Anti-Chlamydia Vaccine: From Bench to Bedside Ashlesh Murthy, M.B.B.S., Ph.D Research Assistant Professor University of Texas at San Antonio

Chlamydia Obligate intracellular pathogen C. trachomatis, C. pneumoniae, C. psittaci, C. pecorum, C. muridarum C. trachomatis multiple serovars: A-K, L1-3

Chlamydia trachomatis Infections Trachoma and Inclusion Conjunctivitis Serovars - A, B, Ba, C Urogenital Infections Serovars - D, Da, E, F, G, Ga, H, I, J, K LymphoGranuloma Venereum Serovars - L1, L2, L3

Global Prevalence of Chlamydial STD

Chlamydial STD Adapted from http://www.cdc.gov/std/stats/tables/table1.htm

Chlamydial STD-Ascending Infection Eskilsden and Gupta et al., In Revision

Chlamydial STD-Ascending Infection PID No Symptoms Dye alone Dye+Chlamydia Vaginitis Cervicitis Endometritis Salpingitis Antimicrobials

Chlamydial STD-Reproductive Damage Pelvic Inflammatory Disease Ectopic pregnancy Infertility Normal

Chlamydial STD- Statistics 90 million cases 50-70% of patients initially asymptomatic: not treated 40% of untreated females – pelvic inflammatory disease 20% of PID cases- infertility Adapted from www.cdc.gov- 2006 STD statistics

Anti-Chlamydial Vaccine

Goals for Chlamydial Vaccine No Symptoms (75%) Immunity Transmission Symptoms (25%) Re-Infection Sequelae Infection Prevent infection (Sterilizing immunity)- ideal Reduce transmission / Duration of shedding- practical Prevent pathological sequelae Protection against multiple serovars

Chlamydial Vaccine Trials of 1960s Formalin-killed whole chlamydial organisms Reduction in incidence for ~ 1 year Comparable/exaggerated ocular pathology in vaccinees Focus on sub-unit vaccines

Chlamydial Developmental Cycle

Vaccines Against Chlamydial STD Major outer membrane protein (MOMP) Chlamydia-purified MOMP Recombinant MOMP MOMP DNA MOMP synthetic peptides MOMP plus outer membrane protein 2 (omp2) Chlamydia-purified MOMP refolded to native configuration Cons: MOMP - serovar-specific Currently no licensed vaccine against C. trachomatis Need for identification of new vaccine candidates

Chlamydial Developmental Cycle

Chlamydial Protease-Like Activity Factor (CPAF) Secreted into host cytosol Nucleus Inclusion CPAF Overlay

CPAF-Human Antibody Responses Sharma et al., Infect Immun. 2006

CPAF-Highly Conserved Dong et al., Infect Immun. 2005

CPAF- A Potential Vaccine Candidate

Mouse Model of Chlamydial Infection C. muridarum 106 Vaginal chlamydial shedding Oviduct dilatation 30 80 Days after challenge

Protocol For Vaccination Studies Intranasal Day –1: IL-12 (0.5 mg) Day 0:rCPAF(15mg)+ IL-12 (0.5 mg) Day +1: IL-12 (0.5 mg) Recombinant CPAF from C. trachomatis L2 cloned into E. coli Day 14: rCPAF (15 mg)+IL-12 (0.5 mg) Day 28: rCPAF (15 mg)+ IL-12 (0.5 mg) 30 day Rest Vaginal challenge Estimation of bacterial shedding Examination of disease pathology Day 60: 5X104 IFU C. muridarum

Early Responses to Immunization

CPAF Vaccine : Robust IFN-g Response Murthy et al., Infect Immun. 2007

CPAF Vaccine: Systemic Antibodies Murthy et al., Infect Immun. 2007

CPAF Vaccine: Mucosal Antibodies Murthy et al., Infect Immun. 2007

CPAF Vaccine: Chlamydial Clearance Murthy et al., Infect Immun. 2007

CPAF+IL-12: Prevents Hydrosalpinx Murthy et al., Infect Immun. 2007

CPAF Vaccine: Preserves Fertility Murthy et al., In Review, 2010

Role of Human HLA-DR Murthy et al., Infect Immun. 2006

Minimal Role for Antibody Murthy et al., FEMS Immunol. Med. Microbiol. 2009

Role of CD4+ T-cells CD4+ T cell adoptive transfer Murphey et al., Cell. Immunol. 2006

Role of IFN-g Murthy et al., J. Immunol. 2008

Summary CPAF vaccine administered with Th1 adjuvant: Enhances clearance of infection Protects against severe pathology Induces robust cellular IFN-g response Induces systemic and mucosal antibody Protection is dependent upon CPAF-specific cellular IFN-g responses but not antibody

Ongoing Collaborative Effort US Patent Application No. 12/243,769: Inventors- Arulanandam, Murthy, Zhong

Bench to Bedside Anti-Chlamydia vaccine Identification of pathogenic mechanisms Vaccine antigen discovery Validation of protection and pathogenesis correlates in clinical samples Routes, Delivery systems Adjuvants, Formulations Non-Human Primates Clinical Trials Anti-Chlamydia vaccine

Acknowledgements Bernard Arulanandam, Ph.D., M.B.A. Weidang Li, M.D., Ph.D. Bharat Chaganty, MS Sangamithra Kamalakaran, B.Tech Kishan Evani, MS Rishein Gupta, Ph.D. Yu Cong, M.D. Madhulika Jupelli, Ph.D. Cathi Murphey, M.S. Guangming Zhong, M.D., Ph.D., UTHSCSA M. Neal Guentzel, Ph.D., STCEID, UTSA Aruna Mittal, Ph.D, IOP, India 1R03AI088342