Käyhty, Helena

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Engineering Conferences International

Helena Käyhty, PhDNational Public Health InstituteHelsinki, FinlandDevelopment of Validated Assays for Measuring the Human Antibody Response to Polysaccharide Conjugate Vaccines2Conjugate vaccines• Haemophilus influenzae type b (Hib)• Neisseria meningitidis (Men)• Streptococcus pneumoniae (Pnc)3The capsules• For all, antibody to capsule has been proven protective via complement mediated mechanisms? Bactericidal activity (lysis)? Opsonizing for phagocytosis4Functional activity of antibodies• Serum bactericidal activity (SBA)? The ability of antibodies to mediate killing of bacteria in the presence of complement = antibody and complement mediated lysis• Serum opsonophagocytic activity (OPA)? The ability of antibodies to mediate opsonization of bacteria for phagocytosis by phagocytosing cells (PMNL, HL-60) in the presence of complement = antibody and complement mediated phagocytosis• OBS! Animal experiments do not (always) correlate with the properties of the conjugate and polysaccharide vaccines humans5Antibody concentration? For all, antibody concentration can be measured by radioimmunoassay (RIA) or enzyme immunoassay (EIA)6Immunological memory• Important for long term protection together with sustaining antibody concentrations• Challenge of memory B cells with a polysaccharide or conjugate vaccine → measure antibodies by EIA• Determination of antibody avidity maturation7Assays need to be• Carefully validated• Standardized and with good QC• Give comparable results in different laboratories? Reagents (e.g. EIA antigens and reagents, complement source)  ? Reference and QC materials? StrainsHib conjugate vaccines9Four different Hib conjugate vaccinesPRP-D HbOC PRP-OMPC PRP-TTrade nameProHIBit HibTiter PedvaxHIBActHib, HiberixManufacturerWyeth Merck sanofi pasteur,GSKCarrier Diphtheriatoxoid (D)Mutant diphtheriaToxin (CRM)Men OMP complexTetanus toxoid (T)PS μg 25 (sized) 10 (OS) 7.5 (native) 10 (native)10WHO Recommendationhttp://www.who.int/biologicals/publications/meetings/areas/vaccines/en/11Evaluation criteria? Primary endpoint: antibody concentration post primary vaccination series • Thresholds 0.15 and 1.0 µg/ml? Functional activity of antibodies measured by SBA (or OPA)? Immunologic memory• Booster response• Avidity12Challenge 1• Radioimmunoassay (RIA) used in 1970-80’s → concentrations associated with protection• Enzyme immunoassays (EIA) replaced RIA? Interlaboratory comparisons? Different protocols? Should give equivalent results to RIA• Reference serum lot 1983 and QC sera from US FDA • EIA antigen (HbOHA) from NIBSC, UK13Search of correlates for Hib PS vaccinesKäyhty ea, JID 198314Challenge 1• Radioimmunoassay (RIA) used in 1970-80’s -> concentrations associated with protection• Enzyme immunoassays (EIA) replaced RIA? Interlaboratory comparisons? Different protocols? Should give equivalent results to RIA• Reference serum lot 1983 and QC sera from US FDA • EIA antigen (HbOHA) from NIBSC, UK15Challenge 2.Continuing need of immunogenicity evaluation• Several Hib conjugate vaccines licensed and in use in developed and developing countries• Licensure of new vaccines based on immunogenicity data and safety• Component of combination vaccines• Schedules• Use in resource poor countries with high risk of Hib disease• Risk groups among adults16Useful addresshttp://www.vaccine.uab.edu/Information on hemophilus and pneumococcal assaysMeningococcal conjugate vaccines18Meningococcal conjugate vaccines• MenC? carriers: mutant diphtheria toxin (CRM) , tetanus toxoid  (T) ? used widely in Europe• MenACW135Y? Carrier: diphtheria toxoid (D)? CRM conjugate in clinical trials? Used in USA for adolescents; clinical trials in infants ongoing• MenA? Planned to be used in African meningitis belt? The Meningitis Vaccine Project 19World Health OrganizationWHO Technical Report Series, No. 926, 2004Recommendations for the production and control of group C meningococcal conjugate vaccinesAPPENDIX 1 Evaluation of the immunogenicity of group C meningococcal conjugate vaccinesWHO/BS/06.2041- 2006 FinalRECOMMENDATIONS TO ASSURE THE QUALITY, SAFETY AND EFFICACY OF GROUP A MENINGOCOCCAL CONJUGATE VACCINES20WHO Expert Committee:Thresholds associated with protection for MenC CVs:• SBA is a gold standard? Baby rabbit complement: titer of ≥8 (rSBA)? Additional indicators: ≥4- fold rise or a titer of ≥4 with human complement (hSBA)21Meningococcal SBA in cases and controlsSera of casesSera of healthy controlsHomologous strain 3/54 (5.6%) 444/540 (82.2%)Goldschneider I et al. J Exp Med 1969; 129: 1307–2622Challenge 1. Human or rabbit complement?• Original Goldschneider study used human complement (hSBA) • Difficult to get and standardize• Baby rabbit complement commercially available... but gives higher SBA titers• UK experience with rSBA and Menc conjugates:? rSBA titer of ≥1:8 in immunized infants and children correlates with effectiveness in post licensure surveillance (Andrews et al. Clin Diag Lab Immunol 2003) 23MenC disease and prevalence of rSBATrotter et al. Vaccine 200324Challenge 2.Serogroups A, W135, Y• Are criteria set up for MenC valid for other serogroups?• Which strains are the relevant to be used in SBA?25Challenge 3:EIA or comparable assays for isotype specific antibody concentrations• Concentration associated with protection not determined• OBS! Should correlate with SBA • To increase correlation use? Pure polysaccharide antigens? Derivatized polysaccahride antigens? Chaotropic agents (thiocyanate) to detach antibodies of low affinity (assumed to be not protective)26Reagents for EIA availableNIBSC, Potters Bar, UK• Polysaccharides• Reference serum• Methylated serum albumin for enhancing the binding of PS to solid phasePneumococcal conjugate vaccines28Pneumococcal conjugate vaccines• PncCRM; Wyeth.? Carrier: mutant diphtheria toxin, CRM197  ? At present 7-valent vaccine lisenced (Prevenar, Prevnar)? 13-valent vaccine in clinical trials• PncPD; GSK? Carrier: H. influenzae Protein D? 11-valent vaccine tested in clinical efficacy trials? 10-valent mixed carrier vaccine filed for licensure• PncOMPC; Merck? Carrier: meningococcal outer membrane protein complex? 7-valent vaccine tested in clinical efficacy trial? Company has stopped the development• PncDT, sanofi pasteur? Carriers: diphtheria and tetanus toxoids? 11-valent vaccine tested in clinical efficacy trial? Company has stopped the development29World Health OrganizationWHO Technical Report Series, No. 927, 2005Annex 2Recommendations for the production and control of pneumococcal conjugate vaccinesAppendixSerological criteria for evaluation and licensure ofnew pneumococcal conjugate vaccine formulationsfor use in infants30Pneumococcal conjugate vaccines• Acceptance of nwe vaccines, schedules, combinations, based mainly on immunological studies• Primary threshold for all serotypes = % subjects with 0.35 µg/ml of antibody measured by EIA1• Secondary thresholds? Demonstration of functional activity of antibodies by testing OPA? Demonstration of immunological memory1http://www.who.int/biologicals/publications/meetings/areas/vaccines/en/31Challenge 1:• Noninferiority to a licensed vaccine • Primary threshold for all serotypes = % subjects with 0.35 µg/ml of antibody measured by EIA• 0.35 µg/ml threshold determined with non-22F EIA32http://www.vaccine.uab.edu/33Problems with EIAs• The commercial antigens are not pure• To gain type specificity absorptions with cell wall polysaccharide (CPS) and 22F PS needed• The effects of extra steps seem to vary in different labs• What to do when 22F is included in the Pnc conjugate vaccine• Danish approach: CPS2 from 22F strain34Antibody to type 11A0,010,1110PCV PS PCV PSinfants adultsug/mlprepostOPA - OPA+11A- 11A+Soininen et al. Clin Diag Lab Imm 2001 35Absorption with CPS or with CPS+22FConcepcion & Frasch, Clin Diag Lab Imm 2001Serum from an unimmunized adult 36Correlation of EIA and OPAR=0.66 R=0.92Concepcion & Frasch, Clin Diag Lab Imm 2001Serum samples from adults, type 437Problems with EIAs• The commercial antigens are not pure• To gain type specificity absorptions with Cell wall polysaccharide (CPS) and 22F PS needed• The effects of extra steps seem to vary in different labs• What to do when 22F is included in the Pnc conjugate vaccine• Danish approach: CPS2 from 22F strain38Reagents for EIA available• Polysaccharides form ATCC• CPS from SSI, Denmark• Reference serum 89S from US FDA• QC sera from NIBSC39Challenge 2:Opsonophagocytosis assay for demonstration of functionality of antibodies• Validated by several groups• Interlaboratory standardization and harmonization in process • Strains, effector cells (HL60), detection, calculation of results• High throughput assays needed40Challenge 3:• Development of immunoassays that would predict protection against other than invasive disease41Challenges for all three• The significance of demonstration of increase in avidity • Standardization of the assays for measuring avidity• Reliable and robust multiplexed assays for measuring concentration and functional activity of antibodies.  ? Should be able to be transfered easily to other labs

Development of Validated Assays for Measuring the Human Antibody Response to Polysaccharide Conjugate Vaccines

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Development of Validated Assays for Measuring the Human Antibody Response to Polysaccharide Conjugate Vaccines

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