Rates of meningococcal disease vary from <1-3/100,000 in developed countries to 10-25/100,000 in less developed countries [1]. Invasive meningococcal disease (IMD) is estimated to cause death in 10-15% of cases [2]. The majority of IMD cases in the Russian Federation occur in children aged up to 2 years, with an incidence of approximately 14/100,000 [3, 4]. In the Russian Federation, the incidence of meningococcal infection in 2007 was reported to have declined from 5.0 per 100,000 population in 1989 through to 1.9 cases per 100,000 population in 2007, but was 8-11/100,000 in children aged <14 years [3]. In India, meningococcal disease surveillance is not enforced [5-7] and several epidemics have been reported. Six meningococcus serogroups,(A, B, C, W, X and Y) cause most cases of IMD [8, 9]. Strain A was associated with all major epidemics in India, with small numbers of serogroup C isolated in one epidemic [5]. In the Russian Federation, serogroups A, B and C are responsible for most IMD cases with serogroup A mainly responsible [10], and Y and W isolated in some cases.

The meningococcal (Groups A, C, Y and W-135) polysaccharide diphtheria toxoid conjugate vaccine (Men-ACYW-D; Menactra, Sanofi Pasteur) is approved for active immunization against IMD in >60 countries [11]. ACYW-D was licensed in the USA in 2005, and is approved for primary vaccination in children aged 9-23 months as a two-dose series, and in individuals aged 2-55 years as a single dose [12, 13]. A rabbit complement serum bactericidal antibody titer of ³1:8 was validated to be seroprotective against serogroup C IMD [14].

When the present study started, no quadrivalent meningococcal conjugated vaccine was licensed for broad-based protection against IMD from early childhood (<2 years) in India or in the Russian Federation, but ACYW-D was licensed for those aged 2-55 years. Previous phase I/II studies showed this vaccine was immunogenic with a good safety profile in infants, toddlers and children [15, 16]. A phase III study was therefore performed to determine the proportion of Indian and Russian infants and toddlers achieving sero-protective titers after immunization with Men-ACYW-D vaccine, and assess any adverse events following immunization.

Infants and toddlers from India and the Russian Federation aged 9-17 months at entry were eligible. Key exclusion criteria included a history (confirmed clinically, serologically, or microbiologically) or high risk (investigators opinion) of meningococcal disease, acute disease/infection (any severity at Russian sites or moderate to severe at Indian sites) according to the investigator or fever (axillary temperature ³37.0°C at Russian sites, and temperature ³38.0°C at Indian sites) on the day of vaccination, bleeding disorders or receipt of anticoagulants in the preceding three weeks, history of known thrombocytopenia, or immunodeficiency conditions. Those receiving immunosuppressive medication, or another vaccine (other than influenza or measles, mumps, rubella [MMR] vaccines, or oral poliomyelitis vaccine [OPV] at the Indian sites) in the four weeks before or after the study vaccination, or those previously vaccinated against meningococcal disease were also excluded.

Participants received two 0.5 mL doses of Men-ACYW-D by intramuscular injection (anterolateral thigh or optionally, deltoid muscle for toddlers aged ³12 months). Dose 2 was given 3-6 months after Dose 1. Each 0.5 mL dose was formulated in sodium phosphate buffered isotonic sodium chloride solution to contain meningococcal capsular polysaccharides (A, C, Y, and W: 4 µg each) conjugated to approximately 48 µg of diphtheria toxoid protein.

Serum samples were collected immediately before Dose 1 and 28 days (+7 days) after Dose 2, frozen and maintained at –20°C until immunogenicity analysis at a central laboratory (Global Clinical Immunology, Sanofi Pasteur, Swiftwater, USA). Meningococcal antibody titers to serogroups A, C, W and Y were determined using the serum bactericidal assay in the presence of human complement (SBA-HC), expressed as SBA-HC geometric mean titers (GMTs) as previously described [12]. Antibody titers ³1:8 were considered seroprotective for each serogroup; although a titer of 1:4 could be considered protective, the higher titer was chosen as a conservative assumption [17]. The number and percentage of participants who achieved a four-fold increase in titers 28 days after the second dose compared with pre-vaccination were calculated.

Statistical analyses: Statistical analyses were performed using SAS Version 9.2 (SAS Institute, Cary, North Carolina, USA). All planned analyses were descriptive with no hypothesis testing. The full analysis set (FAS) consisted of all participants who received ³1 dose of study vaccine; the safety analysis set consisted of all participants in the FAS with safety data available. To calculate the four-fold rises in titers 28 days after the second dose compared with pre-dose, any pre-dose 1 titer reported as <lower limit of quantification (LLOQ) was converted to a value equal to LLOQ, and any post-dose 2 titer reported as <LLOQ was converted to a value of 0.5 LLOQ. Any value >ULOQ was converted to the ULOQ to calculate the four-fold increases. Safety and reactogenicity were assessed on the safety analysis set; the incidences of AEs/ARs were calculated individually, by country, and overall.

At baseline, in the full analysis set, seroprotection (SBA-HC titer of ³8 [1/dil]) rates against meningococcus serogroup A (55.8% in India and 40% in Russia) were higher than for the other serogroups (range 4-7% in both countries; Fig. 1). After Dose 2, 92.9-99.0% and 95.7-99.5% of children achieved seroprotection across the four serogroups in the Russian Federation and India, respectively. In addition, 86.5-97.3% and 80.6-90.8% of participants in India and Russia, respectively, had a ³4-fold increase from baseline to post-Dose 2 in SBA-HC titers across the four serogroups.

Fig. 1 Seroprotection (percentage of participants with SBA-HC titer ³8 (1/dil) [95% confidence intervals]) at baseline and after Dose 2 (Full Analysis Set).

Rates of seroprotection and geometric mean SBA-HC titers by serogroup in India and the Russian Federation are given in Table I. SBA-HC GMTs post-Dose 2 were markedly higher than at baseline for all serogroups.

TABLE I	Rates of Seroprotection and Geometric Mean SBA-HC Titers by Serogroup in India  
and the Russian Federation (Full Analysis set)

No immediate ARs or AEs were reported after any dose of Men-ACYW-D. Solicited reactions occurred in 49.2% of children (Table II). Most solicited injection site reactions and solicited systemic reactions occurred during the three days after injection, were of Grade 1 severity, and resolved within three days. Unsolicited ARs after any study injection were very infrequent (Table II). Unsolicited AEs were reported in approximately 20% of all children (Table II). The most common unsolicited AEs reported occurred at a rate £5% (Table III). One unsolicited non-serious AE (Grade 3 diarrhea at a Russian site, which resolved within one day) was considered by the investigator to be related to the study vaccine. Two other cases of unsolicited non-serious AEs (rhinitis at a Russian site and fever at an Indian site) were of Grade 3 severity. No non-serious AEs led to premature withdrawal from the study.

TABLE II	Safety Overview after any Study Vaccine Injection (Percentage of Participants Experiencing ≥1 Event) 
in the Indian and Russian Federation studies: Safety Analysis Sets 

TABLE III Percentages of Participants Experiencing  ≥1 Unsolicited AE After any Study Vaccine Injection 
(Most Common AEs,  ≥1% in Overall Population) in  the India and Russian Federation studies: Safety Analysis Sets

Four SAEs were reported; one at a Russian site and three at Indian sites: nasopharyngitis, acute respiratory tract infection, lower respiratory tract infection, and forearm abscess (mosquito bite complication at a site unrelated to vaccine injection), which all required hospitalization. None was considered related to study vaccine by the investigators. No deaths occurred during the study.

These studies show that a two-dose series of Men-ACYW-D vaccine administered 3-6 months apart is well tolerated with no safety concerns, and induces seroprotective titers against each of the four meningococcal serogroups in ³93% of Russian and Indian children aged 9-17 months. These results are consistent with other studies of Men-ACYW-D in the similar age groups [12,18,19], and in older age groups [11, 20].