Heterologous Expression of Recombinant Proteins and Their Derivatives Used as Carriers for Conjugate Vaccines.

Carrier proteins that provide an effective and long-term immune response to weak antigens has become a real breakthrough in the disease prevention, making it available to a wider range of patients and making it possible to obtain reliable vaccines against a variety of pathogens. Currently, research is continuing both to identify new peptides, proteins, and their complexes potentially suitable for use as carriers, and to develop new methods for isolation, purification, and conjugation of already known and well-established proteins. The use of recombinant proteins has a number of advantages over isolation from natural sources, such as simpler cultivation of the host organism, the possibility of modifying genetic constructs, use of numerous promoter variants, signal sequences, and other regulatory elements. This review is devoted to the methods of obtaining both traditional and new recombinant proteins and their derivatives already being used or potentially suitable for use as carrier proteins in conjugate vaccines.

High-throughput nanofluidic real-time PCR to discriminate Pneumococcal Conjugate Vaccine (PCV)-associated serogroups 6, 18, and 22 to serotypes using modified oligonucleotides.

Current real-time high-throughput Polymerase Chain Reaction (qPCR) methods do not distinguish serotypes 6A from 6B, 18C from 18A/B and 22F from 22A. We established a nanofluidic real-time PCR (Fluidigm) for serotyping that included Dual-Priming-Oligonucleotides (DPO), a Locked-Nucleic-Acid (LNA) probe and TaqMan assay-sets for high-throughput serotyping. The designed assay-sets target capsular gene wciP in serogroup 6, wciX and wxcM in serogroup 18, and wcwA in serogroup 22. An algorithm combining results from published assay-sets (6A/B/C/D; 6C/D; 18A/B/C; 22A/F) and designed assay-sets for 6A/C; 18B/C/F; 18C/F, 18F and 22F was validated through blind analysis of 1973 archived clinical samples collected from South African children ≤ 5-years-old (2009-2011), previously serotyped with the culture-based Quellung method. All assay-sets were efficient (92-101%), had low variation between replicates (R2 > 0.98), and were able to detect targets at a limit of detection (LOD) of < 100 Colony-Forming-Units (CFU)/mL of sample. There was high concordance (Kappa = 0.73-0.92); sensitivity (85-100%) and specificity (96-100%) for Fluidigm compared with Quellung for serotyping 6A; 6B; 6C; 18C and 22F. Fluidigm distinguishes vaccine-serotypes 6A, 6B, 18C, next-generation PCV-serotype 22F and non-vaccine-serotypes 6C, 6D, 18A, 18B, 18F and 22A. Discriminating single serotypes is important for assessing serotype replacement and the impact of PCVs on vaccine- and non-vaccine serotypes.

An Engineered Receptor-Binding Domain Improves the Immunogenicity of Multivalent SARS-CoV-2 Vaccines.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) protein mediates viral entry into cells expressing angiotensin-converting enzyme 2 (ACE2). The S protein engages ACE2 through its receptor-binding domain (RBD), an independently folded 197-amino-acid fragment of the 1,273-amino-acid S-protein protomer. The RBD is the primary SARS-CoV-2 neutralizing epitope and a critical target of any SARS-CoV-2 vaccine. Here, we show that this RBD conjugated to each of two carrier proteins elicited more potent neutralizing responses in immunized rodents than did a similarly conjugated proline-stabilized S-protein ectodomain. Nonetheless, the native RBD is expressed inefficiently, limiting its usefulness as a vaccine antigen. However, we show that an RBD engineered with four novel glycosylation sites (gRBD) is expressed markedly more efficiently and generates a more potent neutralizing responses as a DNA vaccine antigen than the wild-type RBD or the full-length S protein, especially when fused to multivalent carriers, such as a Helicobacter pylori ferritin 24-mer. Further, gRBD is more immunogenic than the wild-type RBD when administered as a subunit protein vaccine. Our data suggest that multivalent gRBD antigens can reduce costs and doses, and improve the immunogenicity, of all major classes of SARS-CoV-2 vaccines.IMPORTANCE All available vaccines for coronavirus disease 2019 (COVID-19) express or deliver the full-length SARS-CoV-2 spike (S) protein. We show that this antigen is not optimal, consistent with observations that the vast majority of the neutralizing response to the virus is focused on the S-protein receptor-binding domain (RBD). However, this RBD is not expressed well as an independent domain, especially when expressed as a fusion protein with a multivalent scaffold. We therefore engineered a more highly expressed form of the SARS-CoV-2 RBD by introducing four glycosylation sites into a face of the RBD normally occluded in the full S protein. We show that this engineered protein, gRBD, is more immunogenic than the wild-type RBD or the full-length S protein in both genetic and protein-delivered vaccines.

Safety and immunogenicity of a synthetic carbohydrate conjugate vaccine against Shigella flexneri 2a in healthy adult volunteers: a phase 1, dose-escalating, single-blind, randomised, placebo-controlled study.

BACKGROUND: Shigella remains in the top four pathogens responsible for moderate to severe diarrhoea in children below 5 years of age. The shigella O-specific polysaccharide (O-SP) is a promising vaccine target. We developed a conjugate vaccine prototype incorporating a unique well defined synthetic oligosaccharide hapten, chemically designed for optimal antigenic, conformational, structural, and functional mimicry of the O-SP from Shigella flexneri 2a (SF2a). We aimed to assess the safety, tolerability, and immunogenicity of this original synthetic oligosaccharide-based vaccine candidate, SF2a-TT15, conceived to drive the antibody response towards the key protective determinants of the native lipopolysaccharide antigen, in a first-in-human phase 1 study. METHODS: We did a first-in-human, dose-escalating, single-blind, observer-masked, randomised, placebo-controlled study at the Clinical Research Center of Tel Aviv Sourasky Medical Center (Israel). Participants were healthy adults aged 18-45 years with low titres of serum SF2a-specific IgG antibodies. 64 eligible participants were assigned to one of two cohorts. 32 participants in each of the two cohorts were randomly assigned via computer-generated algorithm in a stepwise manner to receive the 2 µg (cohort 1) and 10 µg oligosaccharide dose (cohort 2) of the SF2a-TT15 vaccine candidate non-adjuvanted or adjuvanted with aluminium hydroxide (alum) or matching placebos. The vaccine was administered as three single intramuscular injections into the arm, 28 days apart. The primary outcome was the incidence and severity of adverse events, which were assessed in the intention-to-treat safety population analysis including all participants who were randomly assigned and received at least one vaccine or placebo injection. The immunogenicity endpoints were secondary outcomes and were analysed in all participants who were randomly assigned, received all of the assigned injections before the time of the immunogenicity assessment, and provided blood samples for immunological follow-up (per-protocol immunogenicity analysis). The study is registered with ClinicalStudies.gov, NCT02797236 and is completed. FINDINGS: Of 203 volunteers initially screened, 64 participants were enrolled between Sept 20, 2016, and Sept 26, 2017. In each of the two cohorts, 12 participants received the adjuvanted vaccine, 12 received the non-adjuvanted vaccine and eight received the matching placebo (four each). The SF2a-TT15 glycoconjugate was well tolerated at both doses. No serious or severe adverse events occurred. Overall, seven (88%) of eight to 12 (100%) of 12 in each group of volunteers had one adverse event or more after receiving the study agents with the majority of adverse events, 300 (98%) of 307, considered mild in intensity. Of the seven adverse events defined as moderate in severity, one (nausea) was suspected to be related to the vaccine candidate. At all post-immunisation days and for both oligosaccharide doses, whether adjuvanted or not, SF2a-TT15 induced significantly higher serum IgG anti-SF2a lipopolysaccharide geometric mean titres (GMTs) as compared with baseline or with the corresponding GMTs in placebo recipients (p<0·01). After one injection, the non-adjuvanted 10 µg oligosaccharide dose induced a 27-times increase in IgG GMT (5080 vs 189) and the non-adjuvanted 2 µg oligosaccharide dose induced a five-times increase (1411 vs 283), compared with baseline. Alum enhanced the specific IgG response at 2 µg oligosaccharide dose after the third injection (GMTs 3200 vs 1176, p=0.045). INTERPRETATION: SF2a-TT15 was safe and well tolerated and induced high titres of anti-SF2a LPS IgG antibodies. These results support further evaluation of this original synthetic oligosaccharide-protein conjugate vaccine candidate for safety, immunogenicity, and protective efficacy in target populations. FUNDING: The European Union Seventh Framework Programme.

Short Vi-polysaccharide abrogates T-independent immune response and hyporesponsiveness elicited by long Vi-CRM197 conjugate vaccine.

Polysaccharide-protein conjugates have been developed to overcome the T-independent response, hyporesponsiveness to repeated vaccination, and poor immunogenicity in infants of polysaccharides. To address the impact of polysaccharide length, typhoid conjugates made with short- and long-chain fractions of Vi polysaccharide with average sizes of 9.5, 22.8, 42.7, 82.0, and 165 kDa were compared. Long-chain-conjugated Vi (165 kDa) induced a response in both wild-type and T cell-deficient mice, suggesting that it maintains a T-independent response. In marked contrast, short-chain Vi (9.5 to 42.7 kDa) conjugates induced a response in wild-type mice but not in T cell-deficient mice, suggesting that the response is dependent on T cell help. Mechanistically, this was explained in neonatal mice, in which long-chain, but not short-chain, Vi conjugate induced late apoptosis of Vi-specific B cells in spleen and early depletion of Vi-specific B cells in bone marrow, resulting in hyporesponsiveness and lack of long-term persistence of Vi-specific IgG in serum and IgG+ antibody-secreting cells in bone marrow. We conclude that while conjugation of long-chain Vi generates T-dependent antigens, the conjugates also retain T-independent properties, leading to detrimental effects on immune responses. The data reported here may explain some inconsistencies observed in clinical trials and help guide the design of effective conjugate vaccines.

Glycoconjugate vaccines: current approaches towards faster vaccine design.

Introduction: Over the last decades, glycoconjugate vaccines have been proven to be a successful strategy to prevent infectious diseases. Many diseases remain to be controlled, especially in developing countries, and emerging antibiotic-resistant bacteria present an alarming public-health threat. The increasing complexity of future vaccines, and the need to accelerate development processes have triggered the development of faster approaches to glycoconjugate vaccines design. Areas covered: This review provides an overview of recent progress in glycoconjugation technologies toward faster vaccine design. Expert opinion: Among the different emerging approaches, glycoengineering has the potential to combine glycan assembly and conjugation to carrier systems (such as proteins or outer membrane vesicles) in one step, resulting in a simplified manufacturing process and fewer analytical controls. Chemical and enzymatic strategies, and their automation can facilitate glycoepitope identification for vaccine design. Other approaches, such as the liposomal encapsulation of polysaccharides, potentially enable fast and easy combination of numerous antigens in the same formulation. Additional progress is envisaged in the near future, and some of these systems still need to be further validated in humans. In parallel, new strategies are needed to accelerate the vaccine development process, including the associated clinical trials, up to vaccine release onto the market.

Mucosal vaccine based on attenuated influenza virus and the group B Streptococcus recombinant peptides protected mice from influenza and S. pneumoniae infections.

Although many influenza-related deaths are attributable to secondary bacterial infection with S. pneumoniae, vaccines that simultaneously protect against influenza and pneumococcal infection are currently not developed. The aim of our study was to evaluate the possibility to prevent post-influenza pneumococcal infection using an associated vaccine based on live influenza vaccine (LAIV) combined with recombinant polypeptides derived from superficial factors of Group B streptococcus (GBS) determining pathogenicity. We demonstrated in a model of post-influenza pneumococcal pneumonia that intranasal pneumococcal super-infection seriously complicated the course of A/Shanghai/2/2013(H7N9) CDC-RG virus infection in mice. Associated immunization using LAIV and GBS vaccine (GBSV) prevented post-influenza pneumococcal pneumonia better than mono-LAIV or GBSV immunization. At the same time, parenteral pneumococcal post-influenza infection of immune mice was more severe in the groups immunized using recombinant GBS peptides which can be explained by antibody-dependent enhancement of infection. In this case, the introduction of blockers of histamine receptors type 1 and 2 reduced the burden of secondary pneumococcal infection.

A platform for glycoengineering a polyvalent pneumococcal bioconjugate vaccine using E. coli as a host.

Chemical synthesis of conjugate vaccines, consisting of a polysaccharide linked to a protein, can be technically challenging, and in vivo bacterial conjugations (bioconjugations) have emerged as manufacturing alternatives. Bioconjugation relies upon an oligosaccharyltransferase to attach polysaccharides to proteins, but currently employed enzymes are not suitable for the generation of conjugate vaccines when the polysaccharides contain glucose at the reducing end, which is the case for ~75% of Streptococcus pneumoniae capsules. Here, we use an O-linking oligosaccharyltransferase to generate a polyvalent pneumococcal bioconjugate vaccine with polysaccharides containing glucose at their reducing end. In addition, we show that different vaccine carrier proteins can be glycosylated using this system. Pneumococcal bioconjugates are immunogenic, protective and rapidly produced within E. coli using recombinant techniques. These proof-of-principle experiments establish a platform to overcome limitations of other conjugating enzymes enabling the development of bioconjugate vaccines for many important human and animal pathogens.

Direct Cloning of Bacterial Surface Polysaccharide Gene Cluster for One-Step Production of Glycoconjugate Vaccine.

Bacterial pathogen infections are fast-growing public health threats and worldwide problems. Glycoconjugate vaccines are among the most effective means in combating such infections. Recent advances in bacterial protein glycan coupling technology (PGCT) have revolutionized the production of glycoconjugate vaccines and drawn enormous attention from both researchers and pharmaceutical companies. Cloning of bacterial surface polysaccharide gene cluster is a prerequisite for the application of PGCT. In this study, we applied the RecET direct cloning strategy for rapid and efficient cloning of O-antigen polysaccharide gene clusters from Escherichia coli serotypes O25b, O26, and O55 in a high-fidelity manner. Then, these gene clusters were applied in PGCT to produce corresponding glycoconjugates. Subsequent immunological studies verified the abilities of glycoconjugate vaccine candidates O25-maltose-binding protein (MBP), O26-MBP, and O55-MBP to generate serotype-specific antibodies and confer protection against E. coli infections. The combination of RecET direct cloning and PGCT makes the rapid production of glycoconjugate vaccines against fast-expanding bacterial pathogens possible.

Antimicrobial glycoconjugate vaccines: an overview of classic and modern approaches for protein modification.

Glycoconjugate vaccines obtained by chemical linkage of a carbohydrate antigen to a protein are part of routine vaccinations in many countries. Licensed antimicrobial glycan-protein conjugate vaccines are obtained by random conjugation of native or sized polysaccharides to lysine, aspartic or glutamic amino acid residues that are generally abundantly exposed on the protein surface. In the last few years, the structural approaches for the definition of the polysaccharide portion (epitope) responsible for the immunological activity has shown potential to aid a deeper understanding of the mode of action of glycoconjugates and to lead to the rational design of more efficacious and safer vaccines. The combination of technologies to obtain more defined carbohydrate antigens of higher purity and novel approaches for protein modification has a fundamental role. In particular, methods for site selective glycoconjugation like chemical or enzymatic modification of specific amino acid residues, incorporation of unnatural amino acids and glycoengineering, are rapidly evolving. Here we discuss the state of the art of protein engineering with carbohydrates to obtain glycococonjugates vaccines and future perspectives.

Cyclic OmpC peptidic epitope conjugated to tetanus toxoid as a potential vaccine candidate against shigellosis.

In earlier works we have described that mice immunized with outer membrane protein OmpC survive the challenge with live Shigella flexnerii 3a. We have also identified conformational epitope of this protein, that was recognized by mice antibodies. The aim of current work was to investigate whether synthetic OmpC epitope homologs can elicit immunological response sufficient in protecting mice against shigellosis. Several linear peptides containing RYDERY motif were synthesized and conjugated to poly-lysine. These conjugates appeared to be poor immunogens and to boost the immunological response an addition of the adjuvant (MPL) was required. Unfortunately, the MPL alone caused a very high immunological reaction that was masking response to peptidic epitope. Under those circumstances we used tetanus toxoid (TT) as the carrier protein for the peptides and the agent stimulating immunological response. Series of cyclic peptides, homologs of the OmpC main epitope were synthesized and conjugated to TT. The loop size in cyclic peptides varied by number of glycine residues, i.e., 1-3 residues added to the GLNRYDERYIGK motif. The linear GLNRYDERYIGC-TT was also prepared as the control. The latter conjugate gave the highest immunological response, followed by the cyclic-GGLNRYDERYIGC-TT and cyclic-GLNRYDERYIGC-TT. The third peptide, cyclic-GGGLNRYDERYIGC-TT, gave a very low response, although it was the most resistant to proteolysis. However, antibodies obtained against cyclic-GGLNRYDERYIGC-TT were more potent to recognize both OmpC and Shigella flexnerii 3a cells than the antibodies against linear GLNRYDERYIGC-TT. Furthermore, the monoclonal antibodies raised against linear GLNRYDERYIGC-TT showed 20-fold lower dissociation constant (KD) than the naturally occurring polyclonal antibodies from umbilical cord sera. Monoclonal antibodies also gave a weaker signal in electron microscope than mice and human polyclonal antibodies. In overall, our results point to cyclic peptides as better candidates for a vaccine development, since they are eliciting production of the higher affinity antibodies against Shigella cells and OmpC.

Further Characterization of the Capsule-Like Complex (CLC) Produced by Francisella tularensis Subspecies tularensis: Protective Efficacy and Similarity to Outer Membrane Vesicles.

Francisella tularensis is the etiologic agent of tularemia, and subspecies tularensis (type A) is the most virulent subspecies. The live vaccine strain (LVS) of subspecies holarctica produces a capsule-like complex (CLC) that consists of a large variety of glycoproteins. Expression of the CLC is greatly enhanced when the bacteria are subcultured in and grown on chemically defined medium. Deletion of two genes responsible for CLC glycosylation in LVS results in an attenuated mutant that is protective against respiratory tularemia in a mouse model. We sought to further characterize the CLC composition and to determine if a type A CLC glycosylation mutant would be attenuated in mice. The CLCs isolated from LVS extracted with 0.5% phenol or 1 M urea were similar, as determined by gel electrophoresis and Western blotting, but the CLC extracted with urea was more water-soluble. The CLC extracted with either 0.5% phenol or 1 M urea from type A strains was also similar to the CLC of LVS in antigenic properties, electrophoretic profile, and by transmission electron microscopy (TEM). The solubility of the CLC could be further enhanced by fractionation with Triton X-114 followed by N-Lauroylsarcosine detergents; the largest (>250 kDa) molecular size component appeared to be an aggregate of smaller components. Outer membrane vesicles/tubules (OMV/T) isolated by differential centrifugation and micro-filtration appeared similar to the CLC by TEM, and many of the proteins present in the OMV/T were also identified in soluble and insoluble fractions of the CLC. Further investigation is warranted to assess the relationship between OMV/T and the CLC. The CLC conjugated to keyhole limpet hemocyanin or flagellin was highly protective against high-dose LVS intradermal challenge and partially protective against intranasal challenge. A protective response was associated with a significant rise in cytokines IL-12, IL-10, and IFN-γ. However, a type A CLC glycosylation mutant remained virulent in BALB/c mice, and immunization with the CLC did not protect mice against high dose respiratory challenge with type A strain SCHU S4.

Structural and immunological characterization of E. coli derived recombinant CRM197 protein used as carrier in conjugate vaccines.

It is established that the immunogenicity of polysaccharides is enhanced by coupling them to carrier proteins. Cross reacting material (CRM197), a nontoxic variant of diphtheria toxin (DT) is widely used carrier protein for polysaccharide conjugate vaccines. Conventionally, CRM197 is isolated by fermentation of Corynebacterium diphtheriae C7 (ß197) cultures, which often suffers from low yield. Recently, several recombinant approaches have been reported with robust processes and higher yields, which will improve the affordability of CRM197-based vaccines. Vaccine manufacturers require detailed analytical information to ensure that the CRM197 meets quality standards and regulatory requirements. In the present manuscript we have described detailed structural characteristics of Escherichia coli based recombinant CRM197 (rCRM197) carrier protein. The crystal structure of the E. coli based rCRM197 was found to be identical with the reported crystal structure of the C7 CRM197 produced in C. diphtheriae C7 strain (Protein Data Bank (PDB) ID: 4EA0). The crystal structure of rCRM197 was determined at 2.3 Å resolution and structure was submitted to the PDB with accession number ID 5I82. This is the first report of a crystal structure of E. coli derived recombinant CRM197 carrier protein. Furthermore, the rCRM197 was conjugated to Vi polysaccharide to generate Typhoid conjugate vaccine (Vi-rCRM197) and its immunogenicity was evaluated in Balb/C Mice. The Vi-rCRM197 conjugate vaccine was found to generate strong primary α-Vi antibody response and also showed a booster response after subsequent vaccination in mice. Overall data suggest that E. coli based recombinant CRM197 exhibits structural and immunological similarity with the C7 CRM197 and can be used as a carrier protein in conjugate vaccine development.

Development of experimental GBS vaccine for mucosal immunization.

Streptococcus agalactiae, or group B streptococcus (GBS), is an important pathogen as it is the leading cause of neonatal deaths due to sepsis, meningitis or bacterial pneumonia. Although the development of an effective and safe GBS vaccine is on the agenda of many research labs, there is no GBS vaccine on the market yet. In the present study we attempted to engineer a live vaccine strain based on Bac, a surface protein of GBS, incorporated into a surface fimbrial protein of probiotic Enterococcus. The resulting strain induced specific systemic and local immune responses in mice and provided protection against GBS when administered via the intranasal, oral or intravaginal immunization routes.

Analytical Comparability Assessments of 5 Recombinant CRM197 Proteins From Different Manufacturers and Expression Systems.

Cross-reacting material 197 (CRM197), a single amino acid mutant of diphtheria toxoid, is a commonly used carrier protein in commercial polysaccharide protein conjugate vaccines. In this study, CRM197 proteins from 3 different expression systems and 5 different manufacturers were obtained for an analytical comparability assessment using a wide variety of physicochemical and in vitro antigenic binding assays. A comprehensive analysis of the 5 CRM197 molecules demonstrate that recombinant CRM197's expressed in heterologous systems (Escherichia coli and Pseudomonas fluorescens) are overall highly similar (if not better in some cases) to those expressed in the traditional system (Corynebacterium diphtheriae) in terms of primary sequence/post-translational modifications, higher order structural integrity, apparent solubility, physical stability profile (vs. pH and temperature), and in vitro antigenicity. These results are an encouraging step to demonstrate that recombinant CRM197 expressed in alternative sources have the potential to replace CRM197 expressed in C diphtheriae as a source of immunogenic carrier protein for lower cost polysaccharide conjugate vaccines. The physicochemical assays established in this work to monitor the key structural attributes of CRM197 should also prove useful as complementary characterization methods (to routine quality control assays) to support future process and formulation development of lower cost CRM197 carrier proteins for use in various conjugate vaccines.

Physicochemical characterisation, immunogenicity and protective efficacy of a lead streptococcal vaccine: progress towards Phase I trial.

Globally, group A streptococcal infections are responsible for over 500,000 deaths per year. A safe vaccine that does not induce autoimmune pathology and that affords coverage for most GAS serotypes is highly desired. We have previously demonstrated that a vaccine based on the conserved M-protein epitope, J8 was safe and immunogenic in a pilot Phase I study. We subsequently improved vaccine efficacy by incorporation of a B-cell epitope from the IL-8 protease, SpyCEP, which protected IL-8 and enhanced neutrophil ingress to the site of infection. We have now substituted the carrier protein, diphtheria toxoid with its superior analogue, CRM197 which provides better immunogenicity and is widely used in licenced human vaccines. The new vaccine was compared with the DT conjugate vaccine to confirm that these modifications have not altered the physicochemical properties of the vaccine. This vaccine, when tested in an animal model of GAS infection, demonstrated significant reduction in systemic and local GAS burden, with comparable efficacy to the DT conjugate vaccine. The vaccine was shown to be equally effective in the presence of human plasma and in the presence of pre-existing DT-specific antibodies, thus minimising concerns regarding its potential efficacy in humans.

Biosynthesis of Conjugate Vaccines Using an O-Linked Glycosylation System.

UNLABELLED: Conjugate vaccines are known to be one of the most effective and safest types of vaccines against bacterial pathogens. Previously, vaccine biosynthesis has been performed by using N-linked glycosylation systems. However, the structural specificity of these systems for sugar substrates has hindered their application. Here, we report a novel protein glycosylation system (O-linked glycosylation via Neisseria meningitidis) that can transfer virtually any glycan to produce a conjugate vaccine. We successfully established this system in Shigella spp., avoiding the construction of an expression vector for polysaccharide synthesis. We further found that different protein substrates can be glycosylated using this system and that the O-linked glycosylation system can also effectively function in other Gram-negative bacteria, including some strains whose polysaccharide structure was not suitable for conjugation using the N-linked glycosylation system. The results from a series of animal experiments show that the conjugate vaccine produced by this O-linked glycosylation system offered a potentially protective antibody response. Furthermore, we elucidated and optimized the recognition motif, named MOOR, for the O-glycosyltransferase PglL. Finally, we demonstrated that the fusion of other peptides recognized by major histocompatibility complex class II around MOOR had no adverse effects on substrate glycosylation, suggesting that this optimized system will be useful for future vaccine development. Our results expand the glycoengineering toolbox and provide a simpler and more robust strategy for producing bioconjugate vaccines against a variety of pathogens. IMPORTANCE: Recently, the rapid development of synthetic biology has allowed bioconjugate vaccines with N-linked protein glycosylation to become a reality. However, the difficulty of reestablishing the exogenous polysaccharide synthetic pathway in Escherichia coli hinders their application. Here, we show that an O-linked protein glycosylation system from Neisseria meningitidis, which has a lower structure specificity for sugar substrates, could be engineered directly in attenuated pathogens to produce effective conjugate vaccines. To facilitate the further design of next-generation bioconjugate vaccines, we optimized a novel short motif consisting of 8 amino acids that is sufficient for glycosylation. Our results expand the application potential of O-linked protein glycosylation and demonstrate a simpler and more robust strategy for producing bioconjugate vaccines against different pathogens. In the future, bacterial antigenic polysaccharides could be attached to major histocompatibility complex binding peptides to improve immunological memory or attached to protein subunit vaccine candidates to provide double immune stimulation.

Preparation and immunogenicity-evaluation of typhoid O-specific polysaccharides bio-conjugate vaccines.

Typhoid fever caused by Salmonella Typhi is still a major public health problem in developing countries. In this study, we constructed a genetically modified Salmonella Typhi strain expressing O-specific polysaccharides (OPS) antigen conjugated to a carrier, recombinant Pseudomonas aeruginosa exotoxin A(rEPA N29). The conjugates (OPS-rEPA N29) were further purified and evaluated for their immunogenicity. The results of ELISA showed that the conjugates evoked higher titers of IgG than OPS, suggesting that rEPAN29 increased immunogenicity of OPS significantly as a carrier. Moreover, three injections with 3-week interval evoked slightly higher titers of IgG than three injections with 2-week interval. However, injection of excess conjugates could not evoke higher titers of IgG against lipid polysaccharide (LPS). In summary, our study provides a new strategy for preparing polysaccharides-protein conjugate vaccines as well as similar bio-conjugate vaccines of other Gram-negative pathogens.

Conjugation of ß-glucan markedly increase the immunogencity of meningococcal group Y polysaccharide conjugate vaccine.

Meningococcal disease is a fatal illness of sudden onset caused by Neisseria meningitides. Meningococcal capsular polysaccharide (CPS) is a major virulence factor that generally does not induce immunological memory. Conjugation with a carrier protein can significantly increase the immunogenicity of CPS and induce immunological memory. However, it is highly desired to optimize the CPS-specific immunogenicity of the conjugate vaccine. Although adjuvant has been widely used to improve the immunogenicity of antigens, co-administration and conjugation of adjuvant with the conjugate vaccine has rarely been investigated. As a stimulator of humoral and cellular immunity, ß-glucan can activate macrophages and trigger intracellular processes to secrete cytokines initiating inflammatory reactions. In the present study, a conjugate vaccine (CPS-TT) was generated by conjugation of tetanus toxoid (TT) with meningococcal group Y CPS. CPS-TT was further conjugated with ß-glucan to generate CPS-TT-G. Immunization with CPS-TT-G led to an 8.2-fold increase in the CPS-specific IgG titers as compared with CPS-TT. Presumably, conjugation of ß-glucan ensured the two components to simultaneously reach the antigen presenting cells and stimulate the immune response. In contrast, co-administration of ß-glucan suppressed the CPS-specific immunogenicity of CPS-TT. Thus, conjugation of ß-glucan is an effective strategy to markedly improve the CPS-specific immunogenicity of the conjugate vaccine.

Clinical Studies of Escherichia coli O157:H7 Conjugate Vaccines in Adults and Young Children.

Pediatric immunization has been the most effective measure to prevent and reduce the burden of infectious diseases in children. The recent inclusion of pneumococcal and meningococcal polysaccharide conjugates in infant immunization further reinforces their importance. Currently there is no human vaccine against enterohemorrhagic Escherichia coli (EHEC) infections. This review focuses on the human EHEC vaccine that has been studied clinically, in particular, the polysaccharide conjugate against E. coli O157. The surface polysaccharide antigen, O-specific polysaccharide, was linked to rEPA, recombinant exotoxin A of Pseudomonas aeruginosa. In adults and children 2 to 5 years old, O157-rEPA conjugates, shown to be safe, induced high levels of antilipopolysaccharide immunoglobulin G with bactericidal activities against E. coli O157, a functional bioassay that mimics the killing of inoculum in vivo. A similar construct using the B subunit of Shiga toxin (Stx) 1 as the carrier protein elicited both bactericidal and toxin-neutralizing antibodies in mice. So far there is no clinical study of Stx-based human vaccine. Passive immunization of Stx-specific antibodies with humanized, chimeric, or human monoclonal antibodies, produced in transgenic mice, showed promising data in animal models and offered high prospects. Demonstrations of their safety and effectiveness in treating hemolytic-uremic syndrome or patients with EHEC infections are under way, and results are much anticipated. For future development, other virulence factors such as the nontoxic Stx B subunit or intimin should be included, either as carrier protein in conjugates or as independent components. The additional antigens from O157 may provide broader coverage to non-O157 Stx-producing E. coli and facilitate both preventive and therapeutic treatment.