Clostridium difficile Toxoid Vaccine Candidate Confers Broad Protection against a Range of Prevalent Circulating Strains in a Nonclinical Setting.

Clostridium difficile infection (CDI) is a leading cause of nosocomial and antibiotic-associated diarrhea. A vaccine, based on formalin-inactivated toxins A and B purified from anaerobic cultures of C. difficile strain VPI 10463 (toxinotype 0), has been in development for the prevention of symptomatic CDI. We evaluated the breadth of protection conferred by this C. difficile toxoid vaccine in cross-neutralization assessments using sera from vaccinated hamsters against a collection of 165 clinical isolates. Hamster antisera raised against the C. difficile toxoid vaccine neutralized the cytotoxic activity of culture supernatants from several toxinotype 0 strains and heterologous strains from 10 different toxinotypes. Further assessments performed with purified toxins confirmed that vaccine-elicited antibodies can neutralize both A and B toxins from a variety of toxinotypes. In the hamster challenge model, the vaccine conferred significant cross-protection against disease symptoms and death caused by heterologous C. difficile strains from the most common phylogenetic clades, including the most prevalent toxinotypes.

IscR Regulates Synthesis of Colonization Factor Antigen I Fimbriae in Response to Iron Starvation in Enterotoxigenic Escherichia coli.

UNLABELLED: Iron availability functions as an environmental cue for enteropathogenic bacteria, signaling arrival within the human host. As enterotoxigenic Escherichia coli (ETEC) is a major cause of human diarrhea, the effect of iron on ETEC virulence factors was evaluated here. ETEC pathogenicity is directly linked to production of fimbrial colonization factors and secretion of heat-labile enterotoxin (LT) and/or heat-stable enterotoxin (ST). Efficient colonization of the small intestine further requires at least the flagellin binding adhesin EtpA. Under iron starvation, production of the CFA/I fimbriae was increased in the ETEC H10407 prototype strain. In contrast, LT secretion was inhibited. Furthermore, under iron starvation, gene expression of the cfa (CFA/I) and etp (EtpBAC) operons was induced, whereas transcription of toxin genes was either unchanged or repressed. Transcriptional reporter fusion experiments focusing on the cfa operon further showed that iron starvation stimulated cfaA promoter activity in ETEC, indicating that the impact of iron on CFA/I production was mediated by transcriptional regulation. Evaluation of cfaA promoter activity in heterologous E. coli single mutant knockout strains identified IscR as the regulator responsible for inducing cfa fimbrial gene expression in response to iron starvation, and this was confirmed in an ETEC ΔiscR strain. The global iron response regulator, Fur, was not implicated. IscR binding sites were identified in silico within the cfaA promoter and fixation confirmed by DNase I footprinting, indicating that IscR directly binds the promoter region to induce CFA/I. IMPORTANCE: Pathogenic enterobacteria modulate expression of virulence genes in response to iron availability. Although the Fur transcription factor represents the global regulator of iron homeostasis in Escherichia coli, we show that several ETEC virulence factors are modulated by iron, with expression of the major fimbriae under the control of the iron-sulfur cluster regulator, IscR. Furthermore, we demonstrate that the apo form of IscR, lacking an Fe-S cluster, is able to directly fix the corresponding promoter region. These results provide further evidence implicating IscR in bacterial virulence and suggest that IscR may represent a more general regulator mediating the iron response in enteropathogens.

Deep longitudinal multi-omics analysis of Bordetella pertussis cultivated in bioreactors highlights medium starvations and transitory metabolisms, associated to vaccine antigen biosynthesis variations and global virulence regulation.

Bordetella pertussis is the bacterial causative agent of whooping cough, a serious respiratory illness. An extensive knowledge on its virulence regulation and metabolism is a key factor to ensure pertussis vaccine manufacturing process robustness. The aim of this study was to refine our comprehension of B. pertussis physiology during in vitro cultures in bioreactors. A longitudinal multi-omics analysis was carried out over 26 h small-scale cultures of B. pertussis. Cultures were performed in batch mode and under culture conditions intending to mimic industrial processes. Putative cysteine and proline starvations were, respectively, observed at the beginning of the exponential phase (from 4 to 8 h) and during the exponential phase (18 h 45 min). As revealed by multi-omics analyses, the proline starvation induced major molecular changes, including a transient metabolism with internal stock consumption. In the meantime, growth and specific total PT, PRN, and Fim2 antigen productions were negatively affected. Interestingly, the master virulence-regulating two-component system of B. pertussis (BvgASR) was not evidenced as the sole virulence regulator in this in vitro growth condition. Indeed, novel intermediate regulators were identified as putatively involved in the expression of some virulence-activated genes (vags). Such longitudinal multi-omics analysis applied to B. pertussis culture process emerges as a powerful tool for characterization and incremental optimization of vaccine antigen production.

Different innate signatures induced in human monocyte-derived dendritic cells by wild-type dengue 3 virus, attenuated but reactogenic dengue 3 vaccine virus, or attenuated nonreactogenic dengue 1-4 vaccine virus strains.

DNA microarrays were used to assess the innate gene signature in human myeloid dendritic cells infected with chimeric dengue 1-4 vaccines, a wild-type dengue 3 virus, or a classically attenuated serotype 3 vaccine shown to be reactogenic in humans. We observed a very reproducible signature for each of the 4 chimeric dengue vaccines, involving stimulation of type I interferon and associated genes, together with genes encoding chemokines and other mediators involved in the initiation of adaptive responses. In contrast, wild-typeDEN3 virus induced a predominantly inflammatory profile, while the reactogenic attenuated serotype 3 vaccine appeared to induce a blunted response.

Optimized enzyme-linked immunosorbent assay for detecting cytomegalovirus infections during clinical trials of recombinant vaccines.

BACKGROUND: In clinical trials of cytomegalovirus (CMV) glycoprotein B (gB) vaccines, CMV infection is detected by first depleting serum of anti-gB antibodies and then measuring anti-CMV antibodies with a commercially available enzyme-linked immunosorbent assay (ELISA) kit, with confirmation of positive findings by immunoblot. OBJECTIVES: Identification of CMV immunoantigens for the development of an ELISA that detects specifically CMV infection in clinical samples from individuals immunized with gB vaccines. STUDY DESIGN: Sensitivity and specificity of ELISAs using antigenic regions of CMV proteins UL83/pp65, UL99/pp28, UL44/pp52, UL80a/pp38, UL57, and UL32/pp150 were measured. RESULTS: An IgG ELISA using a UL32/pp150 [862-1048] capture peptide was the most specific (93.7%) and sensitive (96.4%) for detecting CMV-specific antibodies in sera. The ELISA successfully detected CMV-specific antibodies in 22 of 22 sera of subjects who had been vaccinated with a gB vaccine but who had later been infected with CMV. The ELISA was linear over a wide range of CMV concentrations (57-16,814 ELISA units/mL) and was reproducible as indicated by a 5% intra-day and 7% inter-day coefficients of variation. The signal was specifically competed by UL32/pp150 [862-1048] peptide but not by CMV-gB or herpes simplex virus 2 glycoprotein D. Lipid and hemoglobin matrix did not interfere with the assay. CONCLUSION: The UL32/pp150 [862-1048] IgG ELISA can be used for the sensitive and specific detection of CMV infection in gB-vaccinated individuals.

CD4 T cell antigens from Staphylococcus aureus Newman strain identified following immunization with heat-killed bacteria.

Staphylococcus aureus is a commensal bacterium associated with the skin and mucosal surfaces of humans and animals that can also cause chronic infection. The emergence of antibiotic-resistant strains such as methicillin-resistant S. aureus (MRSA) and strains causing chronic intramammary infections (IMI) in cows results in severe human and livestock infections. Conventional approaches to vaccine development have yielded only a few noneffective vaccines against MRSA or IMI strains, so there is a need for improved vaccine development. CD4 T lymphocytes are required for promoting gamma interferon (IFN-γ) mediated immunoglobulin isotype switching in B lymphocytes to produce high-affinity IgG antibodies and IFN-γ-mediated phagocyte activation for an effective resolution of bacterial infection. However, the lack of known CD4 T cell antigens from S. aureus has made it difficult to design effective vaccines. The goal of this study was to identify S. aureus proteins recognized by immune CD4 T cells. Using a reverse genetics approach, 43 antigens were selected from the S. aureus Newman strain. These included lipoproteins, proteases, transcription regulators, an alkaline shock protein, conserved-domain proteins, hemolysins, fibrinogen-binding protein, staphylokinase, exotoxin, enterotoxin, sortase, and protein A. Screening of expressed proteins for recall T cell responses in outbred, immune calves identified 13 proteins that share over 80% sequence identity among MRSA or IMI strains. These may be useful for inclusion in a broadly protective multiantigen vaccine against MRSA or IMI.