Combining proteomics and bioinformatics to explore novel tegumental antigens as vaccine candidates against Echinococcus granulosus infection.

Echinococcus granulosus is the parasite responsible for cystic echinococcosis (CE), an important worldwide-distributed zoonosis. New effective vaccines against CE could potentially have great economic and health benefits. Here, we describe an innovative vaccine design scheme starting from an antigenic fraction enriched in tegumental antigens from the protoscolex stage (termed PSEx) already known to induce protection against CE. We first used mass spectrometry to characterize the protein composition of PSEx followed by Gene Ontology analysis to study the potential Biological Processes, Molecular Functions, and Cellular Localizations of the identified proteins. Following, antigenicity predictions and determination of conservancy degree against other organisms were determined. Thus, nine novel proteins were identified as potential vaccine candidates. Furthermore, linear B cell epitopes free of posttranslational modifications were predicted in the whole PSEx proteome through colocalization of in silico predicted epitopes within peptide fragments identified by matrix-assisted laser desorption/ionization-TOF/TOF. Resulting peptides were termed "clean linear B cell epitopes," and through BLASTp scanning against all nonhelminth proteins, those with 100% identity against any other protein were discarded. Then, the secondary structure was predicted for peptides and their corresponding proteins. Peptides with highly similar secondary structure respect to their parental protein were selected, and those potentially toxic and/or allergenic were discarded. Finally, the selected clean linear B cell epitopes were mapped within their corresponding 3D-modeled protein to analyze their possible antibody accessibilities, resulting in 14 putative peptide vaccine candidates. We propose nine novel proteins and 14 peptides to be further tested as vaccine candidates against CE.

Genome based screening of epitope ensemble vaccine candidates against dreadful visceral leishmaniasis using immunoinformatics approach.

Visceral leishmaniasis (VL) is a dreadful protozoan disease caused by Leishmania donovani that severely affects huge populations in tropical and sub-tropical regions. The present study reports an unbiased genome based screening of 4 potent vaccine antigens against 8023 L. donovani proteins by following the criteria of presence of signal peptides, GPI-anchors and ≤1 transmembrane helix using advanced bioinformatics tools viz. SignalP4.0, PredGPI and TMHMM2.0, respectively. They are designated as genome based predicted signal peptide antigens (GBPSPA). The antigenicity/immunogenicity of chosen vaccine antigens (GBPSPA) with 4 randomly selected known leishmanial antigens (RSKLA) was compared by simulation study employing C-ImmSim software for human immune responses. This revealed better immunological responses. These antigens were further evaluated for the presence of B- and T-cell epitopes using immune epitope database (IEDB) based recommended consensus method of MHC class I and II tools. It was found to forecast CD4+ and CD8+ T-cell responses in genetically diverse human population worldwide as well as different endemic regions through IEDB based predicted population coverage (PPC) analysis tool. The worldwide percent PPC value of combined (HLA class I and II) epitope ensemble forecast was found to be 99.98, 99.96 and 50.04, respectively for GBPSPA, RSKLA and experimentally known epitopes (EKE) of L. donovani. Therefore, these potential antigens/epitope ensembles could favor the design of prospective and novel vaccine constructs like self-assembled epitopes as nano vaccine formulations against VL. Overall, the present study will serve as a model framework that might improve the effectiveness of designed vaccine against L. donovani and other related pathogens.

Potential Vaccine Targets against Rabbit Coccidiosis by Immunoproteomic Analysis.

The aim of this study was to identify antigens for a vaccine or drug target to control rabbit coccidiosis. A combination of 2-dimensional electrophoresis, immunoblotting, and mass spectrometric analysis were used to identify novel antigens from the sporozoites of Eimeria stiedae. Protein spots were recognized by the sera of New Zealand rabbits infected artificially with E. stiedae. The proteins were characterized by matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF/TOF-MS) analysis in combination with bioinformatics. Approximately 868 protein spots were detected by silver-staining, and a total of 41 immunoreactive protein spots were recognized by anti-E. stiedae sera. Finally, 23 protein spots were successfully identified. The proteins such as heat shock protein 70 and aspartyl protease may have potential as immunodiagnostic or vaccine antigens. The immunoreactive proteins were found to possess a wide range of biological functions. This study is the first to report the proteins recognized by sera of infected rabbits with E. stiedae, which might be helpful in identifying potential targets for vaccine development to control rabbit coccidiosis.

Vaccination with Toxoplasma gondii calcium-dependent protein kinase 6 and rhoptry protein 18 encapsulated in poly(lactide-co-glycolide) microspheres induces long-term protective immunity in mice.

BACKGROUND: Toxoplasmosis is a worldwide zoonosis caused by the intracellular parasite Toxoplasma gondii. However, no effective vaccine is yet available. Poly(lactide-co-glycolide) polymers can reduce protein degradation and sustain the release of antigens over a long period, which could generate a long-lasting immune response in vivo. Using a mouse model of toxoplasmosis, we evaluated the protective efficacy of vaccination with two recombinant proteins, which are formulated in biodegradable polymers. METHODS: Two recombinant proteins, rCDPK6 and rROP18, were encapsulated in poly(D,L-lactide-co-glycolide) (PLG), and then injected subcutaneously into Kunming mice. The mice immune responses were evaluated in terms of lympho-proliferation, cytokine expression, and antibodies. The survival of infected mice and brain cyst formation were also evaluated at 6 weeks after challenge with T. gondii RH strain (genotype I) or PRU strain (genotype II). RESULTS: Both protein vaccines induced Th1-biased immune responses, with increased specific antibodies and T cells, high levels of interferon-γ and interleukin 2, and strong lymphocyte proliferative responses. The mice immunized with the various protein vaccines survived slightly longer time than the control groups (P > 0.05) after injection with T. gondii RH strain. There were fewer brain cysts in the mice in all the immunized groups than that in the control groups, and the brain cysts were significantly reduced in mice immunized with proteins + 206, rCDPK6 + PLG and rCDPK6 + rROP18 + PLG (P < 0.05) compared controls. Further comparison of the immune responses to the proteins adjuvanted with PLG or Montanide™ ISA 206 VG 6 weeks after the last immunization revealed that antigens encapsulated in PLG conferred greater protective immunity against challenge. CONCLUSIONS: These findings suggest that the two recombinant T. gondii proteins encapsulated in PLG conferred immunity to T. gondii for an extended period, providing the foundation for the further development of a commercial vaccine against toxoplasmosis.

From genome screening to creation of vaccine against Trypanosoma cruzi by use of immunoinformatics.

Chagas disease is caused by the protozoan parasite Trypanosoma cruzi, and activation of CD8(+) T cells is crucial for a protective immune response. Therefore, the identification of antigens with major histocompatibility complex class I epitopes is a crucial step for vaccine development against T. cruzi. Our aim was to identify novel antigens and epitopes by immunoinformatics analysis of the parasite proteome (12 969 proteins) and to validate their immunotherapeutic potential in infected mice. We identified 172 predicted epitopes, using NetMHC and RANKPEP. The corresponding protein sequences were reanalyzed to generate a consensus prediction, and 26 epitopes were selected for in vivo validation. The interferon γ (IFN-γ) recall response of splenocytes from T. cruzi-infected mice confirmed that 10 of 26 epitopes (38%) induced IFN-γ production. The immunotherapeutic potential of a mixture of all 10 peptides was evaluated in infected mice. The therapeutic vaccine was able to control T. cruzi infection, as evidenced by reduced parasitemia, cardiac tissue inflammation, and parasite burden and increased survival. These findings illustrate the benefits of this approach for the rapid development of a vaccine against pathogens with large genomes. The identified peptides and the proteins from which they are derived are excellent candidates for the development of a vaccine against T. cruzi.

Recent advances in Toxoplasma gondii immunotherapeutics.

Toxoplasmosis is an opportunistic infection caused by the protozoan parasite Toxoplasma gondii. T. gondii is widespread globally and causes severe diseases in individuals with impaired immune defences as well as congenitally infected infants. The high prevalence rate in some parts of the world such as South America and Africa, coupled with the current drug treatments that trigger hypersensitivity reactions, makes the development of immunotherapeutics intervention a highly important research priority. Immunotherapeutics strategies could either be a vaccine which would confer a pre-emptive immunity to infection, or passive immunization in cases of disease recrudescence or recurrent clinical diseases. As the severity of clinical manifestations is often greater in developing nations, the development of well-tolerated and safe immunotherapeutics becomes not only a scientific pursuit, but a humanitarian enterprise. In the last few years, much progress has been made in vaccine research with new antigens, novel adjuvants, and innovative vaccine delivery such as nanoparticles and antigen encapsulations. A literature search over the past 5 years showed that most experimental studies were focused on DNA vaccination at 52%, followed by protein vaccination which formed 36% of the studies, live attenuated vaccinations at 9%, and heterologous vaccination at 3%; while there were few on passive immunization. Recent progress in studies on vaccination, passive immunization, as well as insights gained from these immunotherapeutics is highlighted in this review.

Toward a defined anti-Leishmania vaccine targeting vector antigens: characterization of a protective salivary protein.

Leishmania parasites are transmitted to their vertebrate hosts by infected phlebotomine sand fly bites. Sand fly saliva is known to enhance Leishmania infection, while immunity to the saliva protects against infection as determined by coinoculation of parasites with vector salivary gland homogenates (SGHs) or by infected sand fly bites (Kamhawi, S., Y. Belkaid, G. Modi, E. Rowton, and D. Sacks. 2000. Science. 290:1351-1354). We have now characterized nine salivary proteins of Phlebotomus papatasi, the vector of Leishmania major. One of these salivary proteins, extracted from SDS gels and having an apparent mol wt of 15 kD, was able to protect vaccinated mice challenged with parasites plus SGH. A DNA vaccine containing the cDNA for the predominant 15-kD protein (named SP15) provided this same protection. Protection lasted at least 3 mo after immunization. The vaccine produced both intense humoral and delayed-type hypersensitivity (DTH) reactions. B cell-deficient mice immunized with the SP15 plasmid vaccine successfully controlled Leishmania infection when injected with Leishmania plus SGH. These results indicate that DTH response against saliva provides most or all of the protective effects of this vaccine and that salivary gland proteins or their cDNAs are viable vaccine targets against leishmaniasis.

The genome of the protozoan parasite Cystoisospora suis and a reverse vaccinology approach to identify vaccine candidates.

Vaccine development targeting protozoan parasites remains challenging, partly due to the complex interactions between these eukaryotes and the host immune system. Reverse vaccinology is a promising approach for direct screening of genome sequence assemblies for new vaccine candidate proteins. Here, we applied this paradigm to Cystoisospora suis, an apicomplexan parasite that causes enteritis and diarrhea in suckling piglets and economic losses in pig production worldwide. Using Next Generation Sequencing we produced an ∼84Mb sequence assembly for the C. suis genome, making it the first available reference for the genus Cystoisospora. Then, we derived a manually curated annotation of more than 11,000 protein-coding genes and applied the tool Vacceed to identify 1,168 vaccine candidates by screening the predicted C. suis proteome. To refine the set of candidates, we looked at proteins that are highly expressed in merozoites and specific to apicomplexans. The stringent set of candidates included 220 proteins, among which were 152 proteins with unknown function, 17 surface antigens of the SAG and SRS gene families, 12 proteins of the apicomplexan-specific secretory organelles including AMA1, MIC6, MIC13, ROP6, ROP12, ROP27, ROP32 and three proteins related to cell adhesion. Finally, we demonstrated in vitro the immunogenic potential of a C. suis-specific 42kDa transmembrane protein, which might constitute an attractive candidate for further testing.

Selection strategy of phage-displayed immunogens based on an in vitro evaluation of the Th1 response of PBMCs and their potential use as a vaccine against Leishmania infantum infection.

BACKGROUND: The development of a vaccine for the prevention of visceral leishmaniasis (VL) still represents a significant unmet medical need. A human vaccine can be found if one takes into consideration that many people living in endemic areas of disease are infected but do not develop active VL, including those subjects with subclinical or asymptomatic infection. METHODS: In this study, a phage display was used to select phage-exposed peptides that were specific to immunoglobulin G (IgG) antibodies from asymptomatic and symptomatic VL patients, separating them from non-infected subjects. Phage clones presenting valid peptide sequences were selected and used as stimuli of peripheral blood mononuclear cells (PBMCs) obtained from both patients' groups and controls. Those with higher interferon-gamma (IFN-γ)/interleukin (IL)-10 ratios were further selected for vaccination tests. RESULTS: Among 17 evaluated clones, two were selected, B1 and D11, and used to immunize BALB/c mice in an attempt to further validate their in vivo protective efficacy against Leishmania infantum infection. Both clones induced partial protection against the parasite challenge, which was evidenced by the reduction of parasitism in the evaluated organs, a process mediated by a specific T helper (Th)1 immune response. CONCLUSIONS: To the best of our knowledge, this study is the first to use a rational strategy based on in vitro stimulation of human PBMCs with selected phage-displayed clones to obtain new immunogens against VL.

Identification of Peptide Mimics of a Glycan Epitope on the Surface of Parasitic Nematode Larvae.

Phage display was used to identify peptide mimics of an immunologically protective nematode glycan (CarLA) by screening a constrained C7C peptide library for ligands that bound to an anti-CarLA mAb (PAB1). Characterisation of these peptide mimotopes revealed functional similarities with an epitope that is defined by PAB1. Mimotope vaccinations of mice with three selected individual phage clones facilitated the induction of antibody responses that recognised the purified, native CarLA molecule which was obtained from Trichostrongylus colubriformis. Furthermore, these mimotopes are specifically recognised by antibodies in the saliva of animals that were immune to natural polygeneric nematode challenge. This shows that antibodies to the PAB1 epitope form part of the mucosal polyclonal anti-CarLA antibody response of nematode immune host animals. This demonstrates that the selected peptide mimotopes are of biological relevance. These peptides are the first to mimic the PAB1 epitope of CarLA, a defined larval glycan epitope which is conserved between many nematode species.

Prophylactic and therapeutic DNA vaccines against Chagas disease.

Chagas disease is a zoonosis caused by Trypanosoma cruzi in which the most affected organ is the heart. Conventional chemotherapy has a very low effectiveness; despite recent efforts, there is currently no better or more effective treatment available. DNA vaccines provide a new alternative for both prevention and treatment of a variety of infectious disorders, including Chagas disease. Recombinant DNA technology has allowed some vaccines to be developed using recombinant proteins or virus-like particles capable of inducing both a humoral and cellular specific immune response. This type of immunization has been successfully used in preclinical studies and there are diverse models for viral, bacterial and/or parasitic diseases, allergies, tumors and other diseases. Therefore, several research groups have been given the task of designing a DNA vaccine against experimental infection with T. cruzi. In this review we explain what DNA vaccines are and the most recent studies that have been done to develop them with prophylactic or therapeutic purposes against Chagas disease.

A review of the global burden, novel diagnostics, therapeutics, and vaccine targets for cryptosporidium.

Cryptosporidium spp are well recognised as causes of diarrhoeal disease during waterborne epidemics and in immunocompromised hosts. Studies have also drawn attention to an underestimated global burden and suggest major gaps in optimum diagnosis, treatment, and immunisation. Cryptosporidiosis is increasingly identified as an important cause of morbidity and mortality worldwide. Studies in low-resource settings and high-income countries have confirmed the importance of cryptosporidium as a cause of diarrhoea and childhood malnutrition. Diagnostic tests for cryptosporidium infection are suboptimum, necessitating specialised tests that are often insensitive. Antigen-detection and PCR improve sensitivity, and multiplexed antigen detection and molecular assays are underused. Therapy has some effect in healthy hosts and no proven efficacy in patients with AIDS. Use of cryptosporidium genomes has helped to identify promising therapeutic targets, and drugs are in development, but methods to assess the efficacy in vitro and in animals are not well standardised. Partial immunity after exposure suggests the potential for successful vaccines, and several are in development; however, surrogates of protection are not well defined. Improved methods for propagation and genetic manipulation of the organism would be significant advances.

Influence of immunoprotection on genetic variability of cysteine proteinases from Haemonchus contortus adult worms.

The limitations associated with the use of anthelmintic drugs in the control of gastrotintestinal nematodosis, such as the emergence of anthelmintic resistance, have stimulated the study of the immunological control of many parasites. In the case of Haemonchus contortus, several vaccination trials using native and recombinant antigens have been conducted. A group of antigens with demonstrated immunoprotective value are cathepsin B - like proteolytic enzymes of the cysteine proteinase type. These enzymes, which have been observed in both excretory-secretory products and somatic extracts of H. contortus, may vary among different geographic isolates and on strains isolated from different hosts, or even from the same host, as has been demonstrated in some comparative studies of genetic variability. In the present study, we evaluated the genetic variability of the worms that fully developed their endogenous cycle in immunised sheep and goat in order to identify the alleles of most immunoprotective value. To address these objectives, groups of sheep and goats were immunised with PBS soluble fractions enriched for cysteine proteinases from adult worms of H. contortus from either a strain of H. contortus isolated from goats of Gran Canaria Island (SP) or a strain isolated from sheep of North America (NA). The results confirmed the immunoprophylactic value of this type of enzyme against haemonchosis in both sheep and goats in association with increased levels of specific IgG. The genetic analysis demonstrated that the immunisation had a genetic selection on proteinase-encoding genes. In all the immunised animals, allelic frequencies were statistically different from those observed in non-immunised control animals in the four analysed genes. The reduction in the allelic frequencies suggests that parasites expressing these proteases are selectively targeted by the vaccine, and hence they should be considered in any subunit vaccine approach to control haemonchosis in small ruminants.

Identification of a common Plasmodium epitope (CPE) recognised by a pan-specific inhibitory monoclonal antibody.

A Plasmodium falciparum genomic expression library was screened with a monoclonal antibody produced from mice infected with Plasmodium yoelii. Eleven unique clones were isolated all of which contained the sequence NKND, IKND or KKND. This sequence was confirmed as the epitope of M26-32 by testing a series of overlapping peptides and the allowable substitutions determined by testing the binding of M26-32 to peptides containing all possible single amino acid replacements of NKND. Potential epitopes of M26-32 occur in many plasmodial proteins and this is consistent with the large number of proteins recognised in these parasites by Western blotting. Since this monoclonal antibody shows marked in vitro inhibition of P. falciparum growth, these data suggest that an anti-malarial vaccine may be produced by targeting such common plasmodial epitopes without necessarily identifying the corresponding antigens.

Polymorphism of SPAG-1, a candidate antigen for inclusion in a sub-unit vaccine against Theileria annulata.

SPAG-1, a Theileria annulata sporozoite surface antigen, is a vaccine candidate. Data is presented, based on the clonal segregation of SPAG-1 associated RFLPs, showing that this antigen is encoded by a single copy gene. We have cloned and sequenced a full-length genomic copy of the SPAG-1 gene and a comparison of this with a previously published SPAG-1 cDNA sequence demonstrates a high degree of polymorphism. We infer that these sequences represent two distinct allelic SPAG-1 variants. The deduced polypeptides show an overall identity of 92% with the most variable stretch (60% identity) occurring towards the middle of the molecule. The N and C termini are more conserved with identities of 92% and 97% respectively. The elastin receptor ligand, VGVAPG, present 3 times in the protein sequence derived from the cDNA is not found in that deduced from the genomic copy. Evidence for 2 further SPAG-1 alleles was obtained from PCR based sequences using macroschizont clones containing different SPAG-1 associated RFLPs. In summary we have shown the existence of at least 4 highly polymorphic SPAG-1 alleles. The implications of such polymorphism between and within distinct geographical isolates for the development of a SPAG-1 based subunit vaccine is discussed.

Analysis of sequence diversity in the Plasmodium falciparum merozoite surface protein-1 (MSP-1).

Immunization with the first identified Plasmodium falciparum merozoite surface protein (MSP-1) protected monkeys from an otherwise fatal infection. The question of whether the high degree of diversity in MSP-1 among parasite clones will be an impediment to its development as a vaccine candidate needs to be resolved. We have aligned all published sequences, identifying errors, resequencing a portion of one parasite clone, and identifying probable duplicate sequences of four pairs of parasite clones. The sequences are displayed in a fashion that facilitates the study of variation and its potentially diverse origins. The original dimorphic sequences described by Tanabe et al. have been modified to include only common sequences throughout the entire gene. The extension of the dimorphic region to the 5' end of block 3 brings into question the involvement of intragenic crossover as the major mechanism generating allelic diversity. Additional diversity developed from point mutations and recombination in certain regions of the gene. The regions of variability and conservation should serve as a data base for planning vaccine trials.

Sequence conservation in the C-terminal part of the precursor to the major merozoite surface proteins (MSP1) of Plasmodium falciparum from field isolates.

The C-terminal part of the precursor to the major merozoite surface proteins (MSP1) of Plasmodium falciparum contains potential protective epitopes and two cleavage sites for processing which take place prior to erythrocyte invasion by the merozoite. Since sequences available to date are limited and derived from cultured parasites, we have examined the extent of variations of this important part of the MSP1 gene from natural populations. Our sequence analyses of 1.6-1.7 kb from blocks 13-17 of the gene obtained from 19 Thai wild isolates have identified a deletion of a codon and 18 nucleotide substitutions, all of which are dimorphic substitutions and all but one create amino acid exchanges. However, residues at two cleavage sites for the C-terminus 42 kDa polypeptide and the 19-kDa polypeptide, a subfragment of the former, are conserved. Furthermore, all 12 cysteine residues at the C-terminal 19-kDa polypeptide are perfectly conserved, allowing the formation of 2 epidermal growth factor-like structures. These results indicate that in contrast to extensive variations at the N-terminal part of MSP1, limited variations occur at the C-terminal part.