Rational Design of a Glycoconjugate Vaccine against Group A Streptococcus.

No commercial vaccine is yet available against Group A Streptococcus (GAS), major cause of pharyngitis and impetigo, with a high frequency of serious sequelae in low- and middle-income countries. Group A Carbohydrate (GAC), conjugated to an appropriate carrier protein, has been proposed as an attractive vaccine candidate. Here, we explored the possibility to use GAS Streptolysin O (SLO), SpyCEP and SpyAD protein antigens with dual role of antigen and carrier, to enhance the efficacy of the final vaccine and reduce its complexity. All protein antigens resulted good carrier for GAC, inducing similar anti-GAC IgG response to the more traditional CRM197 conjugate in mice. However, conjugation to the polysaccharide had a negative impact on the anti-protein responses, especially in terms of functionality as evaluated by an IL-8 cleavage assay for SpyCEP and a hemolysis assay for SLO. After selecting CRM197 as carrier, optimal conditions for its conjugation to GAC were identified through a Design of Experiment approach, improving process robustness and yield This work supports the development of a vaccine against GAS and shows how novel statistical tools and recent advancements in the field of conjugation can lead to improved design of glycoconjugate vaccines.

Systematic Review and Meta-analysis of the Prevalence of Group A Streptococcal emm Clusters in Africa To Inform Vaccine Development.

An emm-cluster based system was proposed as a standard typing scheme to facilitate and enhance future studies of group A Streptococcus (GAS) epidemiological surveillance, M protein function, and vaccine development strategies. We provide an evidence-based distribution of GAS emm clusters in Africa and assess the potential coverage of the new 30-valent vaccine in terms of an emm cluster-based approach. Two reviewers independently assessed studies retrieved from a comprehensive search and extracted relevant data. Meta-analyses were performed (random-effects model) to aggregate emm cluster prevalence estimates. Eight studies (n = 1,595 isolates) revealed the predominant emm clusters as E6 (18%; 95% confidence interval [CI], 12.6% to 24.0%), followed by E3 (14%; 95% CI, 11.2% to 17.4%) and E4 (13%; 95% CI, 9.5% to 16.0%). There was negligible variation in emm clusters with regard to regions, age, and socioeconomic status across the continent. Considering an emm cluster-based vaccine strategy, which assumes cross-protection within clusters, the 30-valent vaccine currently in clinical development would provide hypothetical coverage to 80.3% of isolates in Africa. This systematic review indicates the most predominant GAS emm cluster in Africa is E6 followed by E3, E4, and D4. The current 30-valent vaccine would provide considerable coverage across the diversity of emm cluster types in Africa. Future efforts could be directed toward estimating the overall potential coverage of the new 30-valent vaccine based on cross-opsonization studies with representative panels of GAS isolates from populations at highest risk for GAS diseases.IMPORTANCE Low vaccine coverage is of grave public health concern, particularly in developing countries where epidemiological data are often absent. To inform vaccine development for group A Streptococcus (GAS), we report on the epidemiology of the M protein emm clusters from GAS infections in Africa, where GAS-related illnesses and their sequelae, including rheumatic fever and rheumatic heart disease, are of a high burden. This first report of emm clusters across the continent indicates a high probably of coverage by the M protein-based vaccine currently undergoing testing were an emm-cluster based approach to be used.

Group A Streptococcus T Antigens Have a Highly Conserved Structure Concealed under a Heterogeneous Surface That Has Implications for Vaccine Design.

Group A Streptococcus (GAS) (Streptococcus pyogenes) is an important human pathogen associated with significant global morbidity and mortality for which there is no safe and efficacious vaccine. The T antigen, a protein that polymerizes to form the backbone of the GAS pilus structure, is a potential vaccine candidate. Previous surveys of the tee gene, which encodes the T antigen, have identified 21 different tee types and subtypes such that any T antigen-based vaccine must be multivalent and carefully designed to provide broad strain coverage. In this study, the crystal structures of three two-domain T antigens (T3.2, T13, and T18.1) were determined and found to have remarkable structural similarity to the previously reported T1 antigen, despite moderate overall sequence similarity. This has enabled reliable modeling of all major two-domain T antigens to reveal that T antigen sequence variation is distributed along the full length of the protein and shields a highly conserved core. Immunoassays performed with sera from immunized animals and commercial T-typing sera identified a significant cross-reactive antibody response between T18.1, T18.2, T3.2, and T13. The existence of shared epitopes between T antigens, combined with the remarkably conserved structure and high level of surface sequence divergence, has important implications for the design of multivalent T antigen-based vaccines.

Temperature-Induced Self-Assembly of the Group B Streptococcus (GBS) Fusion Antigen GBS-NN.

Group B Streptococcus (GBS) is a leading cause of serious bacterial neonatal infections worldwide, which provides an unmet medical need for a globally effective vaccine. The recombinant GBS fusion antigen GBS-NN contains the N-terminal regions of the GBS Rib and Alpha C proteins. It shows promising immunogenicity eliciting protective immunity in mice and encouraging results in early human clinical trials. Understanding the physical stability of GBS-NN containing conformational B-cell epitopes is crucial to ensure optimal vaccine stability, efficacy, and safety. We initially discovered that GBS-NN is prone to form higher-order structures at elevated temperatures. We therefore investigated the self-assembly behavior of GBS-NN and characterized the higher-order conformational structures as a function of temperature. In the native state, GBS-NN exists as a monomer and has a secondary structure containing α-helix and ß-sheet. Langmuir studies demonstrated that the native protein is highly surface-active and forms a monolayer film at the air-water interface because of its amphipathic properties. The conformational stability of GBS-NN was measured as a function of temperature. GBS-NN has an unusual thermal behavior with a phase transition of approximately 61 °C, which is not accompanied by any major changes in the secondary structure. However, the antigen showed irreversible self-assembly as a function of temperature into higher-order structures with a hydrodynamic diameter of approximately 100 nm. Cryo-transmission electron microscopy analyses demonstrated that these self-assemblies consist of vesicular, ring-like structures with a hollow aqueous interior. Therefore, GBS-NN is a physically stable monomeric protein but is prone to temperature-induced self-assembly above 61 °C.

Population and Whole Genome Sequence Based Characterization of Invasive Group A Streptococci Recovered in the United States during 2015.

Group A streptococci (GAS) are genetically diverse. Determination of strain features can reveal associations with disease and resistance and assist in vaccine formulation. We employed whole-genome sequence (WGS)-based characterization of 1,454 invasive GAS isolates recovered in 2015 by Active Bacterial Core Surveillance and performed conventional antimicrobial susceptibility testing. Predictions were made for genotype, GAS carbohydrate, antimicrobial resistance, surface proteins (M family, fibronectin binding, T, R28), secreted virulence proteins (Sda1, Sic, exotoxins), hyaluronate capsule, and an upregulated nga operon (encodes NADase and streptolysin O) promoter (Pnga3). Sixty-four M protein gene (emm) types were identified among 69 clonal complexes (CCs), including one CC of Streptococcus dysgalactiae subsp. equisimilisemm types predicted the presence or absence of active sof determinants and were segregated into sof-positive or sof-negative genetic complexes. Only one "emm type switch" between strains was apparent. sof-negative strains showed a propensity to cause infections in the first quarter of the year, while sof+ strain infections were more likely in summer. Of 1,454 isolates, 808 (55.6%) were Pnga3 positive and 637 (78.9%) were accounted for by types emm1, emm89, and emm12 Theoretical coverage of a 30-valent M vaccine combined with an M-related protein (Mrp) vaccine encompassed 98% of the isolates. WGS data predicted that 15.3, 13.8, 12.7, and 0.6% of the isolates were nonsusceptible to tetracycline, erythromycin plus clindamycin, erythromycin, and fluoroquinolones, respectively, with only 19 discordant phenotypic results. Close phylogenetic clustering of emm59 isolates was consistent with recent regional emergence. This study revealed strain traits informative for GAS disease incidence tracking, outbreak detection, vaccine strategy, and antimicrobial therapy.IMPORTANCE The current population-based WGS data from GAS strains causing invasive disease in the United States provide insights important for prevention and control strategies. Strain distribution data support recently proposed multivalent M type-specific and conserved M-like protein vaccine formulations that could potentially protect against nearly all invasive U.S. strains. The three most prevalent clonal complexes share key polymorphisms in the nga operon encoding two secreted virulence factors (NADase and streptolysin O) that have been previously associated with high strain virulence and transmissibility. We find that Streptococcus pyogenes is phylogenetically subdivided into loosely defined multilocus sequence type-based clusters consisting of solely sof-negative or sof-positive strains; with sof-negative strains demonstrating differential seasonal preference for infection, consistent with the recently demonstrated differential seasonal preference based on phylogenetic clustering of full-length M proteins. This might relate to the differences in GAS strain compositions found in different geographic settings and could further inform prevention strategies.

Synthesis, Characterization and Immunological Evaluation of Self-Adjuvanting Group†A Streptococcal Vaccine Candidates Bearing Various Lipidic Adjuvanting Moieties.

Four group A streptococcal glycolipopeptide vaccine candidates with different lipidic adjuvanting moieties were prepared and characterized. The immunogenicity of the compounds was evaluated by macrophage and dendritic cell uptake studies and by in vivo quantification of systemic IgG antibody by ELISA. Three of the candidates showed significant induction of the IgG response.

Immunogenicity and prediction of epitopic region of antigen Ag I/II and glucosyltransferase from Streptococcus mutans.

The levels of Streptococcus (S.) mutans infections in saliva were evaluated and a comparison for specific antibody levels among children with different levels of S. mutans infection was made. The promising epitopic regions of antigen AgI/II (PAc) and glucosyltransferase (GTF) for potential vaccine targets related to S. mutans adherence were screened. A total of 94 children aged 3-4 years were randomly selected, including 53 caries-negative and 41 caries-positive children. The values of S. mutans and those of salivary total secretory immunoglobulin A (sIgA), anti-PAc and anti-Glucan binding domain (anti-GLU) were compared to determine the correlation among them. It was found the level of s-IgA against specific antigens did not increase with increasing severity of S. mutans infection, and the complete amino acid sequence of PAc and GTFB was analyzed using the DNAStar Protean system for developing specific anti-caries vaccines related to S. mutans adherence. A significantly positive correlation between the amount of S. mutans and children decayed, missing, and filled teeth index was observed. No significant difference was detected in specific sIgA against PAc or GLU between any two groups. No significant correlation was found between such specific sIgA and caries index. A total of 16 peptides from PAc as well as 13 peptides from GTFB were chosen for further investigation. S. mutans colonization contributed to early children caries as an important etiological factor. The level of sIgA against specific antigens did not increase with increasing severity of S. mutans infection in children. The epitopes of PAc and GTF have been screened to develop the peptide-based or protein-based anti-caries vaccines.

Streptococcus pneumoniae proteomics: determinants of pathogenesis and vaccine development.

Streptococcus pneumoniae is a major pathogen that is responsible for a variety of invasive diseases. The bacteria gain entry initially by establishing a carriage state in the nasopharynx from where they migrate to other sites in the body. The worldwide distribution of the bacteria and the severity of the diseases have led to a significant level of interest in the development of vaccines against the bacteria. Current vaccines, based on the bacterial polysaccharide, have a number of limitations including poor immunogenicity and limited effectiveness against all pneumococcal serotypes. There are many challenges in developing vaccines that will be effective against the diverse range of isolates and serotypes for this highly variable bacterial pathogen. This review considers how proteomic technologies have extended our understanding of the pathogenic mechanisms of nasopharyngeal colonization and disease development as well as the critical areas in developing protein-based vaccines.

Anti-Group B Streptococcus Glycan-Conjugate Vaccines Using Pilus Protein GBS80 As Carrier and Antigen: Comparing Lysine and Tyrosine-directed Conjugation.

Gram-positive Streptococcus agalactiae or group B Streptococcus (GBS) is a leading cause of invasive infections in pregnant women, newborns, and elderly people. Vaccination of pregnant women represents the best strategy for prevention of neonatal disease, and GBS polysaccharide-based conjugate vaccines are currently under clinical testing. The potential of GBS pilus proteins selected by genome-based reverse vaccinology as protective antigens for anti-streptococcal vaccines has also been demonstrated. Dressing pilus proteins with surface glycan antigens could be an attractive approach to extend vaccine coverage. We have recently developed an efficient method for tyrosine-directed ligation of large glycans to proteins via copper-free azide-alkyne [3 + 2] cycloaddition. This method enables targeting of predetermined sites of the protein, ensuring that protein epitopes are preserved prior to glycan coupling and a higher consistency in glycoconjugate batches. Herein, we compared conjugates of the GBS type II polysaccharide (PSII) and the GBS80 pilus protein obtained by classic lysine random conjugation and by the recently developed tyrosine-directed ligation. PSII conjugated to CRM197, a carrier protein used for vaccines in the market, was used as a control. We found that the constructs made from PSII and GBS80 were able to elicit murine antibodies recognizing individually the glycan and protein epitopes on the bacterial surface. The generated antibodies were efficacious in mediating opsonophagocytic killing of strains expressing exclusively PSII or GBS80 proteins. The two glycoconjugates were also effective in protecting newborn mice against GBS infection following vaccination of the dams. Altogether, these results demonstrated that polysaccharide-conjugated GBS80 pilus protein functions as a carrier comparably to CRM197, while maintaining its properties of protective protein antigen. Glycoconjugation and reverse vaccinology can, therefore, be combined to design vaccines with broad coverage. This approach opens a path to a new generation of vaccines. Tyrosine-ligation allows creation of more homogeneous vaccines, correlation of the immune response to defined connectivity points, and fine-tuning of the conjugation site in glycan-protein conjugates.

Structure-informed design of an enzymatically inactive vaccine component for group A Streptococcus.

UNLABELLED: Streptococcus pyogenes (group A Streptococcus [GAS]) causes ~700 million human infections/year, resulting in >500,000 deaths. There is no commercial GAS vaccine available. The GAS surface protein arginine deiminase (ADI) protects mice against a lethal challenge. ADI is an enzyme that converts arginine to citrulline and ammonia. Administration of a GAS vaccine preparation containing wild-type ADI, a protein with inherent enzymatic activity, may present a safety risk. In an approach intended to maximize the vaccine safety of GAS ADI, X-ray crystallography and structural immunogenic epitope mapping were used to inform vaccine design. This study aimed to knock out ADI enzyme activity without disrupting the three-dimensional structure or the recognition of immunogenic epitopes. We determined the crystal structure of ADI at 2.5 Å resolution and used it to select a number of amino acid residues for mutagenesis to alanine (D166, E220, H275, D277, and C401). Each mutant protein displayed abrogated activity, and three of the mutant proteins (those with the D166A, H275A, and D277A mutations) possessed a secondary structure and oligomerization state equivalent to those of the wild type, produced high-titer antisera, and avoided disruption of B-cell epitopes of ADI. In addition, antisera raised against the D166A and D277A mutant proteins bound to the GAS cell surface. The inactivated D166A and D277A mutant ADIs are ideal for inclusion in a GAS vaccine preparation. There is no human ortholog of ADI, and we confirm that despite limited structural similarity in the active-site region to human peptidyl ADI 4 (PAD4), ADI does not functionally mimic PAD4 and antiserum raised against GAS ADI does not recognize human PAD4. IMPORTANCE: We present an example of structural biology informing human vaccine design. We previously showed that the administration of the enzyme arginine deiminase (ADI) to mice protected the mice against infection with multiple GAS serotypes. In this study, we determined the structure of GAS ADI and used this information to improve the vaccine safety of GAS ADI. Catalytically inactive mutant forms of ADI retained structure, recognition by antisera, and immunogenic epitopes, rendering them ideal for inclusion in GAS vaccine preparations. This example of structural biology informing vaccine design may underpin the formulation of a safe and efficacious GAS vaccine.

Synthesis and immunological characterization of modified hyaluronic acid hexasaccharide conjugates.

The synthesis of a tetanus toxoid (TT)-conjugate of a hyaluronic acid (HA) hexasaccharide is described. The compound was intended for use in monitoring HA levels as a disease marker and as a potential vaccine against Group A Streptococcus (GAS) infections. We also report the synthesis of a chemically modified HA-hexasaccharide-TT conjugate in which the N-acetyl moiety of the N-acetyl-D-glucosamine residue is replaced with an N-propionyl unit in order to enhance immunogenicity. The oligosaccharides are synthesized in a convergent manner. The TT-conjugate syntheses rely on the reaction of the amines on the 6-aminohexyl aglycon of the hexasaccharides with diethyl squarate to give the monoethyl squarate adducts. Subsequent reactions with lysine ε-amino groups on TT then give the glycoconjugates containing an average of 8 hexasaccharide haptens per TT molecule. Immunological studies in mice show very similar antibody responses with both conjugates, suggesting that the N-acetyl groups of the glucosaminyl residues of the HA-hexasaccharide are not a critical part of the epitope recognized by the anti-HA polyclonal immune response. Furthermore, it would appear that the N-acyl moieties are not in close contact with the amino acid residues of the antibody combining sites.

An efficient, chemically-defined semisynthetic lipid-adjuvanted nanoparticulate vaccine development system.

A novel vaccine development platform that enables the site-specific conjugation of synthetic lipid adjuvants to recombinant proteins was produced. This technology facilitates the simple and efficient production of homogeneous, chemically-defined, semisynthetic lipoprotein vaccines. Using a polytope 'string-of-beads' approach, a synthetic gene incorporating seven Streptococcus pyogenes M protein strain-specific antigens, and a conserved M protein antigen (J14) was produced, expressed, and attached to a lipoamino acid based adjuvant (lipid core peptide; LCP). Nanoparticles (40 nm diameter) of an optimal size for stimulating antibody-mediated immunity were formed upon the addition of these lipoproteins to aqueous buffer (PBS). Systemic antigen-specific IgG antibodies were raised against all eight antigens in C57BL/6J mice, without the need to formulate with additional adjuvant. These antibodies bound cell surface M proteins of S. pyogenes strains represented within the polytope sequence, with higher antibody levels observed where a dendritic cell targeting peptide (DCpep) was incorporated within the LCP adjuvant. FROM THE CLINICAL EDITOR: In this study, a novel vaccine development system is presented, combining adjuvants with recombinant protein antigens, and presenting the antigen in a nanoparticle system optimized for antibody production. They demonstrate efficient vaccination in a murine model system without the need for additional adjuvants.

Structure-activity relationship for the development of a self-adjuvanting mucosally active lipopeptide vaccine against Streptococcus pyogenes.

Infection with group A streptococcus (GAS) can result in a number of diseases, some of which are potentially life-threatening. The oral-nasal mucosa is a primary site of GAS infection, and a mucosally active vaccine candidate could form the basis of an antidisease and transmission-blocking GAS vaccine. In the present study, a peptide from the GAS M protein (J14) representing a B cell epitope was incorporated alongside a universal T cell helper epitope and a Toll-like receptor 2 targeting lipid moiety to form lipopeptide constructs. Through structure activity studies, we identified a vaccine candidate that induces J14-specific mucosal and systemic antibody responses when administered intranasally without additional adjuvants. The systemic antibodies elicited were capable of inhibiting the growth of GAS. In addition, J14-specific mucosal antibodies corresponded with reduced throat colonization after respiratory GAS challenge. These preclinical experiments show that this lipopeptide could form the basis of an optimal needle-free mucosal GAS vaccine.

Development of a novel mucosal vaccine against strangles by supercritical enhanced atomization spray-drying of Streptococcus equi extracts and evaluation in a mouse model.

Strangles is an extremely contagious and sometimes deadly disease of the Equidae. The development of an effective vaccine should constitute an important asset to eradicate this worldwide infectious disease. In this work, we address the development of a mucosal vaccine by using a Supercritical Enhanced Atomization (SEA) spray-drying technique. Aqueous solutions containing the Streptococcus equi extracts and chitosan were converted into nanospheres with no use of organic solvents. The immune response in a mouse model showed that the nanospheres induced a well-balanced Th1 and Th2 response characterized by a unitary ratio between the concentrations of IgG2a and IgG1, together with IgA production. This strategy revealed to be an effective alternative for immunization against S. equi, and therefore, it may constitute a feasible option for production of a strangles vaccine.

Immunological evaluation of lipopeptide group A streptococcus (GAS) vaccine: structure-activity relationship.

Streptococcus pyogenes (group A streptococcus, GAS) is a Gram-positive bacterial pathogen responsible for a wide variety of diseases. To date, GAS vaccine development has focused primarily on the M-protein. The M-protein is highly variable at the amino (N)-terminus (determining serotype) but is conserved at the carboxyl (C)-terminus. Previously a 29 amino acid peptide (named J14) from the conserved region of the M-protein was identified as a potential vaccine candidate. J14 was capable of eliciting protective antibodies that recognized many GAS serotypes when co-administered with immuno-stimulants. This minimal epitope however showed no immunogenicity when administered alone. In an attempt overcome this immunological non-responsiveness, we developed a self-adjuvanting vaccine candidate composed of three components: the B-cell epitope (J14), a universal helper T-cell epitope (P25) and a lipid moiety consisting of lipoamino acids (Laas) which target Toll-like receptor 2 (TLR2). Immunological evaluation in B10.BR (H-2k) mice demonstrated that the epitope attachment to the point of lipid moiety, and the length of the Laa alkyl chain have a profound effect on vaccine immunogenicity after intranasal administration. It was demonstrated that a vaccine featuring C-terminal lipid moiety containing alkyl chains of 16 carbons, with P25 located at the N-terminus, and J14 attached to the side chain of a central lysine residue was capable of inducing optimal antibody response. These findings have considerable relevance to the development of a broad spectrum J14-based GAS vaccine and in particular provided a rational basis for peptide vaccine design based on this self-adjuvanting lipopeptide technology.

Immunoproteomic analysis of the antibody response obtained in Nile tilapia following vaccination with a Streptococcus iniae vaccine.

Streptococcus iniae is one of the most economically important Gram-positive pathogens in cultured fish species worldwide. The USDA-ARS Aquatic Animal Health Research Unit developed a modified (contains concentrated culture supernatant) S. iniae bacterin that has been demonstrated to be efficacious, and protection is mediated by specific anti-S. iniae antibodies. Although effective, the specific vaccine components important for efficacy are not known. In the present study, an immunoproteomic approach was utilized to identify whole-cell lysate proteins of S. iniae that stimulated specific antibody production in Nile tilapia (Oreochromis niloticus) following vaccination. Groups of tilapia were vaccinated by intraperitoneal injection with the modified S. iniae bacterin or were mock-vaccinated, and at 30 d post-vaccination sera samples were obtained from individual fish. Vaccination of tilapia with the S. iniae vaccine stimulated significantly elevated specific antibody responses against proteins of the bacterium and passive immunization of tilapia with this serum demonstrated the antibodies were highly protective. Whole-cell lysate proteins of S. iniae were separated by 2D-PAGE and were probed with a pooled serum sample from vaccinated tilapia. A total of eleven unique immunogenic proteins were positively identified by mass spectrometry. Based on research conducted on homologous proteins in other Streptococcus spp., antibodies specific for three of the identified proteins, enolase, glyceraldehyde-3-phosphate dehydrogenase, and fructose-bisphosphate aldolase, are likely involved in protection from streptococcosis caused by S. iniae.

Design of three-component vaccines against group A streptococcal infections: importance of spatial arrangement of vaccine components.

Immunological assessment of group A streptococcal (GAS) branched lipopeptides demonstrated the impact of spatial arrangement of vaccine components on both the quality and quantity of their immune responses. Each lipopeptide was composed of three components: a GAS B-cell epitope (J14), a universal CD4(+) T-cell helper epitope (P25), and an immunostimulant lipid moiety that differs only in its spatial arrangement. The best systemic immune responses were demonstrated by a lipopeptide featuring the lipid moiety at the lipopeptide C-terminus. However, this candidate did not achieve protection against bacterial challenge. The best protection (100%) was shown by a lipopeptide featuring a C-terminal J14, conjugated through a lysine residue to P25 at the N-terminus, and a lipid moiety on the lysine side chain. The former candidate features α-helical conformation required to produce protective J14-specific antibodies. Our results highlight the importance of epitope orientation and lipid position in the design of three-component synthetic vaccines.

Surface modified polymeric nanoparticles for immunisation against equine strangles.

The successful development of particulate vaccines depends on the understanding of their physicochemical and biological characteristics. Therefore, the main purpose of this study was to develop and characterise stable surface modified poly(lactic acid) (PLA) nanoparticles, using polyvinyl alcohol (PVA), alginate (ALG) and glycolchitosan (GCS) containing a Streptococcus equi enzymatic extract adsorbed onto the surface. The characterisation of the preparations and a physicochemical study of the adsorption process were performed. The adsorption of S. equi proteins is a rapid process reaching, within 1h, maximum adsorption efficiency values of 75.2+/-1.9% (w/w) for PLA-PVA, 84.9+/-0.2% (w/w) for PLA-GCS and 78.1+/-0.4% (w/w) for PLA-ALG nanoparticles. No protein degradation was detected throughout the formulation procedures. As expected from a complex mixture of proteins, adsorption data suggest a Freundlich-type of equilibrium with regression coefficients (r(2)) of 0.9958, 0.9839 and 0.9940 for PLA-PVA, PLA-GCS and PLA-ALG, respectively. Desorption studies revealed a burst release within the first 6h, for all formulations, followed by a sustained release profile. Nanoparticle surface modification with GCS improved the sustained release profile, as 20% of protein remained attached to the particle surface after 30 days. The results show that adsorption is an alternative method for the production of S. equi antigen carriers for vaccination purposes.

Development of conformational mimetics of conserved Streptococcus pyogenes minimal epitope as vaccine candidates.

One of the factors responsible for the poor immunogenicity of synthetic peptide antigens is the lack of conformational integrity. Embedding the minimal epitopes in helix-promoting peptide sequences has successfully enhanced the immunogenicity of the epitopes derived from the alpha-helical regions of the M protein of group A streptococci (Streptococcus pyogenes, GAS). However, the introduction of "foreign" peptide sequences is believed to have an unfavourable impact on the antigen specificity. In the current study, we employed a non-peptide approach, using topological carbohydrate templates, to induce helical conformation of the peptide antigens. Utilized together with the advances of the lipid core peptide system and chemoselective ligation, five GAS vaccine candidates incorporating the minimal epitope J14i (ASREAKKQVEKALE) were synthesized with high purity. Circular dichroism studies indicated that the template-assembled peptides formed alpha-helix bundles. This atom-economic strategy also reduces the complexity and cost of vaccine production by simply reducing the peptide epitope size.

Investigation toward multi-epitope vaccine candidates using native chemical ligation.

We applied native chemical ligation (NCL) method to the synthesis of highly pure lipid-core peptide (LCP) vaccines to attach various peptide epitopes. In the case of the synthesis of LCP vaccine with two different peptide epitopes, LCP moieties having two free Cys and two protected Cys derivatives (S-acetamidemethyl-Cys, (Cys(Acm)), N-methylsulfonylethyloxycarbonyl-Cys (Msc-Cys), or 1,3-thiazolidine-4-carboxylic acid (Thz)) on oligolysine branches were prepared in order to couple two different epitopes by stepwise NCL. It was found that the difficulty in NCL of first two peptide antigen was associated with the steric hindrance. Using Thz instead of Cys(Acm) and Msc-Cys was important to reduce the steric hindrance and improve NCL yield.