Rotavirus spike protein ΔVP8* as a novel carrier protein for conjugate vaccine platform with demonstrated antigenic potential for use as bivalent vaccine.

Conjugate vaccine platform is a promising strategy to overcome the poor immunogenicity of bacterial polysaccharide antigens in infants and children. A carrier protein in conjugate vaccines works not only as an immune stimulator to polysaccharide, but also as an immunogen; with the latter generally not considered as a measured outcome in real world. Here, we probed the potential of a conjugate vaccine platform to induce enhanced immunogenicity of a truncated rotavirus spike protein ΔVP8*. ΔVP8* was covalently conjugated to Vi capsular polysaccharide (Vi) of Salmonella Typhi to develop a bivalent vaccine, termed Vi-ΔVP8*. Our results demonstrated that the Vi-ΔVP8* vaccine can induce specific immune responses against both antigens in immunized mice. The conjugate vaccine elicits high antibody titers and functional antibodies against S. Typhi and Rotavirus (RV) when compared to immunization with a single antigen. Together, these results indicate that Vi-ΔVP8* is a potent and immunogenic vaccine candidate, thus strengthening the potential of conjugate vaccine platform with enhanced immune responses to carrier protein, including ΔVP8*.

Development of a bivalent conjugate vaccine candidate against rotaviral diarrhea and tuberculosis using polysaccharide from Mycobacterium tuberculosis conjugated to ΔVP8* protein from rotavirus.

Conjugation of carbohydrate antigens with a carrier protein is a clinically proven strategy to overcome the poor immunogenicity of bacterial polysaccharide. In addition to its primary role, which is to help generate a T cell-mediate long-lasting immune response directed against the carbohydrate antigen, the carrier protein in a glycoconjugate vaccine can also play an important role as a protective antigen. Among carrier proteins currently used in licensed conjugate vaccines, non-typeable Haemophilus influenzae protein D has been used as an antigenically active carrier protein. Our previous studies also indicate that some carrier proteins provide B cell epitopes, along with T cell helper epitopes. Herein we investigated the dual role of truncated rotavirus spike protein ΔVP8* as a carrier and a protective antigen. Capsular polysaccharide lipoarabinomannan (LAM), purified from Mycobacterium tuberculosis (M.tb), was chemically conjugated with ΔVP8*. Mouse immunization experiments showed that the resultant conjugates elicited strong and specific immune responses against the polysaccharide antigen, and the responses were comparable to those induced by Diphtheria toxoid (DT)-based conjugates. The conjugate vaccine induced enhanced antibody titers and functional antibodies against ΔVP8* when compared to immunization with the unconjugated ΔVP8*. Thus, these results indicate that ΔVP8* can be a relevant carrier protein for glycoconjugate vaccine and the glycoconjugates consisting of ΔVP8* with LAM are effective bivalent vaccine candidates against rotavirus and tuberculosis.

Spectroscopic characterisation of a series of Salmonella Typhi Vi-diphtheria toxoid glycoconjugate antigens differing in polysaccharide-protein ratio.

Glycoconjugate vaccines consisting of the Salmonella enterica subsp. enterica serovar Typhi (S. Typhi) Vi capsular polysaccharide (PS) covalently attached to a suitable carrier protein have become available to support mass paediatric vaccination campaigns against typhoid. One developmental vaccine from the International Vaccine Institute (IVI) uses diphtheria toxoid (DTx) as the carrier protein. Several investigational conjugates with different PS:protein ratios were prepared, as previously reported by the IVI group, for physicochemical and immunochemical characterisation. We describe here the further spectroscopic characterisation of this series of glycoconjugate immunogen bulks using NMR spectroscopy, circular dichroism and absorption spectroscopy. We have used several mathematical approaches to extract information from the spectroscopic data not previously applied to glycoconjugates. These complementary approaches provide information on (i) the integrity of the carrier protein, (ii) consistency between batches of vaccine components, (iii) the polysaccharide: protein ratio (iv) the O-acetylation of the Vi in the conjugate (v) the stability of the O-acetylation of the Vi, and (vi) the presence of residual process reagents in the bulk. The utility of the data analysis approaches is discussed. Together, these analytical methods provide important characterisation of Vi-DTx conjugates to support development and quality control of commercial products.

A novel method for purification of Vi capsular polysaccharide produced by Salmonella enterica subspecies enterica serovar Typhi.

Vi capsular polysaccharide is the major component of Vi polysaccharide typhoid vaccines. Vi is synthesized during growth of Salmonella enterica subspecies enterica serovar Typhi and is released into the fermentation broth in large quantities. Along with the Vi considerable amounts of impurities consisting of bacterial protein, nucleic acid and lipopolysaccharide (LPS) as well as media components contaminate the fermentation broth. A purification method based on selective precipitation of Vi using the cationic detergent cetavlon was developed to separate impurities from Vi. A novel method for handling the Vi precipitate using 0.2 µm sterilizing grade filters to trap and wash the Vi and then, after re-solubilization, allow the Vi to pass through the filter was developed. Cetavlon selectively precipitates Vi and is the major purification step in the process, however, the conditions must be carefully controlled otherwise LPS will co-precipitate in large quantities. Various diafiltration steps help to remove contaminating protein, nucleic acid and fermentation media components as well as chemicals added during the process to induce precipitation of either Vi or contaminants. The final yield of purified Vi was approximately 45% and the bulk concentrate complied with the specifications defined in the WHO recommendations for Vi polysaccharide vaccine. Analysis of the Vi by size exclusion chromatography revealed a uniform peak with a narrow size distribution. The Nuclear Magnetic Resonance spectrum was similar to Vi produced by other methods. The method developed produces large quantities of Vi using low cost production methods translating into Vi based vaccines that can be produced at affordable prices for use in developing countries.

Immune suppression induced by Vi capsular polysaccharide is overcome by Vi-DT conjugate vaccine.

The influence pre-exposure of mice to Vi capsular polysaccharide, purified from Salmonella enterica Serovar Typhi, on the subsequent immune response induced by a Vi-diphtheria toxoid (Vi-DT) conjugate was evaluated. Vi induced low anti Vi IgG titers with the dominant subclass being IgG3. The Vi-DT conjugate induced high titers of anti Vi IgG with the dominant subclass being IgG1 but with considerable quantities of IgG2a, IgG2b and IgG3. Priming of mice with Vi suppressed the response to a subsequent dose of conjugate and the suppression was overcome by a second dose of conjugate. Priming with conjugate prevented suppression of the anti Vi response and subsequent dosing with Vi raised titers back to previous levels but did not boost to new higher levels. The anti DT IgG response to one dose of conjugate was relatively strong and protracted and continued to rise for 12 weeks, compared to the response to one dose of DT which was poor and peaked at two weeks. The prolonged anti DT response was most likely due to the slow release of DT from the conjugate lattice as it degrades within the mouse resulting in a continuous stimulation of the immune response. The presence of increasing amounts of un-conjugated Vi, up to 50%, administered with the conjugate resulted in increasingly higher levels of both anti Vi and anti DT. Larger amounts of un-conjugated Vi inhibited the anti Vi response. These findings have implications for vaccine quality and a limit for un-conjugated polysaccharide should not exceed 50% and from a vaccine program perspective if the results presented here translate to humans then a Vi conjugate, once it becomes available, should replace Vi polysaccharide vaccines.

Physico-chemical properties of Salmonella typhi Vi polysaccharide-diphtheria toxoid conjugate vaccines affect immunogenicity.

In this study it was demonstrated that the immunogenicity of Vi polysaccharide-diphtheria toxoid conjugates was related to the physical and chemical structure of the conjugate. Conjugates were prepared in two steps, firstly binding adipic acid dihydrazide (ADH) spacer molecules to diphtheria toxoid (DT) carrier protein then secondly binding varying amounts of this derivatized DT to a fixed amount of Vi capsular polysaccharide purified from Salmonella enterica Serovar Typhi. As the amount of DT bound to the Vi increased the size of the conjugate increased but also the degree of cross-linking increased. The immunogenicity of the conjugates was tested in mice and measured by ELISA for anti Vi and anti DT IgG responses, and the results revealed a trend that as the amount of DT bound to the Vi increased the anti Vi responses increased. This study establishes a correlation between physico-chemical characteristics of the conjugate and the magnitude of the anti Vi and anti DT responses.

Oral poliovirus vaccine type 3 from a patient with transverse myelitis is neurovirulent in a transgenic mouse model.

BACKGROUND: It is accepted that oral poliovirus vaccine (OPV) can cause vaccine-associated paralytic poliomyelitis (VAPP) and that wild poliovirus infection can rarely present as transverse myelitis. It is therefore possible that OPV could cause transverse myelitis. We previously reported a case of transverse myelitis that developed in a 6-month-old boy, 7 days after receiving his second dose of OPV. OBJECTIVES: Our aim was to test the virus from this patient with transverse myelitis for neurovirulence in a mouse model. STUDY DESIGN: The TgPVR21 transgenic mouse line, which expresses the human poliovirus receptor CD155, was used to assess neurovirulence of the viruses tested. Neurovirulence was expressed as the PD(50), the dose of virus causing paralysis in 50% of the mice. Four type 3 polioviruses were tested: a prototype wild strain, a fully attenuated polio vaccine virus, a virus from a patient with VAPP and the virus from the patient with transverse myelitis. RESULTS: The PD(50) for the wild poliovirus strain was 3.83 and for the fully attenuated vaccine strain, 7.63. The PD(50) for the two clinical isolates were between these values, > or = 4.96 for the poliovirus known to have caused VAPP and > or = 4.81 for the virus from the patient with transverse myelitis. CONCLUSIONS: The report of an OPV strain from a transverse myelitis case being neurovirulent in an in vivo mouse model provides further evidence for a causal association between OPV and transverse myelitis.

Annual report of the Australian National Poliovirus Reference Laboratory, 2008.

The Australian National Poliovirus Reference Laboratory (NPRL) is accredited by the World Health Organization (WHO) for the testing of stool specimens from cases of acute flaccid paralysis (AFP), a major clinical presentation of poliovirus infection. The NPRL, in collaboration with the Australian Paediatric Surveillance Unit, co-ordinates surveillance for cases of AFP in children in Australia, according to criteria recommended by the WHO. Clinical specimens are referred from AFP cases in children and suspected case of poliomyelitis in persons of any age. The WHO AFP surveillance performance indicator for a polio-free country such as Australia, is 1 non-polio AFP case per 100,000 children less than 15 years of age. In 2008, the Polio Expert Committee (PEC) classified 62 cases as non-polio AFP, or 1.51 non-polio AFP cases per 100,000 children aged less than 15 years. Poliovirus infection is confirmed by virus culture of stool specimens from AFP cases as other conditions that present with acute paralysis can mimic polio. While no poliovirus was reported in Australia from any source in 2008, the non-polio enteroviruses echovirus 25, coxsackievirus B2 and echovirus 11 were isolated from stool specimens of AFP cases. The last report of a wild poliovirus in Australia was due to an importation from Pakistan in 2007. With 4 countries remaining endemic for poliomyelitis--Afghanistan, India, Nigeria and Pakistan--and more than 1,600 confirmed cases of wild poliovirus infection in 18 countries in 2008, Australia continues to be at risk of further importation events.

Optimization of Vi capsular polysaccharide production during growth of Salmonella enterica serotype Typhi Ty2 in a bioreactor.

Vi capsular polysaccharide is synthesized during growth of Salmonella typhi Ty2 and is spontaneously released from the bacterial cells into the culture medium during culture. Vi production was dependent on cell growth and the greater the cell mass the greater the production of Vi. Using fed batch culture to optimize bacterial growth resulted is an increase in cell mass and consequently Vi production. The yield of Vi obtained in fed batch culture was 415 mgl(-1), which was over three times that, obtained in batch culture. A proportion of the Vi remained cell associated in the form of a capsule and at least part of this was released from the bacterial surface by sonication. The size of the Vi polysaccharide produced was consistently high and did not change during the different phases of bacterial growth. The synthesis of Vi was also dependent upon the media components and the fermentation conditions. The presence of high concentrations of glucose at the beginning of growth inhibited the production of Vi, particularly during the stationary phase. At a concentration of 400 mM sodium phosphate the synthesis of Vi was strongly inhibited.