This study was conducted in accordance with the protocol and consensus ethical principles derived from international guidelines including the Declaration of Helsinki and the International Council for Harmonization (ICH) guidelines for Good Clinical Practice (GCP), and all applicable laws, rules, and regulations. Participants turning 18 years old during the study or parents/legal representatives provided informed consent. Ethical approval was obtained from the following committees:

Singapore: NHG Domain Specific Review Boards (DSRB).

Spain: Comité de Ética de la Investigación Con Medicamentos del Hospital General Universitario Gregorio Marañón.

Hungary: Egészségügyi Tudományos Tanács Klinikai Farmakológiai Etikai Bizottság/Medical Research Council Ethics Committee for Clinical Pharmacology.

Italy: Comitato Etico Regionale Liguria Largo Rosanna Benzi; Comitato Etico Milano Area 1—ASST Sacco e Fatebenefratelli; Comitato Etico dell’Area Vasta Emilia Nord; Segreteria Locale di Parma c/o Azienda Ospedaliero; Universitaria di Parma Via Gramsci 14; Comitato Etico Palermo 1; Comitato Etico “Ospedali Riuniti” Di Foggia; Comitato Etico Regione Calabria—Sezione Area Centro.

Participants were recruited from 20 sites in Europe (Spain, Italy, and Hungary) and one site in Asia (Singapore). Healthy adolescents aged 10–17 years who were either MenC vaccine-primed before 2 years of age or MenC-naïve and were covered by health insurance, if required by local regulations, were included. Exclusion criteria included pregnancy, lactation, or not using effective contraception if the individual was of child-bearing age, as well as previous vaccination against meningococcal disease with either the study vaccine or another vaccine, excluding MenC vaccination during infancy. Similarly, participants with any previous history of HPV vaccination or any tetanus, diphtheria, pertussis, or inactivated polio virus vaccine within the last 3 years were excluded. Participants turning 18 years old during the study or parents/legal representatives provided informed consent. Assent forms were completed as per local regulations.

Participants were enrolled and randomized between March 16, 2021 and December 22, 2021 in a 3:3:2 ratio to receive either MenACYW-TT on D01 and 9vHPV + Tdap-IPV on D31 (group 1), MCV4-TT on D01 and 9vHPV + Tdap-IPV on D31 (group 2), or MenACYW-TT and 9vHPV + Tdap-IPV on D01 (group 3) (Fig. 1). Groups 1 and 2 were observer-blind and group 3 was open-label.

Study design. BS blood sample

MenACYW-TT (MenQuadfi, Sanofi Inc., Swiftwater, PA, USA) was presented as a ready-to-use solution in single-dose vials. Each dose contained 10 μg meningococcal capsular polysaccharides from the four serogroups conjugated to approximately 55 μg tetanus toxoid protein carrier. MCV4-TT (Nimenrix, Pfizer Limited, Sandwich, UK) was presented as a lyophilized powder and solvent for resuspension, containing 5 μg meningococcal capsular polysaccharides from the four serogroups conjugated to approximately 44 μg tetanus toxoid protein carrier. 9vHPV (Gardasil® 9, Merck, Sharp & Dohme Limited, Kenilworth, NJ, USA) was presented a ready to use suspension containing: 30 μg HPV type 6 L1 protein; 40 μg HPV type 11 L1 protein; 60 μg HPV type 16 L1 protein; 40 μg HPV type 18 L1 protein; 20 μg HPV type 31 L1 protein; 20 μg HPV type 33 L1 protein; 20 μg HPV type 45 L1 protein; 20 μg HPV type 52 L1 protein; and 20 μg HPV type 58 L1 protein. Tdap-IPV (Repevax®/Triaxis® Polio/Adacel® Polio, Sanofi Limited, Toronto, Canada) was presented as a ready-to-use suspension containing: 5 Lf of tetanus toxoid, 2 Lf of diphtheria toxoid and acellular pertussis antigens [2.5 µg pertussis toxin (PT), 5 µg filamentous hemagglutinin (FHA), 3 µg pertactin, and 5 µg fimbriae], and IPV type 1 (Mahoney strain) 40 D-antigen unit, type 2 (MEF-1 strain) 8 D-antigen unit, and type 3 (Saukett strain) 32 D-antigen unit. All vaccines were given at 0.5 ml per dose intramuscularly (deltoid).

hSBA and baby rabbit complement assay (rSBA) (in a subgroup of participants in each group) were used to measure antibodies against the four meningococcal vaccine serogroups at D0 and D31. Seroprotection against each meningococcal serogroup was defined as hSBA titer ≥ 1:8. [11,12,13, 24, 25]. The proportion of participants with rSBA titers ≥ 1:8 for each meningococcal serogroups was also assessed. The seroprotection thresholds of hSBA and rSBA titers ≥ 1:8 were defined based on previous research on the MenACYW-TT and MCV4-TT vaccines [11,12,13, 24,25,26,27]. Seroresponse to meningococcal vaccination was defined as a post-vaccination hSBA titer ≥ 1:16 in participants with pre-vaccination hSBA titer < 1:8, or a post-vaccination titer at least fourfold greater than the pre-vaccination titer in those with a pre-vaccination titer ≥ 1:8. For rSBA titers, this was defined as a post-vaccination titer ≥ 1:32 for participants with pre-vaccination rSBA titer < 1:8, or a post-vaccination titer ≥ fourfold increase from baseline for participants with pre-vaccination rSBA titer ≥ 1:8. Antibody titers/concentrations against antigens contained in 9vHPV and Tdap-IPV vaccines were measured 30 days after vaccination with 9vHPV and Tdap-IPV. Vaccine seroconversion for each of the HPV types (6, 11, 16, 18, 31, 33, 45, 52, and 58) by D31 (+ 14 days) after vaccination, with seroconversion was defined as change in serostatus from seronegative at D01 to seropositive by D31. A participant with a titer at or above the serostatus cut-off for a given HPV type was considered seropositive for that type. The serostatus cut-offs were 9, 6, 5, 5, 3, 4, 3, 5, and 5 milli-Merck units (mMU)/ml for HPV types 6, 11, 16, 18, 31, 33, 45, 52, and 58, respectively. The rSBA testing was performed at the Vaccine Evaluation Unit, Public Health Laboratory, Manchester, UK. All other antibody assessments were performed at Global Clinical Immunology (GCI) (Swiftwater, PA, USA) or at qualified contract laboratories for GCI. Participants were kept under observation for 30 min after vaccination to ensure their safety and to record any immediate adverse events (AEs) and adverse reactions (ARs). Solicited injection-site reactions were collected from D01 to D08 after each vaccination. AEs were collected from D01 until D31 after each vaccination and serious AEs (SAEs) and AEs of special interest (AESIs) were collected throughout the study. Participants, or their parents or legally acceptable representatives, recorded AEs and SAEs in a diary card specifically designed for this study, and were provided rulers for measuring the size of injection site reactions and standard digital thermometers for measuring daily temperatures prior to the study. Participants or their parents/legally acceptable representatives were interviewed at specified intervals to collect the information recorded in the diary card and to clarify anything that was incomplete or unclear. All clinical study information gathered was reported electronically by the investigators using a web-based case report form. Safety outcomes were classified using "MedDRA", the Medical Dictionary for Regulatory Activities (version 25.1) [28].

The primary objective was to demonstrate non-inferiority of the seroprotection rate achieved with MenACYW-TT after 30 days (day 31 [D31]) post-vaccination (percentage of participants achieving seroprotection, defined as hSBA titers ≥ 1:8 for meningococcal serogroups A, C, W, and Y) to that achieved with MCV4-TT.

Key secondary objectives included describing the immune response of meningococcal serogroups A, C, W, and Y measured by hSBA before and at D31 post-vaccination with MenACYW-TT or MCV4-TT, and additionally, describing the antibody response to serogroup C measured by hSBA and by serum bactericidal assay using rSBA before and at D31 post-vaccination with MenACYW-TT, and according to MenC priming status (MenC-naïve or MenC-primed during infancy). Other key secondary objectives were to describe the immune response of 9vHPV and Tdap-IPV vaccine antigens before and at D31 post-vaccination when administered alone or concomitantly with MenACYW-TT, and to describe the safety profile in each group after each and any vaccination. Observational objectives included describing the kinetics of antibody titers against the four meningococcal serogroups in the first 60 participants in group 3, measured by hSBA before and at D07 and D31 post-vaccination, and describing the antibody response against the four meningococcal serogroups as measured by rSBA, before and at D31 post-meningococcal vaccination, in the first 50 participants in each group.

A total of 464 participants were planned to be enrolled including 174 subjects in groups 1 and 2 and 116 in group 3. For the primary objective, 378 participants were planned to be enrolled in group 1 and group 2 (174 participants per group), to ensure the study had > 90% power (Farrington and Manning formula [29])) to declare the non-inferiority of MenACYW-TT (group 1) versus MCV4-TT (group 2) based on A, C, W, and Y hSBA seroprotection rates, assuming a 10% drop-out rate from the per-protocol analysis set (PPAS), a one-sided alpha level of 2.5% and a non-inferiority margin of 10%. The sample size was arbitrarily set to 116 participants in group 3, as these data were not intended to be used for hypothesis testing. For the secondary and observational objectives, no formal sample size calculations were performed.

For the primary objective, a non-inferiority approach was used to compare the D31 post-vaccination hSBA seroprotection rates in the MenACYW-TT group (group 1) to that in the MCV4-TT group (group 2). Non-inferiority was demonstrated if the lower limit of the 95% confidence interval (CI) of the difference was greater than – 10% for all four serogroups. The two-sided 95% CI was calculated based on the Wilson score method without continuity correction, as described by Newcombe 1998 [30]. For secondary and observational objectives, no hypotheses were tested and analyses were descriptive. In general, categorical variables were summarized and presented by frequency counts, percentages, and CIs. The 95% CIs of percentages were calculated using the exact binomial distribution (Clopper–Pearson method). For antibody geometric mean titers and geometric mean concentrations, 95% CIs of the point estimates were calculated using a normal approximation assuming these were log-normally distributed.

The per-protocol analysis set for meningococcal vaccines using hSBA (hSBA PPASM) and the per-protocol analysis set for meningococcal vaccines using rSBA (rSBA PPASM) were defined as the subset of participants who received a dose of a meningococcal vaccine, excluding those with at least one of the relevant protocol deviations, with a valid hSBA or rSBA result, respectively, on D31. The per-protocol analysis set for concomitant vaccines (PPASC) was defined as the subset of participants who received meningococcal vaccination with concomitant administration of 9vHPV and Tdap-IPV, excluding those with at least one of the relevant protocol deviations. The safety analysis set (SafAS) was defined as participants who received a dose of any vaccine who had safety data available. Safety endpoints are reported for the SafAS overall and at visit 1 (SafAS1) and at visit 2 (SafAS2).