Multiple Antigen Peptide Research Articles

Multiple antigenic peptide vaccine targeting the loop neutralizing determinant of protective antigen from Bacillus anthracis completely protects rabbits from aerosol spore challenge (52.24)

Abstract The loop neutralizing determinant (LND) from domain 2 of Bacillus anthracis protective antigen (PA) has been shown to represent a potentially important target for an epitope-specific vaccine for anthrax. This region in the 2β2-2β3 loop of PA is immunologically inert, since rabbits immunized with PA or otherwise immune to PA do not contain antibody specific for this region. Herein we evaluated the immunogenicity in rabbits of a MAP peptide comprising the T* helper T cell epitope from Plasmodium falciparum colinearly synthesized at either the N- or C-terminus with a.a. 304-319 from PA. In initial studies, the MAP peptides formulated in Freund’s adjuvant were found to be highly immunogenic in rabbits, rapidly eliciting high levels of neutralizing antibody. To more fully evaluate this peptide as an anthrax vaccine, we immunized separate groups of rabbits (n=7) five times at two-week intervals with either of the two LND MAP peptides or with soluble PA in Freund’s adjuvant and then subjected all rabbits to a 200 LD50 aerosol spore challenge with B. anthracis Ames strain. 100% of rabbits immunized with the LND MAP in the B-T orientation and with soluble PA, and 89% of rabbits immunized with the LND MAP in the T-B orientation, survived the spore challenge, while all naïve rabbits died. Analysis of post-challenge serum suggested little or no spore germination in challenged rabbits. We conclude that the LND MAP peptide is a promising epitope-specific prototype vaccine for anthrax.

An M2e-based multiple antigenic peptide vaccine protects mice from lethal challenge with divergent H5N1 influenza viruses

BackgroundA growing concern has raised regarding the pandemic potential of the highly pathogenic avian influenza (HPAI) H5N1 viruses. Consequently, there is an urgent need to develop an effective and safe vaccine against the divergent H5N1 influenza viruses. In the present study, we designed a tetra-branched multiple antigenic peptide (MAP)-based vaccine, designated M2e-MAP, which contains the sequence overlapping the highly conserved extracellular domain of matrix protein 2 (M2e) of a HPAI H5N1 virus, and investigated its immune responses and cross-protection against different clades of H5N1 viruses.ResultsOur results showed that M2e-MAP vaccine induced strong M2e-specific IgG antibody responses following 3-dose immunization of mice with M2e-MAP in the presence of Freunds' or aluminium (alum) adjuvant. M2e-MAP vaccination limited viral replication and attenuated histopathological damage in the challenged mouse lungs. The M2e-MAP-based vaccine protected immunized mice against both clade1: VN/1194 and clade2.3.4: SZ/406H H5N1 virus challenge, being able to counteract weight lost and elevate survival rate following lethal challenge of H5N1 viruses.ConclusionsThese results suggest that M2e-MAP presenting M2e of H5N1 virus has a great potential to be developed into an effective subunit vaccine for the prevention of infection by a broad spectrum of HPAI H5N1 viruses.

Strategies and Limitations in Dendrimeric Immunogen Synthesis. The Influenza Virus M2e Epitope as a Case Study

Dendrimeric platforms such as multiple antigen peptides (MAPs) are regarded as one of the most efficacious approaches for antigenic presentation. Originally described as available by stepwise solid-phase peptide synthesis (SPPS), MAPs have also been prepared by chemical (thioether, oxime, hydrazone) ligation of appropriately functionalized tetra- or octavalent polylysine scaffolds with the peptide antigen to be multiply displayed. In this work, the advantages and limitations of two of the most frequent methods of MAP preparation, namely, chemoselective thioether ligation in solution, and all-solid-phase synthesis, have been tested in the case of a particularly troublesome epitope model, the ectodomain of protein M2 from influenza virus (M2e). The strong tendency of M2e to self-associate is a serious inconvenient for conjugation in solution, which as a result fails to produce the target MAPs with the specified number of M2e copies. In contrast, the fully stepwise SPPS approach is shown to be quite practical, especially when 6-aminohexanoic acid spacer units providing increased internal flexibility are inserted at each branching point.

Murine neutralizing antibody response and toxicity to synthetic peptides derived from E1 and E2 proteins of hepatitis C virus

Introduction The highest estimated prevalence of HCV infection has been reported in Egypt, nearly 12% mostly type 4. Currently, a commercial vaccine to protect this high risk population as well as global HCV infected patients is not available. Objectives In the present study, we aim at: (1) examining the viral binding capacities of purified monospecific polyclonal murine antibodies raised against genetically conserved viral protein sequences, i.e. synthetic peptides derived from those sequences located within envelope proteins and (2) assessment of immunogenic properties and safety parameters of those peptides individually and in a vaccine format in mice. Methods Purified IgG Abs from immunized mice were used in immunocapture RT-PCR experiments to test viral neutralization by Abs raised against each of 4 peptides termed p35 (E1), p36 (E2), p37 (E2) and p38 (E2). Swiss mice were immunized with each of the 3 peptides (p35, p37 and p38) which generated neutralizing antibodies in immunocapture experiments. Antibody responses to corresponding peptides were determined using different routes of administration, different adjuvants, different doses and at different time points post-injection. To explore the dose range for future pharmacological studies, three doses namely 50 ng, 10 μg and 50 μg/25 gm mouse body weight were tested for biochemical and histopathological changes in several organs. Results Murine Abs against p35, p37 and p38 but not p36 showed HCV neutralization in immunocapture experiments. Subcutaneous injection of peptides elicited higher responses than i.m. and i.p. Immunization with Multiple Antigenic Peptide (MAP) form or coupled to Al PO4 elicited the highest Ab responses. Peptide doses of 50 ng/25 gm body weight or less were effective and safe, however dose assessment still requires further study. Histopathological changes were observed in animals that received doses ∼1000 times higher than the potential therapeutic dose. Conclusion Exploration of humoral immunogenicity, neutralization capacity and safety suggested that the peptides presented herein are candidate vaccine components for further preclinical assessment.

Diagnostic and immunoprophylactic applications of synthetic peptides in veterinary microbiology

Chemically synthesized peptides are considered as potential reagents for various applications in biological sciences. They mimic naturally occurring peptides or segments of proteins and have emerged as diagnostic reagents and safe immunogens in animal science. Carefully selected peptides resembling authentic epitopes serve as synthetic antigens in diagnostic tests. Synthetic peptide-based vaccines can elicit antibodies against animal pathogens. The early use of synthetic peptides as a vaccine for foot-and-mouth disease stimulated interest in the development of peptide-based diagnostics and immunoprophylactics. The development of a peptide vaccine for canine parvovirus confirmed the usefulness of peptides as immunoprophylactics. Recently, the advent of the technology for the development of multiple antigenic peptides (MAPs) has provided a well-defined method for the production of highly immunogenic peptides and anti-peptide antibodies. Antibodies raised against major epitopes can be used in the detection of the native antigen (virus) in the enzyme-linked immunosorbent assay (ELISA) and other tests, vindicating the usefulness of peptides for safe, chemically defined, non-infectious diagnostics and immunoprophylactics. This article focuses on the methods for selecting and preparing peptides for the predicted epitopes, their characterization and use, and the application of MAPs.

Synthesis of Highly Immunogenic Multiple Antigenic Peptides for Epitopes of Viral Antigen to Use in ELISA

Synthesis of multiple antigenic peptides (MAPs) for predicted antigenic determinants of a viral antigen is described. The method includes prediction of linear epitopes using predictive computer algorithms, synthesis of peptides for the predicted regions, testing of peptides to find the most reactive sites, synthesis of MAPs and their testing. The procedure involves manual synthesis of MAPs by solid phase peptide synthesis with Wang resin as solid support. The MAPs were prepared in eight copies and used for immunization of rabbits to generate anti-peptide antibodies. Further, the reactivity of MAPs in detecting the native cognate antigen in the whole virus was confirmed by ELISA. The MAP and anti-peptide antibodies could serve as diagnostic tools for viral diseases. MAPs have efficiently been used to confirm the presence of linear antigenic and immunogenic epitopes on viral proteins, for possible use in diagnostic and vaccine. It was suggested that this method could help in epitope mapping of dreadful human or animal pathogens as it involves production of safe, chemically defined and non-infectious materials for use as antigen as well as immunogen.

A Heterologous Helper T-Cell Epitope Enhances the Immunogenicity of a Multiple-Antigenic-Peptide Vaccine Targeting the Cryptic Loop-Neutralizing Determinant ofBacillus anthracisProtective Antigen

We previously showed that a multiple antigenic peptide (MAP) displaying amino acids (aa) 305 to 319 from the 2beta2-2beta3 loop of protective antigen (PA) can elicit high-titered antibody that neutralizes lethal toxin (LeTx) in vitro and that this loop-neutralizing determinant (LND) specificity is absent in PA-immune rabbits. Some immune rabbits were, however, nonresponders to the MAP. We hypothesized that the immunogen elicited suboptimal major histocompatibility complex (MHC) class II-restricted T-cell help and that introduction of a functional helper T-cell epitope would increase MHC-restricted responsiveness and the magnitude and affinity of the antibody responses. In the current study, we characterized the T- and B-cell responses to LND peptides in mice, then designed second-generation MAP immunogens for eliciting LND-specific immunity, and tested them in rabbits. The 305-319 sequence was devoid of helper T-cell epitopes in three strains of mice; however, a T-B peptide comprising aa 305 to 319, colinearly synthesized with the P30 helper epitope of tetanus toxin, elicited robust LeTx-neutralizing immunity in mice. T-B MAPs displaying B-cell epitopes 304 to 319 (MAP304) or 305 to 319 (MAP305) elicited high-titer, durable antibody responses in rabbits which exhibited potent neutralization of LeTx in vitro. All MAP304-immune rabbits demonstrated neutralization titers exceeding that of hyperimmune sera of rabbits immunized with PA in Freund's adjuvant, with peak neutralization titers 23-, 6-, and 3-fold higher than that of the PA antiserum. Overall, immunization with MAPs containing the P30 epitope elicited higher antibody and toxin neutralization titers and peptide-specific affinity than immunization with an LND MAP lacking a helper epitope. P30-containing MAP304 represents a promising LND-specific vaccine for anthrax.

Assessment of synthetic chimeric multiple antigenic peptides for diagnosis of GB virus C infection

The use of synthetic peptides of both structural and nonstructural proteins of GB virus C (GBV-C) has been studied for the development of new systems to diagnose infection caused by this virus. In an attempt to increase the antigenicity of linear peptide sequences, chimeric multiple antigenic peptides (MAPs) containing epitopes from E2, NS4, and NS5 GBV-C proteins have been synthesized. The synthetic constructs were evaluated by ELISA to establish whether the epitopes in chimeric branched peptides are more efficiently recognized by the specific antibodies compared to the monomeric linear sequences. Moreover, we have investigated the application of a commercial biosensor instrument for the detection of antibodies against the GBV-C in human serum samples. The results of the immunoassays reported in this work highlight the usefulness of synthetic tetrameric branched peptides containing sequences from envelope and nonstructural GBV-C proteins for the diagnosis of GBV-C infection. The potential clinical value of the MAP 4(E2-NS5a) for the serodiagnosis of GBV-C infection was demonstrated, thus providing the basis for performing prevalence studies of the infection among the hemodialyzed and hepatitis C virus (HCV)-infected population.

Enhanced immunogenicity and cross-reactivity of HIV-1 V3-peptide and multiple antigen peptides conjugated to distinct carrier proteins

To be effective, vaccines against the highly variable HIV-1 must elicit antibodies to a huge number of clinical isolates. For this purpose, new strategies to overcome this variability are needed. We previously reported a useful immunogenic strategy which consists of conjugating multiple antigen peptides (MAPs) to HBsAg. This vaccine candidate reduces the dose of immunogen required and increases the cross-reactivity towards other HIV strains. In the present study, we have expanded on those results by working with other carrier proteins. Thus, JY1-peptide (V3 regions of gp120 of HIV-1, subtype D) and JY1-MAP8 were synthesized and coupled to several carrier proteins such as KLH, HBsAg and P64k (recombinant meningococcal protein). Mice were immunized with various conjugates and their antigenicity, immunogenicity, and the level of cross-reactivity to a panel of five heterologous V3 peptides were compared. Our results show that conjugate JY1-MAP8 were not only more immunogenic than conjugate, they were also more or equally as immunogenic as 4-fold more JY1-MAP8 alone. Furthermore, conjugates to HBsAg and KLH were more immunogenic than those to P64k. Moreover, conjugates to HBsAg, KLH and P64k showed enhanced cross-reactivity to heterologous V3 peptides compared to JY1-peptide and JY1-MAP8. The analysis showed that conjugate-based immunogens are more prompt to elicit immunogenicity and cross-reactivity. These results can find application in the development of HIV vaccine candidates.

Development, Characterization, and Immunotherapeutic Use of Peptide Mimics of the Thomsen-Friedenreich Carbohydrate Antigen

The tumor-associated carbohydrate Thomsen-Friedenreich antigen (TF-Ag; Galß1-3GalNAcα-O-Ser/Thr) is overexpressed on the cell surface of several types of tumor cells, contributing to cancer cell adhesion and metastasis to sites containing TF-Ag-binding lectins. A highly specific immunoglobulin G3 monoclonal antibody (Ab) developed to TF-Ag (JAA-F11) impedes TF-Ag binding to vascular endothelium, blocking a primary metastatic step and providing a survival advantage. In addition, in patients, even low levels of antibodies to TF-Ag seem to improve prognosis; thus, it is expected that vaccines generating antibodies toward TF-Ag would be clinically valuable. Unfortunately, vaccinations with protein conjugates of carbohydrate tumor-associated Ags have induced clinically inadequate immune responses. However, immunization using peptides that mimic carbohydrate Ags such as Lewis has resulted in both Ab and T-cell responses. Here, we tested the hypothesis that vaccinations with unique TF-Ag peptide mimics may generate immune responses to TF-Ag epitopes on tumor cells, useful for active immunotherapy against relevant cancers. Peptide mimics of TF-Ag were selected by phage display biopanning using JAA-F11 and rabbit anti-TF-Ag Ab and were analyzed in vitro to confirm TF-Ag peptide mimicry. In vitro, TF-Ag peptide mimics bound to TF-Ag-specific peanut agglutinin and blocked TF-Ag-mediated rolling and stable adhesion of cancer cells to vascular endothelium. In vivo, the immunization with TF-Ag-mimicking multiple antigenic peptides induced TFAg- reactive Ab production. We propose that this novel active immunotherapy approach could decrease tumor burden in cancer patients by specifically targeting TF-Ag-positive cancer cells and blocking metastasis.

Synthetic Peptide Vaccine Targeting a Cryptic Neutralizing Epitope in Domain 2 ofBacillus anthracisProtective Antigen

Current evidence suggests that protective antigen (PA)-based anthrax vaccines may elicit a narrow neutralizing antibody repertoire, and this may represent a vulnerability with PA-based vaccines. In an effort to identify neutralizing specificities which may complement those prevalent in PA antiserum, we evaluated whether sequences within the 2beta2-2beta3 loop of PA, which are apparent in the crystal structure of heptameric but not monomeric PA, might represent a target for an epitope-specific vaccine for anthrax and, further, whether antibodies to these sequences are induced in rabbits immunized with monomeric PA. We evaluated the immunogenicity in rabbits of a multiple antigenic peptide (MAP) displaying copies of amino acids (aa) 305 to 319 of this region. Overall, four out of six rabbits vaccinated with the MAP peptide in Freund's adjuvant developed high-titer, high-avidity antibody responses which cross-reacted with the immobilized peptide sequence comprising aa 305 to 319 and with PA, as determined by an enzyme-linked immunosorbent assay, and which displayed potent and durable neutralization of lethal toxin (LeTx) in vitro, with peak titers which were 452%, 100%, 67%, and 41% of the peak neutralization titers observed in positive-control rabbits immunized with PA. Importantly, analysis of sera from multiple cohorts of rabbits with high-titer immunity to PA demonstrated a virtual absence of this potent antibody specificity, and work by others suggests that this specificity may be present at only low levels in primate PA antiserum. These results highlight the potential importance of this immunologically cryptic neutralizing epitope from PA as a target for alternative and adjunctive vaccines for anthrax.

JVRS-100 adjuvanted universal influenza A vaccine based on a multiple antigenic peptide of M2e protects from lethal challenge (132.6)

Abstract Background: The conventional vaccine strategy for control of influenza A is vulnerable to antigenic drift and the emergence of unmatched epidemic strains that cause primary vaccine failure. A vaccine strategy that targets an influenza antigen, which is less susceptible to antigenic variation, would be a major improvement. Methods: Balb/c mice were vaccinated three times with the M2e peptide in the context of a multiple antigenic peptide (MAP) complex and combination with the cationic lipid DNA complex adjuvant (JVRS-100). Antibody titers were monitored over the time course of vaccination and the mice received a lethal challenge with H1N1 (PR/8/34) or H3N2 (HKx31) two weeks after the final vaccination. Results: Vaccination of mice with JVRS-100-MAP4-M2e compared to MAP4-M2e resulted in increased survival following lethal challenge with 6x LD50 PR/8/34 H1N1 (100% vs. 30%, P=0.002) or 10x LD50 HKx31 H3N2 (80% vs. 20%). Recipients of 200µl serum from MAP-4-M2e/JVRS-100 vaccinated mice and challenged with H1N1 (6x LD50 PR/8/34) resulted in survival of 60% (P=0.014) compared with control mice which received 200µl naive serum (0% survival). Furthermore, recipients of 300µl serum from MAP-4-M2e/JVRS-100 vaccinated mice and challenged with H1N1 (2x LD50 PR/8/34) resulted in survival of 100% (P=0.0026) compared with control mice which received 200µl naive serum (0% survival). Conclusion: These studies demonstrate that a simple, synthetic M2e vaccine in a MAP configuration with a strong adjuvant may result in a viable vaccine candidate for universal flu vaccination and the protective immunity is primarily due to an antibody response.

Mimotope vaccination – from allergy to cancer

Background: Mimotopes are peptides mimicking protein, carbohydrates or lipid epitopes and can be generated by phage display technology. When selected by antibodies, they represent exclusively B-cell epitopes and are devoid of antigen/allergen-specific T-cell epitopes. Coupled to carriers or presented in a multiple antigenic peptide form mimotopes achieve immunogenicity and induce epitope-specific antibody responses upon vaccination. Objective/methods: In allergy IgG antibodies may block IgE binding to allergens, whereas other IgG antibody specificities enhance this and support the anaphylactic reaction. In cancer, inhibitory antibody specificities prevent growth signals derived from overexpressed oncogenes, whereas growth-promoting specificities enhance signalling and proliferation. Therefore, the mimotope concept is applicable to both fields for epitope-specific vaccination and analysis of conformational B-cell epitopes for the allergen/antigen. Results/conclusions: Mimotope technology is a relatively young theme in allergology and oncology. Still, proof of concept studies testing allergen and tumour mimotope vaccines suggest that mimotopes are ready for clinical trials.

Prediction of the B cell epitopes of human heparanase protein and determination of their immunogenicity

Objective To predict the secondary structure and the B cell epitopes of human heparanase protein, and to identify its immunogenicity. Methods The flexible regions of secondary structure and the B cell epitopes of human heparanase amino acid sequence were predicted by DNAStar and Bcepred software. The multiple antigenic peptides (MAP) of the epitopes were synthesized in 8-branch form. Rabbits were immunized with the 8-branch MAPs mixed with a universal T-helper epitope human IL-1β peptide (VQGEESNDK, amino acid 163-171 ). The immunogenicity of the synthesized peptides was evaluated by ELISA, Western blot and immunohistochemistry. Results Amino acid 1 -15 ( MAP1), 279-293 (MAP2) and 175-189(MAP3) of large-subunit of human heparanase protein was predicted as the most potential epitopes of human heparanase protein. All the three synthesized MAPs induced high titer of antibodies. ELISA, Western blot and immunohistochemistry analysis showed all the three MAPs could produce high titer serum antibodies, antibodies induced by MAP1 and MAP2 had high specific binding activity , and MAP2 antibody showed the strongest binding activity with liver cancer tissues. Conclusion The large-subunit No. 1-15, 279-293 amino acid of human heparanase protein may be the B cell preponderant epitopes and the strongest immunogenicity may be No. 279-293 peptide, which provided a theoretic basis for the antibody and vaccine development of heparanase subunit peptide. Key words: Heparanase; B cell epitopes; Epitope prediction; Immunogenicity

Screening and identification of novel B cell epitopes in human heparanase and their anti-invasion property for hepatocellular carcinoma.

The aim of this study was to screen and identify novel B cell epitopes within the human heparanase protein and to investigate the impact of self-developed anti-heparanase polypeptide antibodies on growth and invasion of HCCLM6 human hepatocellular carcinoma cells in vitro. The flexible regions of secondary structure and the B cell epitopes of the human heparanase amino acid sequence were predicted by DNAStar and Bcepred software.The multiple antigenic peptides (MAP) of the epitopes were synthesized in eight-branched form. Rabbits were immunized with the eight-branched MAPs mixed with the universal T-helper epitope human IL-1beta peptide (VQGEESNDK, amino acid 163-171). The immunogenicity of the synthesized peptides was evaluated by ELISA, western blot and immunohistochemistry. The impact of the self-developed rabbit anti-heparanase polyclonal antibodies on growth and invasion ability of HCCMLM6 cells were analyzed in a cell culture model. The cells were first treated with one of the three antibodies, respectively, and then measured by using MTT, flow cytometry, plate clone formation, invasion assay and heparan sulfate degrading enzyme assay. The three amino acid sequences 1-15 (MAP1), 279-293 (MAP2), and 175-189 (MAP3) in the large subunit of the human heparanase protein were predicted as its most potential epitopes. ELISA, western blot and immunohistochemistry analysis showed that all three MAPs were capable to induce high titer of serum antibodies. Antibodies induced by MAP1 and MAP2 were high specific. Furthermore, anti-MAP2 antibodies showed the strongest avidity towards liver cancer tissues. Under the treatment with the three anti-heparanase antibodies, respectively, the growth, cell cycle and clone formation of the cells remained unchanged when compared with a treatment with normal rabbit IgG. However, an inhibition of cell invasiveness and heparanase activity could be detected under the treatment with anti-MAP1- or anti-MAP2-antibody (with a terminal concentration of 100 mug/ml). The cell invasiveness was decreased by 54 and 38%, respectively, the heparanase activity by 43 and 39%, respectively. The multiple antigenic peptides MAP1 (AC 1-15) and MAP2 (AC 279-293) may be the dominant B cell epitopes in the human heparanase protein. The induced polypeptide antibodies can effectively inhibit the heparanase activity of HCCLM6 liver cancer cells and therefore influence their invasion ability, which provides a theoretic basis for the development of anti-heparanase antibodies and their clinical use as vaccine.

Refined multivalent display of bacterial spore-binding peptides

A multiple antigen peptide display scaffold was used to create multivalent versions of a heptapeptide selected previously by phage display to bind to Bacillus subtilis spores. A simple flow cytometric assay was developed in which a biotinylated form of the peptide was first bound to fluorescent streptavidin, then the fluorescent streptavidin-peptide complex was bound to spores before introduction into the cytometer. This assay clearly demonstrated that the tetravalent scaffold enhanced the affinity for B. subtilis spores by greater than 1 and 2 orders of magnitude when compared to divalent and monovalent analogues, respectively. However, variations in the number and flexibility of spacer residues within the scaffold did not significantly affect the binding affinity of the tetravalent peptides. Similar to prior reports, these multivalent scaffolds are effective most likely because they mimic the multivalent display of the original peptide library on the phage coat. Moreover, the tetravalent peptides can be readily integrated into a variety of heterogeneous and homogeneous spore-detection assay formats.

Conjugation of Latent Membrane Protein (LMP)-2 Epitope to Gold Nanoparticles as Highly Immunogenic Multiple Antigenic Peptides for Induction of Epstein−Barr Virus-Specific Cytotoxic T-Lymphocyte Responses in Vitro

Nasopharyngeal carcinoma is a neoplasm with a high incidence in Southeast Asia, and it is strongly associated with Epstein-Barr virus (EBV) activation involving the expression of a weakly immunogenic protein, namely, latent membrane protein (LMP)-2. Previous immunological studies already identified the human leukocyte antigen (HLA)-A11 restricted peptide epitope (SSCSSCPLSK) in the LMP-2 antigen. In this work, we prepared gold nanoparticle (AuNP)-peptide conjugate 1 by treating the nanoparticles with the N-cysteinated LMP-2 epitope. The AuNP-peptide conjugates have been characterized by TEM (15-24 nm in diameter) and UV-vis spectroscopy (surface plasmon resonance absorption band at lambda(max) = 520 nm). In the presence of a CALNN capping peptide, the AuNP-peptide conjugates are stable in solution without aggregation at room temperature for at least 48 h. By ELIspot studies, AuNP-peptide conjugate 1 was found to elicit a significantly stronger INF-gamma response [number of spot forming cells (SPC) = 727 +/- 198] from peripheral blood mononuclear cells of healthy HLA-A11 donors when compared to that induced by the unconjugated LMP-2 peptides (SFC = 73 +/- 28). Further studies showed that dendritic cells treated with conjugate 1 can effect CD8+ T-cell activation leading to epitope-specific cytotoxic T lymphocyte killing responses in vitro.

Schistosoma mansoni ex vivo lung-stage larvae excretory–secretory antigens as vaccine candidates against schistosomiasis

Schistosoma mansoni lung-stage larvae are known to be the major target of innate and acquired immunity to schistosomiasis. Lung schistosomula cytosolic or surface membrane antigens are hidden, entirely inaccessible to the host immune system, and hence are not particularly important as vaccine candidates. Conversely, excretory–secretory (E–S) products released from intact, viable, elongated, and contractile schistosomula are ideal potential vaccines, as such molecules can readily play a central role in the induction of local primary and memory immune response effectors that would directly target, surround, and pursue the larvae while negotiating the lung capillaries. Therefore, 6-day-old ex vivo larvae were isolated from mouse or hamster lung cells and used for generation of E–S products, which were shown to elicit strong immune responses and significant ( P < 0.05) protection against challenge infection in BALB/c mice. Proteomic analysis of E–S molecules following 10× concentration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis identified peptides related to innumerable host and about 15 S. mansoni-specific proteins. Selected S. mansoni-specific E–S peptides prepared in a multiple antigen peptide (MAP) or recombinant form were shown to stimulate considerable specific antibody response and peripheral blood mononuclear cell expression of mRNA for several cytokines in immunized C57BL/6 and BALB/c mice. However, highly significant ( P < 0.05 to <0.005) reduction in challenge infection worm burden and egg load was recorded only when the immunization conditions in test mice provided the S. mansoni antigen-specific T helper (Th) type response milieu favorable for each immunogen. That was polarized Th1 for S. mansoni aldolase and thioredoxin peroxidase 1 MAPs, polarized Th2 for recombinant 14-3-3-like protein, mixed Th1/Th17 for calpain MAP, and mixed Th1/Th2 for recombinant p18 protein. The findings together indicated that the immune responses issue is as critical as the nature and source of the antigen for the development of vaccine against schistosomiasis.

Branched Peptides as Therapeutics

The concept of 'magic bullet', initially ascribed to immunoglobulins by Paul Ehrlich at the beginning of the 20th century and strengthened by the hybridoma technology of Kohler and Milstein in the mid 70s, can nowadays be attributed to different target-specific molecules, such as peptides. This attribution is increasingly valid in light of the explosion of new technologies for peptide library construction and screening, not to mention improvements in peptide synthesis and conjugation and in-vivo peptide stability, which make peptide molecules specific bullets for targeting pathological markers and pathogens. Today, hundreds of peptides are being developed and dozens are in clinical trials for a variety of diseases, demonstrating that the general reluctance towards peptide drugs that existed a decade ago has now been overcome. In spite of this progress, the development of new peptide drugs has largely been limited by their short half-life. Branched peptides such as Multiple Antigen Peptides (MAPs) were invented in the 80s by Tam [Tam, J.P., (1998) Proc. Natl. Acad. Sci. USA, 85, 5409] and have been extensively tested to reproduce single epitopes to stimulate the immune system for new vaccine discovery. In our lab we discovered that MAP molecules acquire strong resistance to proteases and peptidases. This resistance renders MAPs very stable and thus suitable for drug development. Here we report our experience with several MAP molecules in different biotechnological applications ranging from antimicrobial and anti toxin peptides to peptides for tumor targeting.

Schistosoma mansoni glyceraldehyde 3-phosphate dehydrogenase is a lung-stage schistosomula surface membrane antigen

We have previously reported that Schistosoma mansoni larvae emerging from host lung at pH 7.5-7.8 and then fixed with diluted formaldehyde (HCHO) readily bind radiation-attenuated cercariae (RA) vaccine serum antibodies, as assessed by indirect membrane immunofluorescence (IF). Here we show that S. mansoni schistosomula emerging from lung pieces under 5% CO2 (pH < or = 7.3) readily bind RA vaccine serum antibodies, provided they have been incubated for 12 h at pH 7.5-7.8 in foetal calf serum-free RPMI medium, and fixed with diluted HCHO. Ex vivo larvae exposed during incubation to GW4869, a specific inhibitor of tegument-bound, neutral sphingomyelinase (nSMase) displayed significantly diminished binding of RA vaccine serum antibodies, thus suggesting that nSMase activity at pH > or = 7.5 leads to exposure of lung-stage larvae surface membrane antigens to specific antibody detection. More importantly, ex vivo larvae readily bound antibodies directed to dipeptidic multiple antigen peptide constructs, based on S. mansoni-specific sequences in S. mansoni glyceraldehyde 3-phosphate dehydrogenase (SG3PDH). Lung-stage schistosomula IF reactivity was diminished following antiserum absorption with recombinant SG3PDH. The data together indicate that intact ex vivo, as well as, 5-day-old in vitro-grown larvae express SG3PDH on their surface membrane. The findings are discussed in relation to the importance of surface membrane proteins as candidate vaccine antigens.