meningococcal vaccines, methods and protocols

Humana Press Meningococcal Vaccines Edited by Andrew J. Pollard, MD, PhD Martin C. J. Maiden, PhD Methods and Protocols Humana Press Meningococcal Vaccines Edited by Andrew J. Pollard, MD, PhD Martin C. J. Maiden, PhD Methods and Protocols M E T H O D S I N M O L E C U L A R M E D I C I N E TM Meningococcal Vaccines and Developments 1 1 From: Methods in Molecular Medicine, vol. 66: Meningococcal Vaccines: Methods and Protocols Edited by: A. J. Pollard and M. C. J. Maiden © Humana Press Inc., Totowa, NJ 1 Meningococcal Vaccines and Vaccine Developments Ian M. Feavers 1. Introduction Despite rapid advances in the diagnosis of bacterial infections and the avail- ability of effective antibiotics, meningococcal disease continues to represent a substantial public health problem for most countries (1–4). Disease usually develops rapidly, is notoriously difficult to distinguish from other febrile ill- nesses, and generally has a high case-fatality rate. The death of an otherwise fit and healthy individual can occur within a very short time from the first appear- ance of symptoms, those who survive frequently suffer from permanent tissue damage and neurological problems (4,5). Consequently, the development and implementation of effective immunoprophylaxis is a sine qua non for the com- prehensive control of meningococcal disease. From an historical perspective, many meningococcal vaccines have been developed and evaluated in clinical trials; unfortunately, no vaccine so far offers comprehensive protection. This overview traces the development of the existing licensed vaccines and exam- ines the prospects of vaccine candidates that are currently under development or subject to clinical evaluation. The challenges faced by the vaccine developer in designing meningococcal vaccines that are safe, comprehensive, and efficacious in the age groups most at risk of disease are a consequence of the complex biology of Neisseria meningitidis. It is a Gram-negative, encapsulated organism that is naturally competent for transformation with DNA. It only thrives in the human host and is not known to colonize any other animal or environmental niches. Meningo- coccal carriage is very much more common than disease (6) and, notwithstand- ing the devastating impact of meningococcal disease, it may be more 2 Feavers appropriate to consider this bacterium as a commensal that rarely causes dis- ease rather than as a strict pathogen. The meningococcus is, therefore, specifi- cally adapted to the colonization of humans and has evolved a battery of mechanisms that enable it to evade the human immune response. Meningococcal meningitis and septicaemia are ostensibly childhood dis- eases, with highest attack rates in infants (7). Carbohydrate antigens, such as capsular polysaccharide or lipopolysaccharide (LPS), are poorly immunogenic in the very young and frequently mimic host cell structures (8–10) posing a dilemma for the vaccine developer: can immunity to a carbohydrate be enhanced in infants and, if so, would such a vaccine elicit an autoimmune response? Protein vaccine candidates present a different problem; they are gen- erally better immunogens than carbohydrates, but the more immunogenic men- ingococcal surface-protein antigens suffer from the disadvantage that they are also antigenically highly variable (11,12). In this case, the vaccine developer is faced with producing a vaccine that offers adequate cross-protection against the majority of virulent meningococci circulating in the population. Besides hiding behind a camouflage of poorly immunogenic and highly vari- able cell-surface structures, meningococci utilize a variety of genetical mech- anisms to facilitate their persistent colonization of humans. These simultaneously provide them with the potential to circumvent anything less than comprehen- sive immune protection. The mosaic structure of the genes and operons that encode major cell surface structures provides evidence of the importance of horizontal genetical exchange, mediated by transformation and recombina- tion, in the generation of meningococcal antigenic diversity (13,14; see also Chapter 24). It has profound implications for both the development and evalu- ation of vaccine candidates, as well as for the implementation of vaccination programs (15), as it provides a mechanism for the reassortment of antigen- encoding genes among meningococcal clones and increases the prospect of meningococci evading host immunity (16,17). In addition, the expression of many antigen genes is tightly regulated so that critical antigens are not con- tinuously expressed in vivo (18–22). Like many other medically important bacteria, the meningococcus has his- torically been characterized serologically on the basis of its surface antigens (23–26). It can synthesize one of a number of polysaccharide capsules that define the serogroup; pathogenic isolates invariably belong to one of five serogroups, A, B, C, W135, or Y. Serogroups are further subdivided into sero- types and serosubtypes on the basis the serological reactivity of major outer membrane proteins (OMPs) and into immunotypes on the basis of differences in LPS structure. Perhaps not surprisingly, the capsular antigens have been critical in the development of the licensed vaccines. Arguably, if it had been Meningococcal Vaccines and Developments 3 possible to produce a pentavalent vaccine based on the capsular polysaccha- ride of the pathogenic serogroups that was safe and effective in infants, com- prehensive control of meningococcal disease through routine immunization would already be possible. However, the use of serogroup B capsule presents particular problems, and as a result many of the other surface antigens are under consideration as potential components of future vaccines (for review, see ref. 27). 1.1. Historical Perspective Historically, attempts to prevent meningococcal disease by immuno- prophylaxis seem to have been inspired by successes in the prevention of other important diseases through vaccination. Following the use of killed whole- cell vaccines for the prevention of typhoid at the turn of the last century (28), numerous studies explored the potential of immunization with heat-killed men- ingococcal cells to prevent disease (29). Many of the clinical trials that were conducted with whole cell formulations were poorly controlled and the effi- cacy of these preparations was at best questionable. This, together with the unacceptable reactogenicity caused by their high endotoxin content, ultimately resulted in the abandonment of the killed whole-cell vaccine approach. In the 1930s, the successful prevention of diphtheria and tetanus by immu- nization with toxoids prompted the search for a meningococcal toxin in cell- free culture supernatants. Kuhns et al. evaluated the vaccine potential of culture filtrates in studies that provided limited evidence for the efficacy of this approach (30,31). Because the culture supernatants would have been contaminated with capsular polysaccharide, endotoxin, and OMPs, it is impossible to attribute the protection observed to a particular antigen. These preliminary observations do not appear to have been pursued further. In common with research on vaccines against other infectious diseases at that time, perhaps the optimism surround- ing the introduction of antibiotics suppressed interest in meningococcal vac- cine development. During the early 1940s, the association of meningococcal disease with the increase in the recruitment of Allied Forces rekindled interest in vaccination to control disease outbreaks. Once again it was a vaccine against another patho- gen that was to provide the inspiration for subsequent developments. Promis- ing results with a multivalent pneumococcal polysaccharide vaccine indicated that capsular polysaccharides may be able to elicit protective immune responses (32). The clinical evaluations of early preparations of meningococcal serogroup A and C polysaccharides were far from encouraging, probably because the capsular material was degraded to low molecular-weight oligosaccharides by the purification methods employed at the time. However, during the 1960s the development of an innovative purification procedure permitted the production 4 Feavers of highly purified, high molecular-weight meningococcal capsular polysaccha- rides (33). Polysaccharides produced in this way have proved to be safe and immunogenic in adults and older children (34–36). They form the basis of the currently licensed meningococcal polysaccharide vaccine formulations. Unfortunately, polysaccharides are usually T-cell independent antigens. Consequently, they are poorly immunogenic in the very young, they fail to stimulate a good anamnestic response, and they often elicit low-avidity anti- body responses. Meningococcal capsular polysaccharides are no exception (37); the currently licensed polysaccharide vaccines are not indicated for chil- dren under 2 yr of age and the vaccines are not used in long-term immunization programs. Recently, the successful introduction of the Hib vaccine into a num- ber of national immunization programs (38) has been followed by the rapid development of meningococcal glycoconjugate vaccines (39–41). These con- sist of partially hydrolyzed, size-fractionated oligosaccharides chemically con- jugated to either tetanus or diphtheria toxoids as carrier proteins. In clinical studies they have proved to be safe, immunogenic, and to give a good anam- nestic response regardless of the age of the vaccinee (42–49). The first such vaccine was licensed in the UK at the end of 1999 and has since been licensed for use in a number of other European countries. Assuming that such glycoconjugate vaccines prove to be effective in infant immunization schedules, the development of safe and effective vaccines that offer protection against serogroup B disease remains a major challenge. Today serogroup B organisms are responsible for most meningococcal disease in developed countries (7). However, attempts to develop vaccines based upon serogroup B polysaccharide have proved unsuccessful (9). Purified B polysac- charide, a polymer of _ 2-8 linked sialic acid, has failed to elicit a significant increase in antibody responses in clinical trials. The lack of response in man may be explained by immunological tolerance to similar sialic-acid structures on human cells and raises the question of whether a serogroup B polysaccha- ride vaccine that overcame tolerance would be acceptable in terms of its safety. 2. Vaccines 2.1. Polysaccharide Vaccines The currently licensed polysaccharide vaccines include two formulations— a bivalent A and C vaccine and a tetravalent formulation containing A, C, W135, and Y polysaccharides—that are produced by a number of European and North American companies. The high molecular size polysaccharides used in these vaccines are produced by essentially the same method as first described by Gotschlich et al. (33). All four polysaccharide components have been shown to be immunogenic in adults and older children (34,50,51), although it has only been possible to demonstrate protective efficacy against infection with Meningococcal Vaccines and Developments 5 serogroup A and C organisms because of the low incidence of W135 and Y disease. In early protective efficacy trials in US military recruits, monovalent serogroup C vaccines were demonstrated to have an efficacy in the region of 90% (35). Similar levels of protection were observed when serogroup A vac- cines were studied in Africa and Finland (36). Serum bactericidal antibodies play a crucial role in the protection of the host against meningococcal disease. The evidence for this includes an association between the lack of serogroup specific bactericidal antibodies and occurrence of disease among military recruits (52) and the susceptibility of individuals, who congenitally lack complement components in the membrane-attack com- plex, to repeated meningococcal infections (53). Although there has been con- siderable debate over the way in which the assay should be performed, the serum bactericidal-antibody titer provides an important immunological surro- gate for protection, without which the subsequent development of glycoconjugate vaccines would have been severely hampered. The size and duration of the immune response is age-dependent, reflecting the fact that meningococcal polysaccharides, like other carbohydrate antigens, are T-independent antigens, and suggests that B-cell maturation is critical for an effective immune response (37,54,55). The serogroup C response was not effective in children under 2 yr of age and the licensed vaccines are con- sequently not indicated for use below this age. Serogroup A polysaccharide appears to be more immunogenic than C polysaccharide in young children but neither is capable of inducing long-term immunological memory. The polysac- charide vaccines are therefore generally not used in routine immunization pro- grams due to the lack of protection that they offer in infancy and the relatively short-lived immune response that they elicit. Nevertheless, they are frequently offered to individuals who are at particular risk of infection including: military recruits, undergraduate students, patients with immunodeficiencies, and trav- elers to the so-called “meningitis belt” countries and the Haj pilgrimage (27,56). They are also used together with chemotherapy to control localized outbreaks of serogroup C disease in schools and colleges in industrialized coun- tries (57). In the meningitis belt, polysaccharide vaccine has proved effective at controlling the spread of serogroup A epidemics (58,59) and recently the World Health Organization (WHO) has established a stock of vaccine that can be dispatched to sub-Saharan Africa at short notice whenever a sudden increase in disease rate indicates the potential onset of an epidemic. 2.2. Glycoconjugate Vaccines The success of the Hib glycoconjugate vaccine has highlighted the advan- tages of converting polysaccharides into T-dependent antigens by chemical conjugation to protein-carrier molecules (38,60,61) and has led to the clinical 6 Feavers development of similar vaccines based on the meningococcal serogroup A and C capsular polysaccharides (41,62). Size-fractionated oligosaccharides derived from purified capsular polysaccharides conjugated to either the nontoxic, cross- reacting mutant of diphtheria toxin, CRM197, or tetanus toxoid have been evaluated for their safety and immunogenicity in clinical trials. The depoly- merization, activation, and conjugation of meningococcal serogroup C polysac- charide to tetanus toxoid is detailed in Chapter 4. Miller and Farrington, in Chapter 6 of this volume, review the rationale behind the conduct of clinical trials and the particular problems encountered in the evaluation of meningococcal vaccines. Generally, meningococcal- conjugate vaccines have been well-tolerated; both local and systemic reactions have been relatively mild and similar to those expected for unconjugated polysaccharide vaccines. They have proved to be highly immunogenic over a wide age range, including very young infants (42–45,47–49). Studies in which infants have received three doses of vaccine at 2, 3, and 4 mo have shown that serogroup C- CRM197 conjugates induce high levels of high-avidity, anti-C polysaccharide antibodies that are bactericidal. Richmond et al. also demon- strated that the immune response of infants primed with the conjugate vaccine was boosted by the administration of serogroup C polysaccharide, confirming that the vaccine induces immunological memory (49). These data indicate the successful induction of a T-cell dependent antibody response by serogroup C-CRM197 conjugate vaccines. Other clinical studies have shown that serogroup C conjugates in which tetanus toxoid has been used as the carrier protein or the C polysaccharide is O-deacetylated to be similarly immunogenic and well-tolerated (46). Three serogroup C conjugate vaccines have been licensed in the UK to date. Given the low incidence of disease caused by serogroup C organisms, it was impractical to conduct controlled protective efficacy studies and the license was granted on the basis that: 1) the conjugate was more immunogenic than the existing licensed polysaccharide vaccine, particularly in the very young; 2) it induced a good anamnestic response; and 3) the success of glycoconjugate vaccine technology in reducing disease had been established with the Hib vac- cine. Careful monitoring of serogroup C disease throughout the phased intro- duction of the vaccine into national immunization schedules should provide some assessment of the effectiveness of these vaccines.* Provided that there is sufficient vaccine coverage, the introduction of serogroup C conjugate vaccine *Recent estimates based on surveillance during the first 9 mo following the introduction of the serogroup C conjugate in England indicate that the short-term efficacy of the vaccine was 97% (95% CI 77–99) for teenagers and 92% (65–98) for toddlers (Ramsay, Andrews, Kaczmarski and Miller, 2001, Lancet 357, 195, 196). Meningococcal Vaccines and Developments 7 can reasonably be expected to parallel the previous success of the Hib vaccine, eventually leading to the eradication of serogroup C disease. Although draw- ing such parallels has been expeditious in the development of the new vaccines this optimism is, however, tempered by the knowledge that certain aspects of meningococcal disease and invasive Haemophilus influenzae type b disease are quite different (15). Type b organisms account for almost all septicaemic isolates of H. influ- enzae, whereas several different meningococcal serogroups cause invasive infections. In addition, there is little evidence that virulent isolates of non- type b H. influenzae arise through the genetical exchange of capsular polysaccha- ride loci (63), whereas there is extensive evidence that virulent meningococci frequently exchange antigen genes, including those encoding their capsular polysaccharides (17,64,65). The licensed serogroup C conjugate vaccines offer no cross-protective immunity to the non-serogroup C meningococci that are responsible for most of the meningococcal disease in industrialized coun- tries, and that may arise as consequence of capsular switching. With the wide- spread use of monovalent serogroup C conjugate vaccines, the associated increase in the level of serogroup C specific salivary antibody together with the induction of immunological memory in the vaccinated population is likely to serve to reduce nasopharyngeal carriage, thereby increasing herd immunity (66). This would represent a important shift in the immunological selection acting on meningococci circulating in the vaccinated population and could ultimately result in an increase in disease caused by the other pathogenic serogroups. Further development of meningococcal glycoconjugate compo- nents will inevitably lead to the availability of more comprehensive formula- tions comprising combinations of serogroup A, C, W135, and Y conjugates, but the development of an effective vaccine offering protection against disease caused by serogroup B organisms clearly remains the decisive obstacle in the elimination of meningococcal disease. The poor immunogenicity of vaccine candidates consisting of native serogroup B polysaccharide conjugated to carrier proteins has been attributed to immunological tolerance associated with the presence of sialylated glyco- peptides in human and animal tissues (10). During embryonic and neonatal development, the neural cell adhesion molecule (N-CAM), which is widely distributed in human tissue, has long polysialic acid chains that are recognized by anti-serogroup B antibodies (67). A number of studies have shown that the sialylation of N-CAM modulates cell-cell interactions during organogenesis and has led to concern that pregnancy or fetal development may be adversely affected by high levels of high avidity cross-reacting antibodies produced in response to a serogroup B conjugate vaccine. Jennings et al. postulated that chemical modification of the polysaccharide might overcome immunological 8 Feavers tolerance and induce a safe and protective immune response (68). A modified B polysaccharide, in which the N-acetyl groups at position C-5 of the sialic acid residues are replaced with N-propionyl groups, conjugated to tetanus tox- oid proved to be immunogenic in mice. More recently, N-propionylated serogroup B polysaccharide conjugated to a recombinant meningococcal outer- membrane protein (rPorB) has been shown to be highly immunogenic in non human primates (69). Importantly, no adverse reactions to the trial vaccine were observed in these studies, providing grounds for optimism, although the absence of an autoimmune response and the overall safety of such a vaccine remain to be substantiated by clinical trials, and it will inevitably take many years to establish its long-term safety. The preparation and characteristics of N-propionylated serogroup B polysaccharide conjugated to tetanus toxoid are described in Chapter 5. 2.3. Protein Vaccines Concern over the safety of vaccines based on the serogroup B capsular polysaccharide has focused attention on alternative cell-surface antigens as vaccine candidates (Table 1). The most advanced of these, in terms of their clinical development, consist of meningococcal outer-membrane vesicles (OMVs) (70–72) or purified outer-membrane proteins (OMPs) (73). Grown in broth culture, N. meningitidis produces substantial quantity of outer-membrane blebs, containing the same complement of OMPs as the organism itself (74). These vesicles can be readily purified from detergent treated meningococcal cultures to form the basis of vaccine formulations (Chapters 6 and 7). Unfortu- nately, such vaccines suffer from significant drawbacks: 1) the most immuno- genic antigens they contain are also the most variable, suggesting that OMV vaccines may not offer comprehensive protection against all meningococci; 2) their protective efficacy in young infants, the group most at risk of meningococcal dis- ease, has not been demonstrated; and 3) protection appears to be short-lived. It has been suggested that mucosally administered OMV formulations may overcome some of these shortcomings and to explore this possibility immunogenicity studies have been performed in human volunteers (see Chapter 16) (75). Efficacy trials have been conducted with both OMV and purified OMP for- mulations. In response to an outbreak of disease in Cuba in the late 1980s, the Finlay Institute produced an OMV vaccine, based on this BϺ4ϺP1.19,15 (ET-5 complex) isolate, that also contained serogroup C capsular polysaccharide. Case controlled studies using the Cuban vaccine in Brazil revealed that protec- tive efficacy was age-dependent; an efficacy of greater than 70% was recorded for children older than four years, while in younger children no efficacy was demonstrated (76). Similarly, an increase in meningococcal disease in Norway caused by a BϺ15ϺP1.7,16 isolate belonging to the ET5 complex prompted the Meningococcal Vaccines and Developments 9 development of an OMV vaccine, the protective efficacy of which proved to be 57% in a double-blind, placebo-controlled trial conducted in secondary- school pupils (71). A serotype-specific outbreak of serogroup B meningococ- cal disease in Iquique, Chile during the 1980s lead to the evaluation of a vaccine consisting of purified meningococcal OMPs noncovalently complexed to serogroup C polysaccharide in a randomized, controlled trial. The vaccine effi- cacy was 70% in the volunteers aged from 5–21 yr, but was not protective in children aged between 1 and 4 yr (73). In all three studies, which used two dose schedules, there was evidence of better protection early after immunization, indicating that protection is short-lived and leading to suggestions that a third dose of vaccine may improve protective efficacy (27). Each of these vaccines was based on a specific meningococcal isolate. Given the antigenic diversity of N. meningitidis isolates, this raises concerns that they cannot be relied upon to offer cross-protection against all virulent meningococci; fears that have been substantiated by immunogenicity studies showing that the ability of OMV vac- cines to elicit cross-protective bactericidal antibodies is limited (77). Table 1 Summary of Protein-Vaccine Candidates That Might Offer Protection Against Serogroup B Disease Vaccine candidate Stage of development Reference Outer membrane vesicle: Finlay Institute Licensed in some Central (70) and Southern American countries NIPH Completed efficacy (phase III) studies in teenagers (71) RIVM Immunogenicity (phase II) (72,85) studies in various age groups Purified outer membrane Efficacy studies (73) proteins Transferrin binding protein B Preliminary clinical studies (87) (TbpB) in adult volunteers Neisseria surface protein Preclinical research (113) (NspA) Transferrin binding protein A Preclinical research (114) (TbpA) FrpB Preclinical research (115) Recombinant PorA Preclinical research (116) Peptides from PorA Preclinical research (117) TspA Preclinical research (118) [...]... Moore, P S., and Broome, C V (1989) Global epidemiology of meningococcal disease Clin Microbiol Rev 2, s118–s124 4 Peltola, H (1983) Meningococcal disease: still with us Rev Infect Dis 5, 71–91 5 Steven, N and Wood, M (1995) The clinical spectrum of meningococcal disease, in Meningococcal Disease (Cartwright, K., ed.), John Wiley and Sons, Chichester, UK, pp 177–205 6 Cartwright, K A V (1995) Meningococcal. .. is increased by cell contactFrom: Methods in Molecular Medicine, vol 66: Meningococcal Vaccines: Methods and Protocols Edited by: A J Pollard and M C J Maiden © Humana Press Inc., Totowa, NJ 23 24 Pollard and Goldblatt dependent transcriptional upregulation of the PilC1 protein that is required for pilin assembly (7) However, tighter adherence between the organism and the epithelial cell is mediated... influenza infection and invasive meningococcal disease and the increased risk of meningococcal disease and carriage associated with exposure to tobacco smoke (19) both suggest that the integrity of the mucosal surface is important in resisting colonization and invasion by meningococci Recent data suggest that the charge and hydrophobicity of the mucosa are affected by exposure to tobacco smoke and that this... Goldschneider, I., Lepow, M L., Draper, T F., and Randolph, M (1978) Carriage of Neisseria meningitidis and Neisseria lactamica in infants and children J Infect Dis 137, 112–121 2 Cartwright, K A., Stuart, J M., and Robinson, P M (1991) Meningococcal carriage in close contacts of cases Epidemiol Infect 106, 133–141 3 Virji, M., Alexandrescu, C., Ferguson, D J., Saunders, J R., and Moxon, E R (1992) Variations in... S., and Hankins, W A (1986) Reactogenicity and immunogenicity of a quadravalent combined meningococcal polysaccharide vaccine in children J Infect Dis 154, 1033–1036 Goldschneider, I., Gotschlich, E C., and Artenstein, M S (1969) Human immunity to the meningococcus I The role of humoral antibodies J Exp Med 129, 1307–1326 Figueroa, J., Andreoni, J., and Densen, P (1993) Complement deficiency states and. .. deficiency states and meningococcal disease Immunol Res 12, 295–311 Lepow, M L., Goldschneider, I., Gold, R., Randolph, M., and Gotschlich, E C (1977) Persistence of antibody following immunization of children with groups A and C meningococcal polysaccharide vaccines Pediatrics 60, 673–680 Goldblatt, D (1998) immunization and the maturation of infant immune responses Dev Biol Stand 95, 125–132 WHO (2000)... al (2000) Immunogenicity and safety of a hexavalent meningococcal outer-membrane-vesicle vaccine in children of 2–3 and 7–8 years of age Vaccine 18, 1456–1466 73 Boslego, J., Garcia, J., Cruz, C., Zollinger, W., Brandt, B., Ruiz, S., et al (1995) Efficacy, safety, and immunogenicity of a meningococcal group B ( 15:P1.3) outer Meningococcal Vaccines and Developments 74 75 76 77 78 79 80 81 82 83 84 85... Cartwright, K A., and Poolman, J T (1992) The lipooligosaccharide immunotype as a virulence determinant in Neisseria meningitidis Microb Pathog 13, 219–224 Zollinger, W D and Moran, E (1991) Meningococcal vaccines—present and future Trans R Soc Trop Med Hyg 85(Suppl 1), 37–43 Estabrook, M., Mandrell, R E., Apicella, M A., and Griffiss, J M (1990) Measurement of the human response to meningococcal lipooligosaccharide... teenagers Reservations over the safety and effectiveness of polysaccharide and OMV vaccines against serogroup B disease have stimulated the search for the “Holy Grail” vaccine candidate that is antigenically highly conserved and yet elicits a safe and protective immune response Most alternative vaccine candidates have not so far progressed beyond preclinical research and development (see Table 1) Only... expression (by slipped-strand mispairing in the polsialyltransferase gene) facilitates adherence and invasion in vivo (12) Methods used in the study of interactions of meningococci with epithelia and endothelial cells are considered in Meningococcal Disease,” edited by A J Pollard and M C J Maiden, (12a) It appears that there are several bacterialsurface structures critical for adhesion to and invasion through . Humana Press Meningococcal Vaccines Edited by Andrew J. Pollard, MD, PhD Martin C. J. Maiden, PhD Methods and Protocols Humana Press Meningococcal Vaccines Edited by Andrew J. Pollard,. Maiden, PhD Methods and Protocols M E T H O D S I N M O L E C U L A R M E D I C I N E TM Meningococcal Vaccines and Developments 1 1 From: Methods in Molecular Medicine, vol. 66: Meningococcal. vol. 66: Meningococcal Vaccines: Methods and Protocols Edited by: A. J. Pollard and M. C. J. Maiden © Humana Press Inc., Totowa, NJ 1 Meningococcal Vaccines and Vaccine Developments Ian M. Feavers 1.