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Standardisation and Validation of Serological Assays for the Evaluation of Immune Responses to Neisseria meningitidis Serogroup A and C Vaccines
Biologicals (2000) 28, 193–197 doi:10.1006/biol.2000.0253, available online at http://www.idealibrary.com on
MEETING REPORT
Standardisation and Validation of Serological Assays for the Evaluation of Immune Responses to Neisseria meningitidis Serogroup A and C Vaccines Luis Jodar1*, Keith Cartwright2 and Ian M. Feavers3 1 World Health Organization, 1211 Geneva 27, Switzerland 2 Public Health Laboratory, Gloucestershire Royal Hospital, Great Western Road, Gloucester GL1 3NN, U.K. 3 National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Herts EN6 3QG, U.K.
Introduction A conservative WHO estimate of the worldwide burden of meningococcal disease is approximately 300 000–350 000 cases per year. Strains causing invasive meningococcal disease are restricted to five meningococcal serogroups (A, B, C, Y and W-135), o#ering a realistic prospect for the elimination of disease if e#ective vaccines can be developed against all of them. All countries su#er from endemic meningococcal disease, primarily in children under the age of five, with an annual attack rate of around 1–3/100 000 of the population. In addition, some countries, predominantly but not exclusively in the developing world, such as those located in the so-called African ‘‘meningitis belt’’ su#er from occasional or even regular epidemics of serogroup A meningococcal disease involving attack rates from about 10/100 000 to as high as 400–800/100 000, yielding tens of thousands of cases.1,2 In 1996 the largest epidemic ever recorded took place in numerous Sub-Saharan African countries and involved more than 180 000 reported cases and around 18 000 deaths.3 Serogroup B and C strains are most prevalent during endemic periods, but they have also been responsible for localised outbreaks.4 Even with optimal antimicrobial therapy, case–fatality ratios remain at 8–12%.5–7 Approximately 10% of those who survive are *To whom correspondence should be addressed: Dr Luis Jodar, World Health Organization, 1211 Geneva 27, Switzerland. E-mail: [email protected] 1045–1056/00/030193+05$30.00/0
left permanently impaired by deafness or mental retardation.8,9 With the exception of serogroup B, plain polysaccharide vaccines that are safe and result in an age-related protection have been available for a number of years. The serogroup A polysaccharide may induce antibody in some children as young as 3 months of age, although a response comparable to that seen among adults is not achieved. The immune response in infants to serogroup C polysaccharide is inferior to that of the serogroup A polysaccharide. Following administration of meningococcal plain polysaccharide vaccines, antibody titres fall rapidly in young children and more slowly in older children and adults. Immunological memory is not induced and e#icacy is likely to be related to persistence of antibody. E#icacy data parallel immunogenicity data. There is proven e#icacy in the short term but there is a drop in e#icacy over time particularly in young children.10,11 These characteristics make the routine use of meningococcal polysaccharides for ongoing population-wide programmes of disease prevention highly problematic. To overcome these and building on the acknowledged success of conjugated Hib vaccines, recent research has focused on the development of meningococcal serogroup A and C glycoconjugate vaccines. An important juncture has been reached in the development and licensing of such meningococal conjugate vaccines. Few, if any, countries experience su#iciently high levels of serogroup C disease 2000 The International Association for Biologicals
194
L. Jodar et al.
to permit comprehensive prospective studies of vaccine protective e#icacy in formal clinical trials. In the United Kingdom in October 1999, the serogroup C conjugate vaccine Meningitec became the first vaccine to be licensed for use in infants for which protective e#icacy was not determined by a phase III clinical trial but inferred from immunogenicity data. Having set this precedent, a similar approach may be adopted for the licensure of new meningococcal conjugate vaccines being developed for prevention of disease caused by the other serogroups. Since decisions on the licensure of novel vaccines and the wider implementation of existing vaccines are critically dependent upon serological data, it was essential to assess whether current serological assays provide appropriate data. A meeting was held in March 1999, under the auspices of the WHO in Geneva, to attempt to clarify and resolve issues relating to laboratory assays for the analysis of human serum for meningococcal serogroup A- and C-specific antibodies. The participants addressed: (a) whether the existing standardised serologic assays provide su#iciently unambiguous data to permit decisions for licensing and public health recommendations of meningococcal serogroup A and C vaccines and (b) what additional studies, if any, needed to be conducted in order to resolve the outstanding issues relating to current assays, and the need, if any, for development of improved assays. Functional assays Serogroup A and C polysaccharide vaccines were first shown to be immunogenic and protective in humans by Gotschlich et al.12,13 Since then antibody responses to these capsular polysaccharide vaccines have been measured by various serological methods. Serum bactericidal antibody (SBA) activity has been shown to correlate well with both natural and vaccine-induced immunity to meningococcal disease. The induction of complement-dependent bactericidal antibodies after vaccination with meningococcal polysaccharide or conjugate vaccines has, therefore, been widely accepted as evidence of the potential e#icacy of these vaccines. Thus, a functional serological surrogate of vaccine e#icacy exists that can be used to inform decisions about the licensure and implementation of meningococcal polysaccharide and conjugate vaccines. The original assays that associated bactericidal antibody with protection used human complement sources from individuals after natural infection,
whereas vaccine licensure was supported using SBA titres obtained with sera before and after vaccination and using baby rabbit complement.12,14 When SBA titres have been compared after using baby rabbit and human serum as a complement source, significant di#erences in antibody titres have been observed; human complement tends to give lower titres.15–17 The ideal exogenous complement is human serum from a patient with untreated agammaglobulinemia and normal complement activity. However, the widespread use of gammaglobulin treatment means that only small amounts of agammaglobulinemic serum from untreated patients are available for this purpose. Normal human serum that lacks intrinsic bactericidal activity would then be the only alternative source. The meeting participants agreed that it would prove di#icult for di#erent laboratories to obtain su#icient quantities of suitable human complement to standardise the assay. Infant rabbit serum provides a uniform and standardisable complement source that gives comparable results in the SBA, but there is concern that the increased sensitivity of the assay using rabbit complement may lead to false positive results. Since easily standardised batches of rabbit complement are commercially available, it was decided to commission a detailed statistical comparison of data from assays using human and rabbit complement with a suitable range of serum samples to determine a threshold titre for SBAs using infant rabbit complement that correlated with protection in man. Antigen binding assays Although the SBA provides a good surrogate of protective immunity, in common with most bioassays, it gives results that have greater variability than well-controlled antigen binding assays such as the ELISA. Bioassays also tend to be less sensitive for detection of antibody and are arguably more time consuming to perform. For these reasons, in addition to the SBA, it is useful to have an ELISA to measure total or isotype-specific serum antibody responses in a larger number of vaccinated subjects. ELISA results provide an accurate assessment of vaccine immunogenicity and also provide information on the type of antibody response that helps distinguish plain polysaccharide from conjugated polysaccharide antigens (i.e., IgG vs. IgM; or IgG2 vs. IgG1 subclass). An ELISA is more convenient for the analysis of large collections of serum samples and may be particularly useful for comparing
Meeting Report
antibody responses to vaccination in di#erent age groups or populations, to compare di#erent vaccines or to assess the manufacturing consistency of di#erent batches of vaccine. The SBA may be reserved for assaying a subset of the serum samples to confirm whether the anticapsular antibody being measured in the ELISA is functionally active and likely to confer protection. Results of two recent multilaboratory studies have demonstrated that reproducible measurements of serogroup A and C anticapsular antibody concentrations may be obtained by a standardised ELISA.18,19 However, data supporting the extrapolation of serum anticapsular antibody concentrations to bactericidal antibody titres (functional antibody activity) are limited. High serum antibody responses to meningococcal polysaccharide by ELISA in the presence of low or undetectable bactericidal antibody have been detected, especially in infants, toddlers, and even in some adults who receive meningococcal polysaccharide vaccines.20–22 The most likely explanation is that the polysaccharide vaccine, as a T-cell independent antigen, does not give rise to antibody a#inity maturation so that the immune response consists principally of low-avidity anticapsular antibodies. These antibodies are detected by ELISA but appear to be less active or inactive in the bactericidal assay. These results are problematic if the IgG ELISA antibody responses are being compared after one or two doses of plain polysaccharide or after one or two doses of protein conjugate vaccine; a single dose of either vaccine can elicit low-avidity antibodies particularly in infants and toddlers.16,17 By including an appropriate concentration of a chaotropic agent, antigen binding assays can be modified to measure only the higher a#inity antibodies.17 Evidence presented at the meeting indicated that an ELISA modified in this way provided measurements that correlated well with the corresponding SBA data, providing an acceptable alternative for the evaluation and comparison of immune responses to meningococcal vaccines. A discussion of the most appropriate coating antigen for the ELISA focused on the use of either a combination of methylated human serum albumin (mHSA) and polysaccharide or the derivatised antigen described by Grano# et al.17 Specificity of antibody binding is determined for each test serum by inhibition of binding by soluble ‘‘native’’ polysaccharide. The so-called ‘‘standard ELISA’’ uses meningococcal C polysaccharide mixed with mHSA as the solid phase of the assay, which is easier to
195
prepare than a derivatised antigen. It was suggested that the mHSA/polysaccharide preparation could be used in ‘‘second generation’’ modified ELISA.
Conclusions and recommendations The meeting participants agreed the following general recommendations: 1. E#icacy studies are not needed for licensure of serogroup A and C meningococcal conjugate vaccines because of compelling data that serum anticapsular antibodies confer protection. 2. The principal but not the sole function of antibody assays is to support licensure. 3. Both serum bactericidal assay (SBA) and ELISA data will be needed for licensure. The specific recommendations listed below also reached consensus and are intended to be applicable to foreseeable combination vaccines and novel formulations: 1. The SBA is the functional assay of choice at this time and results are considered to predict protection. 2. Developmental work should continue on other functional assays. 3. Adoption of standard SBA technique was agreed. 4. The CDC (Maslanka et al., 1997)22 serogroup C SBA assay with a provision for a modification to allow semi-automation of colony counting was adopted as the optimal methodology. 5. Baby rabbit serum (BRS) will be the source of exogenous complement. 6. To avoid overestimating protection with BRS complement, a threshold titre correlating with protection will be determined by reference to SBA data obtained with human complement. 7. A SBA study design for collaborative evaluation of the rabbit and human complement will be developed. 8. ELISA results should predict functional antibody activity. 9. A modified (high avidity) assay was adopted as the best ELISA technique for assessing functional antibody activity to serogroup C polysaccharide. 10. Reference laboratories should standardise on a single high avidity ELISA protocol for serogroup C polysaccharide.
196
L. Jodar et al.
11. Standard ELISA o#ers important additional information on human responses to meningococcal A and C polysaccharide and conjugate vaccines. 12. Reference polysaccharides are available and should be used for standardisation. 13. The CDC 1992 reference serum was regarded as the most important ELISA external quality standard. 14. Data derived from modified ELISAs should be expressed in units that are clearly distinct from, but mapped to, mass units by means of the CDC1992 reference serum. 15. Laboratories are encouraged to use the CDC1992 serum pool as their primary reference serum. 16. For both ELISA and SBA, additional quality control sera with lower antibody levels are required, as are sera containing low avidity antibody (one inter-laboratory comparison will be required). 17. Adherence to ICH guidelines on validation of assay methods is recommended. 18. Modifications to current serogroup A SBAs and ELISAs need to be evaluated. 19. A source of human complement for a serogroup A SBA is needed. 20. More data are needed on serogroup A infections: population immunity, priming and memory to polysaccharide and antibody avidity. 21. Additional epidemiological studies are needed to link clinical e#icacy with serological correlates of protection against serogroup A and C disease. Three of the laboratories represented at the meeting, each with extensive experience in serological assays, agreed to analyse a set of data based on sera that had been examined in both types of SBA and reach a consensus on the threshold titre for BRSbased assays that correlates with protection. Although a consensus has been achieved on the threshold titre, there are di#erent arguments with respect to the analysis of the data and their interpretation. This issue will be addressed later this year by the WHO Expert Committee on Biologicals and Standardisation (ECBS) which will arrive at a decision on the advice of an expert panel. The expert panel, including permanent members of the ECBS, FDA and MCA representatives, and other independent advisors will consider the three
submitted analyses and the latest e#ectiveness data available from the U.K. at that time.
List of participants Dr P. Anderson (U.S.A.); Dr R. Burrow (Manchester Public Health Laboratory, U.K.); Dr G. Carlone (Centers for Disease Control and Prevention, U.S.A.); Dr K. Cartwright (Public Health Laboratory Service, U.K.); Dr P. Densen (University of Iowa, U.S.A.); Dr I. Feavers (National Institute for Biological Standards and Control, U.K.); Dr Carl Frasch (Center for Biologics Evaluation and Research, US Food and Drug Administration, U.S.A.); Dr D. Granoff (Children’s Hospital Oakland Research Institute, U.S.A.); Dr M. Granstrom (Karolinska Hospital, Sweden); Dr J. Holst (National Institute of Public Health, Norway); Dr H. Ka¨yhty (National Public Health Institute, Finland); Dr A. Michaelides (LCDC, Canada); Dr M. H. Nahm (University of Rochester, U.S.A.); Dr B. Perkins (Centers for Disease Control and Prevention, U.S.A.); Drs A. Bartolini, P. Constantino, J. Donnelly, B. Fritzell (Chiron Vaccines); Dr K. Mayer (Chiron Behring GmbH & Co.); Drs M. Bybel, C. Ethevenaux, R. P. Ryall (Aventis Pasteur); Drs M. Hohenboken, Laferriere, G. Metz (SmithKline Beecham Biologicals); Dr P. Fusco (North American Vaccines); Dr D. Madore (Wyeth-Lederle Vaccines and Pediatrics); Dr L. van Alphen (RIVM, The Netherlands); Drs E. Griffiths, L. Jo´dar, J. Wenger (WHO).
References 1. The World Health Report 1998. World Health Organization Geneva, p. 45. 2. Tikhomirov E. Present risk of epidemics of meningococcal disease throughout the world. In: Proceedings of the 5th International Congress for Infectious Diseases, Nairobi, Kenya, June 7–11 1992. 113: (abstract 397). 3. Response to epidemic meningitis in Africa. Weekly Epidemiol Rec 1997; 72: 313–320. 4. Control of epidemic meningococcal disease. WHO practical guidelines, 1995. WHO/Fondation Marcel Merieux. Lyon, Edition Fondation Marcel Merieux, p. 6. 5. Havens PL, Garland JS, Brook MM, Dewitz BA, Stremski ES, Troshynski TJ. Trends in mortality in children hospitalized with meningococcal infections, 1957 to 1987. Pediatr Infect Dis J 1989; 8: 8–11. 6. Veeken H, Ritmeijer K, Hausman B. Priority during a meningitis epidemic: vaccination or treatment? Bull WHO 1998; 76: 135–141.
Meeting Report
7. Greenwood BM, Bradley AK, Smith AW, Wall RA. Mortality from meningococcal disease during an epidemic in The Gambia, West Africa. Trans R Soc Trop Med Hyg 1987; 81: 536–538. 8. Smith AW, Bradley AK, Wall RA et al. Sequelae of epidemic meningococcal meningitis in Africa. Trans R Soc Trop Med Hyg 1988; 82: 312–320. 9. Varaine F, Caugant DA, Riou JY et al. Meningitis outbreaks and vaccination strategy. Trans R Soc Trop Med Hyg 1997; 91: 3–7. 10. Kayhty H, Karanko V, Peltola H, Sarna S, Makela PH. Serum antibodies to capsular polysaccharide vaccine of group A Neissera meningitidis followed for thrre years in infants and children. J Infect Dis 1980; 142: 861–868. 11. King WJ, MacDonald NE, Wells G et al. Total and functional antibody response to a quadrivalent meningococcal polysaccharide vaccine among children. J Pediatr 1996; 128: 196–202. 12. Gotschlich EC, Goldschneider I, Artenstein MS. Human immunity to the meningococcus IV. Immunogenicity of serogroup A and serogroup C polysaccharides in human volunteers. J Exp Med 1969; 129: 1367–1384. 13. Goldschneider I, Gotschlich EC, Artenstein MS. Human immunity to the meningococcus. I. The role of humoral antibodies. J Exp Med 1969; 129: 1307–1326. 14. World Health Organization 1976. Requirements for meningococcal polysaccharide vaccine. World Health Organization Technical Report Series, no. 594, Annex 2, World Health Organization, Geneva. 15. Maslanka SE, Gheesling LL, LiButti DE et al. Standardization and a multilaboratory comparison of Neisseria meningitidis serogroup A and C serum bactericidal assays. The Multilaboratory Study Group. Clin Diagn Lab Immunol 1997; 4: 156–167. 16. Borrow R, Richmond P, Fox AJ et al. Induction of immunological memory in UK infants by a meningo-
17.
18.
19.
20.
21.
22.
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coccal A/C conjugate vaccine. Abstracts of the 11th International Pathogenic Neisseria Conference, Nice 1998; 156 (Abstract). Grano# DM, Maslanka SE, Carlone GM et al. A modified enzyme-linked immunosorbent assay for measurement of antibody responses to meningococcal C polysaccharide that correlate with bactericidal responses. Clin Diagn Lab Immunol 1998; 5: 479–485. Carlone GM, Frasch CE, Siber GR et al. Multilaboratory comparison of levels of antibody to the Neisseria meningitidis group A capsular polysaccharide measured by using an enzyme-linked immunosorbent assay. J Clin Microbiol 1992; 30: 154–159. Gheesling LL, Carlone GM, Pais LB et al. Multicenter comparison of Neisseria meningitidis serogroup C anti-capsular polysaccharide antibody levels measured by a standardized enzyme-linked immunosorbent assay. J Clin Microbiol 1994; 32: 1475–1482. Maslanka SE, Tappero JW, Plikaytis BD et al. Agedependent Neisseria meningitidis serogroup C classspecific antibody concentrations and bactericidal titers in sera from young children from Montana immunized with a licensed polysaccharide vaccine. Infect Immun 1998; 66: 2453–2459. Lieberman JM, Chiu SS, Wong VK et al. Safety and immunogenicity of a serogroups A/C Meisseria meningitidis oligosaccharide-protein conjugate vaccine in young children. A randomized controlled trial. JAMA 1996; 275: 1499–1503. Maslanka SE, Gheesling LL, LiButti DE et al. Standardization and a multilaboratory comparison of Neisseria meningitidis serogroup A and C serum bactericidal assays. The Multilaboratory Study Group. Clin Diagn Lab Immunol 1997; 4: 156–167.
Received for publication 2 June 2000; accepted 2 June 2000
MEETING REPORT
Standardisation and Validation of Serological Assays for the Evaluation of Immune Responses to Neisseria meningitidis Serogroup A and C Vaccines Luis Jodar1*, Keith Cartwright2 and Ian M. Feavers3 1 World Health Organization, 1211 Geneva 27, Switzerland 2 Public Health Laboratory, Gloucestershire Royal Hospital, Great Western Road, Gloucester GL1 3NN, U.K. 3 National Institute for Biological Standards and Control, Blanche Lane, South Mimms, Potters Bar, Herts EN6 3QG, U.K.
Introduction A conservative WHO estimate of the worldwide burden of meningococcal disease is approximately 300 000–350 000 cases per year. Strains causing invasive meningococcal disease are restricted to five meningococcal serogroups (A, B, C, Y and W-135), o#ering a realistic prospect for the elimination of disease if e#ective vaccines can be developed against all of them. All countries su#er from endemic meningococcal disease, primarily in children under the age of five, with an annual attack rate of around 1–3/100 000 of the population. In addition, some countries, predominantly but not exclusively in the developing world, such as those located in the so-called African ‘‘meningitis belt’’ su#er from occasional or even regular epidemics of serogroup A meningococcal disease involving attack rates from about 10/100 000 to as high as 400–800/100 000, yielding tens of thousands of cases.1,2 In 1996 the largest epidemic ever recorded took place in numerous Sub-Saharan African countries and involved more than 180 000 reported cases and around 18 000 deaths.3 Serogroup B and C strains are most prevalent during endemic periods, but they have also been responsible for localised outbreaks.4 Even with optimal antimicrobial therapy, case–fatality ratios remain at 8–12%.5–7 Approximately 10% of those who survive are *To whom correspondence should be addressed: Dr Luis Jodar, World Health Organization, 1211 Geneva 27, Switzerland. E-mail: [email protected] 1045–1056/00/030193+05$30.00/0
left permanently impaired by deafness or mental retardation.8,9 With the exception of serogroup B, plain polysaccharide vaccines that are safe and result in an age-related protection have been available for a number of years. The serogroup A polysaccharide may induce antibody in some children as young as 3 months of age, although a response comparable to that seen among adults is not achieved. The immune response in infants to serogroup C polysaccharide is inferior to that of the serogroup A polysaccharide. Following administration of meningococcal plain polysaccharide vaccines, antibody titres fall rapidly in young children and more slowly in older children and adults. Immunological memory is not induced and e#icacy is likely to be related to persistence of antibody. E#icacy data parallel immunogenicity data. There is proven e#icacy in the short term but there is a drop in e#icacy over time particularly in young children.10,11 These characteristics make the routine use of meningococcal polysaccharides for ongoing population-wide programmes of disease prevention highly problematic. To overcome these and building on the acknowledged success of conjugated Hib vaccines, recent research has focused on the development of meningococcal serogroup A and C glycoconjugate vaccines. An important juncture has been reached in the development and licensing of such meningococal conjugate vaccines. Few, if any, countries experience su#iciently high levels of serogroup C disease 2000 The International Association for Biologicals
194
L. Jodar et al.
to permit comprehensive prospective studies of vaccine protective e#icacy in formal clinical trials. In the United Kingdom in October 1999, the serogroup C conjugate vaccine Meningitec became the first vaccine to be licensed for use in infants for which protective e#icacy was not determined by a phase III clinical trial but inferred from immunogenicity data. Having set this precedent, a similar approach may be adopted for the licensure of new meningococcal conjugate vaccines being developed for prevention of disease caused by the other serogroups. Since decisions on the licensure of novel vaccines and the wider implementation of existing vaccines are critically dependent upon serological data, it was essential to assess whether current serological assays provide appropriate data. A meeting was held in March 1999, under the auspices of the WHO in Geneva, to attempt to clarify and resolve issues relating to laboratory assays for the analysis of human serum for meningococcal serogroup A- and C-specific antibodies. The participants addressed: (a) whether the existing standardised serologic assays provide su#iciently unambiguous data to permit decisions for licensing and public health recommendations of meningococcal serogroup A and C vaccines and (b) what additional studies, if any, needed to be conducted in order to resolve the outstanding issues relating to current assays, and the need, if any, for development of improved assays. Functional assays Serogroup A and C polysaccharide vaccines were first shown to be immunogenic and protective in humans by Gotschlich et al.12,13 Since then antibody responses to these capsular polysaccharide vaccines have been measured by various serological methods. Serum bactericidal antibody (SBA) activity has been shown to correlate well with both natural and vaccine-induced immunity to meningococcal disease. The induction of complement-dependent bactericidal antibodies after vaccination with meningococcal polysaccharide or conjugate vaccines has, therefore, been widely accepted as evidence of the potential e#icacy of these vaccines. Thus, a functional serological surrogate of vaccine e#icacy exists that can be used to inform decisions about the licensure and implementation of meningococcal polysaccharide and conjugate vaccines. The original assays that associated bactericidal antibody with protection used human complement sources from individuals after natural infection,
whereas vaccine licensure was supported using SBA titres obtained with sera before and after vaccination and using baby rabbit complement.12,14 When SBA titres have been compared after using baby rabbit and human serum as a complement source, significant di#erences in antibody titres have been observed; human complement tends to give lower titres.15–17 The ideal exogenous complement is human serum from a patient with untreated agammaglobulinemia and normal complement activity. However, the widespread use of gammaglobulin treatment means that only small amounts of agammaglobulinemic serum from untreated patients are available for this purpose. Normal human serum that lacks intrinsic bactericidal activity would then be the only alternative source. The meeting participants agreed that it would prove di#icult for di#erent laboratories to obtain su#icient quantities of suitable human complement to standardise the assay. Infant rabbit serum provides a uniform and standardisable complement source that gives comparable results in the SBA, but there is concern that the increased sensitivity of the assay using rabbit complement may lead to false positive results. Since easily standardised batches of rabbit complement are commercially available, it was decided to commission a detailed statistical comparison of data from assays using human and rabbit complement with a suitable range of serum samples to determine a threshold titre for SBAs using infant rabbit complement that correlated with protection in man. Antigen binding assays Although the SBA provides a good surrogate of protective immunity, in common with most bioassays, it gives results that have greater variability than well-controlled antigen binding assays such as the ELISA. Bioassays also tend to be less sensitive for detection of antibody and are arguably more time consuming to perform. For these reasons, in addition to the SBA, it is useful to have an ELISA to measure total or isotype-specific serum antibody responses in a larger number of vaccinated subjects. ELISA results provide an accurate assessment of vaccine immunogenicity and also provide information on the type of antibody response that helps distinguish plain polysaccharide from conjugated polysaccharide antigens (i.e., IgG vs. IgM; or IgG2 vs. IgG1 subclass). An ELISA is more convenient for the analysis of large collections of serum samples and may be particularly useful for comparing
Meeting Report
antibody responses to vaccination in di#erent age groups or populations, to compare di#erent vaccines or to assess the manufacturing consistency of di#erent batches of vaccine. The SBA may be reserved for assaying a subset of the serum samples to confirm whether the anticapsular antibody being measured in the ELISA is functionally active and likely to confer protection. Results of two recent multilaboratory studies have demonstrated that reproducible measurements of serogroup A and C anticapsular antibody concentrations may be obtained by a standardised ELISA.18,19 However, data supporting the extrapolation of serum anticapsular antibody concentrations to bactericidal antibody titres (functional antibody activity) are limited. High serum antibody responses to meningococcal polysaccharide by ELISA in the presence of low or undetectable bactericidal antibody have been detected, especially in infants, toddlers, and even in some adults who receive meningococcal polysaccharide vaccines.20–22 The most likely explanation is that the polysaccharide vaccine, as a T-cell independent antigen, does not give rise to antibody a#inity maturation so that the immune response consists principally of low-avidity anticapsular antibodies. These antibodies are detected by ELISA but appear to be less active or inactive in the bactericidal assay. These results are problematic if the IgG ELISA antibody responses are being compared after one or two doses of plain polysaccharide or after one or two doses of protein conjugate vaccine; a single dose of either vaccine can elicit low-avidity antibodies particularly in infants and toddlers.16,17 By including an appropriate concentration of a chaotropic agent, antigen binding assays can be modified to measure only the higher a#inity antibodies.17 Evidence presented at the meeting indicated that an ELISA modified in this way provided measurements that correlated well with the corresponding SBA data, providing an acceptable alternative for the evaluation and comparison of immune responses to meningococcal vaccines. A discussion of the most appropriate coating antigen for the ELISA focused on the use of either a combination of methylated human serum albumin (mHSA) and polysaccharide or the derivatised antigen described by Grano# et al.17 Specificity of antibody binding is determined for each test serum by inhibition of binding by soluble ‘‘native’’ polysaccharide. The so-called ‘‘standard ELISA’’ uses meningococcal C polysaccharide mixed with mHSA as the solid phase of the assay, which is easier to
195
prepare than a derivatised antigen. It was suggested that the mHSA/polysaccharide preparation could be used in ‘‘second generation’’ modified ELISA.
Conclusions and recommendations The meeting participants agreed the following general recommendations: 1. E#icacy studies are not needed for licensure of serogroup A and C meningococcal conjugate vaccines because of compelling data that serum anticapsular antibodies confer protection. 2. The principal but not the sole function of antibody assays is to support licensure. 3. Both serum bactericidal assay (SBA) and ELISA data will be needed for licensure. The specific recommendations listed below also reached consensus and are intended to be applicable to foreseeable combination vaccines and novel formulations: 1. The SBA is the functional assay of choice at this time and results are considered to predict protection. 2. Developmental work should continue on other functional assays. 3. Adoption of standard SBA technique was agreed. 4. The CDC (Maslanka et al., 1997)22 serogroup C SBA assay with a provision for a modification to allow semi-automation of colony counting was adopted as the optimal methodology. 5. Baby rabbit serum (BRS) will be the source of exogenous complement. 6. To avoid overestimating protection with BRS complement, a threshold titre correlating with protection will be determined by reference to SBA data obtained with human complement. 7. A SBA study design for collaborative evaluation of the rabbit and human complement will be developed. 8. ELISA results should predict functional antibody activity. 9. A modified (high avidity) assay was adopted as the best ELISA technique for assessing functional antibody activity to serogroup C polysaccharide. 10. Reference laboratories should standardise on a single high avidity ELISA protocol for serogroup C polysaccharide.
196
L. Jodar et al.
11. Standard ELISA o#ers important additional information on human responses to meningococcal A and C polysaccharide and conjugate vaccines. 12. Reference polysaccharides are available and should be used for standardisation. 13. The CDC 1992 reference serum was regarded as the most important ELISA external quality standard. 14. Data derived from modified ELISAs should be expressed in units that are clearly distinct from, but mapped to, mass units by means of the CDC1992 reference serum. 15. Laboratories are encouraged to use the CDC1992 serum pool as their primary reference serum. 16. For both ELISA and SBA, additional quality control sera with lower antibody levels are required, as are sera containing low avidity antibody (one inter-laboratory comparison will be required). 17. Adherence to ICH guidelines on validation of assay methods is recommended. 18. Modifications to current serogroup A SBAs and ELISAs need to be evaluated. 19. A source of human complement for a serogroup A SBA is needed. 20. More data are needed on serogroup A infections: population immunity, priming and memory to polysaccharide and antibody avidity. 21. Additional epidemiological studies are needed to link clinical e#icacy with serological correlates of protection against serogroup A and C disease. Three of the laboratories represented at the meeting, each with extensive experience in serological assays, agreed to analyse a set of data based on sera that had been examined in both types of SBA and reach a consensus on the threshold titre for BRSbased assays that correlates with protection. Although a consensus has been achieved on the threshold titre, there are di#erent arguments with respect to the analysis of the data and their interpretation. This issue will be addressed later this year by the WHO Expert Committee on Biologicals and Standardisation (ECBS) which will arrive at a decision on the advice of an expert panel. The expert panel, including permanent members of the ECBS, FDA and MCA representatives, and other independent advisors will consider the three
submitted analyses and the latest e#ectiveness data available from the U.K. at that time.
List of participants Dr P. Anderson (U.S.A.); Dr R. Burrow (Manchester Public Health Laboratory, U.K.); Dr G. Carlone (Centers for Disease Control and Prevention, U.S.A.); Dr K. Cartwright (Public Health Laboratory Service, U.K.); Dr P. Densen (University of Iowa, U.S.A.); Dr I. Feavers (National Institute for Biological Standards and Control, U.K.); Dr Carl Frasch (Center for Biologics Evaluation and Research, US Food and Drug Administration, U.S.A.); Dr D. Granoff (Children’s Hospital Oakland Research Institute, U.S.A.); Dr M. Granstrom (Karolinska Hospital, Sweden); Dr J. Holst (National Institute of Public Health, Norway); Dr H. Ka¨yhty (National Public Health Institute, Finland); Dr A. Michaelides (LCDC, Canada); Dr M. H. Nahm (University of Rochester, U.S.A.); Dr B. Perkins (Centers for Disease Control and Prevention, U.S.A.); Drs A. Bartolini, P. Constantino, J. Donnelly, B. Fritzell (Chiron Vaccines); Dr K. Mayer (Chiron Behring GmbH & Co.); Drs M. Bybel, C. Ethevenaux, R. P. Ryall (Aventis Pasteur); Drs M. Hohenboken, Laferriere, G. Metz (SmithKline Beecham Biologicals); Dr P. Fusco (North American Vaccines); Dr D. Madore (Wyeth-Lederle Vaccines and Pediatrics); Dr L. van Alphen (RIVM, The Netherlands); Drs E. Griffiths, L. Jo´dar, J. Wenger (WHO).
References 1. The World Health Report 1998. World Health Organization Geneva, p. 45. 2. Tikhomirov E. Present risk of epidemics of meningococcal disease throughout the world. In: Proceedings of the 5th International Congress for Infectious Diseases, Nairobi, Kenya, June 7–11 1992. 113: (abstract 397). 3. Response to epidemic meningitis in Africa. Weekly Epidemiol Rec 1997; 72: 313–320. 4. Control of epidemic meningococcal disease. WHO practical guidelines, 1995. WHO/Fondation Marcel Merieux. Lyon, Edition Fondation Marcel Merieux, p. 6. 5. Havens PL, Garland JS, Brook MM, Dewitz BA, Stremski ES, Troshynski TJ. Trends in mortality in children hospitalized with meningococcal infections, 1957 to 1987. Pediatr Infect Dis J 1989; 8: 8–11. 6. Veeken H, Ritmeijer K, Hausman B. Priority during a meningitis epidemic: vaccination or treatment? Bull WHO 1998; 76: 135–141.
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Received for publication 2 June 2000; accepted 2 June 2000