Safety and immunogenicity of New Zealand strain meningococcal serogroup B OMV vaccine in healthy adults: Beginning of epidemic control

Vaccine 24 (2006) 1395–1400

Safety and immunogenicity of New Zealand strain meningococcal serogroup B OMV vaccine in healthy adults: Beginning of epidemic control V. Thornton a , D. Lennon a,∗ , K. Rasanathan a , J. O’Hallahan c , P. Oster d , J. Stewart a , S. Tilman d , I. Aaberge e , B. Feiring e , H. Nokleby e , E. Rosenqvist e , K. White a , S. Reid f , K. Mulholland g , M.J. Wakefield d , D. Martin b a

The University of Auckland, Meningococcal B Project, P.O. Box 98847, South Auckland Mail Centre, Auckland, New Zealand b The Institute of Environmental Science and Research Ltd. (ESR), Wellington, New Zealand c The Ministry of Health, Private Bag, Wellington, New Zealand d Chiron Vaccines S.r.l, Via Fiorentina, 1 53100 Siena, Italy e Norwegian Institute of Public Health, P.O. Box 4404, Nydalen, No0403 Oslo, Norway f Ropata Village Medical Centre, 577 High Street, Lower Hutt, New Zealand g University of Melbourne, Royal Children’s Hospital, Flemington Rd, Parkville, Vic. 3051, Australia Received 1 February 2005; received in revised form 25 May 2005; accepted 9 September 2005 Available online 4 October 2005

Abstract As the first step towards control of a strain specific epidemic of meningococcal disease in New Zealand (NZ), this study, an observer-blind, randomised controlled trial in 75 healthy adults, evaluated safety and immunogenicity of two different dosages of a meningococcal group B vaccine administered in a three dose regime. The “tailor-made” outer membrane vesicle (OMV) vaccine (candidate vaccine) developed using a New Zealand meningococcal group B strain (B:4:P1.7b,4) was well tolerated with no vaccine related serious adverse events. Similar local and systemic reactions were observed in those receiving the New Zealand candidate vaccine and the control parent Norwegian vaccine (MenBvacTM ). A four-fold rise in serum bactericidal antibodies (SBAb) against the vaccine strain 4–6 weeks after the third vaccination was achieved in 100% of New Zealand candidate vaccine 2519 ␮g participants and in 87% of 50 ␮g participants. The safety and immunogenicity profile observed in this study of healthy adults enabled studies in children to be initiated using 25 ␮g dosage. © 2005 Elsevier Ltd. All rights reserved. Keywords: Meningococcal group B; OMV vaccine; Phase I/II trial

1. Introduction In 2002, New Zealand (NZ) entered its 12th year of a widespread epidemic of group B meningococcal disease dominated by a clone of strains with a single Por A subtype which is estimated to account for approximately 85% of reported cases [1,2]. The incidence of reported cases of meningococcal disease in 2002 was 14.9 per 100,000 [2]. More than 80% of cases occur in those under 20 years of age. ∗

Corresponding author. Tel.: +64 9 263 3988; fax: +64 9 263 9467. E-mail address: [email protected] (D. Lennon).

0264-410X/$ – see front matter © 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.vaccine.2005.09.043

The greatest risk is in less than one year old at an age-specific rate of 153.7 per 100,000. There was no indication that this epidemic was abating [2,3]. Group B outer membrane vesicle (OMV) vaccines produced in Norway and Cuba have been assessed in efficacy trials and have been found to be effective in teenagers [4,5]. The large scale use of the group B OMV vaccines, mostly in Latin America, has increased the possibility of evaluating vaccine effectiveness in ways other than a randomised trial [6]. The persistence of the epidemic and of the subtype in New Zealand, the increasing confidence that serum bactericidal antibodies (SBAb), as measured by the serum bactericidal

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assay (SBA), may provide an indication of protection from invasive meningococcal disease and a demonstrated robust serologic response against the homologous strain (vaccine strain specific) in infants [7] enabled a strategy of developing a strain specific “tailor-made” vaccine followed by clinical trials to be designed [8]. This was supported by the United Kingdom approach to meningococcal group C disease control where vaccines were licensed based on a serological correlate accompanied by immunogenicity and safety data [9]. While the use of the SBAb level achieved as a correlate for meningococcal vaccine efficacy is best shown by group C polysaccharide vaccine studies [10–12], SBAb has also become the primary indicator used to assess protective immunity induced by serogroup B meningococcal vaccine candidates [7,13–22]. New Zealand strain vaccine production is similar to that of the parent vaccine MenBvacTM [23], with a strain change; thus establishing a similarity between the vaccine both at the developmental level and in clinical trials. This will enable linkage to the large database of safety data available for the parent vaccine. New Zealand’s approach, through a consortium of the Ministry of Health, Chiron Vaccines and a research team lead from The University of Auckland, is to conduct safety and immunogenicity trials in adults, school students, toddlers and infants. It is assumed these studies will lead to eventual vaccine licensure and the ability to offer the vaccine to all under 20-year-old in New Zealand. The aim is to achieve rapid control of the epidemic and reduce the incidence of invasive meningococcal group B disease particularly in younger children who carry the burden of disease. This is the first use of this vaccine in humans although the similarity to the Norwegian parent vaccine MenBvacTM allowed a phase I/II trial. High immunogenicity with induction of bactericidal antibodies against the B:4:P1.7b,4 meningococcal strains from NZ was found in mice, and no unexpected toxicity or adverse events were observed [24]. This phase I/II study in healthy adults is the first in a series of studies to establish safety and demonstrate immunogenicity of the NZ candidate vaccine developed and produced at the Norwegian Institute of Public Health in collaboration with Chiron Vaccines. This will enable further studies in children. Two dosages were used (25 and 50 ␮g), to assist with defining an appropriate dose to be used in further studies. The parent Norwegian vaccine MenBvacTM (25 ␮g) was the comparator.

2. Methods 2.1. Study design A phase I/II randomised, controlled, observer-blind study of OMV vaccines among healthy adult (18–50 years inclusive) volunteers was undertaken. Recruitment of hospital staff took place in Auckland, New Zealand.

Ethics approval from the Ministry of Health Ethics Committee (Auckland region) was obtained and 75 participants provided written informed consent before enrolment. Participants were excluded by medical history and/or physical examination if they were pregnant had a hypersensitivity to previous vaccine, or an acute or chronic systemic illness. Other reasons for exclusion from the study were systemic antibiotic use in the 14 days prior to consent, or if they had received blood products within three months. Intimate household or day care centre exposure to Neisseria meningitidis serogroup B disease or receipt of any vaccination within the last 50 days (except the influenza or adult diphtheria–tetanus vaccines) were also reasons for exclusions. 2.2. Vaccination and specimen collection The NZ candidate vaccine was made with reference to the parent vaccine, the Norwegian meningococcal group B vaccine, MenBvacTM [23]. The study vaccine was prepared from a B:4:P1.7b,4 New Zealand meningococcal case strain (NZ98/254) by fermented growth and extraction with the detergent deoxycholate to yield OMVs which were adsorbed to aluminium hydroxide.A synthetic medium, with no material of bovine origin, was used as a culture medium for the NZ candidate vaccines [24]. Each vaccine dose contained either 25 or 50 ␮g per 0.5 ml of OMV antigen for the NZ vaccine. The vaccine vials contained up to 0.8 ml. The vaccines were all approved for use in this trial by the New Zealand Standing Committee on Therapeutic Trials. The 75 volunteers were randomised by random number table in a 1:1:1 ratio to one of the three vaccine groups. The vaccine was administered intramuscularly, by an unblinded study staff member, to the non-dominant deltoid, through a 25 mm 23-gauge needle in a three-dose regimen, at six weekly intervals. The dosing interval was based on previous experience with the parent Norwegian vaccine MenBvacTM [5,18]. Post vaccination each participant was observed for 30 min. Blood samples were obtained, prior to vaccination by venepuncture on Day 1 and 4–6 weeks after each vaccination. After centrifugation on the same day, the sample was stored between +2 and +8 ◦ C. The samples were couriered to the ESR laboratory in Wellington for subsequent analysis. The serum was stored at −75 ◦ C. 2.3. Outcome measures For 7 days following the vaccination participants recorded daily, on a diary card, the presence and severity of local and systemic reactions. Swelling, erythema and induration were measured. Pain, headache, nausea, malaise, myalgia and arthralgia were ranked and reported on a standardized form. Analgesic use and absence from work was recorded. Sublingual temperature was recorded daily. Active telephone surveillance occurred at 24–48 h post vaccination. The diary card was collected between Days 8 and 13 with assessment of clinical status at that time and verification of the diary card.

V. Thornton et al. / Vaccine 24 (2006) 1395–1400

The primary immunogenicity outcome was whether the vaccines induced an immune response as measured by enzyme-linked immunosorbent assay (ELISA) and/or SBA. A sero-responder was defined as a participant showing at least a four-fold increase in SBAb levels at 4–6 weeks post vaccination as compared to pre-vaccination against the vaccine strain NZ98/254. SBAb titres were expressed as reciprocal values of serum dilution giving ≥50% kill of the target strain, defining a titre of <2 as 1. ELISA antibody levels were expressed on a continuous scale. 2.4. Laboratory methods Serum bactericidal antibodies were determined by a modification of the serum bactericidal assay [14] using as target strains the NZ candidate vaccine strain (NZ98/254), the parent Norwegian strain (44/76-SL) and two selected other “wild-type” patient isolates (NZ94/167, NZ02/09) representative of the epidemic strain in New Zealand (B:4:P1.7b,4). All serum samples from the same subject were tested at the same time under the same test conditions using human complement. Serum IgG concentrations were also tested by enzyme-linked immunosorbent assay using NZ OMV vaccine vesicles as the antigen source [18].

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by exponentiating (base 10) the least square means of the logarithmically transformed (base 10) titres.

3. Results Seventy-five participants were enrolled in the study conducted between May and October 2002. The mean age of participants was 34.6 years (range 20–50 years) with 52 (69%) participants female. Seventy-three participants completed the three-dose regimen and provided four blood samples. One participant from each of the NZ candidate vaccine groups withdraw at the time of the second vaccination. These withdrawals were unrelated to the vaccine. Five participants were excluded on one occasion from SBAb analysis due to concurrent antibiotic use. Participants received an estimated 0.5–0.6 ml of vaccine per dose. 3.1. Adverse events The vaccines were well tolerated and there were no serious vaccine related adverse events. The local and systemic reactions are shown in Table 1. The majority of reactions were mild in nature. The study was not powered to detect a difference in adverse events between vaccine arms.

2.5. Data analysis This phase I/II study was designed as a descriptive study. Incidences of local and systemic reactions were reported as those occurring during the 7 days post vaccination and summarised by dose maximal severity and vaccine group. The percentage of seroresponders against each of the four meningococcal strains measured by SBA and/or vaccine strain vesicles measured by ELISA at Day 1 and 6 weeks after each vaccination were determined. GMT’s were constructed

3.1.1. Local reactions The local reactions were reported at approximately the same frequency in the NZ candidate vaccine groups and the MenBvacTM group. Pain at the site of injection was the most commonly experienced local reaction occurring after at least one dose in 100% of the NZ candidate vaccine groups and 96% of the MenBvacTM group (Table 1). Most of the pain was mild in nature (Table 2). Mild pain, after at least one dose, persisted at Day 7 in 44% of the 25 ␮g NZ candidate vac-

Table 1 Local and systemic reactions following immunisation of healthy adults by vaccinea NZ candidate vaccine, 25 ␮g vaccine (N = 25)

NZ candidate vaccine, 50 ␮g vaccine (N = 24)

MenBvacTM , 25 ␮g vaccine (N = 26)

Local reactions Pain at site of injection Erythema Swelling Induration

25 (100%) 10 (40%) 5 (20%) 11 (44%)

24 (100%) 16 (67%) 14 (58%) 11 (46%)

25 (96%) 8 (31%) 9 (35%) 7 (27%)

Systemic reactions Nausea Malaise Myalgia Arthralgia Headache Fever ≥38.5 ◦ C

8 (32%) 12 (48%) 10 (40%) 2 (8%) 11 (44%) 0

6 (25%) 11 (46%) 14 (58%) 4 (17%) 12 (50%) 1 (4%)

10 (38%) 14 (54%) 11 (42%) 3 (12%) 14 (54%) 1 (4%)

Others Analgesics Stayed at home due to reaction

10 (40%) 1 (4%)

14 (58%) 4 (17%)

17 (65%) 2 (8%)

a

All reactions considered to be present in a participant if they occurred after at least one of the vaccinations.

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Table 2 Local and systemic reactions following immunisation of healthy adults with NZ Candidate25 ␮g vaccine by dose Reactions

Dose 1 (n = 25)

Dose 2 (n = 24)

Dose 3 (n = 24)

Milda

Moderateb

Severec

Milda

Moderateb

Severec

(%)

(%)

(%)

(%)

(%)

(%)

Milda (%)

Local Pain Erythema Swelling Induration

64 16 4 8

28 4 4 4

0 0 8 4

54 4 0 13

46 4 4 4

0 0 4 0

83 17 0 21

8 4 0 4

4 0 4 0

Systemic Nausea Malaise Myalgia Arthralgia Headache Fever (≥38.5 ◦ C)

16 24 8 4 16 0

0 4 4 0 4 0

0 0 0 0 0 0

5 13 17 4 17 0

8 4 4 0 8 0

0 0 0 0 0 0

9 8 21 0 17 0

4 13 4 0 4 0

0 0 4 0 0 0

Moderateb (%)

Severec (%)

Mild, moderate and severe are the severity of adverse reactions. a Mild reaction was defined as the symptom being transient with no limitation to normal activity, or erythema/induration/swelling being 10–25 mm in diameter. b Moderate reaction was defined as the symptom causing some limitation to normal activity, or erythema/induration/swelling of 26–50 mm in diameter. c Severe reaction was defined as the symptom resulting in the participant being unable to perform normal activities, or erythema/induration/swelling of >50 mm. (Severe = maximum possible response.)

cine group, in 58% of the 50 ␮g NZ candidate vaccine group and 65% of the parent vaccine group. Erythema, swelling and induration for each group of vaccines were considerably less common than pain (Table 1). Frequency of most local reactions decreased over the 7 days post vaccination (Fig. 1). Adverse reactions following each dose appeared to be similar (data shown for the 25 ␮g NZ candidate vaccine group in Table 2).

candidate vaccine and two after MenBvacTM vaccine. Almost all were absent for 1 day only, and following only one dose. The exceptions were one recipient of the Norwegian vaccine who was absent for 2 days and two recipients of 50 ␮g New Zealand candidate vaccine who were both away after two doses, one for 2 days on one occasion.

3.1.2. Systemic reactions The overall frequency of the systemic reactions in the NZ candidate vaccine group was similar to the MenBvacTM vaccine group (Table 1). Approximately 20% of the participants experienced malaise, myalgia or headache after any one dose of vaccine (Table 2). Most systemic reactions were mild or moderate and resolved quickly (Fig. 1). Only two participants experienced fever (sub-lingual temperature between 38.50 and 39.50 ◦ C). Seven different participants had at least 1 day of absence from work following at least one vaccination, one after 25 ␮g NZ candidate vaccine, four after the 50 ␮g NZ

3.2.1. NZ candidate vaccines Almost all participants who received the 25 and 50 ␮g NZ candidate vaccines showed a four-fold or greater increase in SBAb to the meningococcal strain NZ98/254, and IgG antibodies in ELISA, between baseline and 6 weeks following the third dose. The percentage of seroresponders after three doses of vaccine was 100% in the 25 ␮g group and 87% in the 50 ␮g group (Table 3) by SBA against NZ98/254. After two doses the respective responses were 87 and 78%. The percentage seroresponders after three doses against the heterologous strain H44/76-SL was 48% in the vaccine 25 ␮g group and 43% in the 50 ␮g group. The anti-NZ98/254 OMV IgG (ELISA) antibody levels rose at least four-fold in 88% of the 25 ␮g group and 78% of the 50 ␮g group. Serum bactericidal antibody titres against the NZ 98/254 strain in the 25 ␮g group following the three vaccine doses showed wide individual variability (Fig. 2) with a GMT of 14, 28, and 49 after dose 1, 2 and 3, respectively. Following dose 3 of 25 ␮g of NZ candidate vaccine all participants showed a SBAb titre of at least 1:8 against the vaccine strain (range 8–430). Both NZ candidate vaccine groups showed an increase in SBAb titres similar to the response to the vaccine strain NZ98/254 against the other two “wild-type” NZ strains (NZ02/09 and NZ94/167) tested (Table 3).

Fig. 1. Adverse reactions of healthy adults following immunisation with NZ MenB OMV 25 ␮g vaccine, by day post vaccination (*).

3.2. Immunogenicity

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Table 3 Serum bactericidal antibody seroresponders to selected meningococcal group B strains in healthy adults by strain and vaccinea Vaccine

Post dose

Number

Strain NZ98/254 (%)

NZ candidate vaccine 25 ␮g

63b

NZ02/09 (%)

NZ94/167 (%)

H44/76 (%)

1 2 3

24 23 23

87b 100b

46 78 87

38 70 83

29 43 48

NZ candidate vaccine 50 ␮g

1 2 3

23 23 23

65 78 87

48 70 83

57 78 78

30 35 43

MenBvacTM 25 ␮g

1 2 3

24 25 26

33 44 42

21 28 35

42 52 54

54b 52b 65b

a A seroresponder is defined as a participant who shows at least a four-fold increase in serum bactericidal antibody compared to their baseline (pre-vaccination) measure, with a titre <2, i.e. below the detection limit, assigned a value of one and using a continuous scale of titre values.(interpolated titres). b The response measured to the strain from which the vaccine was derived.

Fig. 2. Interpolated serum bactericidal antibody to NZ98/254 in healthy adults following immunisation with 25 ␮g NZ candidate vaccine.

3.2.2. MenBvacTM vaccine Sixty-five percent of participants who received MenBvac produced a four-fold rise in SBAb against strain 44/76-SL after the third dose (Table 3). Eighty-one percent produced a corresponding four-fold rise in ELISA antibody levels (IgG) after the third dose. The response to the heterologous strain NZ98/254 in the MenBvac vaccine group was 42% attaining a four-fold SBAb rise and 88% a four-fold ELISA (IgG) rise.

4. Discussion This is the first study of the New Zealand meningococcal P1.7b, 4 vaccine in humans and the beginning of the pathway

to epidemic control. The safety and immunogenicity profile observed in this study of healthy adults enabled studies in children to be initiated using the 25 ␮g dosage. The results confirmed the New Zealand candidate vaccine has a reactogenicity profile similar to MenBvacTM [25]. Furthermore, there were no serious vaccine related adverse events. The incidence of local and systemic reactions to the NZ candidate vaccine is similar to MenBvacTM [5,7,25,26]. Most people experienced mild or moderate symptoms which resolved reasonably quickly. The systemic symptoms were less common than local reactions and were predominantly mild to moderate in nature. Systemic reactions rarely persisted beyond Day 7 and were mild and similar in frequency in all vaccine groups. A four-fold rise in SBAb against strain NZ98/254 after the third vaccination when compared to pre-vaccination values was achieved in 100% of New Zealand candidate vaccine 25 ␮g participants and in 87% of 50 ␮g participants. The results in this study compare well to previous group B meningococcal vaccine studies using similar laboratory methodologies with minor differences where a four-fold rise in titre was seen in high percentages of recipients after the third dose [7,21,26]. Four-fold SBAb rise as measured by serum bactericidal assay against NZ98/254 occurred in only 42% of those given MenBvacTM . The study was not powered to conclude whether different dosages of vaccine (i.e., 25 or 50 ␮g) influenced the percentage of participants attaining a SBAb titre rise. Recipients of the NZ candidate vaccine had comparable antibody titres against the NZ98/254 vaccine strain and the two “wild-type” strains. Other studies have found three doses to sustain the immune response and therefore, potential for protection [18,21]; thus three doses were given in this study. An extension to this study will be undertaken to elicit ongoing kinetics of immune response to NZ candidate vaccines in the trial participants. This study has provided the basis for further vaccine trials in children, toddlers and babies using the NZ candidate 25 ␮g vaccine. It is hoped that the widespread use of the vaccine will

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lead to the eventual control of the group B meningococcal epidemic in New Zealand.

Acknowledgements We thank the study participants for their altruism in taking part. We also thank the research co-ordinators Sarah Douglas and Robyn Beckerleg, the study nurses, the laboratory workers at ESR Anne Glennie, Nicola Ruijne, Lisa McCallum and the study staff at Chiron Vaccines. We acknowledge the assistance of the District Health Boards. This study was funded by the New Zealand Ministry of Health and Chiron Vaccines.

References [1] Martin DR, Walker SJ, Baker MG, Lennon DR. New Zealand epidemic of meningococcal disease identified by a strain with phenotype B:4:P1.4. J Infect Dis 1998;177:497–500. [2] Ministry of Health. The epidemiology of meningococcal disease in New Zealand in 2002 (http://www.moh.govt.nz). [3] Bremner C, Lennon D, Martin D, Baker M, Rumke H. Epidemic meningococcal disease in New Zealand: epidemiology and potential for prevention by vaccine. N Z Med J 1999;112(1091):257–9. [4] Sierra GVG, Campa HC, Varcacel NM, Sarcia IL, Izqierdo PL, Sotolongo PF, et al. Vaccine against group B Neisseria meningitidis: protection trial and mass vaccination results in Cuba. NIPH Ann 1991;14(2):195–207. [5] Bjune G, Hoiby EA, Gronnesby JK, Arnesen O, Fredriksen JH, Lindbak AK, et al. Effect of an outer membrane vesicle vaccine against group B meningococcal disease in Norway. Lancet 1991;338: 1093–6. [6] Wenger JD. Serogroup B meningococcal disease: new outbreaks, new strategies. JAMA 1999;281(16):1541–3. [7] Tappero JW, Lagos R, Ballesteros AM, Pikaytis B, Williams D, Dykes J, et al. Immunogenicity of 2 Serogroup B outer-membrane protein meningococcal vaccines: a randomised controlled trial in chile. JAMA 1999;281(16):1520–7. [8] Holst J, Feiring B, Meyer Naess L, Norheim G, Kristiansen P, Hoiby EA, et al. The concept of “tailor-made”, protein based outer membrane vesicle vaccines against meningococcal disease. Vaccine 2005;23:2202–5. [9] Miller E, Salisbury D, Ramsay M. Planning, registration, and implementation of an immunisation campaign against meningococcal serogroup C disease in the UK: a success story. Vaccine 2002;20(Suppl. 1):S58–67. [10] Goldschneider I, Gotschlich E, Artenstein M. Human immunity to the meningococcus. I The role of humoral antibodies. J Exp Med 1969;129:1307–26. [11] Goldschneider I, Gotschlich E, Artenstein M. Human immunity to the meningococcus. II Development of natural immunity. J Exp Med 1969;129:1327–48. [12] Gotschlich E, Goldschneider I, Artenstein M. Human immunity to the meningococcus. IV Immunogenicity of group A and group C meningococcal polysaccharides in human volunteers. J Exp Med 1969;129:1367–84.

[13] Boslego J, Garcia J, Cruz C, Zollinger W, Brandt B, Martinez M, et al. Efficacy, safety, and immunogenicity of a meningococcal group B (15:P1.3) outer membrane protein vaccine in Iquique, Chile. Vaccine 1995;13(9):821–9. [14] Hoiby EA, Rosenqvist E, Froholm LO, Bjune G, Feiring B, Nokleby H, et al. Bactericidal antibodies after vaccination with the Norwegian meningococcal serogroup B outer membrane vesicle vaccine: a brief survey. NIPH Ann 1991;14(2):147–55 (discussion 155–6). [15] Rosenqvist E, Hoiby EA, Wedege E, Kusecek B, Achtman M. The 5C protein of Neisseria meningitidis is highly immunogenic in humans and induces bactericidal antibodies. J Infect Dis 1993;167:1065–73. [16] Milagres LC, Ramos SR, Saachi CT, Melles CEA, Vieira VSD, Sato H, et al. Immune response of brazilian children to a neisseria meningitidis serogroup B outer membrane protein vaccine: comparison with efficacy. Infect Immun 1994;62(10):4419–24. [17] Aase A, Bjune G, Hoiby EA, Rosenqvist E, Pedersen AK, Michaelsen TE. Comparison among opsonic activity, antimeningococcal immunoglobulin g response, and serum bactericidal activity against meningococci in sera from vaccines after immunization with a serogroup b outer membrane vesicle vaccine. Infect Immun 1995;63(9):3531–6. [18] Rosenqvist E, Hoiby EA, Wedege E, Bryn K, Kolber J, Klem A, et al. Human antibody responses to meningococcal outer membrane antigens after three doses of the norwegian group B meningococcal vaccine. Infect Immun 1995;63(12):4642–52. [19] Peeters CCAM, Rumke HC, Sundermann LC, Van der Voort ER, Meulenbelt J, Schuller M, et al. Phase I clinical trial with a hexavalent PorA containing meningococcal outer membrane vesicle vaccine. Vaccine 1996;14(10):1009–15. [20] Van der Voort ER, Van der Ley P, Van der Biezen J, George S, Tunnela O, Van Dijken H, et al. Specificity of human bactericidal antibodies against porA P1.7, 16 induced with a hexavalent meningococcal outer membrane vesicle vaccine. Infect Immun 1996;64:2745–51. [21] Perkins BA, Jonsdottir K, Briem H, Griffiths E, Pikaytis BD, Hoiby E, et al. Immunogenicity of two efficacious outer membrane proteinbased serogroup B meningococcal vaccines among young adults in Iceland. J Infect Dis 1998;177:683–91. [22] Holst J, Feiring B, Fuglesang JE, Hoiby E, Nokleby H, Aaberge I, et al. Serum bactericidal activity correlates with the vaccine efficacy of outer membrane vesicle vaccines against Neisseria meningitidis serogroup B disease. Vaccine 2003;21(7-8):734–7. [23] Frasch CE, van Alphen L, Holst J, Poolman JT, Rosenqvist E. Outer membrane protein vesicle vaccines for meningococcal disease. In: Pollard AJ, Maiden MCJ, editors. Methods in molecular medicine meningococcal vaccines: methods and protocols, vol. 66. Totowa, New Jersey: Humana Press Inc.; 2001. p. 81–107. [24] Rosenqvist E, Bryn K, Harbak K, Holst J, Hoiby E, Kristiansen P. Development of a tailor-made outer membrane vesicle vaccine against the group B meningococcal epidemic in New Zealand. In: Abstracts of the 13th international pathogenic neisseria conference. AS, Oslo: Nordberg Aksidenstrykkeri; 2002. p. 64. [25] Nokleby H, Feiring B. The Norwegian meningococcal group B outer membrane vesicle vaccine: side effects in phase II trials. NIPH Ann 1991;14(2):95–101 (discussion 101–2). [26] Fischer M, Carlone GM, Holst J, Williams D, Stephens DS, Perkins BA. Neisseria meningitidis serogroup B outer membrane vesicle vaccine in adults with occupational risk for meningococcal disease. Vaccine 1999;17(19):2377–83.