Strategies toward developing a universal ExPEC vaccine

capable of broad protectionMelha Mellata, PhD.

Center for Infectious Diseases and Vaccinology, Arizona State University,

Tempe, AZ, 85287-5401, USAMelha.mellata@asu.edu

Valencia, September 24-26-2014

Background

Escherichia coliNon-Pathogenic

E. coli

Pathogenic E. coli

Intestinal tropism Extra-Intestinal tropism (ExPEC)

EPEC EHECO157:H7

EIEC

Sepsis

EAECETEC

Diarrhea

UPEC NMEC APEC

Septicemia

90% in humans, 56% in wild mammals, 23% in birds 10% in reptiles

Meningitis

UTI-pyelonephritis

AirsaculitisPericarditis

PerihepatitisCellulitis

MeningitisSepticemia

Treatment failures due to ATB resistance increase the cost of care and results in prolonged morbidity for patients.

As the proportion of elderly and immunocompromised patients is increasing rapidly, the number of E. coli infections will undoubtedly increase, and their treatment will be more challenging

vaccines, are needed to manage these infections in the future

The difficulty of developing an effective vaccine against ExPEC is related to their diversity.

Mellata, 2013, Foodborne Pathog Dis

Contrary to commensal E. coli, ExPEC strains have acquired specific virulence attributes that confer an ability to survive in different niches outside of their normal intestinal habitat in both mammals and birds: Colonization, Invasion, Iron uptake, Serum-complement resistance, Antiphagocytic activity, Regulation

Efficacious vaccines should target virulence factors involved in key steps of pathogenesis and should be broadly distributed among these bacteria.

Table 1. Antigens included in our vaccineVF Antigens Relevant characteristics RolesAdhesin EcpA Major shaft subunit of E. coli common pilus (ECP). Adhesion, invasion, biofilm

EcpD Polymerized tip adhesin subunit of E. coli common pilus (ECP).Siderophores IutA A ferric aerobactin receptor used for iron acquisition.. Iron-uptake

IroN Salmochelin uptake receptor used for iron acquisition.

rAntigens were tested in mice either as : Individual antigens (IutA, IroN, EcpA, EcpD), Two antigens (IutA/IroN and EcpA/EcpD), All four antigens [at high dose (80 g) or low dose (40 g)]

Schematic diagram of the immunization and challenge studies

Do antigens elicit antibodies?Are they protective?

IgG against EcpA and EcpD were detected in mice sera as early as day 21 post-vaccination

IgG against IutA and IroN were only detected three weeks after the second vaccination

Depending on the nature of the antigens included in the vaccine, their specific total IgG were elicited differently in vaccinated mice

Iron-uptake antigensAdhesin antigens

Sera tested were pooled from 3 mice from the same group. Data are representative of 2 experiments of similar design

Total IgG antibodies titres in vaccinated mice

The levels of antigen-specific IgG elicited in vaccinated mice depended on:

the nature of the antigens, their combinations with other antigens

the concentration of proteins

in the vaccine dose injected

Levels of antigen-specific IgG elicited Sera tested were from individual mice. The values are means ± standard deviations for 10 mice from each group.

Profile of IgG1 and IgG2a antibody responses in mice Both humoral and cell mediated responses are important in a vaccine against ExPEC, as T-cell response is

needed for the clearance of the intracellular bacteria. The determination of IgG2a and IgG1 isotypes as markers for Th1 (cell mediated immunity) and Th2

(antibody mediated immunity) lymphocytes respectively, was investigated in vaccinated mice.

Our data show an induction of both humoral- and cell-mediated immunity in vaccinated mice.

EcpA and IutA equally stimulated Th1 and Th2

immune responses

EcpD elicited a predominantly Th2-

type response

IroN elicited a predominantly Th1-

type response

Expression of antigens in different growth conditions

CFT073, isolated from the blood and urine of a hospitalized patient with acute pyelonephritis

Are elicited Antibodies protective?Challenge experiments

Lethal challenge

Elicited Antibodies are protective!!!!

Mice were regularly monitored for up 48 hours after infections for deaths

Mice vaccinated with EcpA+EcpD, all antigens at high dose, EcpD, and EcpA respectively had better survivability to the bacterial CFT073 challenge (2.2 x 108 CFU) than the non-vaccinated group

All vaccinated groups had better protection than the PBS group

100% protectionEcpD, EcpA+EcpD, and all antigens at high dose

70% protectionEcpA, IutA+IroN, and all antigens at low dose

50% protectionIroN and IutA

Non-Lethal challenge Vaccinated and non-vaccinated mice were IP. challenged with ~5.5 107 CFT073 grown in DMEM +

Mannose + 2’2’ dipyridyl standing for 48 hours Bacterial loads were determined in organs (spleen, liver) and blood at 24 hours post-challenge

Mice vaccinated had significantly less bacteria in spleen, liver, and blood compared to the non-vaccinated group

Conclusions

In this study, selected E. coli antigens were tested in different vaccine formulas for their ability to elicit specific humoral responses (Th1/Th2) and provide protection against sepsis E. coli challenge in two sepsis mouse models.

A protective vaccine against ExPEC would have outstanding public health advantages. The significant increases in humoral immunity and decreases in CFU recovery in vaccinated mice depict a first step towards the development of a broadly protective vaccine against ExPEC.

In future studies, the same vaccines could be tested in other animal models of human and chicken ExPEC infections, including female adult mice (urethral infection) and newborn rats (meningitis infection) to determine if our vaccine could have a universal application.

Roy Curtiss IIINatalie Mitchell

Financial supports

NIH : R21 AI090416 USDA/NIFA: 2011-67005-30182

Scientific advisorsTechnical assistance

Kristen MorrowFlorian Schödel

Acknowledgments

Undergraduate student

Professor and director, Center for Infectious Diseases and Vaccinology and Center for Microbial Genetic

Engineering

Research Technologist

Independent pharmaceutical consultant and owner at

Philimmune LLC, consultant/adviser for vaccines construction.