Preclinical safety and immunogenicity of Streptococcus pyogenes (Strep A) peptide vaccines.

We have developed two candidate vaccines to protect against multiple strains of Strep A infections. The candidates are combinatorial synthetic peptide vaccines composed of a M protein epitope (J8 or p*17) and a non-M protein epitope (K4S2). To enhance immunogenicity, each peptide is conjugated to the carrier protein CRM197 (CRM) and formulated with aluminium hydroxide adjuvant Alhydrogel (Alum) to make the final vaccines, J8-CRM + K4S2-CRM/Alum and p*17-CRM + K4S2-CRM/Alum. The safety and toxicity of each vaccine was assessed. Sprague Dawley rats were administered three intramuscular doses, over a six-week study with a 4-week recovery period. A control group received CRM only formulated with Alum (CRM/Alum). There was no evidence of systemic toxicity in the rats administered either vaccine. There was an associated increase in white blood cell, lymphocyte and monocyte counts, increased adrenal gland weights, adrenocortical hypertrophy, and increased severity of granulomatous inflammation at the sites of injection and the associated inguinal lymph nodes. These changes were considered non-adverse. All rats administered vaccine developed a robust and sustained immunological response. The absence of clinical toxicity and the development of an immunological response in the rats suggests that the vaccines are safe for use in a phase 1 clinical trial in healthy humans.

Protein adhesins as vaccine antigens for Group A Streptococcus.

Group A Streptococcus (GAS) is a globally important human pathogen that causes a broad spectrum of disease ranging from mild superficial infections to severe invasive diseases with high morbidity and mortality. Currently, there is no vaccine available for human use. GAS produces a vast array of virulence factors including multiple adhesin molecules. These mediate binding of the bacteria to host tissues and are essential in the initial phases of infection. Prophylactic vaccination with adhesins is a promising vaccine strategy and many GAS adhesins are currently in development as vaccine candidates. The most advanced candidates, having entered clinical trials, are based on the M protein, while components of the pilus and a number of fibronectin-binding proteins are in pre-clinical development. Adhesin-based vaccines aim to induce protective immunity via two main mechanisms: neutralisation where adhesin-specific antibodies block the ability of the adhesin to bind to host tissue and opsonisation in which adhesin-specific antibodies tag the GAS bacteria for phagocytosis. This review summarises our current knowledge of GAS adhesins and their structural features in the context of vaccine development.

Preclinical safety study of a recombinant Streptococcus pyogenes vaccine formulated with aluminum adjuvant.

A recombinant vaccine composed of a fusion protein formulated with aluminum hydroxide adjuvant is under development for protection against diseases caused by Streptococcus pyogenes. The safety and local reactogenicity of the vaccine was assessed by a comprehensive series of clinical, pathologic and immunologic tests in preclinical experiments. Outbred mice received three intramuscular injections of 1/5th of the human dose (0.1 ml) and rabbits received two injections of the full human dose. Control groups received adjuvant or protein antigen. The vaccine did not cause clinical evidence of systemic toxicity in mice or rabbits. There was a transient increase of peripheral blood neutrophils after the third vaccination of mice. In addition, the concentration of acute phase proteins serum amyloid A and haptoglobin was significantly increased 1 day after injection of the vaccine in mice. There was mild transient swelling and erythema of the injection site in both mice and rabbits. Treatment-related pathology was limited to inflammation at the injection site and accumulation of adjuvant-containing macrophages in the draining lymph nodes. In conclusion, the absence of clinical toxicity in two animal species suggest that the vaccine is safe for use in a phase I human clinical trial. Copyright © 2016 John Wiley & Sons, Ltd.

Preclinical immunogenicity and safety of a Group A streptococcal M protein-based vaccine candidate.

Streptococcus pyogenes (group A streptococcus, GAS) causes a wide range of clinical manifestations ranging from mild self-limiting pyoderma to invasive diseases such as sepsis. Also of concern are the post-infectious immune-mediated diseases including rheumatic heart disease. The development of a vaccine against GAS would have a large health impact on populations at risk of these diseases. However, there is a lack of suitable models for the safety evaluation of vaccines with respect to post-infectious complications. We have utilized the Lewis Rat model for cardiac valvulitis to evaluate the safety of the J8-DT vaccine formulation in parallel with a rabbit toxicology study. These studies demonstrated that the vaccine did not induce abnormal pathology. We also show that in mice the vaccine is highly immunogenic but that 3 doses are required to induce protection from a GAS skin challenge even though 2 doses are sufficient to induce a high antibody titer.

Homologous whole bacterin vaccination is not able to reduce Streptococcus suis serotype 9 strain 7997 transmission among pigs or colonization.

Streptococcus suis (S. suis) is an important porcine pathogen worldwide, and antibiotics are often applied to treat or prevent clinical signs. Vaccination could be an alternative measure to reduce the abundant use of antimicrobials. The aim of this study was to determine the effect of vaccination with homologues whole bacterin vaccine containing S. suis serotype 9 strain 7997 on transmission of this serotype among pigs and on mucosal colonization. Caesarean derived, colostrum deprived pigs (N=50) were housed pair wise. Thirteen pairs were vaccinated intramuscularly with 2-3×10(9) colony forming units (CFU) inactivated S. suis serotype 9 per dose and α-tocopherolactetaat as adjuvant at 3 and 5 weeks of age; twelve pairs served as non-vaccinated controls. At 7 weeks of age, one pig of each pair was intranasally inoculated with 1-2×10(9)CFU of the homologues strain, whereas the other pig of each pair was contact-exposed. Tonsil brushings and saliva swabs were collected for 4 weeks, and tested for the presence of S. suis by bacteriological culture. No differences in number of S. suis in the tonsils or saliva samples or in clinical signs were observed between vaccinated and control pigs. In all pairs, transmission between inoculated and contact exposed pigs occurred, and no difference was observed in rate at which this occurred. The estimated transmission rate parameter ß between vaccinated pigs was ß(v)=5.27/day, and for non-vaccinated pigs ß(nv)=2.77/day (P=0.18). It was concluded that vaccination against S. suis serotype 9 did not reduce transmission, nor colonization and that there were no indications that protection against clinical signs was induced.

A vaccine against S. pyogenes: design and experimental immune response.

Streptococcus pyogenes causes severe invasive infections: the post-streptococcal sequelae of acute rheumatic fever (RF) and rheumatic heart disease (RHD), acute glomerulonephritis, and uncomplicated pharyngitis and pyoderma. Efforts to produce a vaccine against S. pyogenes began several decades ago, and different models have been proposed. Here, we describe the methodology used in the development of a new vaccine model, consisting of both T and B protective epitopes constructed as synthetic peptides and recombinant proteins. Two adjuvants were tested in an experimental inbred mouse model: a classical Freund's adjuvant and a new adjuvant (AFCo1) that induces mucosal immune responses and is obtained by calcium precipitation of a proteoliposome derived from the outer membrane of Neisseria meningitides B. The StreptInCor vaccine epitope co-administrated with AFCo1 adjuvant induced mucosal (IgA) and systemic (IgG) antibodies as preferential Th1-mediated immune responses. No autoimmune reactions were observed, suggesting that the vaccine epitope is safe.

Synthesis of a Streptococcus pyogenes vaccine candidate based on the M protein PL1 epitope.

Group A streptococcus is a Gram-positive bacteria that causes a range of infectious diseases. Targeting the bacteria, a new self-adjuvanting vaccine candidate, incorporating a carbohydrate carrier and an amino acid-based adjuvant, was synthesised utilising carbohydrate chemistry and solid-phase peptide synthesis procedures. Characterisation of the candidate was achieved using reverse-phase HPLC and electrospray ionisation mass spectrometry. The successful synthesis and characterisation of the vaccine candidate may contribute to the discovery of a therapeutically and clinically viable vaccine against group A streptococcus.

Structure-activity relationship of a series of synthetic lipopeptide self-adjuvanting group a streptococcal vaccine candidates.

The development of 16 self-adjuvanting group A streptococcal vaccine candidates, composed of (i) a universal helper T-cell epitope (P25), (ii) a target GAS B-cell epitope (J14), and (iii) a lipid moiety, is described. Systemic J14-specific IgG antibodies were detected following subcutaneous immunization of BALB/c (H-2 (d)) mice with each construct without the need for an additional adjuvant. The effect of changing the order of P25, J14, and lipid moiety attachment or incorporation of P25 and J14 into a lipid-core peptide system on antibody titers was assessed. The point of lipid moiety attachment had the greatest influence on systemic J14-specific IgG antibody titers. Overall, the best vaccines featured a C-terminal lipid moiety, conjugated through a lysine residue to P25 at the N-terminus, and J14 on the lysine side chain.

Association between immunogenicity and adsorption of a recombinant Streptococcus pneumoniae vaccine antigen by an aluminum adjuvant.

The impact on immunogenicity of the degree of adsorption of three Streptococcus pneumoniae (Sp) vaccine antigens to aluminum adjuvants was studied. The three antigens evaluated (Sp1, Sp2 and Sp3) were highly adsorbed by aluminum hydroxide adjuvant, but not adsorbed by aluminum phosphate adjuvant. All of the Sp antigens adjuvanted with aluminum hydroxide elicited higher antibody responses in mice than formulations prepared with aluminum phosphate or non-adjuvanted antigen. Varying the percent aluminum-bound Sp antigen in the formulated vaccine affected the observed antibody responses. These observations suggest that the antibody response observed for Sp antigens in this study is stimulated by a depot effect of the antigen bound to an aluminum adjuvant.

Method for the synthesis of multi-epitopic Streptococcus pyogenes lipopeptide vaccines using native chemical ligation.

The aim of this study was to investigate methods for the synthesis of highly pure, well-characterized analogues of the lipid core peptide (LCP) system. Difficulties synthesizing and purifying conventional LCP systems have led to the requirement for a technique to produce highly pure, LCP-based vaccines for potential use in human clinical trials. The current study describes methods for the attachment of lipophilic adjuvants onto multi-epitopic peptide vaccines. Described is the synthesis, using native chemical ligation, of a highly pure, tri-epitopic, group A streptococcal (GAS) lipopeptide vaccine candidate. Intranasal immunization of the described tri-epitopic GAS lipopeptide with the mucosal adjuvant cholera toxin B subunit induced high serum IgG antibody titers specific for each of the incorporated peptide epitopes.

Identification and assessment of new vaccine candidates for group A streptococcal infections.

Group A Streptococcus (GAS) is a human-specific pathogen responsible for a wide variety of human diseases. Numerous GAS surface antigens interact with the human immune system and only some of these proteins have been studied in depth. A few of these may elicit protective response against GAS infection. In this study, we have used an in silico approach to identify antigenic peptides from GAS surface proteins. Putative GAS surface proteins from the M1 GAS genome were identified by the presence on LPxTG cell-wall anchoring motif and an export signal sequence. This technique identified 17 proteins of known or putative function, and another 11 which do not have known homologues. Peptides derived from predicted antigenic sequences near the amino terminus of six of these proteins, and another seven peptides derived from the two known surface proteins, GRAB and MtsA, were conjugated to keyhole lymphocyanin (KLH), and investigated for their capacity to induce opsonic antibody responses in outbred Quackenbush mice. All peptide-KLH antisera demonstrated opsonic capacity against both 88/30 and M1 GAS. However, KLH sera alone was also able to induce opsonic antibodies, suggesting that anti-KLH antibodies contributed to the opsonisation seen in the peptide-KLH antisera. KLH is therefore a promising carrier molecule for potential GAS peptide vaccines.

Effects of various adjuvants on efficacy of a vaccine against Streptococcus bovis and Lactobacillus spp in cattle.

OBJECTIVE: To determine efficacy of vaccines incorporating QuilA, alum, dextran combined with mineral oil, or Freund adjuvant for immunization of feedlot cattle against Streptococcus bovis and Lactobacillus spp. ANIMALS: 24 steers housed under feedlot conditions. PROCEDURE: Steers were randomly assigned to 4 experimental groups and a control group. Animals in experimental groups were inoculated on days 0 and 26 with vaccines containing Freund adjuvant (FCA), QuilA, dextran combined with mineral oil (Dex), or alum as adjuvant. Serum anti-S bovis and anti-Lactobacillus IgG concentrations were measured, along with fecal pH, ruminal fluid pH, and number of S bovis and Lactobacillus spp in ruminal fluid. RESULTS: Throughout the study, serum anti-S bovis and anti-Lactobacillus IgG concentrations for animals in the Dex, QuilA, and alum groups were similar to or significantly higher than concentrations for animals in the FCA group. Serum anti-S bovis and anti-Lactobacillus IgG concentrations were significantly increased on days 26 through 75 in all 4 experimental groups, and there was a linear relationship between anti-S bovis and anti-Lactobacillus IgG concentrations. For animals in the QuilA and Dex groups, mean pH of feces throughout the period of experiment were significantly higher and numbers of S bovis and Lactobacillus spp in ruminal fluid on day 47 were significantly lower than values for control cattle. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggest that immunization of feedlot steers against S bovis and Lactobacillus spp with vaccines incorporating Freund adjuvant, QuilA, dextran, or alum as an adjuvant effectively induced high, long-lasting serum anti-S bovis and anti-Lactobacillus IgG concentrations. Of the adjuvants tested, dextran may be the most effective.