SPECIALFOCUSRESEARCHPAPER HumanVaccines&Immunotherapeutics8:7,881–887;July2012;G2012LandesBioscience The investigational meningococcal serogroups A, C, W-135 and Y tetanus toxoid conjugate vaccine (ACWY-TT) and the seasonal influenza virus vaccine are immunogenic and well-tolerated when co-administered in adults Mari Rose Aplasca-De Los Reyes,1,* Efren Dimaano,2 Noel Macalalad,3 Ghassan Dbaibo,4 Veronique Bianco,5 Yaela Baine6 and Jacqueline Miller6 1ResearchInstituteforTropicalMedicine;FilinvestCorporateCity;Alabang,MuntinlupaCity,Philippines;2SanLazaroHospitalSanLazaroCompound;Sta.Cruz,Manila,Philippines; 3DeLaSalleUniversityMedicalCenterCongressionalRoad;Dasmarinas,Cavite,Philippines;4AmericanUniversityofBeirutMedicalCenter;RiadElSolh,Beirut,Lebanon; 5GlaxoSmithKlineBiologicals;Wavre,Belgium;6GlaxoSmithKlineBiologicals;KingofPrussia,Philadelphia,PAUSA Keywords: Neisseria meningitidis, vaccine, ACWY vaccine, influenza vaccine, co-administration, immunogenicity, polysaccharide vaccine, adult Abbreviations:ACWY–TT,TetravalentserogroupsA,C,W-135andYvaccinewithallserogroupsconjugatedtothetetanustoxoidcarrier protein;(S)AE,(serious)adverseevent;ATPcohort,according-to-protocolcohort;CI,confidenceinterval;CHMP,CommitteeonHuman MedicinalProducts;GMT,geometricmeanantibodytiter;GSK,GlaxoSmithKlineBiologicals;HI,hemagglutinininhibition; MenPS,meningococcaltetravalentpolysaccharidevaccine;rSBA,meningococcalbactericidaltitersusingrabbitcomplementasexogenous complementsource Co-administrationofmeningococcalserogroupsA,C,W-135andYconjugatevaccine(ACWY-TT)withseasonalinfluenza vaccinewasinvestigatedinasubsetofadultsenrolledinalargerstudyevaluatinglot-to-lotconsistencyofACWY-TTand non-inferiority to licensed tetravalent meningococcal polysaccharide vaccine (MenPS). Subjects in this sub-study were randomized (3:1:1) to receive ACWY-TT alone (ACWY-TT group) or with seasonal influenza vaccine (Coad), or licensed MenPS alone. Serum bactericidal antibodies (rSBA) and serum haemagglutination-inhibition (HI) antibody titers were measured pre- and 1 mo post-vaccination. Non-inferiority of the Coad group compared with ACWY-TT group was demonstratedintermsofrSBAgeometricmeanantibodytiters(GMTs)toserogroupsA,W-135andY.ForserogroupC the pre-defined non-inferiority limit was marginally exceeded. Post-vaccination rSBA GMTs were significantly higher (exploratory analysis) in the Coad group compared with the MenPS group for serogroups A, W-135, and Y and were similartotheMenPSgroupforserogroupC.Overall,.97%ofsubjectsachievedrSBAtiters$1:128forallserogroups. The Coad group met all criteria defined by the Committee on Human Medicinal Products (CHMP) for seroprotection, seroconversionandseroconversionfactorforHIantibodiesforallthreeinfluenzastrains.Grade3solicitedlocal/general symptomswerereportedby#1.9%ofsubjectsinanygroup.Thesedatasupporttheco-administrationofACWY-TTwith seasonalinfluenza vaccine whenprotection isneeded againstboth diseases. Thisstudy is registered atclinicaltrials.gov NCT00453986 Introduction incidence of IMD is highest in infants, but disease occurs in all age groups, with a substantial proportion of cases that occur in Infection with Neisseria meningitidis causes serious, potentially adults.3 In older age groups case fatality increases with increasing life-threatening disease. Approximately 10% of invasive menin- age.3 Adult populations particularly at risk of IMD include gococcal infections are fatal, despite appropriate antibiotic travelers to meningococcal endemic regions. As global travel treatment and supportive care.1 The majority of invasive activity continues to rise, regional differences in IMD incidence meningococcal disease (IMD) is caused by six serogroups: A, B, and serogroup distribution pose increasing risk for international C, W-135, Y and X, whose distribution varies globally.1,2 The travelers to acquire and facilitate the intercontinental spread of *Correspondenceto:MariRoseAplasca-DeLosReyes;Email:[email protected] Submitted:12/07/11;Revised:03/26/12;Accepted:03/30/12 http://dx.doi.org/10.4161/hv.20212 www.landesbioscience.com HumanVaccines&Immunotherapeutics 881 meningococcal disease. In particular, travelers to the Hajj face an group) was # 2.0]. For serogroup C the upper limit of 2.0 for increased risk of meningococcal disease, and meningococcal the ratio of rSBA GMTs was marginally exceeded (by 0.03) vaccination against serogroups A, C, W-135 and Y is now (Table 1). When compared with the MenPS control group, an requiredpriortoHajjattendanceforallpilgrimsover2yofage.4,5 exploratory analysis showed that the upper limit of the 2-sided Travel also has an important role in disseminating influenza 95% CI on the GMT ratio (ACWY-TT/Men-PS group) was # outbreaks,asevidentduringtherecentinfluenzapandemic.6Prior 2.0 for all serogroups (Table 1). to travel it is often necessary to administer multiple vaccines A vaccine response definedas an rSBAtiter$1:32 ininitially simultaneously. Given the global endemicity of both seronegative subjects [pre-vaccination titer , 1:8], or a $ 4-fold N. meningitidis and influenza virus, immunogenicity and safety increase in titer for initially seropositive subjects [pre-vaccination data of co-administered meningococcal conjugate and inactivated titer $ 1:8] against individual serogroups was observed in 76.5– influenza vaccines are desirable. 88.7%ofsubjectsintheCoadgroup,80.6–92.0%intheACWY- The investigational tetravalent polysaccharide conjugate TT group and 63.6–87.3% in the MenPS group. Exploratory vaccine against N. meningitidis serogroups A, C, W-135 and Y, analyses did not detect any statistically significant differences using tetanus toxoid as the carrier protein [ACWY-TT, GlaxoSmithKline Biologicals (GSK) Belgium] is immunogenic Table1. Evaluation of primary non-inferiority hypotheses and exploratory in toddlers, children and adolescents.7-13 Immunogenicity and analyses:Coadgroupvs.ACWY-TTandMenPSgroups(ATPinfluenzacohort safety of co-administration of ACWY-TT and seasonal influenza forimmunogenicity) vaccine (FluarixTM, GSK Biologicals) was assessed in adults Antibody N Adjusted N Adjusted AdjustedGMT 18–55yofageasasub-studynestedinapartiallydouble-blinded, GMT GMT ratio(95%CI) controlled, non-inferiority study. Within the sub-study (the ACWY-TT Coad (ACWY-TT/Coad)** Influenzacohort),subjectswererandomized1:1:1:1:1toreceivea rSBA-MenA 263 3895.9 85 2860.8 1.36(1.04;1.78) single dose of ACWY-TT (one of three manufacturing lots); rSBA-MenC 293 10299.7 97 6908.0 1.49(1.10;2.03) or MencevaxTM ACWY (MenPS group), or ACWY-TT co- rSBA-MenW-135 299 5848.2 101 4770.5 1.23(0.91;1.65) administered with the seasonal influenza vaccine (Coad group). Results of the “influenza” cohort are reported here. In the same rSBA-MenY 300 7331.2 102 5617.2 1.31(1.00;1.70) study, manufacturing consistency using three different manufac- MenPS Coad (MenPS/Coad) turing lots was established and pooled serological results were rSBA-MenA 88 1794.3 85 2860.8 0.63(0.45;0.87) compared against the tetravalent polysaccharide vaccine control rSBA-MenC 101 9032.1 97 6908.0 1.31(0.90;1.90) (MenPS: MencevaxTM ACWY, GSK Biologicals). These results rSBA-MenW-135 102 2639.6 101 4770.5 0.55(0.39;0.80) are reported separately. rSBA-MenY 100 3385.8 102 5617.2 0.60(0.43;0.84) Results Vaccine Difference response betweengroups A total of 520 adults were included in the total vaccinated N* %VR N* %VR %(95%CI) Influenza cohort (ACWY-TT, Lot A, n = 311; Coad, n = 105; Coad ACWY-TT (Coadminus MenPS, n = 104). Three subjects (all in the ACWY-TT group) ACWY-TT) were eliminated from the according to protocol (ATP) influenza rSBA-MenA 85 76.5 263 80.6 -4.14(-15.12;5.27) cohort for immunogenicity: two subjects received a vaccine rSBA-MenC 97 88.7 293 89.1 -0.42(-8.85;6.04) forbidden by the protocol (one subject received an inactivated rSBA-MenW-135 101 88.1 299 92.0 -3.85(-12.05;2.32) influenza vaccine and one subject received a tetanus toxoid rSBA-MenY 102 81.4 300 86.3 -4.96(-14.30;2.79) vaccine within the 30 d period post-vaccination) and one subject Coad MenPS (Coadminus hadnopost-vaccinationbloodsample,leaving517subjectsinthe MenPS) ATP influenza cohort for immunogenicity. More male subjects rSBA-MenA 85 76.5 88 63.6 12.83(-0.88;26.13) than female subjects were enrolled (56.9% vs. 43.1%). The predominanceofmaleswasmorepronouncedintheCoadgroup rSBA-MenC 97 88.7 101 87.1 1.53(-7.94;10.97) (61.9% males vs. 55.9% and 54.8% in the ACWY-TT and rSBA-MenW-135 101 88.1 102 87.3 0.86(-8.52;10.26) MenPSgroups,respectively).Themeanageofsubjectswas35.6y rSBA-MenY 102 81.4 100 73.0 8.37(-3.26;19.96) (standard deviation 10.57 y, range 18–55). All but one subject N, number of subjects with availableresults; N*, numberof subjectswith was of South East Asian descent. No subject withdrew during both pre and post results available; % VR, percentage of subjects with a the study. vaccine response defined as: For initially seronegative subjects, post- Thepre-defined statistical criterion ofnon-inferiority of serum vaccination antibody titer $ 1:32; For initially seropositive subjects, post- bactericidal activity geometric mean titers (using rabbit comple- vaccinationtiter$4-foldthepre-vaccinationtiter.95%CI,95%confidence interval;bold,upperlimitof95%CIisbelowthepre-definedlimitof2.0for ment as the exogenous complement source: rSBA GMTs) the adjusted GMT ratios—(ANCOVA model: adjustment for age strata, (ACWY-TT group over Coad group) was met for serogroups A, baselinetiter-pooledvariancewithmorethantwogroups);**primarynon- W-135 and Y [Table 1, the upper limit of the two-sided 95% inferiorityobjective,allothercomparisonswereexploratory.AdjustedGMT, confidence interval (CI) on the GMT ratio (ACWY-TT/Coad geometricmeanantibodytiteradjustedforagestrataandbaselinetiter. 882 HumanVaccines&Immunotherapeutics Volume8Issue7 between groups in terms of vaccine response rates (Table 1). All onset5daftervaccinationthatwasconsideredbytheinvestigator initially seronegative subjects mounted a booster response to toberelatedtovaccination.Attheconclusionofthe6-mosafety vaccination for each serogroup with the following exception: in follow-up, no subject in the Influenza cohort reported rash, an the ACWY-TT group, one subject for serogroup A (2.2%) and emergency room visit or new onset of chronic disease. one subject for serogroup Y (4.2%), in the MenPS group, one subject for serogroup W-135 (5.6%) and one subject for Discussion serogroup C (5.9%), and in the Coad group one subject for serogroup W-135 (6.2%). ACWY-TT co-administered with seasonal influenza vaccine Overall, at least 99.0% and 97.1% of subjects in all vaccine induced robust immune responses, with at least 76.5% of groups achieved rSBA titers $ 1:8 and $ 1:128 respectively, for subjects having a vaccine response to each of the meningococcal each of the four serogroups (data not shown). No differences serogroups,andaseroconversionrateofatleast61.9%toallthree between groups were observed in terms of the percentage of influenza strains. The statistical criterion of non-inferiority of subjects reaching the 1:8 and 1:128 cut-offs (data not shown). Coad compared with ACWY-TT was met for serogroups A, Exploratory analysis showedthatrSBA GMTs adjusted forage W-135 and Y, but was not achieved for serogroup C by a small strataandpre-vaccinationtiterwerestatisticallysignificantlylower margin (0.03). Nevertheless, a high percentage of subjects intheCoadgroupthanintheACWY-TTgroupforserogroupsA achieved post-vaccination titers $ 1:128 (98.1%), a level con- and C, and statistically significantly higher in the Coad group sidered a conservative threshold of protection.15-18 Exploratory than in the MenPS group for serogroups A, W-135 and Y analyses did not detect any statistically significant differences (Fig.1).Reversecumulativecurves(RCCs)showingpreandpost- between the Coad group and the ACWY-TT group in the vaccination rSBA titers indicate robust increases in GMTs in all percentages of subjects with titers $ 1:8 and $ 1:128, or in the treatmentgroupsforallfourserogroupsaftervaccination(Fig.2). vaccineresponserateforanymeningococcalserogroup,indicating The Coad group met all pre-defined statistical criteria set out that both groups exhibited robust immune responses to by the European Medicines Agency Committee for Proprietary meningococcal vaccination. Furthermore, no statistically signifi- Medicinal Products (CHMP) for antibody responses against cant difference in rSBA-MenC GMTs between the Coad group influenza antigens A/H1N1, A/H3N2 and B (Table 2). and the MenPS control group were observed (exploratory The most frequently reported solicited local and general analysis), demonstrating a comparable immune response after Adverse Events (AEs) in the Coad, ACWY-TT and MenPS co-administrationofACWY-TTandseasonalinfluenzavaccineto groupswereinjectionsitepainandheadache,respectively(Fig.3). the standard of care in the Philippines at the time of the study. Grade 3 solicited local/general AEs were reported by no more Finally, the post-vaccination rSBA-MenC GMT in the Coad than 1.9% of subjects in any vaccination group. group was within the range of GMTs previously reported Four subjects in the Influenza cohort reported seven serious following administration of ACWY conjugate vaccines to AEs(SAEs)(allintheACWY-TTgroup)duringthestudyperiod. adolescent and adult populations, in studies employing the same Of these, one subject reported abdominal pain and gastritis with rSBA.7,19 The magnitude of the post-vaccination rSBA GMT was lower in the Coad group than the ACWY-TT group for all meningococcal vaccine serogroups (sig- nificant only for serogroups A and C), suggesting the possibility of immune interference when ACWY-TT andinfluenzavaccineareco-administered.However,all butonesubjectintheCoadgroupachievedrSBAtiters $ 1:8 and no more than 2.9% of subjects failed to achieve rSBA titers $ 1:128, for any one vaccine serogroup.Additionally,rSBAGMTsweresignificantly higher intheCoadgroupthantheMenPSgroupfor3 out of 4 vaccine serogroups. Although the data from this study suggest some reduction in meningococcal rSBA titers when ACWY-TT is co-administered with trivalentinactivatedinfluenzavaccine,theavailabledata alsosuggestthatanypotentialinterferenceisunlikelyto have clinical consequences, given the robust immune responsesasillustratedbytheRCCcurves,andthefact that the post-vaccination titers are within previously- Figure1.rSBAGMTsineachgrouponemonthaftervaccination(ATPInfluenza cohortforimmunogenicity).*Statisticallysignificantdifferencebetween reported ranges. This, taken with the observation that theindicatedgroupandtheCoadgroup:differencesbetweengroupsweredone all 3 CHMP criteria defining an acceptable immune onGMTvaluesadjustedforpre-vaccinationmeasurementsandagestrata, response to influenza vaccine were met for all 3 tested exploratoryanalysis,whereastheGMTsdisplayedareunadjusted. influenza strains suggests that ACWY-TT and seasonal www.landesbioscience.com HumanVaccines&Immunotherapeutics 883 Figure2.ReversecumulativecurvesshowingrSBAtitersforN.meningitidisserogroupsA,C,W-135andY.ACWY+F,Coadgroup;ACWY_F,ACWY-TT group;MenPS_F,MenPSgroup. influenza vaccine can be co-administered, while maintaining alone is justified based on the manner in which regulatory acceptable immune responses to both vaccines. agenciesdeterminewhethertheyearlychangeininfluenzavaccine We identified only one other study where a meningococcal formulation can be marketed in a given year. Additionally, vaccine (MenC-CRM ) was co-administered with an influenza because ACWY-TT was administered intramuscularly while 197 vaccine.20 The study was designed to assess the impact of an MenPS was given subcutaneously, the study was conducted immuno-modulating agent on responses to co-administered open-blind with respect to ACWY-TT and Men-PS administra- MenC-CRM and influenza vaccines. The response to both tion.Potentialbiasintheanalysisofimmunogenicitywasreduced 197 vaccines in these individuals appeared adequate. However, by blinding laboratory personnel to age and group. The open comparison with the current study is precluded by use of design may have influenced attribution of the relationship of differentassay methods, andinterpretationoftheresultsinterms adverse events to vaccination, but reporting bias would more of the immunogenicity of co-administration cannot be made in likely be against the investigational product. the absence of a group that received MenC-CRM alone. ACWY-TT had an acceptable safety profile in healthy adults, 197 The present study is potentially limited by the absence of which was not affected by co-administration with influenza controlgroupstoevaluateMenPSco-administeredwithinfluenza vaccine. Co-administration of vaccines reduces the number of vaccine, or influenza vaccine alone. However, the criteria for visits required to complete vaccination and improves coverage of clinical acceptability of the immune response were based on the co-administered antigens, which may be particularly impor- CHMP objective criteria for the yearly registration of inactivated tant in travelers who need to be vaccinated against multiple influenzavaccine,andnotonanon-inferioritycomparison.Thus, antigens in a short duration of time. The administration of thedecisionnottoincludeagroupthatreceivedinfluenzavaccine seasonal influenza vaccine may coincide with that of ACWY-TT 884 HumanVaccines&Immunotherapeutics Volume8Issue7 Table2.Influenzahumoralimmuneresponsesonemonthaftervaccination(ATPInfluenzacohortforimmunogenicity) Influenza Time Seroconversionrate Seroconversionfactor Anti-haemagglutinationinhibitionantibodies strain point N % (95%CI) N % (95%CI) N %$1:40 (95%CI) GMT (95%CI) A/H1N1 Pre - - - - - - 105 64.8 (54.8;73.8) 54.1 (43.0;68.1) Post 105 71.4 (61.8;79.8) 105 9.9 (7.5;13.1) 105 99.0 (94.8;100) 537.2 (446.9;645.8) A/H3N2 Pre - - - - - - 105 54.3 (44.3;64.0) 31.5 (25.1;39.6) Post 105 61.9 (51.9;71.2) 105 5.6 (4.4;7.2) 105 97.1 (91.9;99.4) 177.8 (150.0;210.7) B Pre - - - - - - 103 42.7 (33.0;52.8) 20.9 (17.0;25.7) Post 103 75.7 (66.3;83.6) 103 9.1 (7.1;11.6) 104 96.2 (90.4;98.9) 192.7 (156.4;237.6) N,numberofsubjectswithavailableresults(forseroconversionrateandseroconversionfactorN,thenumberofsubjectswithpre-andpost-vaccination resultsavailable).%,percentageofsubjectstheindicatedendpoint;95%CI,95%confidenceinterval;Seroconversion:Forinitiallyseronegativesubjects(i.e., anti-HItiters,1:10),antibodytiter$1:40aftervaccination.Forinitiallyseropositivesubjects,antibodytiteraftervaccination$4-foldthepre-vaccination antibodytiter.Seroconversionfactor,geometricmeanratio{mean[log (post-vaccinationGMT/prevaccinationGMT)]}.Pre,priortovaccination,Post,one 10 monthpostvaccination.CHMPcriteriaforsuccess14:thelowerlimitofthetwo-sidedexact95%CIintheseroconversionrateis.40%orthelowerlimitof thetwo-sidedstandardizedasymptotic95%CIinthefoldincreaseinGMTis.2.5,orthelowerlimitofthetwo-sidedexact95%CIintheproportionof subjectsachievinganHItiter$40is.70. in adults 18–55 y of age annually for an approximately four- by ethics committees at each study center. Written informed month season. This is the first reported study describing the consent was obtained from subjects before study entry. co-administration of an ACWY conjugate vaccine with seasonal Enrolledhealthyadults18–55yofagereceivedasingledoseof influenza vaccine. The data support co-administration for one of three manufacturing lots of ACWY-TT (ACWY-TT combined vaccination against invasive meningococcal disease group,lotsA,BandC),orMencevaxTMACWY(MenPSgroup), and seasonal influenza in healthy adult travelers. or ACWY-TT Lot A co-administered with the seasonal influenza vaccine, (FluarixTM: GSK Biologicals, Coad group), respectively. Materials and Methods Thefullstudy consistedofthreecohortswithseparateobjectives: (1) lot-to-lot consistency of three clinical lots of ACWY-TT, Study design. The study was a phase III, randomized, partially which will be reported separately, (2) immunogenicity and safety double-blinded, controlled, non-inferiority study conducted at of ACWY-TT vs. MenPS vaccine, which is reported separately one study center in Lebanon and in three centers in the and (3) safety and immunogenicity when ACWY-TT and Philippines (109067/NCT00453986) between April 2007 and seasonal influenza vaccine were co-administered as compared May 2008. The study was conducted according to good clinical with ACWY-TT alone as assessed in the “influenza” cohort, practice and inaccordance with theDeclaration ofHelsinki.The and reported here. Subjects in the Influenza cohort were healthy protocol and associated documents were reviewed and approved adults enrolled at two sites in the Philippines randomized Figure3.Percentageofsubjectsreportingsolicitedlocalandgeneralsymptomsduringthe4-daypost-vaccinationperiod(totalvaccinatedInfluenza cohort).Note:FortheCoadgroup,localsymptomsrefertothepercentageofsubjectswithatleastonelocalsymptomateitherinjectionsite. Gastrointestinalsymptomsincludednausea,vomiting,diarrheaand/orabdominalpain.Grade3:rednessandswelling.50mm,fever.39.5°C, preventingnormaleverydayactivityforallothersymptoms. www.landesbioscience.com HumanVaccines&Immunotherapeutics 885 1:1:1:1:1 to the ACWY-TT group (pooled lots A, B and C), Statistical analysis. The analysis of immunogenicity was MenPS or Coad groups. conducted on the ATP immunogenicity cohort that included all Study objectives. The co-primary study objectives were: (1) vaccinated subjects who complied with protocol-defined proce- demonstration of non-inferiority of ACWY-TT co-administered dures. For the purposes of the analysis, immunogenicity and withseasonalinfluenzavaccinecomparedwithACWY-TTalone, safety results from the three ACWY-TT lots were pooled. in terms of serum bactericidal antibodies geometric mean titers The95%CIsoftherSBAGMTratiosbetweenvaccinegroups (rSBA GMTs) for serogroups A, C, W-135 and Y 1 mo post- were calculated using an ANCOVA model on the log 10 vaccination,and(2)demonstrationofacceptableimmunogenicity transformation of the titers, using the pre-vaccination log 10 ofseasonal influenza vaccineco-administered with ACWY-TTin transformation of the titers and age strata as covariates. Antibody termsofhaemagglutinationinhibition(HI)antibodytiters,based titers below the cut-off of the assay were given an arbitrary value on criteria defined by the CHMP.14 of half the cut-off for the purpose of GMT calculation. Vaccine Non-inferiority was prospectively defined as the upper limit of groups were considered significantly different if the 95% CI for the two-sided 95% CI on the GMT ratio (ACWY-TT/Coad theGMTratiobetweengroupsdidnotcontainthevalue1,or,if group) adjusted for pre-vaccination rSBA titers being # 2.0. the asymptotic standardized 95% CI for the difference in Immunogenicity 1 mo post-vaccination with respect to influenza threshold rates or vaccine response rates between groups did not antigens (A/H1N1, A/H3N2, B) was pre-defined as acceptable if contain the value 0. Due to the multiplicity of endpoints, oneofthefollowingCHMPstandardcriteriafortheevaluationof statistically significant findings from the exploratory analyses inactivated influenza vaccines was satisfied: the lower limit of the should be interpreted with caution. two-sidedexact95%CIintheseroconversionrateforanti-HIwas The analysis of safety was performed on the total vaccinated .40%;thelowerlimitofthetwo-sidedstandardizedasymptotic cohort that included all vaccinated subjects. The incidence and 95% CI in the fold increase in anti-HI GMT was . 2.5; the intensity of symptoms were calculated with exact 95% CI for lower limit of the two-sided exact 95% CI in the proportion of each group. subjects with anti-HI $ 1:40 was . 70%. The power of the AnalyseswereperformedusingSAS1softwareversion9.1(SAS study to meet both objectives was . 96.7%. Institute Inc.) and Proc StatXact 7.0. Immunogenicity assessment. Blood samples were collected FLUARIX and MENCEVAX are trademarks of the fromallsubjectspriortoandonemonthaftervaccination.Pre-and GlaxoSmithKline group of companies. post-vaccinationseraweretestedforrSBAforeachmeningococcal serogrouptomeasurefunctionalantibodylevelsfollowingACWY- Disclosure of Potential Conflicts ofInterest TT vaccination as previously described.21 The assay works by M.R.A.D.L.R., G.D. and E.D. have received consulting fees and killing the target meningococcal strain in the presence of honorariafromGSKwithinthepastthreeyears.N.M.declaresno complement and serial dilutions of vaccinated subject sera. Both conflict of interest. V.B., Y.B. and J.M. are employees of GSK rabbit and human complement have been used as the exogenous Biologicals. Y.B. and J.M. report ownership of GSK Biologicals complement source; there is currently no international consensus stocks and stock options. on the standard source of human complement to be used.22 The serumbactericidaltiterisexpressedasthereciprocalserumdilution Sources ofSupport yielding $ 50% killing as compared with the number of viable GlaxoSmithKline Biologicals was the funding source and was cells prior to incubation. The cut-off of the rSBA assay was a 1:8 involved in all stages of the study conduct and analysis. GSK dilution and was considered indicative of seroprotection.15,16 A Biologicals also funded all costs associated with the development conservativecut-off of1:128wasalso considered. andthepublishingofthepresentmanuscript.Thecorresponding Pre and post-vaccination samples in the Coad group were also author had full access to the data. testedforHIantibodytitersagainstinfluenzavirusvaccinestrains. HI is dependent on the ability of serial dilutions of vaccinated Acknowledgments subject sera to inhibit the interaction between the virus and The authors thank the individuals who participated in the study, erythrocyte sialic acid. The HI titer is defined as the dilution and all investigators involved in conducting the study. The factor of the highest dilution that completely inhibits hemagglu- authors also thank Sally Gatchalian, Sameh Anis, Aline Stukkens tination. Seroconversion was defined as antibody titer $ 1:40 and Jane Nappa for their assistance in coordination of the study; after vaccination in initially seronegative subjects (anti-HI , Dr Fotios Vikas (GSK) for assistance in preparation of study 1:10),andasa$4-foldincreaseininitiallyseropositive subjects. reports; Laurence Fissette (GSK) for performing the statistical Theseroconversionfactorwasdefinedasthegeometricmeanratio analysis and Dr Brigitte Cheuvart (GSK) for statistical input {mean [log (post-vaccination GMT/pre-vaccination GMT)]}. intheprotocolandstudyset-up;DrDominiqueBoutriauandDr 10 Safetyassessment.LocalandgeneralAEsweresolicitedfor4d Peter Vink for input into protocol development; Dr Pascal after vaccination. Other (unsolicited) non-serious AEs were Lestrate and Koen Maleux for conducting the laboratory assays; recorded for 31 d after vaccination, and specific AEs (rash, new Dr Joanne Wolter (on behalf of GSK) for preparation of the onset of chronic disease and AEs leading to emergency room first draft of the manuscript and Virginie Durbecq and Juliette visits)andSAEswerecollectedthrough6moaftervaccinationvia Gray (Xpe Pharma and Science) for coordination and editorial scripted phone call. assistance. 886 HumanVaccines&Immunotherapeutics Volume8Issue7 References 10. VesikariT,KarvonenA,BiancoV,VanderWielenM, 17. CampbellH,AndrewsN,BorrowR,TrotterC,MillerE. MillerJ.TetravalentmeningococcalserogroupsA,C, Updatedpostlicensuresurveillanceofthemeningococcal 1. Pollard AJ. 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