Development of an automated platform for the optimal production of glycoconjugate vaccines expressed in Escherichia coli.

Protein Glycan Coupling Technology (PGCT) uses purposely modified bacterial cells to produce recombinant glycoconjugate vaccines. This vaccine platform holds great potential in this context, namely due to its modular nature, the simplified production process in comparison to traditional chemical conjugation methods, and its amenability to scaled-up operations. As a result, a considerable reduction in production time and cost is expected, making PGCT-made vaccines a suitable vaccine technology for low-middle income countries, where vaccine coverage remains predominantly low and inconsistent. This work aims to develop an integrated whole-process automated platform for the screening of PGCT-made glycoconjugate vaccine candidates. The successful translation of a bench scale process for glycoconjugate production to a microscale automated setting was achieved. This was integrated with a numerical computational software that allowed hands-free operation and a platform adaptable to biological variation over the course of a production process. Platform robustness was proven with both technical and biological replicates and subsequently the platform was used to screen for the most favourable conditions for production of a pneumococcal serotype 4 vaccine candidate. This work establishes an effective automated platform that enabled the identification of the most suitable E. coli strain and genetic constructs to be used in ongoing early phase research and be further brought into preclinical trials.

Biosynthesis of Glycoconjugate Virus-like Particles (VLPs).

The outermost surface of bacterial pathogens consists primarily of complex carbohydrate structures-polysaccharides, glycolipids, and glycoproteins. To raise a long-lasting and effective immune response against carbohydrate antigens, they generally require covalent attachment to an immunogenic carrier protein-a so-called glycoconjugate vaccine. One hurdle to the development of glycoconjugate vaccines is that carbohydrate antigens remain inaccessible to recombinant production. Thus, the carbohydrate antigen is typically purified from the pathogen and then chemically conjugated to an immunogenic protein. Recent developments in the field of bacterial glycoengineering have opened the opportunity for total recombinant production of glycoconjugate vaccines. In this method, we describe the production of proteinaceous, virus-like particles (VLPs) bearing the conserved N-glycan of Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumoniae.

Novel adjuvant Alum-TLR7 significantly potentiates immune response to glycoconjugate vaccines.

Although glycoconjugate vaccines are generally very efficacious, there is still a need to improve their efficacy, especially in eliciting a strong primary antibody response. We have recently described a new type of vaccine adjuvant based on a TLR7 agonist adsorbed to alum (Alum-TLR7), which is highly efficacious at enhancing immunogenicity of protein based vaccines. Since no adjuvant has been shown to potentiate the immune response to glycoconjugate vaccines in humans, we investigated if Alum-TLR7 is able to improve immunogenicity of this class of vaccines. We found that in a mouse model Alum-TLR7 greatly improved potency of a CRM197-MenC vaccine increasing anti-MenC antibody titers and serum bactericidal activity (SBA) against MenC compared to alum adjuvanted vaccine, especially with a low dose of antigen and already after a single immunization. Alum-TLR7 also drives antibody response towards Th1 isotypes. This adjuvant was also able to increase immunogenicity of all polysaccharides of a multicomponent glycoconjugate vaccine CRM197-MenACWY. Furthermore, we found that Alum-TLR7 increases anti-polysaccharide immune response even in the presence of a prior immune response against the carrier protein. Finally, we demonstrate that Alum-TLR7 adjuvant effect requires a functional TLR7. Taken together, our data support the use of Alum-TLR7 as adjuvant for glycoconjugate vaccines.

Prevention of Meningococcal Infection in the United States: Current Recommendations and Future Considerations.

Neisseria meningitidis is a common cause of bacterial meningitis and septicemia that can lead to permanent sequelae or death. N meningitidis is classified into serogroups based on the composition of the capsular polysaccharide, with serogroups A, B, C, W, X, and Y recognized as the major disease-causing organisms. The unpredictability of infection coupled with the poor prognosis for some patients suggests immunization as an effective preventive strategy. Importantly, four of the six disease-causing serogroups (A, C, Y, and W) may be prevented with available quadrivalent capsular polysaccharide-protein conjugate vaccines; these vaccines have been successfully implemented into immunization programs in the United States. Unfortunately, quadrivalent conjugate vaccines are not effective against serogroup B, now the most common cause of invasive meningococcal disease. Two recombinant protein vaccines recently were licensed for prevention of serogroup B disease. Recommendations for use of these serogroup B vaccines in the United States have been made by the Advisory Committee on Immunization Practices. This article will discuss all available meningococcal vaccines, current recommendations for use, lessons learned from previous experiences, and future considerations, with the hope of further understanding how use of these vaccines may help reduce incidence of meningococcal disease in the United States.

Characterization of Citrobacter sp. line 328 as a source of Vi for a Vi-CRM(197) glycoconjugate vaccine against Salmonella Typhi.

INTRODUCTION: Salmonella enterica serovar Typhi is the causative agent of typhoid fever with over 22 million cases and over 200,000 deaths reported annually. A vaccine is much needed for use in young children and the Novartis Vaccines Institute for Global Health (NVGH) is developing a conjugate vaccine which targets S. Typhi Vi capsular polysaccharide. METHODOLOGY: Here we describe a method suitable for industrial scale production of the Vi antigen based on expression by a Citrobacter line. We optimized the production of Vi by selecting a suitable Citrobacter strain (Citrobacter 328) that yields high and stable expression of Vi in chemically defined medium under industrial-scale fermentation conditions. RESULTS: Vi-CRM197 made using Vi from Citrobacter 328 elicited high anti-Vi antibody levels in mice and rabbits. CONCLUSIONS: Citrobacter 328 is a suitable strain for production of Vi for conjugate anti-Typhi vaccines. Being a BSL-1 organism, which grows in defined medium and stably produces high yields of Vi, it offers excellent potential for safe production of inexpensive vaccines for populations at risk of typhoid fever.

Prescripción de vacunas no incluidas en el calendario vacunal en la Comunitat Valenciana durante el período 2004-2009 / Public health service prescriptions of vaccines not included in systematic vaccination programs in valencian community, Spain, during the period 2004-2009

Fundamentos: En el marco de las políticas de uso racional del medicamento, y al objeto de conseguir una gestión eficiente de los programas de vacunaciones, el objetivo de este trabajo es conocer el número de envases de las vacunas prescritas no incluidas en los programas de vacunación en la Comunitat Valenciana y en sus departamentos de salud, así como el gasto que produjeron en 2009, y analizar la evolución desde 2004, centrando el análisis en la vacuna heptavalente conjugada frente al Streptococcus pneumoniae en menores de dos años. Método: Estudio descriptivo retrospectivo de las vacunas prescritas mediante receta en la Comunitat Valenciana durante el año 2009 y su evolución desde 2004. Variables: número de envases, tipo de beneficiario (activo/pensionista), departamento y gasto generado. Fuentes: Gestor de Prestación Farmacéutica (GAIA) y Sistema Información Poblacional (SIP). Resultados: En 2009 la prescripción mediante receta de vacunas no incluidas en los programas de vacunación generó un gasto de 683.445,71 ] correspondiente a 17.353 envases, lo que supuso el 87! del total del gasto en vacunas recetadas. La vacuna frente al S. pneumoniae generó el 72! del gasto total de las vacunas no incluidas en el calendario. La evolución 2004-2009 muestra un aumento del gasto de 735.334 ] (24,66!) en 2005 a partir del cual se produjo un descenso acusado y paulatino que alcanzó los 1.562.650,67 ] (-228.64!). El gasto por departamentos para la vacuna del neumococo conjugada heptavalente por mil niños/as menores de dos años osciló entre 17.377 y 324 ]. Conclusiones: La tendencia descendente del gasto en recetas prescritas se mantuvo durante 2009, fundamentalmente de vacunas conjugadas frente a neumococo. No obstante, se observó gran variabilidad interdepartamental en las tasas de prescripción que debe ser corregida(AU)

Background: In the context of the policies of rational use of medicine, and in order to achieve an efficient management of the vaccinations programs, we expect to know the number of packings and cost of prescribed vaccines not included in the vaccination programs of Valencian Community and its departments during 2009 and to analyze its evolution since 2004, focusing on an analysis of Heptavalent pneumococcal conjugate vaccine in children under two years old. Methods: Retrospective descriptive study to analyze the prescriptions of vaccines in Valencian Community during 2009 and its evolution since 2004. Variables: vaccine availability, number of packings, group of beneficiary (actives/pensioners), department, and cost of prescriptions. Data sources: Gestor de Prestación Farmacéutica (GAIA) and Sistema Información Poblacional (SIP). Results: In 2009 prescribed vaccines on official national health system prescription forms that are not included in vaccination programs, supposed a cost of 683.445,71 ] corresponding to 17.353 packings (87! of the total prescribed vaccines). Heptavalent pneumococcal conjugate vaccine generated 72! of the total cost of vaccines not included in the vaccination programs. The trend from 2004 to 2009 shows an increase in expenditure of 735.334 ] (24,66!) in 2005 from which there takes place a marked and gradual decrease that reaches 1.562.650,67 ] (-228.64!). The cost by departments of prescriptions per 1000 children under two years old of pneumococcal conjugate vaccine ranges between 17.377 and 324 ]. Conclusions: The declining trend of prescriptions, mainly of pneumococcal conjugate vaccines, continues during 2009. A great interdepartmental variability is observed, nevertheless, in rates of prescription that should be corrected(AU)

Production of glycoprotein vaccines in Escherichia coli.

BACKGROUND: Conjugate vaccines in which polysaccharide antigens are covalently linked to carrier proteins belong to the most effective and safest vaccines against bacterial pathogens. State-of-the art production of conjugate vaccines using chemical methods is a laborious, multi-step process. In vivo enzymatic coupling using the general glycosylation pathway of Campylobacter jejuni in recombinant Escherichia coli has been suggested as a simpler method for producing conjugate vaccines. In this study we describe the in vivo biosynthesis of two novel conjugate vaccine candidates against Shigella dysenteriae type 1, an important bacterial pathogen causing severe gastro-intestinal disease states mainly in developing countries. RESULTS: Two different periplasmic carrier proteins, AcrA from C. jejuni and a toxoid form of Pseudomonas aeruginosa exotoxin were glycosylated with Shigella O antigens in E. coli. Starting from shake flask cultivation in standard complex medium a lab-scale fed-batch process was developed for glycoconjugate production. It was found that efficiency of glycosylation but not carrier protein expression was highly susceptible to the physiological state at induction. After induction glycoconjugates generally appeared later than unglycosylated carrier protein, suggesting that glycosylation was the rate-limiting step for synthesis of conjugate vaccines in E. coli. Glycoconjugate synthesis, in particular expression of oligosaccharyltransferase PglB, strongly inhibited growth of E. coli cells after induction, making it necessary to separate biomass growth and recombinant protein expression phases. With a simple pulse and linear feed strategy and the use of semi-defined glycerol medium, volumetric glycoconjugate yield was increased 30 to 50-fold. CONCLUSIONS: The presented data demonstrate that glycosylated proteins can be produced in recombinant E. coli at a larger scale. The described methodologies constitute an important step towards cost-effective in vivo production of conjugate vaccines, which in future may be used for combating severe infectious diseases, particularly in developing countries.

Preparation of bacterial polysaccharide-protein conjugates: analytical and manufacturing challenges.

A conjugate can be a polysaccharide (PS) covalently attached to a protein, which provides T cell epitopes for a normally T cell independent antigen. To produce a conjugate vaccine, the purified PS must first be chemically modified to generate reactive groups that can link to the protein. Two commonly used methods for PS activation are periodate oxidation at vicinal hydroxyls and cyanylation of hydroxyls. The PS should be of known molecular size before and after activation. Low molecular weight impurities in the protein may result in inefficient conjugation. Two critical measures after conjugation and purification are the PS to protein ratio and the percent non-conjugated saccharide (free saccharide). Yield and conjugate stability are critical considerations. Typically, considerably less than 20% of the activated PS becomes conjugated. Yield can be improved using newer conjugation methods, whereby highly reactive groups are generated on both the PS and carrier protein with yields approaching 50%. Two major measures used to follow vaccine stability are changes in molecular size and percent free (unbound) PS.

LPS-based conjugate vaccines composed of saccharide antigens of smooth-type Salmonella enteritidis and rough-type S. gallinarum 9R bound to bovine serum albumin.

Lipopolysaccahrides (LPSs) from Salmonella enteritidis, S. gallinarum, and S. enterica Typhimurium showed an identical electrophoretic banding pattern and serological cross-reactions among each other. LPSs from wild-type Salmonella enteritidis and rough mutant S. gallinarum 9R were detoxified by cleavage of lipid A moieties using mild acid hydrolysis. The non-toxic saccharide moieties from both strains were coupled directly to bovine serum albumin (BSA). The conjugates were injected in mice in combination with monophosphory lipid A (MPL), Freund, and Alum adjuvants. The highest IgM and IgG titres were obtained when the conjugates were emulsified with MPL adjuvant, followed by Freund adjuvant. The antisera raised against the conjugates in combination with MPL and Freund adjuvants showed high complement-mediated lysis to the homologous strains. A correlation was observed between IgG titres and bactericidal activities against homologous strains. Low in vivo protection was obtained when mice immunized with the conjugates were challenged with 10 times the LD50 of the wild S. enteritidis.

Monitoring activation sites on polysaccharides by GC-MS.

A method has been developed to determine the location and order of activation for potential saccharide antigens used in conjugate vaccine development. Saccharides were monitored for activation by sodium periodate oxidation and subsequent analysis by gas chromatography-mass spectrometry (GC-MS). Pneumococcal serotype polysaccharides 7F and 18C were evaluated as polysaccharides containing multiple potential sites for activation. Sialyllactose was used as a model oligosaccharide compound to evaluate oxidation of terminally linked sialic acids and reducing sugar residues. Oxidized saccharides were analyzed by monosaccharide composition and/or linkage analysis to elucidate specific activation of cis versus trans diols, as well as diols containing primary versus secondary alcohols, at specified levels of periodate. Samples (100-500 microg) were sequentially oxidized, reduced, methanolyzed, and derivatized in a single reaction vial for routine analysis by GC-MS.

Capsular polysaccharide conjugate vaccines against contagious bovine pleuropneumonia: Immune responses and protection in mice.

The immunogenicity of Mycoplasma mycoides subsp. mycoides small colony biotype (MmmSC) vaccines was investigated in BALB/c mice. Groups of mice were vaccinated with either (1) unconjugated capsular polysaccharide (CPS), (2) CPS covalently conjugated to ovalbumin via a carbodiimide reaction, (3) CPS non-covalently bound to latex microspheres, (4) CPS non-covalently complexed with rabbit anti-CPS IgG, and (5) whole inactivated, ultrasonically disrupted (WID) MmmSC. Only mice immunized with the CPS-ovalbumin conjugate exhibited a significant (P<0 small middle dot001) antibody response against CPS. Mice immunized with WID vaccine exhibited a high ELISA antibody titre against non-CPS (protein) antigens only. Mice given WID vaccine were immune against challenge with live MmmSC, and exhibited a significantly reduced degree of mycoplasmaemia (both in incidence and duration) as compared with non-vaccinated controls (P<0 small middle dot001). Mice immunized with the CPS-ovalbumin conjugate did not exhibit a reduction in mycoplasmaemia. The bactericidal activity of rabbit MmmSC-antiserum in an in-vitro growth inhibition test was related to the CPS antibody titre. This was not observed with antisera from the vaccinated mice. None of the mouse antisera exhibited growth inhibiting activity, irrespective of a high CPS or protein antibody titre (CPS-ovalbumin or WID vaccine groups, respectively). Thus, it would seem that protection against an MmmSC-induced mycoplasmaemia in the mouse is based upon cell-mediated rather than humoral immunity. The results suggest that conjugation to ovalbumin significantly increases the antibody response to CPS in the mouse; the lack of bactericidal activity of mouse anti-CPS as compared with rabbit anti-CPS in vitro suggests either that the titre of growth inhibiting antibodies is lower in the mouse or that the mechanism of growth inhibition differs between antibodies of the two species.