Strategies toward developing a universal ExPEC vaccine capable of broad protection

1. Strategies toward developing a universal ExPEC vaccine capable of broad protection Melha Mellata, PhD. Center for Infectious Diseases and Vaccinology, Arizona State University, Tempe, AZ, 85287-5401, USA Melha.mellata@asu.edu Valencia, September 24-26-2014

2. Background 90% in humans, 56% in wild mammals, 23% in birds 10% in reptiles Non-Pathogenic E. coli Escherichia coli Pathogenic E. coli Intestinal tropism Extra-Intestinal tropism (ExPEC) EPEC EHEC O157:H7 ETEC EIEC EAEC UPEC APEC NMEC Sepsis Diarrhea UTI-pyelonephritis Meningitis Airsaculitis Pericarditis Perihepatitis Cellulitis Meningitis Septicemia Septicemia

3. Mellata, 2013, Foodborne Pathog Dis • 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.

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

5. Schematic diagram of the immunization and challenge studies Do antigens elicit antibodies? Are they protective?

6. Total IgG antibodies titres in vaccinated mice Sera tested were pooled from 3 mice from the same group. Data are representative of 2 experiments of similar design Depending on the nature of the antigens included in the vaccine, their specific total IgG were elicited differently in vaccinated mice Adhesin antigens Iron-uptake antigens IgG against IutAand IroN were only detected three weeks after the second vaccination IgG against EcpAand EcpD were detected in mice sera as early as day 21 post-vaccination

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

8. 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

9. Are elicited Antibodies protective? Expression of antigens in different growth conditions Challenge experiments CFT073, isolated from the blood and urine of a hospitalized patient with acute pyelonephritis

10. Lethal challenge 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% protection EcpD, EcpA+EcpD, and all antigens at high dose 70% protection EcpA, IutA+IroN, and all antigens at low dose 50% protection IroN and IutA Elicited Antibodies are protective!!!!

11. 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

12. 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 ExPECinfections, including female adult mice (urethral infection) and newborn rats (meningitis infection) to determine if our vaccine could have a universal application.

13. Acknowledgments Financial supports NIH : R21 AI090416 USDA/NIFA: 2011-67005-30182 Scientific advisors Technical assistance Roy Curtiss III Florian Schödel Natalie Mitchell Kristen Morrow Independent pharmaceutical consultant and owner at Philimmune LLC, consultant/adviser for vaccines construction. Professor and director, Center for Infectious Diseases and Vaccinology       and Center for Microbial Genetic Engineering Undergraduate student Research Technologist