B cell epitopes in the intrinsically disordered regions of neuraminidase and hemagglutinin proteins of H5N1 and H9N2 avian influenza viruses for peptide-based vaccine development.

Avian influenza viruses (AIV) are very active in several parts of the globe and are the cause of huge economic loss for the poultry industry and also human fatalities. Three dimensional modeling was carried out for neuraminidase (NA) and hemagglutinin (HA) proteins of AIV. The C-score, estimated TM-Score, and estimated root-mean-square deviation (RMSD) score for NA of H5N1 were -1.18, 0.57 ± 0.15, and 9.8 ± 7.6, respectively. The C-score, estimated TM-Score, and estimated RMSD score for NA of H9N2 were -1.43, 0.54 ± 0.15, and 10.5 ± 4.6, respectively. The C-score, estimated TM-Score, and estimated RMSD score for HA of H5N1 were -0.03, 0.71 ± 0.12, and 7.7 ± 4.3, respectively. The C-score, estimated TM-Score, and estimated RMSD score for HA of H9N2 were -0.57, 0.64 ± 0.13, and 8.9 ± 4.6, respectively. Intrinsically disordered regions were identified for the NA and HA proteins of H5N1 and H9N2 with the use of PONDR program. Linear B cell epitope was predicted using BepiPred 2 program for NA and HA of H5N1 and H9N2 avian influenza strains. Discontinuous epitopes were predicted by Discotope 2 program. The linear epitopes that were considered likely to be immunogenic and within the intrinsically disordered region for the NA of H5N1 was TKSTNSRSGFEMIWDPNGWTGTDSSFSVK, and for H9N2 it was VGDTPRNDDSSSSSNCRDPNNERGAP. In the case of HA of H5N1, it was QRLVPKIATRSKVNGQSG and ATGLRNSPQRERRRKK; for H9N2 it was INRTFKPLIGPRPLVNGLQG and SLKLAVGLRNVPARSSR. The discontinuous epitopes of NA of H5N1 and H9N2 were identified at various regions of the protein structure spanning from amino acid residue positions 90 to 449 and 107 to 469, respectively. Similarly, the discontinuous epitopes of HA of H5N1 and H9N2 were identified in the amino acid residue positions 27 to 517 and 136 to 521, respectively. This study has identified potential and highly immunogenic linear and conformational B-cell epitopes towards developing a vaccine against AIV both for human and poultry use.

Identification and Design of Synthetic B Cell Epitopes for Carbohydrate-Based Vaccines.

Synthetic oligosaccharide-based vaccines are promising alternatives to conventional antibacterial carbohydrate vaccines prepared with isolated polysaccharides. Unlike polysaccharides, synthetic glycans are well defined, contaminant-free, and accessible even for pathogens that cannot be fermented or show limited carbohydrate biosynthesis in vitro. However, identifying synthetic glycan B cell epitopes that induce protective immunity has traditionally been a time-consuming trial-and-error process, as predicting the immunogenicity of an oligosaccharide by means of structure alone is not straightforward. We here describe how synthetic oligosaccharide epitopes for candidate vaccines can be rationally identified prior to preclinical immunogenicity studies. Epitopes are selected on the basis of their recognition by antibodies associated with protection from disease in humans or small animals. In addition, we show how murine antibody responses to a large oligosaccharide can inform the identification of a minimal B cell epitope that may help designing easy to synthesize vaccine candidates. The procedures, exemplified with a surface carbohydrate of Clostridium difficile, may serve as a guideline for selecting protective oligosaccharide epitopes for vaccines against infectious and malignant diseases.

Multiple B-cell epitope vaccine induces a Staphylococcus enterotoxin B-specific IgG1 protective response against MRSA infection.

No vaccine against methicillin-resistant Staphylococcus aureus (MRSA) has been currently approved for use in humans. Staphylococcus enterotoxin B (SEB) is one of the most potent MRSA exotoxins. In the present study, we evaluated the efficacy and immunologic mechanisms of an SEB multiple B-cell epitope vaccine against MRSA infection. Synthetic overlapping peptide ELISA identified three novel B-cell immunodominant SEB epitopes (in addition to those previously known): SEB31-48, SEB133-150, and SEB193-210. Six B-cell immunodominant epitopes (amino acid residues 31-48, 97-114, 133-150, 193-210, 205-222, and 247-261) were sufficient to induce robust IgG1/IgG2b-specific protective responses against MRSA infection. Therefore, we constructed a recombinant MRSA SEB-specific multiple B-cell epitope vaccine Polypeptides by combining the six SEB immunodominant epitopes and demonstrated its ability to induce a robust SEB-specific IgG1 response to MRSA, as well as a Th2-directing isotype response. Moreover, Polypeptides-induced antisera stimulated synergetic opsonophagocytosis killing of MRSA. Most importantly, Polypeptides was more effective at clearing the bacteria in MRSA-infected mice than the whole SEB antigen, and was able to successfully protect mice from infection by various clinical MRSA isolates. Altogether, these results support further evaluation of the SEB multiple B-cell epitope-vaccine to address MRSA infection in humans.

New approaches to transcutaneous immunotherapy: targeting dendritic cells with novel allergen conjugates.

PURPOSE OF REVIEW: This review summarizes recent preclinical and human studies evaluating allergen-specific immunotherapy via the transcutaneous route, and provides a rationale for the application of modified allergens with reduced allergenicity. Furthermore, it covers approaches to generate hypoallergenic conjugates for specific dendritic cell targeting. RECENT FINDINGS: Efficacy and safety of specific immunotherapy by application of allergens to the skin have been demonstrated in both animal models as well as clinical trials. However, localized adverse events have been reported, and delivery of antigens via barrier-disrupted skin has been linked to the induction of unwanted T helper 2-biased immune responses and allergic sensitization. Coupling of carbohydrates to allergens has been shown to induce formation of nanoparticles, which can specifically target dendritic cells and potentiate immune responses, and by masking B-cell epitopes, can render the molecules hypoallergenic. SUMMARY: Due to its abundance of immunocompetent cells, the skin represents an attractive target tissue for novel and enhanced immunotherapeutic approaches. However, in order to avoid adverse events and therapy-induced sensitizations, transcutaneous immunotherapy requires the use of formulations with reduced allergenic potential. Combining novel hypoallergenic conjugates with painless transcutaneous immunization techniques may provide an efficient and patient-friendly alternative to the standard specific immunotherapy practices.

Synthesized multiple antigenic polypeptide vaccine based on B-cell epitopes of human heparanase could elicit a potent antimetastatic effect on human hepatocellular carcinoma in vivo.

AIMS: The aim of this study was to investigate the antimetastatic effect of multiple antigenic polypeptide (MAP) vaccine based on B-cell epitopes of heparanase (HPSE) on human hepatocellular carcinoma (HCC) in vivo. METHODS: The antiserum against B-cell epitopes of HPSE was isolated, purified and characterized after immunizing white-hair-black-eye (WHBY) rabbit with freshly synthesized MAP vaccine. Tumor-bearing murine models of orthotopic implants using HCC-97H cell line were built in BALB/c nude mice. Anti-MAP polyclonal antibodies induced by MAP vaccine were administrated to the models. The impact on metastasis was assessed, the expressions of VEGF/bFGF in hepatoma tissues and in murine sera were evaluated, and the micro-vessel density (MVD) was counted as well. In addition, the possible impairments of the HPSE MAP vaccine on certain HPSE positive normal organs and blood cells were investigated. RESULTS: The antiserum was harvested, purified and identified. The antibodies induced by MAP vaccine could specifically react with the dominant epitopes of both precursor protein and large subunit monomer of HPSE, markedly decrease HPSE activity, suppress the expressions of both VEGF and bFGF, and reduce the MVD. Pulmonary metastasis was also attenuated significantly by the anti-MAP polyclonal antibodies. In addition, no obvious impairment could be observed in certain HPSE positive organs and cells. CONCLUSION: MAP vaccine based on B-cell epitopes of HPSE is capable of alleviating HCC metastasis in vivo, mainly through inhibiting the HPSE activity and tumor associated angiogenesis, by virtue of the specific anti-MAP polyclonal antibodies. Furthermore, these HPSE-specific antibodies do not cause obvious abnormalities on certain HPSE positive blood cells and organs. Our study provides theoretical evidences for the clinical use of the synthesized MAP vaccine based on B-cell epitopes of HPSE in preventing HCC metastasis.

Design and evaluation of a multi-epitope peptide against Japanese encephalitis virus infection in BALB/c mice.

Epitope-based vaccination is a promising means to achieve protective immunity and to avoid immunopathology in Japanese encephalitis virus (JEV) infection. Several B-cell and T-cell epitopes have been mapped to the E protein of JEV, and they are responsible for the elicitation of the neutralizing antibodies and CTLs that impart protective immunity to the host. In the present study, we optimized a proposed multi-epitope peptide (MEP) using an epitope-based vaccine strategy, which combined six B-cell epitopes (amino acid residues 75-92, 149-163, 258-285, 356-362, 373-399 and 397-403) and two T-cell epitopes (amino acid residues 60-68 and 436-445) from the E protein of JEV. This recombinant protein was expressed in Escherichia coli, named rMEP, and its protective efficacy against JEV infection was assessed in BALB/c mice. The results showed that rMEP was highly immunogenic and could elicit high titer neutralizing antibodies and cell-mediated immune responses. It provided complete protection against lethal challenge with JEV in mice. Our findings indicate that the multi-epitope vaccine rMEP may be an attractive candidate vaccine for the prevention of JEV infection.

Prototype Alzheimer's disease vaccine using the immunodominant B cell epitope from beta-amyloid and promiscuous T cell epitope pan HLA DR-binding peptide.

Immunization of amyloid precursor protein transgenic mice with fibrillar beta-amyloid (Abeta) prevents Alzheimer's disease (AD)-like neuropathology. The first immunotherapy clinical trial used fibrillar Abeta, containing the B and T cell self epitopes of Abeta, as the immunogen formulated with QS21 as the adjuvant in the vaccine. Unfortunately, the clinical trial was halted during the phase II stage when 6% of the participants developed meningoencephalitis. The cause of the meningoencephalitis in the patients that received the vaccine has not been definitively determined; however, analysis of two case reports from the AN-1792 vaccine trial suggest that the meningoencephalitis may have been caused by a T cell-mediated autoimmune response, whereas production of anti-Abeta Abs may have been therapeutic to the AD patients. Therefore, to reduce the risk of an adverse T cell-mediated immune response to Abeta immunotherapy we have designed a prototype epitope vaccine that contains the immunodominant B cell epitope of Abeta in tandem with the synthetic universal Th cell pan HLA DR epitope, pan HLA DR-binding peptide (PADRE). Importantly, the PADRE-Abeta(1-15) sequence lacks the T cell epitope of Abeta. Immunization of BALB/c mice with the PADRE-Abeta(1-15) epitope vaccine produced high titers of anti-Abeta Abs. Splenocytes from immunized mice showed robust T cell stimulation in response to peptides containing PADRE. However, splenocytes from immunized mice were not reactivated by the Abeta peptide. New preclinical trials in amyloid precursor protein transgenic mouse models may help to develop novel immunogen-adjuvant configurations with the potential to avoid the adverse events that occurred in the first clinical trial.