CE17 - Recherche translationnelle en santé

TnSeq Vaccinology: from pathophysiology studies to multivalent vaccine design – Seq-N-Vac

TnSeq Vaccinology: from pathophysiology studies to multivalent vaccine design

In developed countries, E. coli K1, is the leading cause of meningitis in premature neonates and the second cause, after group B streptococci in term neonates. Despite the use of advanced antibiotics, the morbidity and mortality rates associated with E. coli K1 meningitis remain unchanged over the last few decades. Therefore, new modes of prevention and treatment are warranted. It will require a better understanding of E. coli K1 pathogenesis and disease pathophysiology.

Development of a multivalent vaccine against E. coli K1 neonatal meningitis.

Using saturated transposon (Tn) mutagenesis and high-throughput sequencing (TnSeq), a powerful tool to investigate host-pathogen interactions, we will fill-in-the-gaps in our knowledge about the contribution to overall fitness of the multitude of genes in E. coli K1 needed to cause neonatal meningitis. In this project, we propose an approach to (i) comprehensively evaluate the virulence factors of E. coli K1 in neonatal meningitis and (ii) systematically identify the antigens synthesized by this pathogen that can be targeted by immunotherapy (TnSeq Vaccinology). Using P. aeruginosa as proof of concept, three different in vivo settings relevant to P. aeruginosa pathogenesis were previously tested by TnSeq: infection of the lung, colonization of the gastro-intestinal (GI) tract and systemic dissemination to the spleen. In these three settings, Tn-inserts into almost all of the genes encoding known virulences factors of P. aeruginosa resulted in decreased in vivo fitness, but, importantly, many new ones, including new candidate targets for immunotherapy, were also detected. In the research project proposed here, we will extend our expertise with the TnSeq technology to analyze E. coli K1 meningitis pathogenesis, and will also implement an original aspects of TnSeq, the TnSeq-vaccinology, to provide a unique view of meningitis from basic pathogenesis to the prospect of a multivalent vaccine. This vaccine development strategy, if effective, could also target multi or pan-antibiotic resistant strains and contribute to the decrease in antibiotic use and reduce selective pressures for emergence of antibiotic resistance.

TnSeq: In order to investigate E. coli K1 pathophysiology, we established two mouse models of neonatal infection. The first in vivo model used 2 to 3 day-old mice given an intraperitoneal (IP) challenge with 5x10e6 Colony Forming Units (CFU)/mouse of an E. coli K1 Tn-bank that generated a high level of bacteremia. After 24h, the neonatal mice were sacrificed, and the livers, spleens, brains and meninges harvested to determine systemic dissemination followed by brain infection (above 10e8 CFU/organ). The second model was designed to mimic the steps of pathogenesis of neonatal meningitis caused by E. coli K1. Neonatal mice were challenged by oral gavage, as the gastro-intestinal (GI) tract is likely the main site of initial colonization and origin for meningitis pathogens in neonates. After 24h, the neonatal mice were sacrificed and the livers, spleens, brains and meninges harvested. These two models allow determinations of (i) genes important for systemic dissemination, following either IP infection or after GI tract colonization (both routes are seen in human infection), and (ii) genes important for brain infection following the route of infection thought to occur in the human disease (from the GI tract to the brain).

TnVaccine: the E. coli K1 bank of mutants will be under the selective pressure of polyclonal antibodies against E. coli K1, in order to detect the escape mutants in the genes encoding for the antigens of interest.

Among the candidates identified by TnSeq, we selected for further study the conserved surface polysaccharide Poly-ß-(1-6)-N-Acetyl Glucosamine (PNAG), as a strong candidate for vaccine development. We found that PNAG was a virulence factor in our animal model. We showed that both passive and active immunization successfully prevented and/or treated meningitis caused by E. coli K1 in neonatal mice. We found an excellent opsonophagocytic killing activity of the antibodies to PNAG and in vitro these antibodies were also able to decrease binding, invasion and crossing of E. coli K1 through two blood brain barrier cell lines. Finally, to reinforce the potential of PNAG as a vaccine candidate in bacterial neonatal meningitis, we demonstrated that Group B Streptococcus, the main cause of neonatal meningitis in developed countries, also produced PNAG and that antibodies to PNAG could protect in vitro and in vivo against this major neonatal pathogen.

Altogether, these results indicate the utility of a high-throughput DNA sequencing method to identify potential immunotherapy targets for a pathogen, including in this study a potential broad-spectrum target for prevention of neonatal bacterial infections.

N/A

Among infectious diseases, neonatal bacterial meningitis continues to be a serious infection and despite treatment with appropriate antibiotics, mortality rates over 10% still occur and up to 30% of survivors exhibit neurological sequela such as hearing and neurologic impairment, and hydrocephalus. In industrialized Escherichia coli K1 is a major of neonatal meningitis. The goal of the proposed research is to use cutting edge, high throughput sequencing technology (TnSeq) and a new applications (TnSeq Vaccinology) to (i) identify and studythe genes and regulatory genetic elements contributing to optimal fitness E. coli K1 in major steps leading to bacterial meningitis in neonates: systemic dissemination and crossing of the blood brain barrier and, (ii), identify and develop new vaccine candidates.

Project coordination

David Skurnik (Institut National de la Santé et de la Recherche Médicale)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

INSTITUT PASTEUR
INSERM Institut National de la Santé et de la Recherche Médicale
INSERM Institut Nationale de la Santé et de la Recherche Médicale

Help of the ANR 549,662 euros
Beginning and duration of the scientific project: February 2021 - 48 Months

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