, we have shown that clinical isolate-based recombinant mumps viruses lacking expression of either the V protein (rMuVΔV) or the SH protein (rMuVΔSH) are attenuated in a neurovirulence test using newborn rat brains (P. Xu et al., Virology 417:126-136, 2011, http://dx.doi.org/10.1016/j.virol.2011.05.003; P. Xu et al., J. Virol. 86:1768-1776, 2012, http://dx.doi.org/10.1128/JVI.06019-11) and may be good candidates for vaccine development. In this study, we examined immunity induced by rMuVΔSH and rMuVΔV in mice. Furthermore, we generated recombinant mumps viruses lacking expression of both the V protein and the SH protein (rMuVΔSHΔV). Analysis of rMuVΔSHΔV indicated that it was stable in tissue culture cell lines. Importantly, rMuVΔSHΔV was immunogenic in mice, indicating that it is a promising candidate for mumps vaccine development.

Structural Insights into Ail-Mediated Adhesion in Yersinia pestis

Ail is an outer membrane protein from Yersinia pestis that is highly expressed in a rodent model of bubonic plague, making it a good candidate for vaccine development. Ail is important for attaching to host cells and evading host immune responses, facilitating rapid progression of a plague infection. Binding to host cells is important for injection of cytotoxic Yersinia outer proteins. To learn more about how Ail mediates adhesion, we solved two high-resolution crystal structures of Ail, with no ligand bound and in complex with a heparin analog called sucrose octasulfate. We identified multiple adhesion targets, including laminin and heparin, and showed that a 40 kDa domain of laminin called LG4-5 specifically binds to Ail. We also evaluated the contribution of laminin to delivery of Yops to HEp-2 cells. This work constitutes a structural description of how a bacterial outer membrane protein uses a multivalent approach to bind host cells.

Molecular Evolution of Rickettsia Surface Antigens: Evidence of Positive Selection

The Rickettsia genus is a group of obligate intracellular parasitic alpha-proteobacteria that includes human pathogens responsible for the typhus disease and various types of spotted fevers. rOmpA and rOmpB are two members of the "surface cell antigen" (Sca) autotransporter (AT) protein family that may play key roles in the adhesion of the Rickettsia cells to the host tissue. These molecules are likely determinants for the pathogenicity of the Rickettsia and represent good candidates for vaccine development. We identified the 17 members of this family of outer-membrane proteins in nine fully sequenced Rickettsia genomes. The typical architecture of the Sca proteins is composed of an N-terminal signal peptide and a C-terminal AT domain that promote the export of the central passenger domain to the outside of the bacteria. A characteristic of this family is the frequent degradation of the genes, which results in different subsets of the sca genes being expressed among Rickettsia species. Here, we present a detailed analysis of their phylogenetic relationships and evolution. We provide strong evidence that rOmpA and rOmpB as well as three other members of the Sca protein family--Sca1, Sca2, and Sca4--have evolved under positive selection. The exclusive distribution of the predicted positively selected sites within the passenger domains of these proteins argues that these regions are involved in the interaction with the host and may be locked in "arms race" coevolutionary conflicts.

Antigenic Structure of Coxiella burnetii

The antigenic structure of Coxiella burnetii is being investigated by identifying both external and internal cellular epitopes of the morphologic cell types. Both the phase I lipopolysaccharide (LPSI) and several surface proteins are candidates for the development of subunit multivalent vaccines. The protective efficacy of purified LPSI was demonstrated in A/J mice. The purified LPSI preparations contained residual peptides detected by amino acid analysis. Therefore, the protection afforded by LPSI may be, in part, due to the presence of peptides. The purification of proteins free of LPSI must be accomplished before the protective efficacy of proteins or peptides can be established. We have identified three proteins that are both antigenic and immunogenic, as indicated by either enzyme immunoassay, radioimmunoprecipitation, immunoblot assay, or lymphocyte transformation. A 62-kDa protein antigen encoded by the htpB gene of C. burnetii was analyzed for immunogenicity. The purified protein antigen was immunogenic, as it elicited specific antibodies and performed as recall antigen in lymphocyte stimulation assays. The antigen was not detected on the surface of phase I cells but was highly represented on the surface of phase II cells. Therefore, the protein may not be a good candidate for vaccine development. The diagnostic utility of the 62-kDa protein antigen lies in the fact that convalescent and chronic Q fever sera from human patients reacted with the antigen, whereas acute sera did not. Although the 62-kDa protein is a "common antigen," specific peptide-based diagnostic reagents may be useful in the detection of Q fever disease progression. A major surface protein (P1) of roughly 29.5 kDa was purified from the phase I Nine Mile (clone 7) strain. No LPSI was detected in the P1 preparation by three different LPSI monoclonal antibodies. Monoclonal antibodies prepared against P1 were effective in localizing the protein on the cell surface, in the cell wall, and associated with the peptidoglycan of large cells of C. burnetii. Small, pressure-resistant cells did not contain P1. Mice immunized with two 25-micrograms injections of LPSI produced antibodies against LPSI and phase I whole cells. No antibody was detected against phase II whole cells. Immunization with P1 induced antibody against the LPSI fraction and phase I and phase II whole cells. P1 was more effective than LPSI in reducing the number of infectious C. burnetii in the spleens of challenged mice. The gene encoding another protein (P2) recognized by P1 monoclonal antibodies was cloned and sequenced.(ABSTRACT TRUNCATED AT 400 WORDS)