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

2018 ◽  
Vol 38 (5) ◽  
Author(s):  
Ravi P.N. Mishra ◽  
Ravi S.P. Yadav ◽  
Christopher Jones ◽  
Salvatore Nocadello ◽  
George Minasov ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Conjugate Vaccine ◽  
Vaccine Development ◽  
Structural Characteristics ◽  
Data Bank ◽  
Carrier Protein ◽  
Regulatory Requirements ◽  
Conjugate Vaccines ◽  
E Coli

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 (β197) 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.

scholarly journals Outer Membrane Protein Complex of Meningococcus Enhances the Antipolysaccharide Antibody Response to Pneumococcal Polysaccharide–CRM197Conjugate Vaccine

2011 ◽  
Vol 18 (5) ◽  
pp. 724-729 ◽  
Author(s):  
Zengzu Lai ◽  
John R. Schreiber
Keyword(s):  
T Cell ◽  
Membrane Protein ◽  
Outer Membrane ◽  
Conjugate Vaccine ◽  
Outer Membrane Protein ◽  
Protein Complex ◽  
Carrier Protein ◽  
Conjugate Vaccines ◽  
Membrane Protein Complex ◽  
Content Type

ABSTRACTBacterial polysaccharides (PS) are T cell-independent antigens that do not induce immunologic memory and are poor immunogens in infants. Conjugate vaccines in which the PS is covalently linked to a carrier protein have enhanced immunogenicity that resembles that of T cell-dependent antigens. TheHaemophilus influenzaetype b (Hib) conjugate vaccine, which uses the outer membrane protein complex (OMPC) from meningococcus as a carrier protein, elicits protective levels of anti-capsular PS antibody (Ab) after a single dose, in contrast to other conjugate vaccines, which require multiple doses. We have previously shown that OMPC robustly engages Toll-like receptor 2 (TLR2) and enhances the early anti-Hib PS Ab titer associated with an increase in TLR2-mediated induction of cytokines. We now show that the addition of OMPC to the 7-valent pneumococcal PS-CRM197conjugate vaccine during immunization significantly increases the anti-PS IgG and IgM responses to most serotypes of pneumococcus contained in the vaccine. The addition of OMPC also increased the likelihood of anti-PS IgG3 production against serotypes 4, 6B, 9V, 18C, 19F, and 23F. Splenocytes from mice who had received OMPC with the pneumococcal conjugate vaccine produced significantly more interleukin-2 (IL-2), IL-4, IL-6, IL-10, tumor necrosis factor alpha (TNF-α), and gamma interferon (IFN-γ) than splenocytes from mice who received phosphate-buffered saline (PBS) plus the conjugate vaccine. We conclude that OMPC enhances the anti-PS Ab response to pneumococcal PS-CRM197conjugate vaccine, an effect associated with a distinct change in cytokine profile. It may be possible to reduce the number of conjugate vaccine doses required to achieve protective Ab levels by priming with adjuvants that are TLR2 ligands.

scholarly journals Purification and Biochemical Characterization of theMycobacterium tuberculosisβ-Ketoacyl-acyl Carrier Protein Synthases KasA and KasB

2001 ◽  
Vol 276 (50) ◽  
pp. 47029-47037 ◽  
Author(s):  
Merrill L. Schaeffer ◽  
Gautam Agnihotri ◽  
Craig Volker ◽  
Howard Kallender ◽  
Patrick J. Brennan ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Synthase ◽  
Acyl Carrier Protein ◽  
Acid Synthesis ◽  
Mycolic Acid ◽  
Carrier Protein ◽  
Mycolic Acids ◽  
E Coli ◽  
Long Chain

Mycolic acids are vital components of theMycobacterium tuberculosiscell wall, and enzymes involved in their formation represent attractive targets for the discovery of novel anti-tuberculosis agents. Biosynthesis of the fatty acyl chains of mycolic acids involves two fatty acid synthetic systems, the multifunctional polypeptide fatty acid synthase I (FASI), which performsde novofatty acid synthesis, and the dissociated FASII system, which consists of monofunctional enzymes, and acyl carrier protein (ACP) and elongates FASI products to long chain mycolic acid precursors. In this study, we present the initial characterization of purified KasA and KasB, two β-ketoacyl-ACP synthase (KAS) enzymes of theM. tuberculosisFASII system. KasA and KasB were expressed inE. coliand purified by affinity chromatography. Both enzymes showed activity typical of bacterial KASs, condensing an acyl-ACP with malonyl-ACP. Consistent with the proposed role of FASII in mycolic acid synthesis, analysis of various acyl-ACP substrates indicated KasA and KasB had higher specificity for long chain acyl-ACPs containing at least 16 carbons. Activity of KasA and KasB increased with use ofM. tuberculosisAcpM, suggesting that structural differences between AcpM andE. coliACP may affect their recognition by the enzymes. Both enzymes were sensitive to KAS inhibitors cerulenin and thiolactomycin. These results represent important steps in characterizing KasA and KasB as targets for antimycobacterial drug discovery.

scholarly journals Preclinical characterization of immunogenicity and efficacy against diarrhea from MecVax, a multivalent enterotoxigenic E. coli vaccine candidate

2021 ◽  
Author(s):  
Hyesuk Seo ◽  
Carolina Garcia ◽  
Xiaosai Ruan ◽  
Qiangde Duan ◽  
David A Sack ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Developing Countries ◽  
Vaccine Development ◽  
Vaccine Candidate ◽  
Preclinical Study ◽  
Watery Diarrhea ◽  
E Coli ◽  
Heat Stable ◽  
Protective Antibodies

There are no vaccines licensed for enterotoxigenic Escherichia coli (ETEC), a leading cause of diarrhea for children in developing countries and international travelers. Virulence heterogeneity among strains and difficulties identifying safe antigens for protective antibodies against STa, a potent but poorly immunogenic heat-stable toxin which plays a key role in ETEC diarrhea, are challenges in ETEC vaccine development. To overcome these challenges, we applied toxoid fusion strategy and novel epitope- and structure-based multiepitope-fusion-antigen (MEFA) vaccinology platform to construct two chimeric multivalent proteins, toxoid fusion 3xSTaN12S-mnLTR192G/L211A and adhesin CFA/I/II/IV MEFA, and demonstrated that proteins induced protective antibodies against STa and heat-labile toxin (LT) produced by all ETEC strains or the seven most important ETEC adhesins (CFA/I, CS1 to CS6) expressed by the ETEC strains causing 60-70% diarrheal cases and moderate-to-severe cases. Combining two proteins, we prepared a protein-based multivalent ETEC vaccine, MecVax. MecVax was broadly immunogenic; mice and pigs intramuscularly immunized with MecVax developed no apparent adverse effects but robust antibody responses to the target toxins and adhesins. Importantly, MecVax-induced antibodies were broadly protective, demonstrated by significant adherence inhibition against E. coli bacteria producing any of the seven adhesins and neutralization of STa and CT enterotoxicity. Moreover, MecVax protected against watery diarrhea, and over 70% or 90% any diarrhea from an STa+ or an LT+ ETEC strain in a pig challenge model. These results indicated that MecVax induces broadly protective antibodies and prevents diarrhea preclinically, signifying MecVax potentially an effective injectable vaccine for ETEC. IMPORTANCE: Enterotoxigenic Escherichia coli (ETEC) bacteria are a top cause of children’s diarrhea and travelers’ diarrhea and are responsible for over 220 million diarrheal cases and more than 100,000 deaths annually. A safe and effective ETEC vaccine can significantly improve public health, particularly in developing countries. Data from this preclinical study showed that MecVax induces broadly protective anti-adhesin and antitoxin antibodies, becoming the first ETEC vaccine candidate to induce protective antibodies inhibiting adherence of the seven most important ETEC adhesins and neutralizing enterotoxicity of LT but also STa toxin. More importantly, MecVax is shown to protect against clinical diarrhea from STa+ or LT+ ETEC infection in a pig challenge model, recording protection from antibodies induced by protein-based injectable subunit vaccine MecVax against ETEC diarrhea and perhaps the possibility of IM administered protein vaccines for protection against intestinal mucosal infection.

scholarly journals Biosynthesis of Conjugate Vaccines Using an O-Linked Glycosylation System

mBio ◽  
2016 ◽  
Vol 7 (2) ◽  
Author(s):  
Chao Pan ◽  
Peng Sun ◽  
Bo Liu ◽  
Haoyu Liang ◽  
Zhehui Peng ◽  
...  
Keyword(s):  
Major Histocompatibility Complex ◽  
Neisseria Meningitidis ◽  
Conjugate Vaccine ◽  
Vaccine Development ◽  
Rapid Development ◽  
Protein Glycosylation ◽  
Major Histocompatibility ◽  
Protein Subunit ◽  
Conjugate Vaccines ◽  
Histocompatibility Complex

ABSTRACT 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 viaNeisseria meningitidis) that can transfer virtually any glycan to produce a conjugate vaccine. We successfully established this system inShigellaspp., 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 theO-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.IMPORTANCERecently, 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 inEscherichia colihinders their application. Here, we show that an O-linked protein glycosylation system fromNeisseria 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.

scholarly journals Purification and characterization of [acyl-carrier-protein] acetyltransferase from Escherichia coli

1988 ◽  
Vol 250 (3) ◽  
pp. 789-796 ◽  
Author(s):  
P N Lowe ◽  
S Rhodes
Keyword(s):  
Escherichia Coli ◽  
Acyl Carrier Protein ◽  
Carrier Protein ◽  
E Coli ◽  
Step Procedure ◽  
Ping Pong ◽  
Protein Acetyltransferase ◽  
Enzyme Intermediate ◽  
Specific Reagent

A multi-step procedure has been developed for the purification of [acyl-carrier-protein] acetyltransferase from Escherichia coli, which allows the production of small amounts of homogeneous enzyme. The subunit Mr was estimated to be 29,000 and the native Mr was estimated to be 61,000, suggesting a homodimeric structure. The catalytic properties of the enzyme are consistent with a Bi Bi Ping Pong mechanism and the existence of an acetyl-enzyme intermediate in the catalytic cycle. The enzyme was inhibited by N-ethylmaleimide and more slowly by iodoacetamide in reactions protected by the substrate, acetyl-CoA. However, the enzyme was apparently only weakly inhibited by the thiol-specific reagent methyl methanethiosulphonate. The nature of the acetyl-enzyme intermediate is discussed in relationship to that found in other similar enzymes from E. coli, yeast and vertebrates.

scholarly journals Vaccines against Meningococcal Diseases

2020 ◽  
Vol 8 (10) ◽  
pp. 1521
Author(s):  
Mariagrazia Pizza ◽  
Rafik Bekkat-Berkani ◽  
Rino Rappuoli
Keyword(s):  
Conjugate Vaccine ◽  
Meningococcal Disease ◽  
Vaccine Development ◽  
Health Concern ◽  
Global Public Health ◽  
Public Health Concern ◽  
Conjugate Vaccines ◽  
Vaccination Programs ◽  
Meningitis Belt ◽  
Life Threatening

Neisseria meningitidis is the main cause of meningitis and sepsis, potentially life-threatening conditions. Thanks to advancements in vaccine development, vaccines are now available for five out of six meningococcal disease-causing serogroups (A, B, C, W, and Y). Vaccination programs with monovalent meningococcal serogroup C (MenC) conjugate vaccines in Europe have successfully decreased MenC disease and carriage. The use of a monovalent MenA conjugate vaccine in the African meningitis belt has led to a near elimination of MenA disease. Due to the emergence of non-vaccine serogroups, recommendations have gradually shifted, in many countries, from monovalent conjugate vaccines to quadrivalent MenACWY conjugate vaccines to provide broader protection. Recent real-world effectiveness of broad-coverage, protein-based MenB vaccines has been reassuring. Vaccines are also used to control meningococcal outbreaks. Despite major improvements, meningococcal disease remains a global public health concern. Further research into changing epidemiology is needed. Ongoing efforts are being made to develop next-generation, pentavalent vaccines including a MenACWYX conjugate vaccine and a MenACWY conjugate vaccine combined with MenB, which are expected to contribute to the global control of meningitis.

scholarly journals Eliminating Meningococcal Epidemics From the African Meningitis Belt: The Case for Advanced Prevention and Control Using Next-Generation Meningococcal Conjugate Vaccines

2019 ◽  
Vol 220 (Supplement_4) ◽  
pp. S274-S278 ◽  
Author(s):  
Mark R Alderson ◽  
F Marc LaForce ◽  
Ajoke Sobanjo-ter Meulen ◽  
Angela Hwang ◽  
Marie-Pierre Preziosi ◽  
...  
Keyword(s):  
Conjugate Vaccine ◽  
Prevention And Control ◽  
Vaccine Development ◽  
Next Generation ◽  
Meningococcal Infections ◽  
Conjugate Vaccines ◽  
Meningitis Belt ◽  
And Control ◽  
New Vaccines

AbstractThe introduction and rollout of a meningococcal serogroup A conjugate vaccine, MenAfriVac, in the African meningitis belt has eliminated serogroup A meningococcal infections for >300 million Africans. However, serogroup C, W, and X meningococci continue to circulate and have been responsible for focal epidemics in meningitis belt countries. Affordable multivalent meningococcal conjugate vaccines are being developed to prevent these non-A epidemics. This article describes the current epidemiologic situation and status of vaccine development and highlights questions to be addressed to most efficiently use these new vaccines.

scholarly journals Conjugate vaccines for enteric fever: proceedings of a meeting organized in New Delhi, India in 2009

2010 ◽  
Vol 4 (06) ◽  
pp. 404-411 ◽  
Author(s):  
Audino Podda ◽  
Allan James Saul ◽  
Rashmi Arora ◽  
Zulfiqar Bhutta ◽  
Anju Sinha ◽  
...  
Keyword(s):  
South Asia ◽  
Conjugate Vaccine ◽  
Enteric Fever ◽  
Vaccine Development ◽  
Severe Disease ◽  
Age Groups ◽  
New Combination ◽  
Conjugate Vaccines ◽  
Vaccination Programs ◽  
Young Infants

Enteric fever is responsible for significant morbidity in South Asia and high prevalence of severe disease is seen in children under two years of age. Effective typhoid vaccines are available, but they cannot be used for children under two years of age and also have some limitations in older age groups. Participants supported development of a Salmonella Typhi conjugate vaccine able to induce effective, long-lasting immunity in young children. The role of Salmonella Paratyphi A as a cause of enteric fever was discussed and consensus reached that a bivalent S. Typhi-S. Paratyphi A conjugate vaccine is highly desirable; however, considering disease epidemiology and the advanced status of vaccine development, rapid introduction of monovalent S. Typhi conjugate vaccine into vaccination programs of South Asia was recommended. Prevention should be emphasized, available vaccines used, and efforts toward improving sanitation continued. Success of the new vaccine will depend on several factors, including delivery costs and governmental ability to adopt and implement suitable immunization programs. To ensure good immunization coverage, the conjugate vaccine could be administered either to young infants, concomitantly with infant EPI vaccines, or to older infants, concomitantly with measles vaccine, currently given at 9 to 12 months. The need for new combination vaccines, containing both EPI and typhoid antigens, was discussed as a tool to increase coverage and reduce the number of injections and priority conflicts in a crowded infant vaccination schedule. However, stand-alone enteric fever conjugate vaccines would allow more flexibility to immunize different age groups and therefore should be rapidly developed.

scholarly journals Characterization of Glycosylation-Specific Systemic and Mucosal IgA Antibody Responses to Escherichia coli Mucinase YghJ (SslE)

2021 ◽  
Vol 12 ◽  
Author(s):  
Saman Riaz ◽  
Hans Steinsland ◽  
Mette Thorsing ◽  
Ann Z. Andersen ◽  
Anders Boysen ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Vaccine Development ◽  
Fold Increase ◽  
Antibody Responses ◽  
E Coli ◽  
Flow Cytometric ◽  
Median Proportion ◽  
Iga Response ◽  
Iga Antibody

Efforts to develop broadly protective vaccines against pathogenic Escherichia coli are ongoing. A potential antigen candidate for vaccine development is the metalloprotease YghJ, or SslE. YghJ is a conserved mucinase that is immunogenic, heavily glycosylated, and produced by most pathogenic E. coli. To develop efficacious YghJ-based vaccines, there is a need to investigate to what extent potentially protective antibody responses target glycosylated epitopes in YghJ and to describe variations in the quality of YghJ glycosylation in the E. coli population. In this study we estimated the proportion of anti-YghJ IgA antibodies that targeted glycosylated epitopes in serum and intestinal lavage samples from 21 volunteers experimentally infected with wild-type enterotoxigenic E. coli (ETEC) strain TW10722. Glycosylated and non-glycosylated YghJ was expressed, purified, and then gycosylation pattern was verified by BEMAP analysis. Then we used a multiplex bead flow cytometric assay to analyse samples from before and 10 days after TW10722 was ingested. We found that 20 (95%) of the 21 volunteers had IgA antibody responses to homologous, glycosylated YghJ, with a median fold increase in IgA levels of 7.9 (interquartile range [IQR]: 7.1, 11.1) in serum and 3.7 (IQR: 2.1, 10.7) in lavage. The median proportion of anti-YghJ IgA response that specifically targeted glycosylated epitopes was 0.45 (IQR: 0.30, 0.59) in serum and 0.07 (IQR: 0.01, 0.22) in lavage. Our findings suggest that a substantial, but variable, proportion of the IgA antibody response to YghJ in serum during ETEC infection is targeted against glycosylated epitopes, but that gut IgA responses largely target non-glycosylated epitopes. Further research into IgA targeting glycosylated YghJ epitopes is of interest to the vaccine development efforts.

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