Brain temperature as an indicator of neuroinflammation induced by typhoid vaccine: Assessment using whole-brain magnetic resonance spectroscopy in a randomised crossover study.

Prior studies indicate a pathogenic role of neuroinflammation in psychiatric disorders; however, there are no accepted methods that can reliably measure low-level neuroinflammation non-invasively in these individuals. Magnetic resonance spectroscopic imaging (MRSI) is a versatile, non-invasive neuroimaging technique that demonstrates sensitivity to brain inflammation. MRSI in conjunction with echo-planar spectroscopic imaging (EPSI) measures brain metabolites to derive whole-brain and regional brain temperatures, which may increase in neuroinflammation. The validity of MRSI/EPSI for measurement of low level neuroinflammation was tested using a safe experimental model of human brain inflammation - intramuscular administration of typhoid vaccine. Twenty healthy volunteers participated in a double-blind, placebo-controlled crossover study including MRSI/EPSI scans before and 3 h after vaccine/placebo administration. Body temperature and mood, assessed using the Profile of Mood States, were measured every hour up to four hours post-treatment administration. A mixed model analysis of variance was used to test for treatment effects. A significant proportion of brain regions (44/47) increased in temperature post-vaccine compared to post-placebo (p < 0.0001). For temperature change in the brain as a whole, there was no significant treatment effect. Significant associations were seen between mood scores assessed at 4 h and whole brain and regional temperatures post-treatment. Findings indicate that regional brain temperature may be a more sensitive measure of low-level neuroinflammation than whole-brain temperature. Future work where these measurement techniques are applied to populations with psychiatric disorders would be of clinical interest.

Development of a Specific and Sensitive HPAEC-PAD Method for Quantification of Vi Polysaccharide Applicable to other Polysaccharides Containing Amino Uronic Acids.

Typhoid fever is a major cause of morbidity and mortality in developing countries. Vaccines based on the Vi capsular polysaccharide are licensed or in development against typhoid fever. Vi content is a critical quality attribute for vaccines release, to monitor their stability and to ensure appropriate immune response. Vi polysaccharide is a homopolymer of α-1,4-N-acetylgalactosaminouronic acid, O-acetylated at the C-3 position, resistant to the commonly used acid hydrolysis for sugar chain depolymerization before monomer quantification. We previously developed a quantification method based on strong alkaline hydrolysis followed by High Performance Anion Exchange Chromatography-Pulsed Amperometric Detection analysis, but with low sensitivity and use for quantification of an unknown product coming from polysaccharide depolymerization. Here we describe the development of a method for Vi polysaccharide quantification based on acid hydrolysis with concomitant use of trifluoroacetic and hydrochloric acids. A Design of Experiment approach was used for the identification of the optimal hydrolysis conditions. The method is 100-fold more sensitive than the previous one, and specifically, resulting in the formation of a known product, confirmed to be the Vi monomer both de-O- and de-N-acetylated by mono- and bidimensional Nuclear Magnetic Resonance spectroscopy and mass spectrometry. Accuracy and precision were determined, and chromatographic conditions were improved to result in reduced time of analysis. This method will facilitate characterization of Vi-based vaccines. Furthermore, a similar approach has the potential to be extended to other polysaccharides containing 2-amino uronic acids, as already verified here for Shigella sonnei O-antigen, Streptococcus pneumoniae serotype 12F, and Staphylococcus aureus types 5 and 8 capsular polysaccharides.

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.

Production of a conjugate vaccine for Salmonella enterica serovar Typhi from Citrobacter Vi.

A conjugate vaccine for Salmonella enterica serovar Typhi was produced by chemically linking Vi, purified from Citrobacter, to the non-toxic mutant diphtheria toxin CRM(197) via an adipic dihydrazide spacer using N-(3-Dimethylaminopropyl)-N'-ethylcarbodiimide coupling chemistry. The polysaccharide purification process was developed based on Vi precipitation from culture supernatant with cetyl trimethylammonium bromide (CTAB), solubilization of the CTA-polysaccharide salt with ethanol followed by exchange of the CTA(+) counter ion with Na(+). The purified Vi polysaccharide was fully O-acetylated and with high purity. The conjugation process was optimized to obtain a scalable process that has been used for GMP production at pilot scale of vaccine currently in clinical trials.

Oral immunisation with live aroA attenuated Salmonella enterica serovar Typhimurium expressing the Yersinia pestis V antigen protects mice against plague.

Bubonic and pneumonic plague are caused by the bacterium Yersinia pestis. The V antigen of Y. pestis is a protective antigen against plague. In this study, an aroA attenuated strain of Salmonella enterica serovar Typhimurium (SL3261) has been used to deliver the Y. pestis V antigen as a candidate oral plague vaccine. SL3261 was transformed with the expression plasmid pTrc-LcrV, containing the lcrV gene encoding V antigen. Immunoblot analysis showed V antigen expression in SL3261 in vitro and intragastric immunisation of mice with the recombinant Salmonella resulted in the induction of V antigen-specific serum antibody responses and afforded protection against Y. pestis challenge. However, the antibody responses induced by the recombinant Salmonella did not correlate with the protection afforded, indicating that immune responses other than antibody may play a role in the protection afforded against plague by this candidate vaccine.

Oral immunization of swine with attenuated Salmonella typhimurium aroA SL3261 expressing a recombinant antigen of Mycoplasma hyopneumoniae (NrdF) primes the immune system for a NrdF specific secretory IgA response in the lungs.

Salmonella typhimurium SL3261 (aroA mutant) expressing a recombinant Mycoplasma hyopneumoniae antigen was used to orally immunize swine against porcine enzootic pneumonia. This construct, designated S. typhimurium aro A SL3261 (pKF1), expressed a recombinant protein containing the carboxy-terminal 11 kDa of a 42 kDa M. hyopneumoniae NrdF ribonucleotide reductase R2 subunit protein. Here we demonstrate that this antigen is present in all seven geographically diverse strains of M. hyopneumoniae tested, and is recognized by the swine immune system after experimental infection with the virulent M. hyopneumoniae Beaufort strain. The immune response of swine orally immunized twice with S. typhimurium SL3261 (pKF1) on day 0 and day 14 was evaluated. Oral immunization with S. typhimurium SL3261 (pKF1) primed the immune system to elicit a significant (P<0.05) secretory IgA response against the 15 kDa NrdF antigen in the respiratory tract of swine, post-challenge, compared to control groups. Blood lymphocytes from swine immunized with S. typhimurium SL3261 (pKF1) proliferated significantly (P<0.05) following stimulation with M. hyopneumoniae whole-cell extracts compared to control groups 14 days post-vaccination. Following challenge with virulent M. hyopneumoniae, swine immunized with S. typhimurium SL3261 (pKF1) showed higher average daily weight gains and reduced lung pathology compared to control groups.

Adjuvant effect of DEAE-dextran and tetanus toxoid on whole cell heat inactivated phenol preserved typhoid vaccine.

Active mouse protection test (AMPT) and enzyme linked immunosorbent assay (ELISA) were used to determine the immunogenicity of whole cell typhoid vaccine when administered in conjunction with either tetanus toxoid (TT) or DEAE-Dextran (DD). Immunization of mice with whole cell typhoid vaccine showed enhanced potency either when administered in conjunction with TT or DD and values were statistically significant (p < 0.05) in comparison to conventional or standard typhoid vaccines. For ELISA, the mice were immunized with 2 different schedules, one in which a single dose of 0.25 ml subcutaneously (s/c) was administered and in another two doses of 0.25 ml each s/c, 14 days apart. In case of single dose schedule of immunization D vaccine (Whole cell typhoid + 5 mg/ml DD) showed significant increase of immune response (3.201 log10) as compared to plain vaccine (2.550 log10). Two dose schedule further increased the titres to 3.856 log10. DD adjuvanted vaccine showed higher potency by AMPT as compared to the TT adjuvanted vaccine or plain vaccine. The present study clearly demonstrates that a single dose of 0.25 ml which is equivalent to half of the conventionally used single human dose of typhoid vaccine adjuvanted with DD can significantly improve the immunogenicity of the vaccine.