Immunogenicity of a Tetravalent Meningococcal Glycoconjugate Vaccine in Infants: Title and subTitle BreakA Randomized Controlled Trial FREE

It is estimated that each year 1400 to 2800 cases of invasive meningococcal disease occur in the United States and that 10% to 14% of individuals experiencing disease will die.¹ Among survivors, up to 20% have significant sequelae, including neurologic disability, amputation, and hearing loss.^{2 - 3} Highest rates of disease are among infants younger than 1 year (9.2/100 000 during 1991-2002)¹ ; however, a second peak of disease is observed in adolescent years, and 75% of infections in these age groups are caused by serogroups C, W-135, or Y.⁴ Accordingly, the US Advisory Committee on Immunization Practices now advises immunization with a tetravalent(serogroups A, C, W-135, and Y) meningococcal glycoconjugate vaccine for all 11- to 18-year-olds.⁵ This vaccine contains saccharides derived from the capsules of Neisseria meningitidis serogroups A, C, W-135, and Y individually conjugated to diphtheria toxoid carrier proteins. Unlike the tetravalent plain polysaccharide vaccine,^{6 - 7} it is anticipated that the glycoconjugate vaccine will induce relatively long-lasting protection and, through a reduction in oropharyngeal carriage–related transmission, result in herd immunity.

However, in common with the polysaccharide vaccine, the licensed tetravalent glycoconjugate vaccine was poorly immunogenic in infants⁸ and is therefore not licensed for use in children younger than 2 years in the United States⁹ and Canada.¹⁰ This poor immunogenicity in younger children is in contrast with other licensed glycoconjugate vaccines such as the monovalent N meningitidis serogroup C glycoconjugate vaccines, which have been successfully used from infancy in many developed countries.¹¹ Therefore, despite the highest rates of invasive meningococcal disease occurring in children younger than 2 years,¹ no vaccine is licensed in the United States for the prevention of meningococcal serogroups A, C, W-135, or Y disease in this age group. Similarly, no vaccine is available to prevent serogroups A, Y, or W-135 infections in countries using the monovalent serogroup C glycoconjugate vaccines.

Recent developments such as the emergence of serogroup Y in North America in the late 1990s and epidemics of serogroup W-135 in sub-Saharan Africa and the Middle East have emphasized the unpredictable temporal variations in the serogroup epidemiology of meningococcal disease. In the absence of a vaccine that is able to protect against meningococcal serogroup B, a tetravalent meningococcal vaccine offers the broadest possible protection against the emergence of meningococcal disease.

A novel tetravalent meningococcal glycoconjugate vaccine (MenACWY) has therefore been developed. Unlike the currently licensed vaccine, in which a chemically detoxified diphtheria toxoid is used as the carrier protein, MenACWY uses CRM-₁₉₇, a natural mutant of the diphtheria toxin. Other differences include the use of an aluminum phosphate adjuvant and the quantity and lengths of the saccharide chains selected. We report here the results of a randomized controlled multicenter trial of the safety, reactogenicity, and immunogenicity of this novel vaccine in infants.

A phase 2, open-label, randomized controlled trial was conducted in Oxford, England, and Halifax and Vancouver, Canada, from August 2004 to September 2006. UK participants were recruited by information letters sent via child health computer departments (responsible for the mail-out of appointments for routine immunizations) to the parents of all 6-week-old infants in Oxfordshire, Buckinghamshire, and Berkshire. Participants in Vancouver were recruited via either invitation letters sent to all new parents in the Vancouver lower mainland or distribution of information letters on the postnatal wards at the Women's and Children's Hospital. At Halifax, participants were recruited through information letters mailed out through consenting local physicians and by advertising leaflets. The study recruited healthy 2-month-olds (55 to 89 days inclusive). Infants with previous exposure to or infection with diphtheria, tetanus, Haemophilus influenzae type b (Hib), pertussis, polio, hepatitis B, meningococcal serogroup C, or pneumococcus were excluded, as were those previously immunized against these organisms. Other exclusion criteria were previous anaphylactic reactions to vaccine components, severe acute or chronic disease, immune dysfunction, receipt of blood products, bleeding or seizure disorders, recent receipt of antibiotics, and receipt of antipyretics within the 6 hours before enrollment. Written informed consent was obtained from the parents or legal guardians of all enrolled infants. Ethical approval was obtained from the Oxfordshire Research Ethics Committee in the United Kingdom, the University of British Columbia Ethics Board and the Children's and Women's Hospital Ethics Committee in Vancouver, and the Research Ethics Board of the IWK Health Centre in Halifax.

The study vaccine (MenACWY) consisted of N meningitidis serogroups A, C, W-135, and Y capsular saccharides (10 μg of serogroup A; 5 μg each of serogroups C, W-135, and Y) individually conjugated to a CRM₁₉₇ carrier protein with aluminum phosphate as an adjuvant. The control vaccine was a monovalent N meningitidis serogroup C glycoconjugate vaccine (MenC; Menjugate; Novartis Vaccines, Emeryville, California) containing 10 μg of serogroup C oligosaccharide conjugated to CRM₁₉₇. A one-fifth dose of a plain polysaccharide meningococcal A, C, W-135, and Y vaccine (MenPS; Menomune; Sanofi-Pasteur, Swiftwater, Pennsylvania), containing 10 μg of serogroups A, C, W-135, and Y saccharides, was administered to a subset of participants at 12 months. MenACWY and MenC were administered intramuscularly via a 25-gauge, 25-mm needle, whereas MenPS was administered subcutaneously via a 25-gauge, 16-mm needle. Consistent batches of these vaccines were used throughout the study.

As shown in Table 1 the study design allowed for the assessment of 3 primary MenACWY schedules: 2, 3, and 4 months (UK234); 2, 4, and 6 months (CA246); and 2 and 4 months (UK24 and CA24). A control group (UKMenC) received MenC at 2 and 4 months. At age 12 months, all UK groups received a booster dose of MenACWY, as did 50% of those in CA24. MenPS was administered at 12 months to 50% of Canadian participants (CA246[PS] and CA24[PS]) as a probe for the induction of immunologic memory by MenACWY. Participants in CA246− received no further meningococcal vaccine at 12 months.

In the infant stage of the study, UK participants were randomized 2:2:1 to UK234, UK24, and UKMenC, respectively, whereas Canadian participants were randomized 1:1 to CA246 and CA24. At 12 months of age, Canadian participants were further randomized 1:1 to their toddler-stage subgroups. Randomization block size for the primary phase was 5 in the United Kingdom, whereas in Canada randomization block size for primary and toddler phases was 2; researchers were blinded to the randomization block size during the conduct of the study. Assignation of participants to their randomized group was achieved by opening of an opaque envelope by the study nurse or physician after the participant's enrollment to the study.

In addition to the study vaccine, UK participants received concomitant immunization with the combined diphtheria toxoid, tetanus toxoid, acellular pertussis, Hib, and inactivated polio vaccine (Pediacel; Sanofi-Pasteur MSD, Maidenhead, UK) at 2, 3, and 4 months of age and were offered a measles, mumps, and rubella vaccine at 13 months of age after the final study blood draw. Canadian participants received concomitant immunization with diphtheria toxoid, tetanus toxoid, acellular pertussis, Hib, and inactivated polio vaccine (Pentacel; Sanofi-Pasteur), hepatitis B vaccine (Recombivax; Merck and Co, Whitehouse Station, New Jersey), and 7-valent pneumococcal glycoconjugate vaccine (Prevnar; Wyeth Vaccines, Philadelphia, Pennsylvania) at 2, 4, 6, and 12 months of age and measles, mumps, and rubella (MMR II; Merck and Co) at 12 months of age.

After each immunization, participants were observed for 15 minutes for anaphylactic reactions. For the week after administration of each study vaccine, parents recorded local reactions at the site of the vaccination (tenderness, erythema, and induration) and systemic reactions (fever [axillary temperature ≥38°C], irritability, persistent crying, vomiting, diarrhea, sleepiness, and anorexia). Local reactions were classified by the parents as grade 1 (minimal discomfort when leg touched, erythema or induration of 1-25 mm), grade 2 (obvious discomfort when leg touched, erythema or induration of 26-50 mm), or grade 3 (tenderness causing pain on movement of leg, erythema or induration >50 mm). Eliciting of adverse events was enhanced by telephoning parents in the week after study vaccinations, as well as monthly between the infant and toddler stages of the study and 6 months after the final study immunization. Any serious adverse event occurring through the duration of the study was recorded; this included illnesses for which a child was admitted to the hospital or required emergency outpatient treatment at a local hospital after referral by a physician. The determination of the relationship of adverse events to the study vaccine was made by the study investigators according to criteria of temporal relationship and biological plausibility.

Blood samples were collected at baseline and 1 month after the final primary immunization (age 5 months for all groups except CA246, which was collected at 7 months) and before and after toddler immunization (age 12 and 13 months). Blood samples were centrifuged within 24 hours, and the serum obtained was maintained below −18°C until the analysis. Serum bactericidal assays (human complement serum bactericidal activity [hSBA]) for meningococcus serogroups A, C, W-135, and Y were performed at the laboratories of Novartis Vaccines, Marburg, Germany. The reference strains used for the relevant serogroups were serogroup A, F8238; C, C 11; W-135, M01-240070; and Y, 860800. hSBA titers were expressed as interpolated titers according to the reciprocal serum dilution's yielding 50% or greater killing of the target strain after 60 minutes of incubation compared with growth at time 0. Depending on availability of serum, polyribylribitol phosphate, tetanus, and diphtheria–specific IgG concentrations were determined for all groups except UKMenC by enzyme-linked immunosorbent assays (ELISAs) at Novartis Vaccines Laboratory in Marburg, as were the total immunoglobulin levels for hepatitis B. The serum concentrations of IgG specific for the 7-valent pneumococcal glycoconjugate vaccine–related pneumococcus serotypes were measured for Canadian participants by ELISA at the Immunobiology Unit, Institute for Child Health, London, England. The following standard serologic correlates of protection were used: hSBA titers greater than or equal to 1:4 for meningococcus serogroups A, C, W-135, and Y^{12 - 13} ; IgG level greater than or equal to 0.35 μg/mL for pneumococcus serotypes; IgG level greater than or equal to 0.1 IU/mL for tetanus and diphtheria; IgG level greater than or equal to 0.15 μg/mL and 1.0 μg/mL for short- and long-term protection against Hib, respectively; and IgG level greater than or equal to 10 mIU/mL for hepatitis B. Laboratory staff were blinded to the participant's group.

The primary objective of this study was to assess the percentage of participants whose serum, 1 month after MenACWY at 2, 3, and 4 or 2, 4, and 6 months of age, demonstrated hSBA titers greater than or equal to 1:4 against meningococcal serogroups A, C, W-135, and Y. The null hypothesis was that, for at least 1 serogroup, the lower limit of the 2-sided 95% confidence interval (CI) of this percentage would be less than 70%. The percentage of participants with hSBA titers greater than 1:4 and 1:8 was calculated for all serogroups at all the blood sampling times, and the 2-sided 95% CIs were determined with the Clopper-Pearson method. In addition, hSBA titers were log transformed and their geometric mean titers and 2-sided 95% CIs were calculated.

In a post hoc analysis, an assessment of the response to MenPS was made by calculating the percentage of participants achieving a 4-fold or greater increase in hSBA titers, and the 95% CI for this value was calculated by means of the Clopper-Pearson method.

For the UK groups, post hoc analyses were performed to compare the percentage of responders between groups by using the exact logistic regression and to compare the hSBA geometric mean titers with the analysis of variance model. These comparisons were also made for CA246(PS) and CA24(PS) at 13 months, but not for CA246 and CA24 in the infant stage because their blood was drawn at different ages. For the safety data, results were reported descriptively because no formal statistical analyses were made.

The prespecified primary population for immunogenicity analysis was the per-protocol population. Participants were excluded from this population if they did not receive all designated vaccines or they received prohibited concomitant medications (ie, antibiotics, corticosteroids, or nonprotocol vaccines). Exclusions were also made from infant-stage immunogenicity analysis if no blood samples were drawn at 2 months or after completion of primary immunizations and, for the toddler stage, if no blood was drawn after primary immunizations, at 12 months, or at 13 months. Exclusions were also made if these blood samples were drawn more than 56 days after vaccination or, for the 12-month blood draws, more than 56 days after the participant's first birthday (late blood draw). A strict intention-to-treat analysis was not possible because of the exclusion of participants during the study for protocol violations; however, the demographic and immunogenicity data for participants included and excluded from the per-protocol analysis were comparable. Also, in addition to the per-protocol analysis we performed a post hoc analysis that used all available data and included imputed values to replace missing data (the multiple imputation approach). Each missing value was replaced with a set of plausible values that represent the uncertainty about the right values to impute. Multiple imputation was carried out separately for each randomized group, with the variables included in each model dependent on the serologic measures available, plus age, sex, weight, and height. The 95% CIs of percentage of responders were performed with the score test.¹⁴ The population for analysis of vaccine safety was individuals who received at least 1 dose of vaccine. Demographic analysis was performed on the entire enrolled population.

Data were analyzed using SAS version 8.2 (per-protocol analysis) (SAS Institute Inc, Cary, North Carolina). Exact logistic regression and multiple imputation analyses were performed with Stata version 10 (StataCorp, College Station, Texas).

Assuming a 5% level of significance, with a dropout rate of 11% and an assumed 87% response rate to each serogroup, a sample size of 90 participants in each of the UK234 and CA246 groups would give 80% power to reject the null hypothesis.

A total of 421 participants were enrolled (225 UK; 196 Canada), of whom 392 (93%) completed the study (208 [92%] UK; 184 [94%] Canada) (Figure 1 and Figure 2). Participants' demographic details are displayed in Table 2 and were similar between groups. The numbers of participants included in the per-protocol immunogenicity analysis were 381 for the infant stage and 348 for the 13-month analysis.

Primary Objective. After immunization with MenACWY at 2, 3, and 4 months of age, the percentage of participants with hSBA titers greater than or equal to 1:4 was 92% or above for all 4 serogroups in both the per-protocol and imputed analyses (Table 3). Immunization at 2, 4, and 6 months of age resulted in a similarly high percentage of participants achieving the correlate of protection for serogroups C, W-135, and Y; however, for serogroup A this percentage was lower (81% [95% CI, 71%-89%] for the per-protocol analysis; 83% [95% CI, 74%-89%] for the imputed analysis). Because the lower limit of the 95% CIs for percentage of participants with hSBA titers greater than or equal to 1:4 was above 70% for all serogroups in UK234 and CA246, the null hypothesis was rejected.

Secondary Objectives. The percentage of participants receiving MenACWY at 2 and 4 months of age and achieving hSBA titers greater than or equal to 1:4 was at least 84% for serogroups C, W-135, and Y for both analyses; responses to the serogroup A component were lower, at 60% (UK24) and 66% (CA24). hSBA geometric mean titers declined by 12 months of age but were successfully increased by a 12-month dose of MenACWY, such that for both analyses the percentage of participants in groups primed and boosted with MenACWY (UK234, UK24, and CA24[ACWY]), achieving hSBA titers greater than or equal to 1:4, was at least 95% of participants for each of serogroups C, W-135, and Y and 84% for serogroup A (Table 4).

Similar trends were observed when the more conservative threshold of hSBA titers of greater than or equal to 1:8 was applied. One month after a 2-, 3-, and 4-month course of MenACWY, a per-protocol analysis revealed that 88%, 92%, 88%, and 93% of participants achieved an hSBA titer of greater than or equal to 1:8 for serogroups A, C, W, and Y, respectively; after a 2-, 4-, and 6-month course, these percentages were 76%, 98%, 96%, and 89%. One month after a 2- and 4-month course of MenACWY, 76% or more participants achieved an hSBA greater than or equal to 1:8 for serogroups C, W-135, and Y, whereas this correlate was achieved for serogroup A by 54% and 58% of participants in UK24 and CA24, respectively.

In those subgroups randomized to receive an immune challenge with the plain polysaccharide vaccine at 12 months (Table 5), the percentage of participants achieving at least a 4-fold increase in hSBA titers ranged from 65% (CA246[PS], serogroup C) to 95% (CA24[PS], serogroups W-135 and Y) in the per-protocol analysis and 64% (CA246[PS], serogroup C) to 96% (CA24[PS], serogroup Y) in the imputed analysis.

One month after primary immunization, the percentage of participants included in the per-protocol analysis and achieving diphtheria toxoid IgG concentrations greater than or equal to 0.1 IU/mL was 97% (UK234), 100% (CA246), and 96% (UK24). For Hib (polyribylribitol phosphate IgG ≥0.15 μg/mL), these percentages were 97%, 100%, and 97%, respectively, whereas 100% of participants in these groups achieved tetanus toxoid IgG concentrations greater than or equal to 0.1 IU/mL. (Participants in CA24 donated serum at 5 months, before their final dose of diphtheria toxoid, tetanus toxoid, acellular pertussis, Hib and inactivated poliovirus vaccine, hepatitis B, and pneumococcal glycoconjugate vaccine. Concomitant vaccine immunogenicity was not evaluated for children in the UKMenC group.) Of the participants in CA246, 97% achieved a hepatitis B IgG concentration of greater than or equal to 10 IU, and a pneumococcal serotype–specific IgG of greater than or equal to 0.35 μg/mL was observed in 100% (serotype 4), 90% (6B), 97% (9V), 100% (14), 93% (18C), 100% (19F), and 93% (23F) of individuals.

Reactogenicity and Safety. The percentages of participants experiencing any reaction after immunization in the infant and toddler stages are displayed in Table 6 and Table 7, respectively. Although the percentage of participants experiencing grade 3 local tenderness after at least 1 of their primary immunizations was 7% in UK234 and 10% in CA246, no more than 1% experienced grade 3 erythema or induration. A temperature of greater than or equal to 40°C was observed within a week of MenACWY in only 1 participant. Reaction rates between those who received 2 doses of MenACWY (UK24 and CA24) were comparable to those of individuals receiving 2 doses of MenC (UKMenC). Grade 3 local tenderness was observed in 2% of UK MenACWY 2- and 4-month recipients and 4% of MenC 2- and 4-month recipients; temperature of 38°C or greater was observed in 4% and 2% of these groups, respectively.

During the 16-month study duration, a total of 66 serious adverse events were experienced by 56 participants (20 in UK234, 17 in UK24, 6 in UKMenC, 9 in CA246, and 4 in CA24). Of these, only 2 were thought to be related to the study vaccines. The first was an episode of spontaneously resolving idiopathic thrombocytopenic purpura, with onset 7 days after the 12-month dose of MenACWY (UK234). This child had experienced a self-limited viral-like illness with oral ulcerations and rash 2 weeks before study vaccination. The second was an episode of supraventricular tachycardia occurring 6 hours after the second dose of MenACWY (UK24). We determined that this child had a history of recurrent neonatal supraventricular tachycardias and was enrolled in violation of the study's exclusion criteria. Of the remaining 64 serious adverse events, the majority (40) were respiratory, gastrointestinal, or generalized viral infections not related to the study vaccine.

In this study, we have demonstrated that a primary immunization course of the novel tetravalent meningococcal glycoconjugate vaccine MenACWY was well tolerated and immunogenic for serogroups A, C, W-135, and Y when given to healthy infants at either 2, 3, and 4 months or 2, 4, and 6 months of age. Although the 2-dose primary series given at 2 and 4 months of age resulted in lower seroprotection rates for serogroup A, administration of a booster dose of MenACWY at 12 months of age to participants receiving a 2-dose priming regimen resulted in at least 95% of participants achieving seroprotection against each of the serogroups C, W-135, and Y and at least 84% for serogroup A. The serogroup C hSBA geometric mean titers were lower after the primary immunization course for MenACWY recipients than for MenC recipients. This is perhaps not surprising, given that MenACWY contained 5 μg of serogroup C capsular saccharide as opposed to 10 μg in the monovalent MenC. The clinical significance of this is uncertain because the rate of participants with serogroup C hSBA titers greater than or equal to 1:4 after primary immunization was still at least 84% in the MenACWY groups.

As has been observed after primary MenC immunization,¹⁵ a waning of hSBA geometric mean titers was observed for all serogroups (in particular, serogroup A) by 12 months of age. The increase in hSBA titers after the booster dose at 12 months is reassuring, and it is likely that, as with MenC in the United Kingdom,¹⁶ regardless of the primary immunization schedule used, a booster dose of MenACWY at 12 months will be required to provide sustained protection against these organisms. The serogroup-specific hSBA geometric mean titers observed after the 12-month dose of MenACWY were significantly lower for serogroups A, C, and Y in individuals primed with 2 rather than 3 doses of MenACWY; the significance of this for long-term persistence of protective antibody is yet to be determined.

Nevertheless, the 2-, 4-, and 12-month vaccination regimen may be considered preferable to the 2-, 4-, 6-, and 12-month vaccination regimen because it would be more easily incorporated into the routine primary immunization schedule of developed countries where no 6-month vaccination visit exists. Most participants receiving this reduced-dose schedule achieved seroprotection for the 4 serogroups at 13 months of age. Similarly, because at least 84% of participants in the control group (primed with MenC and receiving a single 12-month dose of MenACWY) achieved seroprotection against serogroups C, W-135, and Y at 13 months of age, such a regimen could also be appropriate in areas with a relatively low incidence of serogroup A disease.

The immunogenicity in infancy of MenACWY is in contrast to the currently licensed tetravalent glycoconjugate vaccine. There are a number of possible reasons for this apparent difference, including the different carrier protein used and the use of oligosaccharide sizing to select saccharide chains of a particular quantity and length in the study vaccine. The chemistry used to link the oligosaccharides and protein carrier also differs between the 2 vaccines and, unlike the licensed vaccine, the study vaccine contains an aluminum phosphate adjuvant. A subsequent study showed no reduction in immunogenicity when this adjuvant was removed, however.¹⁷

In this study, the response to a reduced dose of plain polysaccharide vaccine was used as a probe to assess the induction of immunologic memory by the infant schedule. Historical controls demonstrate that only 10% to 17% of 12-month-old recipients of a full dose of MenPS develop a 4-fold increase in (rabbit complement) SBA titers against meningococcal serogroups A, C, W-135, and Y.¹⁸ The higher rates of 4-fold hSBA increases in MenACWY-primed participants in this study (64%-98%) suggest that MenACWY does induce immunologic memory.

Only 2 serious adverse events that were considered possibly related to the study vaccine occurred in this study, both of which resolved spontaneously. The majority of the remaining 64 serious adverse events were observed in children with respiratory and gastrointestinal infections who were treated at their local hospital as either inpatients or, if they had been referred to a hospital by their physician, outpatients. It is possible that the timing of this study contributed to the relatively large number of these non–study vaccine–related serious adverse events experienced by UK participants, most of whom were recruited shortly before the beginning of the northern hemisphere winter. It is also possible that a lower threshold of referral to the hospital by physicians in the United Kingdom compared with those in Canada contributed to this discrepancy.

There were a number of limitations to this study. Most important is that the number of participants was too small to draw firm conclusions regarding the safety of this vaccine, and therefore further studies will be required. In addition, this study was not designed to directly compare the immunogenicity of MenACWY with or without concomitant pneumococcal glycoconjugate vaccine. It is, however, of interest that the hSBA geometric mean titers observed for all 4 serogroups were similar for the UK and Canadian participants receiving MenACWY at 2 and 4 months of age, despite the latter receiving 2 doses of concomitant pneumococcal glycoconjugate vaccine. Although this suggests that pneumococcal glycoconjugate vaccine did not adversely affect the immunogenicity of MenACWY, given the incomplete immunization course with pneumococcal glycoconjugate vaccine and the multiple variables between the UK and Canadian groups (in particular, their different concomitant primary immunizations against diphtheria toxoid, tetanus toxoid, acellular pertussis, Hib, and inactivated poliovirus vaccine), this cannot be stated with certainty until specifically evaluated in appropriately designed studies. Similarly, although direct comparisons of pneumococcal glycoconjugate vaccine immunogenicity with and without concomitant MenACWY were not possible in this study, it is reassuring that for each pneumococcal serogroup more than 90% of participants receiving a full course of the 7-valent pneumococcal glycoconjugate vaccine achieved the correlate of protection. Seroprotection rates after hepatitis B, diphtheria, tetanus, and Hib immunization were also reassuringly high.

In conclusion, MenACWY was well tolerated and immunogenic in the first year of life. This vaccine therefore extends the immune protection provided by the monovalent MenC vaccine to serogroups A, W-135, and Y in infancy.