ACTIVE vs PASSIVE IMMUNIZATION: ADVANTAGES AND DISADVANTAGES OF EACH APPROACH Arturo Casadevall Albert Einstein College of Medicine Bronx, New York DEFINITIONS • ACTIVE IMMUNIZATION = VACCINES • PASSIVE IMMUNIZATION = PREFORMED ANTIBODY ADMINISTRATION ACTIVE IMMUNIZATION - VACCINES ADVANTAGES DISADVANTAGES • LONG PROTECTION • DEPENDENT ON HOST • LESS COSTLY (TO ADMINISTER) IMMUNE SYSTEM – NOT EVERYONE MAY BE PROTECTED • PROTECTIVE RESPONSE TAKES TIME • MAY BE IMPRACTICAL FOR NOSOCOMIAL INFECTION • LONG DEVELOPMENT TIME • ? EFFECTS ON MICROFLORA PASSIVE IMMUNIZATION ADVANTAGES DISADVANTAGES • IMMEDIATE IMMUNITY • COSTLY • PHARMACOLOGIC CONTROL • TEMPORARY IMMUNITY • RAPID DEVELOPMENT TIME • REQUIRES IV ADMINISTRATION • DOSE-RESPONSE UNCERTAINTY REAGENTS FOR PASSIVE THERAPY: mAbs ADVANTAGES DISADVANTAGES • HIGH SPECIFIC ACTIVITY • COST • PRECISE PHARMACOLOGIC • SINGLE SPECIFITY DEFINITION – SELECTION FOR ESCAPE MUTANTS – EFFECTIVENESS α 1/ANTIGENIC • SINGLE ISOTYPE DIVERSITY • LOT TO LOT CONSTANCY • SINGLE ISOTYPE – LIMITED EFFECTOR FUNCTION – LIMITED FcR ENGAGEMENT REAGENTS FOR PASSIVE THERAPY: POLYCLONAL SERA ADVANTAGES DISADVANTAGES • MULTIPLE SPECIFICITIES • LOW SPECIFIC ACTIVITY • MULTIPLE ISOTYPES • LOT TO LOT VARIATION • REQUIREMENT FOR IMMUNIZED DONORS • COST • CONCERN ABOUT INADVERTENT TRANSMISSION OF DISEASE NEW INSIGHTS INTO ANTIBODY-MEDIATED PROTECTION FROM STUDIES WITH A FUNGAL PATHOGEN Or 20 years of work in 18 minutes C. NEOFORMANS – ONLY ENCAPSULATED EUKARYOTIC PATHOGEN OF HUMANS CAPSULE IS THE MAJOR VIRULENCE FACTOR POLYSACCHARIDE CAPSULE ? GLUCURONOXYLOMANNAN MAJOR VIRULENCE FACTOR WEILER HOSPITAL, NEW YORK CITY SUMMER 1988 30-SOMETHING CAMBODIAN MAN WITH C. NEOFORMANS MENINGITIS DIES AGONIZING DEATH DESPITE RECEIVING LARGE DOSES OF AMPHOTERICIN B DURING A PERIOD OF 3 MONTHS… COULD ANTIBODY THERAPY BE DEVELOPED? CIRCA 1990 ADVICE • WORK ON RELEVANT IMMUNOLOGY – CELL MEDIATED IMMUNITY • IF YOU WANT A SCIENTIFIC CAREER DON’T WORK ON USEFUL THINGS •GET A DIFFERENT PROJECT – YOU ARE GOING TO DISCOVER WHAT WE KNOW ABOUT PNEUMOCOCCUS IMMUNOLOGICALTHE GREAT IMMUNOLOGICAL PARADIGMS CATASTROPHE 1970-1990s 1891-1910 1910-1940 1940-1960s HUMORAL IMMUNITY HUMORAL IMMUNITY VIRUSES VIRUSES TOXINS TOXINS ENCAPSULATED BACTERIA ENCAPSULATED BACTERIA “T CELLS ARE USEFUL ONLY FOR STIMULATING B CELLS” HUMORAL ANTIBODY Th2 VS PROTECTS PHAGOCYTIC AGAINST THEORIES ALL MICROBES Th1 ‘TISSUE’ IMMUNITY CELLULAR IMMUNITY MYCOBACTERIA MYCOBACTERIA FUNGI FUNGI INTRACELLULAR PATHOGENS INTRACELLULAR PATHOGENS FOUR ‘HUMORAL “ANTIBODIES ARE USEFUL ONLY IN ELISAs” ‘HUMORS’ IMMUNITY GALEN METHODS FOR ESTABLISHING USEFULNESS OF ANTIBODY-MEDIATED PROTECTION 1. PASSIVE TRANSFER EXPERIMENTS (1891) IMMUNE SERA + INFECTIOUS PROTECTION? SUSCEPTIBLE HOST CHALLENGE LOGICAL ERROR: 2. CORRELATEJUST ANTIBODY BECAUSE THE TITER ASSAY WITH SUGGESTED RESISTANCE THAT (1900s)ANTIBODY WAS NOT SUSCEPTIBILITY 1/titerPROTECTIVE DID NOT MEAN THAT ANTIBODY COULD 3. ASSOCIATE SUSCEPTIBILITY WITHNOT BE ANTIBODY PROTECTIVE DEFICIT (1940s) ERROR INVOLVED MAKING A POSITIVE INFERENCE FROM PROBLEM: THESE METHODSNEGATIVE SUGGEST DATA SPECIFIC ANTIBODY IS NOT IMPORTANT IN DEFENSE AGAINST MANY PATHOGENS e.g. Mycobacterium tuberculosis Cryptococcus neoformans Listeria monocytogenes Many others…. PROBLEM: EXPERIMENTS WITH POLYCLONAL SERA ARE INCONCLUSIVE A DIFFERENT APPROACH TT IMMUNIZE GXM MAKE Y Y Y Y Y 100 mAbs IgG2a 80 60 40 PROTECTION STUDIES of Survival % 20 0 0 20 40 60 80 100 120 140 160 Days NON-PROTECTIVE mAb PROTECTIVE mAb YYYYYY YYYYYY NON-PROTECTIVE mAbs INHIBITED EFFICACY OF PROTECTIVE mAbs ANTIBODY-MEDIATED PROTECTION AGAINST M. TUBERCULOSIS CONTROL CONFIRMATION (5 independent labs): PETHE ET AL. NATURE 2001:412;190 HAMASUR ET AL. VACCINE 2003:20:4081 WILLIAMS ET AL. IMMUNOLOGY 2004:111;328 CHAMBERS ET AL. FEMS IMMUNOL.MED.MICROBIOL. 2004:41;93 PROC.NATL.ACAD.SCI 1998:95;15688 HAMESURE ET AL. CLIN.EXP.IMMUNOLmAb 9d8 2005: 138:30-8 THE CMI vs. HUMORAL PARADIGM UNTENABLE PROTECTIVE MAbs MADE TO: HUMORAL IMMUNITY VIRUSES CRYPTOCOCCUS NEOFORMANS (FUNGUS) TOXINS ENCAPSULATED BACTERIA CANDIDA ALBICANS (FUNGUS) LISTERIA MONOCYTOGENES (INTRACELLULAR BACTERIA) HISTOPLASMA CAPSULATUM (INTRACELLULAR FUNGUS) MYCOBACTERIUM TUBERCULOSIS (INTRACELLULAR BACTERIA) NEW APPROACH USE MAbs EHRLICHIA CHAFFENSIS (INTRACELLULAR BACTERIA) LEISHMANIA MEXICANA (INTRACELLULAR PARASITE) CELLULAR IMMUNITY MYCOBACTERIA FUNGI TWO EMERGING EXPLANATIONS SOME BACTERIA VIRUSES ‘GOOD & BAD’ INADEQUATE AMOUNTS TOXINS (SUPERANTIGENS) C. neoformans L. monocytogenes C. albicans H. capsulatum M. tuberculosis WHY IS IT SO HARD TO PROTECT AGAINST SOME MICROBES WITH ANTIBODY? THE THINGS THAT MATTER…. • SPECIFICITY • ISOTYPE • AMOUNT IgM IgG • DEPENDENCE ON HOST PROTECTIVE NON- PROTECTIVE NON- PROTECTIVE PROTECTIVE CELL-MEDIATED IMMUNITY PATTERN ANNULAR PUNCTATE ANNULAR ANNULAR GENETICS OPSONIC YES NO YES YES INHIBITS YES NO YES YES PS RELEASE ISOTYPE NA NA IgG1, IgG2a, IgG3 IgG2b NEED C3 YES NO NO NO TWO STORIES: IgM – EFFICACY REQUIRES ‘RIGHT’ SPECIFICITY AND COMPLEMENT IgG - EFFICACY REQUIRES ‘RIGHT’ ISOTYPE AND HOST WHAT IS THE DIFFERENCE BETWEEN PROTECTIVE AND NON- PROTECTIVE ANTIBODIES? PARENTAL mAb 12A1 B CELL mAb 13F1 9F11 IgG1 7G7 IgM 4G9 IgG1 16E4 PROTECTIVE IgM NON-PROTECTIVE 12A1 15E8 IgM IgM 12F4 IgG1 14E1 13F1 IgG1 IgM CLINICAL 18B7 25G12 IgA TRIAL IgG1 USED BY VH7183 JH2 BALB/c 7 aa D NZB/NZW Casadevall & Scharff J Exp Med. 1991;174(1):151-60 C3H/He Mukherjee et al J Exp Med. 1993;177(4):1105-16 HUMAN USE V 3 Mukherjee et al J Exp Med. 1995 Jan 1;181(1):405-9 H Nussbaum et al J Exp Med. 1997 Feb 17;185(4):685-94. Vk5.1 Jk1 POSITIONS 33 AND 57 IN PROTECTIVEAND NON-PROTECTIVE CLASS II MAbS TO C. NEOFORMANS POLYSACCHARIDE mAb 33 57 PROTECTIVE IF 3B10 F N YES ANNULAR 2D10 F R YES ANNULAR 2H1 F K YES ANNULAR 3E5 F N YES ANNULAR 18G9 F K YES ANNULAR 10F10 F S YES ANNULAR 12A1 F N YES ANNULAR 17E12 F K YES ANNULAR 18B7 F K YES ANNULAR 471 L K YES ANNULAR 3C2 L K YES ANNULAR 13F1 Y S NO PUNCTATE 21D2 Y S NO PUNCTATE ANTIBODY BINDING SITE FROM X-RAY CRYSTAL STRUCTURE 12A1 N Y F M S W V M I N I N G N N T Y Y P D T V K G D R D G T F G N Y Y A M ANNULAR S31N S - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - ANNULAR F33Y - - Y - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - ANNULAR M50A - - - - - - - A - - - - - - - - - - - - - - - - - - - - - - - - - - - - ANNULAR I53S - - - - - - - - - - S - - - - - - - - - - - - - - - - - - - - - - - - - ANNULAR D56G - - - - - - - - - - - - - G - - - - - - - - - - - - - - - - - - - - - - ANNULAR N57S - - - - - - - - - - - - - - S - - - - - - - - - - - - - - - - - - - - - ANNULAR D80Y - - - - - - - - - - - - - - - - - - - - - - - - Y - - - - - - - - - - - ANNULAR TYROSINE PHENYLALANINE G103Y - - - - - - - - - - - - - - - - - - - - - - - - - - - - - Y - - - - - - ANNULAR F33Y,N57S - - Y - - - - - - - - - - - S - - - - - - - - - - - - - - - - - - - - - PUNCTATE 13F1 S - Y - - - - A - - - - - G S - - - - - - - - - Y - - - - Y - - - - - - PUNCTATE Young et al. J. Mol.Biol. 1997 Nakouzi et al Infect Immun. 2001;69(5):3398-409 COMPLEMENT-INDEPENDENT IgM-MEDIATED PHAGOCYTOSIS OF C. NEOFORMANS CD18 BINDING SITE mAb 13F1 mAb 12A1 CR3 CR3 MACROPHAGE Taborda et al. Immunity 2002 PROTECTIVE AND NON-PROTECTIVE IgM mAbs MEDIATE C3 LOCALIZATION IN DIFFERENT PARTS OF CAPSULE LIGHT IgM C3 MERGE FUNGAL CELLS IN VIVO COATED WITH COMPLEMENT IS DEPOSITED IgM IN ‘PUNCTATE PATTERN IN VIVO AWAY FROM SURFACE PROTECTIVE Ce NON- PROTECTIVE Zaragoza et al. Eur. J. Immunol 2003 Zaragoza et al Infect Immun 2004 Zaragoza et al. Cell Micro 2006 C. neoformans BIOFILM FORMATION 2 h 4 h 8 h 1.6 1.4 B3501 24067 1.2 H99 I23 1 S. cerevisiae C. albicans 50 µm 0.8 OD 492 nm OD 492 0.6 0.4 0.2 0 2 4 8 24 48 Time (h) 16 h 24 h 48 h Adhesion Microcolony Maturation 5 µm PROTECTIVE ANTIBODIES BLOCK BIOFILM FORMATION No Ab MAb 18B7 MAb 3671 (control) 50 µm 50 µg/ml 50 µg/ml BCIP Y Vectastain Y Biotin-GAM-IgM 2D10 100 µg/ml 100 µg/ml 1.6 B3501 1.4 100 24067 B3501 H99 1.2 80 1 60 C. neoformans C. p<0.05 0.8 40 OD 492 nm OD 492 0.6 biofilmsmetabolic activity 20 * Percentage of Percentage * 0.4 0 No Ab 50 100 50 100 50 100 50 100 ug/ml ug/ml ug/ml ug/ml ug/ml ug/ml ug/ml ug/ml 0.2 12A1 12A1 13F1 13F1 21D2 21D2 5C11 5C11 0 PROTECTIVE NON-PROTECTIVE IRRELEVANT No Ab 50 ug/ml 18B7 100 ug/ml 18B7 50 ug/ml 3671 100 ug/ml 3671 Exopolymeric matrix No Ab Y Ab Y Y Ab cross-links Y Y Biofilm Y capsule Interference with Y biofilm formation HOW DOES ANTIBODY INTERFERE WITH FUNGAL PHYSIOLOGY? IgG AND C3 OPSONIZATION HAVE DIFFERENT OUTCOMES IN EXIT DISPERSION IgG OPSONIZATION C3 OPSONIZATION ANTIBODY BINDING CAN ALTER MICROBIAL GENE EXPRESSION PROTECTIVE IgG1 PROTECTIVE IgM NON-PROTECTIVE IgM RELATIVE TO RELATIVE TO RELATIVE TO IRRELEVANT CONTROL IRRELEVANT CONTROL IRRELEVANT CONTROL McClelland, Nicola, Prados, Casadevall J. Clin Invest 2010 JANOFF & FRANK, JCI 2010 (IN PRESS) COMMENTARY MTb RESPONSE UPON CAPSULAR MAB TREATMENT Mid-log phase H37Rv defined MM culture 10 mg/ml Mab 45 min Expression ratio: Capsular Mab/isotype IgG MORE IS NOT BETTER: PROZONE-LIKE EFFECTS 100 90 80 70 60 50 40 70 30 0.05 20 PercentSurvival 10 0.1 60 0 0.2 Culture (ml) 50 0.4 0.2 0.1 0.4 0.05 0.025 40 0.0125 0.00625 0.00156 30 0.003125 Horse Immune Sera (ml) 20 Average Survival (d) Survival Average 3 10 10 PNEUMOCOCCUS 1935 4 10 5 0 10 Innoculum7 A SINGLE mAb CAN BE PROTECTIVE, 1 6 0.5 10 0.1 NON-PROTECTIVE OR DISEASE control mAb 12A1 (mg/mouse) ENHANCING DEPENDING ON ITS CONCENTRATION AND INOCULUM C. NEOFORMANS