CRM197 Carrier Protein Research Articles

Semisynthetic Glycoconjugates as Potential Vaccine Candidates Against Haemophilus influenzae Type a.

Glycoconjugate vaccines are based on chemical conjugation of pathogen-associated carbohydrates with immunogenic carrier proteins and are considered a very cost-effective way to prevent infections. Most of the licensed glycoconjugate vaccines are composed of saccharide antigens extracted from bacterial sources. However, synthetic oligosaccharide antigens have become a promising alternative to natural polysaccharides with the advantage of being well-defined structures providing homogeneous conjugates. Haemophilus influenzae (Hi) is responsible for a number of severe diseases. In recent years, an increasing rate of invasive infections caused by Hi serotype a (Hia) raised some concern, because no vaccine targeting Hia is currently available. The capsular polysaccharide (CPS) of Hia is constituted by phosphodiester-linked 4-β-d-glucose-(1→4)-d-ribitol-5-(PO4→) repeating units and is the antigen for protein-conjugated polysaccharide vaccines. To investigate the antigenic potential of the CPS from Hia, we synthesized related saccharide fragments containing up to five repeating units. Following the synthetic optimization of the needed disaccharide building blocks, they were assembled using the phosphoramidite approach for the installation of the phosphodiester linkages. The resulting CPS-based Hia oligomers were conjugated to CRM197 carrier protein and evaluated in vivo for their immunogenic potential, showing that all glycoconjugates were capable of raising antibodies recognizing Hia synthetic fragments.

Using Surface Immunogenic Protein as a Carrier Protein to Elicit Protective Antibody to Multiple Serotypes for Candidate Group B Streptococcal Glycan Conjugate Vaccines.

Group B Streptococcus (GBS) is a life-threatening opportunistic pathogen, particularly in pregnant women, infants, and the elderly. Currently, maternal vaccination is considered the most viable long-term option for preventing GBS mother-to-infant infection, and two polysaccharide conjugate vaccines utilizing CRM197 as a carrier protein have undergone clinical phase II trials. Surface immunogenic protein (Sip), present in all identified serotypes of GBS strains so far, is a protective surface protein of GBS. In this study, the type Ia capsular polysaccharide (CPS) of GBS was utilized as a model to develop candidate antigens for a polysaccharide conjugate vaccine by coupling it with the Sip of GBS and the traditional carrier protein CRM197. Serum analysis from immunized New Zealand rabbits and CD1 mice revealed that there was no significant difference in antibody titers between the Ia-Sip group and Ia-CRM197 group; however, both were significantly higher than those observed in the Ia polysaccharide group. Opsonophagocytosis and passive immune protection results using rabbit serum indicated no significant difference between the Ia-Sip and Ia-CRM197 groups, both outperforming the Ia polysaccharide group. Furthermore, serum from the Ia-Sip group had a cross-protective effect on multiple types of GBS strains. The challenge test results in CD1 mice demonstrated that the Ia-Sip group provided complete protection against lethal doses of bacteria and also showed cross-protection against type III strain. Our study demonstrates for the first time that Ia-Sip is immunogenic and provides serotype-independent protection in glycan conjugate vaccines, which also indicates Sip may serve as an excellent carrier protein for GBS glycan conjugate vaccines and provide cross-protection against multiple GBS strains.

Preclinical validation of an Escherichia coli O-antigen glycoconjugate for the prevention of serotype O1 invasive disease.

The Escherichia coli serotype O1 O-antigen serogroup is a common cause of invasive bloodstream infections (BSI) in populations at risk such as newborns and the elderly. Sequencing of US BSI isolates and structural analysis of O polysaccharide antigens purified from strains that are representative of genotypic sub-groups confirmed the relevance of the O1a subtype as a vaccine antigen. O polysaccharide was purified from a strain engineered to produce long-chain O1a O-antigen and was chemically conjugated to CRM197 carrier protein. The resulting glycoconjugate elicited functional antibodies and was protective in mice against lethal challenges with virulent K1-encapsulated O1a isolates.

Immunological Exploration of Helicobacter pylori Serotype O2 O‐Antigen by Using a Synthetic Glycan Library

Comprehensive SummaryHelicobacter pylori (H. pylori) infection is a threat to human health. The lipopolysaccharide (LPS) O‐antigen holds promise for developing vaccines. It is meaningful to explore the immunological activity of oligosaccharides with different lengths and frameshifts for antigen development. Herein, a glycan library of H. pylori O2 O‐antigen containing eight fragments is constructed. After screening with anti‐H. pylori O2 LPS sera and patients’ sera by glycan microarray, the disaccharide HPO2G‐2b and trisaccharide HPO2G‐3a show strong antigenicity and then are separately conjugated with carrier protein CRM197. Both glycoconjugates elicit a robust immunoglobulin G (IgG) immune response in rabbits. The anti‐HPO2G‐3a IgG antibodies possess a much stronger binding affinity with the LPS and bacteria of H. pylori O2 than the anti‐HPO2G‐2b IgG antibodies. There is no cross‐reaction between both sera IgG antibodies with LPS and bacteria of H. pylori O1, O6, and E. coli. The results demonstrate the trisaccharide HPO2G‐3a is a promising candidate for H. pylori vaccine development.

Evaluation of structural modification induced activation of pneumococcal polysaccharide by GC-MS for the conjugate vaccine

Polysaccharide (Ps) activation evaluation is an imperative quality attribute in a conjugate vaccine. Pneumococcal polysaccharide (PnPs) serotypes 5, 6B, 14, 19A and 23F were cyanylated for 3 and 8 min. The cyanylated and non-cyanylated polysaccharides were methanolysed and derivatized to assess the activation of each sugar by GC-MS. The activation of 22 and 27% serotype 6B and 11 and 36% in serotype 23 F Ps at 3 and 8 min respectively showed controlled conjugation kinetics with CRM197 carrier protein estimated by SEC-HPLC and optimal absolute molar mass by SEC-MALS. The Glc and Gal are the most commonly activated sugars of all PnPs serotypes while N-acetyl sugars PneuNAc, GalNAc and Rha in serotypes 5, 14 and 19A respectively showed >50% activation which contributes to conjugate aggregate formation at 8 min compared to 3 min cyanylation. The GC-MS analysis of structural modifications at functional groups entails important information to characterize the activated polysaccharide for consistent conjugate vaccine manufacturing.

Cancer Vaccines Based on Fluorine-Modified KH-1 Elicit Robust Immune Response.

KH-1 is a tumor-associated carbohydrate antigen (TACA), which serves as a valuable target of antitumor vaccines for cancer immunotherapies. However, most TACAs are thymus-independent antigens (TD-Ag), and they tend to induce immunological tolerance, leading to their low immunogenicity. To overcome these problems, some fluorinated derivatives of the KH-1 antigen were designed, synthesized, and conjugated to the carrier protein CRM197 to form glycoconjugates, which were used for immunological studies with Freund's adjuvant. The results showed that fluorine-modified N-acyl KH-1 conjugates can induce higher titers of antibodies, especially IgG, which can recognize KH-1-positive cancer cells and can eliminate cancer cells through complement-dependent cytotoxicity (CDC). The trifluoro-modified KH-1-TF-CRM197 showed great potential as an anticancer vaccine candidate.

Preclinical evaluation of an investigational 21-valent pneumococcal conjugate vaccine, V116, in adult-rhesus monkey, rabbit, and mouse models.

Despite the widespread effectiveness of pneumococcal conjugate vaccines on the overall incidence of invasive pneumococcal disease, the global epidemiological landscape continues to be transformed by residual disease from non-vaccine serotypes, thus highlighting the need for vaccines with expanded disease coverage. To address these needs, we have developed V116,an investigational 21-valent non-adjuvanted pneumococcal conjugate vaccine (PCV),containingpneumococcal polysaccharides (PnPs) 3, 6A, 7F, 8, 9N, 10A, 11A,12F, 15A, 16F, 17F, 19A, 20, 22F, 23A, 23B, 24F, 31, 33F, 35B, anda de-O-acetylated 15B(deOAc15B) individually conjugated to the nontoxic diphtheria toxoid CRM197 carrier protein. Preclinical studies evaluated the immunogenicity of V116 inadult monkeys, rabbits, and mice. Following one dose, V116 was found to be immunogenic in preclinical animal species and induced functional antibodies for all serotypes included in the vaccine, in addition to cross-reactive functional antibodies to serotypes 6C and 15B. In these preclinical animal studies, the increased valency of V116 did not result in serotype-specific antibody suppression when compared to lower valent vaccines V114 or PCV13. In addition, when compared with naïve controls, splenocytes from V116 to immunized animals demonstrated significant induction of CRM197-specific T cells in both IFN-γ and IL-4 ELISPOT assays, as well as Th1 and Th2 cytokine induction through in vitro stimulation assays, thus suggesting the ability of V116 to engage T cell dependent immune response pathways to aid in development of memory B cells. V116 also demonstrated significant protection in mice from intratracheal challenge with serotype 24F, a novel serotype not contained in any currently licensed vaccine.

CRM197-conjugated multi antigen dominant epitope for effective human cytomegalovirus vaccine development

Human cytomegalovirus (HCMV) infection is a major cause of neonatal neurodevelopmental disorders and serious complications in organ transplantation. Previous HCMV vaccines focused on humoral immunity but had limited effect on viral infection. T-cell responses are essential to prevent HCMV infection, indicating that effective vaccines require T cells activation. In this study, we designed a novel polypeptides vaccine conjugated to a CRM197 carrier protein, encoding 15 CD8+ T-cell epitopes, five CD4+ T-cell epitopes, and four B-cell epitopes from gB287–320 and pp150311–325 of HCMV to induce T-cell immune responses. To evaluate the effectiveness of vaccines, we subsequently measured the expression of surface molecule markers and proinflammatory cytokines from antigen presenting cells in vivo and in vitro as well as the activation of T cells and antibodies. The results demonstrated that this polypeptide vaccine could activate innate immunity including up-regulating MHCI, II, CD80, CD86, and cytokine expression through the TLR4/NF-κB pathway. Meanwhile, vaccinations elicited potent neutralizing antibody and cellular immune responses producing TNF-α, INF-γ and IL-2, indicating Th1-biased polarization. This finding underlines that CRM197-conjugated polypeptide vaccines facilitate a synergism of humoral and cellular immunity, providing enhanced protection against HCMV, which could be a potential strategy to prevent CMV-associated diseases.

Site-Specific Multi-Functionalization of the Carrier Protein CRM197 by Disulfide Rebridging for Conjugate Vaccine Development.

Conjugation of an antigen to a carrier protein is widely used for vaccine development. To develop the next generation of conjugate vaccines, we describe here a method for the controlled multi‐functionalization of the widely employed carrier protein CRM197 with a carbohydrate‐based antigen and an immune potentiator. The approach is based on the selective reduction of one of the disulfides of CRM197 followed by disulfide rebridging employing an appropriately functionalized dibromopyridazinedione. Efficient protein modification required that the reduction and functionalization with a dibromopyridazinedione was performed as a one‐step procedure with control over the reaction temperature. Furthermore, ligations were most successful when dibromopyridazinediones were employed having a functional entity such as a TLR7/8 agonist and a cyclooctyne for further modification. Site‐specific conjugation avoids modification of T‐epitopes of the carrier protein and covalent attachment of an immune potentiator will ensure that cytokines are produced where the vaccine interacts with relevant immune cells resulting in efficient immune potentiation.

Semisynthetic Glycoconjugate Vaccine Candidates against Escherichia coli O25B Induce Functional IgG Antibodies in Mice.

Extraintestinal pathogenic Escherichia coli (ExPEC) is a major health concern due to emerging antibiotic resistance. Along with O1A, O2, and O6A, E. coli O25B is a major serotype within the ExPEC group, which expresses a unique O-antigen. Clinical studies with a glycoconjugate vaccine of the above-mentioned O-types revealed O25B as the least immunogenic component, inducing relatively weak IgG titers. To evaluate the immunological properties of semisynthetic glycoconjugate vaccine candidates against E. coli O25B, we here report the chemical synthesis of an initial set of five O25B glycan antigens differing in length, from one to three repeat units, and frameshifts of the repeat unit. The oligosaccharide antigens were conjugated to the carrier protein CRM197. The resulting semisynthetic glycoconjugates induced functional IgG antibodies in mice with opsonophagocytic activity against E. coli O25B. Three of the oligosaccharide–CRM197 conjugates elicited functional IgGs in the same order of magnitude as a conventional CRM197 glycoconjugate prepared with native O25B O-antigen and therefore represent promising vaccine candidates for further investigation. Binding studies with two monoclonal antibodies (mAbs) revealed nanomolar anti-O25B IgG responses with nanomolar KD values and with varying binding epitopes. The immunogenicity and mAb binding data now allow for the rational design of additional synthetic antigens for future preclinical studies, with expected further improvements in the functional antibody responses. Moreover, acetylation of a rhamnose residue was shown to be likely dispensable for immunogenicity, as a deacylated antigen was able to elicit strong functional IgG responses. Our findings strongly support the feasibility of a semisynthetic glycoconjugate vaccine against E. coli O25B.

Synthesis and Immunological Evaluation of Pentamannose-Based HIV-1 Vaccine Candidates.

Dense glycosylation and the trimeric conformation of the human immunodeficiency virus-1 (HIV-1) envelope protein limit the accessibility of some cellular glycan processing enzymes and end up with high-mannose-type N-linked glycans on the envelope spike, among which the Man5GlcNAc2 structure occupies a certain proportion. The Man5GlcNAc2 glycan composes the binding sites of some potent broadly neutralizing antibodies, and some lectins that can bind Man5GlcNAc2 show HIV-neutralizing activity. Therefore, Man5GlcNAc2 is a potential target for HIV-1 vaccine development. Herein, a highly convergent and effective strategy was developed for the synthesis of Man5 and its monofluoro-modified, trifluoro-modified, and S-linked analogues. We coupled these haptens to carrier protein CRM197 and evaluated the immunogenicity of the glycoconjugates in mice. The serological assays showed that the native Man5 conjugates failed to induce Man5-specific antibodies in vivo, while the modified analogue conjugates induced stronger antibody responses. However, these antibodies could not bind the native gp120 antigen. These results demonstrated that the immune tolerance mechanism suppressed the immune responses to Man5-related structures and the conformation of glycan epitopes on the synthesized glycoconjugates was distinct from that of native glycan epitopes on gp120.

Development of Klebsiella pneumoniae Capsule Polysaccharide-Conjugated Vaccine Candidates Using Phage Depolymerases.

Klebsiella pneumoniae is an important pathogen associated with nosocomial infection and has developed increasing resistance to antibiotics such as extended-spectrum β-lactams and carbapenem. In recent years, K. pneumoniae isolates have emerged as a major cause of global community-acquired infections such as pneumonia and pyogenic liver abscess. Although serotypes K1 and K2 have been identified as the predominant capsular types associated with invasive infections, no K. pneumoniae vaccine is commercially available, probably due to immunogenicity loss in the traditional depolymerization method to obtain capsule polysaccharide (CPS) for the preparation of conjugated vaccine. In this study, we successfully retained immunogenicity by using K1 (K1-ORF34) and K2 (K2-ORF16) CPS depolymerases that were identified from phages to cleave K1 and K2 CPSs into intact structural units of oligosaccharides with intact modifications. The obtained K1 and K2 oligosaccharides were separately conjugated with CRM197 carrier protein to generate CPS-conjugated vaccines. Immunization experiments of mice showed both K1 and K2 CPS-conjugated vaccines induced anti-CPS antibodies with 128-fold and 64-fold increases of bactericidal activities, respectively, compare to mice without vaccinations. Challenge tests indicated that K1 or K2 CPS-conjugated vaccine and divalent vaccine (a mixture of K1 and K2 CPS-conjugated vaccines) protected mice from subsequent infection of K. pneumoniae by the respective capsular type. Thus, we demonstrated K1 and K2 CPS-conjugated vaccines prepared by CPS depolymerases is a promising candidate for developing vaccines against human K. pneumoniae infections.

Multi-attribute characterization of pneumococcal conjugate vaccine by Size-exclusion chromatography coupled with UV-MALS-RI detections

Streptococcus pneumoniae bacterial infection can cause serious diseases. Among more than 90 known streptococcus pneumoniae serotypes, more than 30 can cause invasive pneumococcal diseases that could lead to morbidity and mortality. Initially, a 23-valent polysaccharide vaccines (PPSV) PNEUMOVAX®23, was developed to generate an antigen-specific immune response and prevent diseases caused by these pneumoniae serotypes. Later, pneumococcal conjugate vaccines (PCV), such as PREVNAR® and VAXNEUVANCE™ have been developed to offer a more robust immune response in the pediatric population.In our effort to develop novel pneumococcal conjugate vaccines, each serotype of pneumococcal polysaccharide (Ps) is conjugated to a detoxified diphtheria toxin carrier protein CRM197 to form a monovalent conjugate (MVC). MVCs from multiple serotypes are formulated with vaccine adjuvant to form a multi-valent vaccine drug product. During the product development, critical attributes including conjugate molecular weight (Mw), protein and polysaccharide concentration, have been used to monitor process and product quality. To measure these attributes, a size-exclusion chromatography (SEC) method was developed with a series of in-line detectors including UV, multi-angle light scattering (MALS) and refractive index (RI). This SEC-UV-MALS-RI method is employed to characterize and monitor process intermediates and product during process development and for product release and stability testing. With this, we have expanded the multi-attribute SEC method to a 15-valent pneumococcal conjugate vaccine.

Total Synthesis of the Tetrasaccharide Haptens of Vibrio vulnificus MO6-24 and BO62316 and Immunological Evaluation of Their Protein Conjugates.

Vibrio vulnificus is a human pathogen that can cause fatal septicemia and necrotizing infections with a high lethal rate exceeding 50%. V. vulnificus MO6-24 and BO62316 are two predominant virulent strains associated with approximately one-third of the clinical infections. The capsular polysaccharides (CPSs) of V. vulnificus consist of several structurally unique sugars and are excellent targets for developing effective glycoconjugate vaccines. This article describes the first total synthesis of the challenging tetrasaccharide repeating units of V. vulnificus MO6-24 and BO62316 CPSs. A key feature of this synthesis was the assembly of the tetrasaccharide skeleton using a 3,4-branched trisaccharide as the glycosyl donor. A modified TEMPO/BAIB oxidation protocol was developed to directly convert α-d-GalN into α-d-GalAN in not only disaccharides but also tri- and tetrasaccharides. The synthetic haptens were covalently coupled with CRM197 carrier protein via a bifunctional linker. Preliminary immunological studies of the resultant glycoconjugates in mice revealed their high efficacy to induce robust T-cell-dependent immune responses, and the IgG antibodies elicited by each glycoconjugate showed weak cross-reactivity with the other synthetic tetrasaccharide.

Synthetic Pseudaminic-Acid-Based Antibacterial Vaccine Confers Effective Protection against Acinetobacter baumannii Infection.

Acinetobacter baumannii exhibits resistance to most first-line antibiotics; thus, development of new antibacterial agents is urgently required. Pseudaminic acid exists as the surface glycan of A. baumannii. In this study, we chemically synthesized pseudaminic acid, conjugated it to carrier protein CRM197 using the OPA (ortho-phthalaldehyde) chemistry, and obtained three Pse–CRM197 conjugates with different Pse loadings. These Pse–CRM197 conjugates were found to stimulate high immune responses in mice, which protected the vaccinated mice from infections caused by Pse-producing A. baumannii. Our data indicate that chemically synthesized Pse–CRM197 conjugates can be developed into vaccines against Pse-bearing pathogens, thus offering a feasible alternative for the control of clinical infections caused by multidrug-resistant (MDR) A. baumannii, for which current treatment options are extremely limited.

A Porphyromonas gingivalis Capsule-Conjugate Vaccine Protects From Experimental Oral Bone Loss.

Periodontal diseases are chronic inflammatory diseases of the periodontium that result in progressive destruction of the soft and hard tissues supporting the teeth, and it is the most common cause of tooth loss among adults. In the US alone, over 100 million individuals are estimated to have periodontal disease. Subgingival bacteria initiate and sustain inflammation, and, although several bacteria have been associated with periodontitis, Porphyromonas gingivalis has emerged as the key etiological organism significantly contributing to the disease. Currently, intensive clinical maintenance strategies are deployed to mitigate the further progression of disease in afflicted individuals; however, these treatments often fail to stop disease progression, and, as such, the development of an effective vaccine for periodontal disease is highly desirable. We generated a conjugate vaccine, comprising of the purified capsular polysaccharide of P. gingivalis conjugated to eCRM®, a proprietary and enhanced version of the CRM197 carrier protein with predetermined conjugation sites (Pg-CV). Mice immunized with alum adjuvanted Pg-CV developed robust serum levels of whole organism-specific IgG in comparison to animals immunized with unconjugated capsular polysaccharide alone. Using the murine oral bone loss model, we observed that mice immunized with the capsule-conjugate vaccine were significantly protected from the effects of P. gingivalis-elicited oral bone loss. Employing a preclinical model of infection-elicited oral bone loss, our data support that a conjugate vaccine incorporating capsular polysaccharide antigen is effective in reducing the main clinical endpoint of periodontal disease—oral bone destruction. Further development of a P. gingivalis capsule-based conjugate vaccine for preventing periodontal diseases is supported.

Simultaneous Evaluation of a Vaccine Component Microheterogeneity and Conformational Integrity Using Native Mass Spectrometry and Limited Charge Reduction.

Analytical characterization of extensively modified proteins (such as haptenated carrier proteins in synthetic vaccines) remains a challenging task due to the high degree of structural heterogeneity. Native mass spectrometry (MS) combined with limited charge reduction allows these obstacles to be overcome and enables meaningful characterization of a heavily haptenated carrier protein CRM197 (inactivated diphtheria toxin conjugated with nicotine), a major component of a smoking cessation vaccine. The extensive conjugation results in a near-continuum distribution of ionic signal in electrospray ionization (ESI) mass spectra of haptenated CRM197 even after size-exclusion chromatographic fractionation. However, supplementing the ESI MS measurements with limited charge reduction of ionic populations selected within narrow m/z windows gives rise to well-resolved charge ladders, from which both masses and charge states of the ionic species can be readily deduced. Application of this technique to a research-grade material of CRM197/H7 conjugate not only reveals its marginal conformational stability (manifested by the appearance of high charge-density ions in ESI MS) but also establishes a role of the extent of haptenation as a major factor driving the loss of the higher order structure integrity. The unique information provided by native MS used in combination with limited charge reduction provides a strong argument for this technique to become a standard/required tool in the analytical arsenal in the field of biotechnology and biopharmaceutical analysis, where protein conjugates are becoming increasingly common.

Adjuvanted bacteriophage vaccine targeting Pseudomonas aeruginosa

Abstract Pseudomonas aeruginosa (Pa) is a Gram-negative bacterium which is associated with chronic diabetic wounds as well as potentially deadly lung infections in cystic fibrosis patients. Approximately 51,000 cases of Pa infections occur annually; of these cases, 13% are resistant to antibiotics which results in the deaths of ~400 individuals every year. Diversity between Pa strains presents a challenge in developing a vaccine or therapeutic capable of resolving infections caused by different strains. Pf phage (a filamentous bacteriophage) has been found in Pa from chronic diabetic wound isolates and has been show to increase virulence of Pa. Recent findings by our team indicate that a Pf phage-targeted vaccine or monoclonal antibody provide protection from the establishment of Pa infection in mice. To further improve this innovative new vaccine targeting Pa, adjuvant systems were used to enhance humoral response to the vaccine. A consensus peptide from Pf Phage coat protein was conjugated to the carrier protein CRM197 and combined with novel adjuvants and delivery systems. Selected adjuvants significantly enhanced humoral immunity to the Pf Phage peptide in a dose-dependent manner. Combination adjuvant systems were also used to further enhance antigen-specific immunity to Pf Phage and cell-mediated immunity to the carrier protein. The development of an adjuvanted Pf phage vaccine to protect against Pa infections is a new and innovative strategy with the potential to protect patients at risk for opportunistic bacterial infections.

Molecular modeling provides insights into the loading of sialic acid-containing antigens onto CRM197: the role of chain flexibility in conjugation efficiency and glycoconjugate architecture.

Vaccination is the most cost-effective way to control disease caused by encapsulated bacteria; the capsular polysaccharide (CPS) is the primary virulence factor and vaccine target. Neisseria meningitidis (Nm) serogroups B, C, Y and W all contain sialic acid, a common surface feature of human pathogens. Two protein-based vaccines against serogroup B infection are available for human use while four tetravalent conjugate vaccines including serogroups C, W and Y have been licensed. The tetravalent Menveo® conjugate vaccine is well-defined: a simple monomeric structure of oligosaccharides terminally conjugated to amino groups of the carrier protein CRM197. However, not only is there a surprisingly low limit for antigen chain attachment to CRM197, but different serogroup saccharides have consistently different CRM197 loading, the reasons for which are unclear. Understanding this phenomenon is important for the long-term goal of controlling conjugation to prepare conjugate vaccines of optimal immunogenicity. Here we use molecular modeling to explore whether antigen flexibility can explain the varying antigen loading of the conjugates. Because flexibility is difficult to separate from other structural factors, we focus on sialic-acid containing CPS present in current glycoconjugate vaccines: serogroups NmC, NmW and NmY. Our simulations reveal a correlation between Nm antigen flexibility (NmW > NmC > NmY) and the number of chains attached to CRM197, suggesting that increased flexibility enables accommodation of additional chains on the protein surface. Further, in silico models of the glycoconjugates confirm the relatively large hydrodynamic size of the saccharide chains and indicate steric constraints to further conjugation.

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.