Efficacy, immunogenicity, and safety of a quadrivalent inactivated influenza vaccine in children aged 6–35 months: A multi-season randomised placebo-controlled trial in the Northern and Southern Hemispheres

Efficacy, immunogenicity, and safety of a quadrivalent inactivated influenza vaccine in children aged 6–35 months: A multi-season randomised placebo-controlled trial in the Northern and Southern Hemispheres Stephanie Pepin a, ⇑ , Martin Dupuy a , Charissa Fay Corazon Borja-Tabora b , May Montellano c , Lulu Bravo d , Jaime Santos e , Jo-Anne de Castro f , Doris Maribel Rivera-Medina g , Clare Cutland h , Miguel Ariza i , Javier Diez-Domingo j , Celia Diaz Gonzalez k , Federico Martinón-Torres l,m , Efimia Papadopoulou-Alataki n , Maria Theodoriadou o , Marie Pierre Kazek-Duret a , Sanjay Gurunathan p , Iris De Bruijn a , for the GQM05 Study Group a Sanofi Pasteur, Marcy-l’Étoile, France b Research Institute for Tropical Medicine, Muntinlupa City, Philippines c Mary Chiles General Hospital, Manila, Philippines d University of the Philippines Manila – National Institutes of Health, Manila, Philippines e Philippine Children’s Medical Center, Quezon City, Philippines f DLSUMC De La Salle Health Sciences Institute, Dasmariñas, Cavite, Philippines g Inversiones en Investigación Médica S.A, Tegucigalpa, Honduras h Chris Hani Baragwanath Hospital, Johannesburg, South Africa i Caimed Dominicana A.S. Investigacion en Salud, Santo Domingo, Dominican Republic j FISABIO-Public Health, Valencia, Spain k Hospital Infantil La Paz, Madrid, Spain l Translational Pediatrics and Infectious Diseases, Universitario de Santiago de Compostela, Santiago de Compostela, Spain m Genetics, Vaccines and Infectious Diseases Research Group (GENVIP), Instituto de Investigación Sanitaria de Santiago, Universidad de Santiago, Spain n School of Medicine, Aristotle University of Thessaloniki, Thessaloniki, Greece o Children’s Hospital Aghia Sofia, Athens, Greece p Sanofi Pasteur, Swiftwater, PA, USA article info Article history: Received 9 July 2018 Received in revised form 9 October 2018 Accepted 26 November 2018 Available online 14 December 2018 Keywords: Quadrivalent inactivated influenza vaccine Children Efficacy Safety Immunogenicity abstract Background: A quadrivalent split-virion inactivated influenza vaccine (VaxigripTetra TM , Sanofi Pasteur; IIV4) containing two A strains (H1N1 and H3N2) and B strains from both lineages (Victoria and Yamagata) was approved in Europe in 2016 for individuals aged 3 years. This study examined the efficacy and safety of IIV4 in children aged 6–35 months. Methods: This was a phase III randomised controlled trial conducted in Latin America, Asia, Africa, and Europe during the Northern Hemisphere 2014/2015 and 2015/2016 and Southern Hemisphere 2014 and 2015 influenza seasons. Healthy children aged 6–35 months not previously vaccinated against influenza were randomised to receive two full doses 28 days apart of IIV4, placebo, the licensed trivalent splitvirion inactivated vaccine (IIV3), an investigational IIV3 containing a B strain from the alternate lineage. The primary objective was to demonstrate efficacy against influenza illness caused by any strain or vaccine-similar strains. Results: The study enrolled 5806 participants. Efficacy, assessed in 4980 participants completing the study according to protocol, was demonstrated for IIV4. Vaccine efficacy was 50.98% (97% CI, 37.36– 61.86%) against influenza caused by any A or B type and 68.40% (97% CI, 47.07–81.92%) against influenza caused by vaccine-like strains. Safety profiles were similar for IIV4, placebo, and the IIV3s, although https://doi.org/10.1016/j.vaccine.2018.11.074 0264-410X/Ó2018 The Authors. Published by Elsevier Ltd. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). ⇑ Corresponding author at: Sanofi Pasteur, 1541 Avenue Marcel Mérieux, 69280 Marcy-l’Étoile, France. E-mail addresses: [email protected],[email protected] (S. Pepin), [email protected] (M. Dupuy), [email protected] (J. Santos), [email protected] (C. Cutland), [email protected] (C.D. Gonzalez), [email protected] (F. Martinón-Torres), [email protected] (E. Papadopoulou-Alataki), [email protected] (M. Theodoriadou), [email protected] (M.P. Kazek-Duret), [email protected] (S. Gurunathan), Iris. [email protected] (I. De Bruijn). Vaccine 37 (2019) 1876–1884 Contents lists available at ScienceDirect Vaccine journal homepage: www.elsevier.com/locate/vaccine injection-site reactions were slightly more frequent for IIV4 than placebo. Conclusions: IIV4 was safe and effective for protecting children aged 6–35 months against influenza illness caused by vaccine-similar or any circulating strains. Clinical trial registration: EudraCT no. 2013-001231-51. Ó2018 The Authors. Published by Elsevier Ltd. ThisisanopenaccessarticleundertheCCBYlicense(http:// creativecommons.org/licenses/by/4.0/). 1. Introduction Influenza is a highly contagious viral infection and a significant burden especially for children due to an increased risk for severe illness and hospitalisation [1,2]. Although influenza A has historically been considered more important, influenza B is now known to be a significant cause of influenza-related illness, hospitalisation, and death [3] and to cause epidemics every 2–4 years [3,4]. Influenza B is a particular concern in young children in whom it causes a disproportionate amount of severe illness and hospitalisation [3,5]. In the 1980s, influenza B viruses diverged into two immunologically distinct lineages, Victoria and Yamagata, which now cocirculate worldwide [6]. Because trivalent influenza vaccines contain only a single B-lineage strain and because circulation varies between seasons and regions, differences between the vaccine and dominant circulating B-lineages are common [7,8]. Due to limited cross-lineage protection [9,10], especially in young children [11], quadrivalent influenza vaccines containing both B lineages may reduce the risk of influenza illness and its associated morbidity and mortality [12]. A quadrivalent split-virion inactivated influenza vaccine (IIV4 1 ; VaxigripTetra TM , Sanofi Pasteur) has been available in Europe since 2016 for individuals aged 3 years. Phase III clinical trials in individuals 3 years demonstrated that IIV4 was as immunogenic as the comparator trivalent inactivated influenza vaccine (IIV3) for each of the three shared influenza strains and superior for the additional B strain [13–16]. These trials also showed that IIV4 has a similar safety and reactogenicity profile as the licensed IIV3 (Vaxigrip Ò , Sanofi Pasteur). Because few studies had reported efficacy of inactivated influenza vaccines in very young children, in 2012, the World Health Organization (WHO) stated that they had only moderate confidence in their efficacy for this population [17]. To assess IIV4 in young children and to help reduce this evidence gap, we performed a phase III placebo-controlled clinical trial in which the primary objective was to demonstrate efficacy in children aged 6–35 months. Importantly, the vaccine used in this study contained a full dose of antigen (15 mg hemagglutinin [HA] per strain) rather than a half dose (7.5 mg HA per strain), which has been used in this age group for >30 years. A half dose of antigen was originally intended to reduce the risk of convulsions associated with earlier whole-virus influenza vaccines [18], but more recent findings suggest that a full dose can be used in children <3 years to improve immunogenicity without increasing fever or other reactions [19–21]. 2. Materials and methods 2.1. Study design This was a phase III, randomised, placebo-controlled trial conducted between March 2014 and July 2016 at 49 centres in Asia, Latin America, Europe, and Africa (EudraCT no. 2013-00123151). 2 The study included healthy children aged 6–35 months who had not been previously vaccinated for or infected with influenza according to participants’ parents or guardians. Children aged < 24 - months had to be born at full term (37 weeks) or with a birth weight 2.5 kg. Further exclusion criteria are listed in Table S1. The primary objective was to demonstrate the clinical efficacy of two full doses (15 mg HA/strain) of IIV4 to prevent laboratoryconfirmed influenza illness caused by any influenza A or B types or caused by vaccine-similar strains. Key secondary objectives were to demonstrate non-inferiority of hemagglutination inhibition (HAI) geometric mean titres (GMTs) for strains shared by IIV4 and IIV3; demonstrate superiority of HAI GMTs for B-lineage strains not shared between IIV4 and IIV3; and describe the immunogenicity and safety of all vaccines. The study design, including the influenza seasons, countries, randomisation schemes, participants included in the analyses for the different outcomes, and data monitoring are summarised in Fig. 1. During the efficacy evaluation period, participants were randomised to receive two 0.5-ml doses 28 days apart (window +7 days) of IIV4 containing the WHO-recommended A(H3N2), A (H1N1), B/Yamagata-lineage, and B/Victoria-lineage strains; an investigational IIV3 containing the WHO-recommended A strains and a strain from the B lineage not recommended by the WHO (Victoria) (IIV3-1); the licensed IIV3 containing the WHOrecommended A and B (Yamagata lineage) strains (IIV3-2); or placebo (saline). Efficacy was assessed only in participants vaccinated with IIV4 or placebo, whereas blood samples were taken for immunogenicity testing from all participants 28 days after the second vaccination. A randomly selected subset of participants who had received IIV4 or placebo during the 2014/2015 season was asked to return during the 2015/2016 season to be vaccinated with IIV4 (revaccination period). Those who had received IIV4 during the 2014/2015 season were vaccinated with a single 0.5-ml dose of IIV4, whereas those who had received placebo were vaccinated with two 0.5-ml doses of IIV4 28 days apart (window ±14 days). Blood samples were taken for immunogenicity testing 28 days after each vaccination. 2.2. Randomisation and blinding Randomisation lists were generated with the permuted block method and were communicated via an interactive voice or webresponse system. The study was single (observer)-blinded for the IIV4 and placebo groups and, due to differences in study procedures, open label for IIV3-1 and IIV3-2. 1 Abbreviations: CI, confidence interval; GMT, geometric mean titre; HA, hemagglutinin; HAI, hemagglutination inhibition; IIV3-1, investigational trivalent splitvirion inactivated vaccine containing the B lineage (Victoria) not recommended by the World Health Organization; IIV3, split-virion inactivated vaccine; IIV3-2, licensed trivalent split-virion inactivated vaccine containing the B lineage (Yamagata) recommended by the World Health Organization; IIV4, quadrivalent split-virion inactivated influenza vaccine; NA, neuraminidase; RT-PCR, reverse transcription-polymerase chain reaction; VE, vaccine efficacy; WHO, World Health Organization. 2 The protocol for this clinical trial is available at https://www.clinicaltrialsregister. eu/. S. Pepin et al./Vaccine 37 (2019) 1876–1884 1877 2.3. Ethics The study was approved by the independent ethics committee or institutional review board for each study site and was conducted in accordance with Good Clinical Practice and the Declaration of Helsinki. Written informed consent was provided by the parents or legal representatives of all children participating in this trial. 2.4. Vaccines All vaccines were thimerosal-free, inactivated, split-virion, and contained 15 l g of HA from each strain per 0.5-ml dose. Vaccines and placebo were presented in 0.5-ml prefilled syringes and were administered by intramuscular or deep subcutaneous injection into the deltoid region or the thigh. Vaccine formulations and strains used in the study are summarised in Table S2. 2.5. Detection of influenza and analysis of similarity to vaccine strains The occurrence of influenza-like illness was followed from 14 days after the second vaccine dose until the end of October for Asia and Africa and the end of April for Europe and Latin America. Participants were considered to have influenza-like illness if they had a fever 38 °C lasting 24 h concurrently with cough, nasal congestion, rhinorrhoea, pharyngitis, otitis, vomiting, or diarrhoea. For participants diagnosed with influenza-like illness, a nasopharyngeal swab was taken for laboratory confirmation of influenza within 10 days after onset. Nasopharyngeal swab samples from subjects with influenzalike illness were used to inoculate and infect influenza virussusceptible tissue culture cell lines. Influenza positive cultures were confirmed by direct immunofluorescence techniques with influenza type-specific (i.e., for influenza A and influenza B) antibodies. For culture confirmation of influenza, three different culture cell lines were utilised for each nasopharyngeal sample: classic influenza A and B culture using Madin-Darby canine kidney cells; classic influenza A and B culture using rhesus monkey kidney cells; and R Mix (a mixed monolayer of human A549 lung carcinoma and mink lung cells). Clinical samples collected during the study period underwent an extraction procedure to isolate the viral RNA from nasopharyngeal swabs prior to testing. The initial molecular test was a reverse Fig. 1. Study design. The efficacy evaluation period was conducted in three cohorts: during the SH 2014 season in South Africa and The Philippines, participants were randomised 1:1 to IIV4 or placebo; during the NH 2014/2015 season in France, Greece, Italy, Spain, and Honduras, participants were randomised 2:2:1:1 to IIV4, placebo, IIV31, or IIV3-2; and during the NH 2014/2015 (Dominican Republic), SH 2015 (Philippines), and NH 2015/2016 (Italy, Spain, and Romania) seasons, participants were randomised 1:1 to IIV4 or placebo. During the revaccination period, a randomly selected subset of participants who had been vaccinated with IIV4 or placebo during the NH 2014/2015 season were invited to come back the following (2015/2016) season to be vaccinated with IIV4. Influenza cases and efficacy data were reviewed periodically by an independent data monitoring committee, and blinded safety data were reviewed by an internal safety management team. Abbreviations: AE, adverse event; HAI, hemagglutination inhibition; IIV3-1, trivalent influenza vaccine containing the B-lineage strain not recommended by the World Health Organization; IIV3-2, trivalent influenza vaccine containing the B-lineage strain recommended by the World Health Organization; IIV4, quadrivalent split-virion inactivated influenza vaccine; ILI, influenzalike illness; NH, Northern Hemisphere; Rep, Republic; SH, Southern Hemisphere. 1878 S. Pepin et al./Vaccine 37 (2019) 1876–1884 transcription-polymerase chain reaction (RT-PCR)-based assay (eSensor Respiratory Viral Panel, GenMark) to determine if influenza A, influenza A H1 seasonal subtype, influenza A H3 seasonal subtype, influenza A 2009 H1N1 subtype, or influenza B were present in the clinical sample. For samples that were positive for influenza based on viral culture or RT-PCR, further testing by Sanger sequencing of the HA and neuraminidase (NA) full-gene segments was performed to identify the specific type or sub-type of the influenza strain. For genetic sequencing of HA and NA gene segments, total nucleic acid was extracted from the specimen/samples on an automated extraction system. Regions of both HA and NA genes were amplified by RTPCR using H1N1, H3N2 and B strain-specific primers. Amplified products were purified by plate filtration, and cycle sequencing reactions were performed on purified amplicons using fluorescently labelled dideoxy terminators. Cycle sequencing reaction products were purified by precipitation and analysed by agarose gel electrophoresis on an automated genetic analyser. The raw genetic sequence of each positive sample was compared with a database of known sequences corresponding to the vaccine and major circulating strains from 2005 up to the time of testing. Upon completion of sequencing runs, consensus sequences were compared to the Sanofi Pasteur reference BLAST database to determine closely related sequences. The best match had to be 95% alignment length and 99% identity. If a sequence did not meet these criteria, it was used as a query against the current version of National Center for Biotechnology Information Influenza Virus Resource Database. The strain responsible for laboratory-confirmed influenza was based on the Sanger sequencing results of the full HA gene, although if the RT-PCR results indicated A/H1N1 2009 pandemic influenza, the strain was considered to be A/California/7/2009. The strain responsible for a laboratory-confirmed influenza case was considered as similar to a strain contained in the vaccine if the best match strain was the same as any of the vaccine strains. 2.6. HAI titres Serum HAI titres were measured as described previously [22] for a randomly selected subset of participants included in the efficacy evaluation period (see Fig. 1) and for all participants included in the revaccination period. Seroconversion was defined as a HAI titre <10 on day 0 and a HAI titre 40 measured 28 days after the last vaccination. A significant increase was defined as a HAI titre 10 on day 0 and a 4-fold increase from baseline in HAI titre 28 days after the last vaccination. 2.7. Solicited reactions and adverse events Solicited reactions were collected by parents and legal guardians, and unsolicited adverse events were recorded by investigators (see Fig. 1 for subsets included). Solicited injection-site reactions included tenderness for participants aged 6–23 months; pain for participants 24–35 months; and erythema, swelling, induration, and ecchymosis for all participants. Solicited systemic reactions included vomiting, abnormal crying, drowsiness, loss of appetite, and irritability for participants aged 6–23 months; headache, malaise, myalgia, and shivering for participants aged 24– 35 months; and fever for all participants. Investigators also recorded serious adverse events and adverse events of special interest up to 6 months after each injection. Adverse events and serious adverse events were recorded according to International Conference for Harmonization guidelines [23]. 2.8. Statistical analysis The co-primary outcomes were vaccine efficacy (VE) in preventing laboratory-confirmed influenza caused by (i) any A or B strain and (ii) vaccine-similar strains. Both were assessed in all randomised participants in the IIV4 and placebo groups without relevant protocol violations and according to the vaccine to which they were randomised. Sensitivity analysis was performed in the full set of randomised patients who received two doses of vaccine. VE was calculated as 100% (1 [number of confirmed influenza cases in the IIV4 group/total number of participants in the IIV4 group]/ [number of influenza cases in the placebo group/total number of participants in the placebo group]). Confidence intervals (CIs) for VE were calculated by an exact method assuming a binomial distribution of the number of cases in each vaccine group conditional on the total number of cases. The co-primary endpoints were assessed using a one-sided 0.015 nominal alpha, resulting in a two-sided 97% CI. For each of the co-primary outcomes, efficacy was considered demonstrated if the lower-bound of the 97% CI for the corresponding VE was >20%. The primary objective was considered to have been met if efficacy was demonstrated for either of the coprimary outcomes. Approximately 553 evaluable influenza cases were needed to provide approximately 80% power to draw a conclusion of VE (primary objective) assuming that the true VE for IIV4 is 45% against vaccine-similar influenza strains and 0% against other strains; an adjusted one-sided type I error rate of 0.01247; a lower-bound of the CI of VE >20% for at least one primary outcome; and an allocation ratio of IIV4 to placebo of 1:1. Considering an overall influenza attack rate of 9% for the occurrence of an influenza case in the placebo group and 90% of enrolled participants evaluable for the primary outcome, a total of 8536 participants were estimated to be needed to be enrolled for influenza surveillance to reach the 553 expected evaluable influenza cases. Non-inferiority of HAI titres was assessed according to the vaccine received in randomised participants who completed the vaccination schedule, had a blood sample drawn 28–35 days after the last dose, and completed the efficacy evaluation period according to protocol. For each strain, non-inferiority was demonstrated if the two-sided 95% CI of the ratio of the geometric mean HAI titre (GMT) between IIV4 and IIV3 containing the same B-lineage strain, calculated using a normal approximation of log-transformed titres, was >2/3. Superiority was assessed in randomised participants who received at least one dose of vaccine and had a blood sample drawn 28–35 days after the last dose. For each B strain, superiority was demonstrated if the two-sided 95% CI of the ratio of the GMT between IIV4 and IIV3 containing the alternate B-lineage strain, calculated using a normal approximation of log-transformed titres, was >1. There were to be approximately 464 subjects assessed for HAI immunogenicity in the IIV4 group and 232 subjects in each IIV3 group. This was to produce an overall power of 90% to demonstrate: (a) non-inferiority of IIV4 vs. IIV3 in terms of HAI GMTs with a one-sided alpha level of 2.5%, a non-inferiority margin of 1.5, a theoretical ratio of 1:1 between groups, assuming a standard deviation of log 10 -transformed titres of 0.6 for A strains and 0.5 for B strains, and 80% subjects evaluable; and (b) superiority of IIV4 vs. each IIV3 group for the B-lineage strain it did not contain assuming that the IIV4 induced at least a 2-fold increase in the IIV3 response to the B strain it did not contain, a standard deviation of log 10 - transformed titres of 0.5, and 90% subjects evaluable. Statistical analysis was performed by Sanofi Pasteur (Marcy L’Etoile, France) using SAS Ò version 9.4 (SAS Institute, Cary, NC, USA). Missing data were not replaced. S. Pepin et al./Vaccine 37 (2019) 1876–1884 1879 2.9. Data monitoring, interim analysis, and stopping guidelines Two interim analyses by an independent data monitoring committee were planned to determine if efficacy had been demonstrated. The first was conducted when 200 confirmed influenza cases had been detected. The second, planned for when 375 confirmed influenza cases had been detected, was not conducted because efficacy was demonstrated at the first interim analysis. Enrolment was to be stopped at either interim analysis if the primary objective of efficacy was met, if the predictive power to demonstrate the primary objective at the end of the ongoing season was high, or if the probability to demonstrate the primary objective at the end of the trial was too low. 3. Results 3.1. Participants The study enrolled 5806 participants aged 6 to 35 months of which 5805 were randomised to receive two injections 28 days apart of IIV4 (n = 2721), placebo (n = 2715), IIV3-1 (n = 183), or IIV3-2 (n = 186) (Fig. 2). The study was conducted over four influenza seasons (Southern Hemisphere 2014 and 2015 and Northern Hemisphere 2014/2015 and 2015/2016) so that enrolment spanned 18 months from March 12, 2014 to December 4, 2015. Enrolment was stopped before the planned study size (8536 participants) was reached because efficacy was demonstrated during a planned interim analysis. A random subset of participants who had received IIV4 (n = 213) or placebo (n = 41) during the efficacy evaluation period were vaccinated with IIV4 the following season. Sex, age, and prevalence of at-risk conditions were similar in the four study groups (Table 1). Regions and ethnicities were similar for the IIV4 and placebo groups but different from the IIV3 groups as a result of the study design (see Fig. 1). No participants left the study due to a vaccine-related adverse event. 3.2. Efficacy vs. laboratory-confirmed influenza illness Of the 5436 participants in the IIV4 and placebo groups who completed the efficacy evaluation period, 456 were not included in the per-protocol efficacy analysis because of protocol violations or deviations. Thus, efficacy was assessed in 4980 participants. Laboratory-confirmed influenza illness was detected in 365 participants, including 120 (4.8%) in the IIV4 group and 245 (9.8%) in the placebo group. Influenza was due to vaccine-similar strains for 100 of the 365 participants with laboratory-confirmed illness (24 for IIV4 and 76 for placebo). By strain, confirmed influenza cases were most commonly due to influenza B (n = 164) and A/H3N2 (n = 156). Influenza A/H1N1 was relatively uncommon (n = 55). The strains detected and their similarity to the vaccine strains are listed in Table S3. The primary objective of efficacy was demonstrated for IIV4, with a VE vs. influenza due to any A or B type of 50.98% (97% CI, 37.36–61.86%) and vs. influenza due to a vaccine-like strain of 68.40% (97% CI, 47.07–81.92%) (Table 2). Results were similar when assessed in the full set of participants vaccinated with two doses of vaccine (Table S4). VE values for IIV4 were also similar when calculated separately for RT-PCR-confirmed influenza and cultureconfirmed influenza and when analysed separately for influenza A, A(H1N1), A(H3N2), B, and B/Yamagata, although not for B/Victoria (Table 2). For the four influenza seasons included, VE ranged Fig. 2. Participant disposition. Abbreviations: AE, adverse event; IIV3-1, trivalent influenza vaccine containing the B-lineage strain not recommended by the World Health Organization; IIV3-2, trivalent influenza vaccine containing the B-lineage strain recommended by the World Health Organization; IIV4, quadrivalent split-virion inactivated influenza vaccine; SAE, serious adverse event. 1880 S. Pepin et al./Vaccine 37 (2019) 1876–1884 from 41.2% to 59.6%, and for the two IIV4 formulations used, VE was 45.8% and 57.9% (Table S5). 3.3. Immunogenicity At baseline, most participants were seronegative for each vaccine strain in IIV4 (Tables S6 and S7), with the exception of participants in Asia during the Southern Hemisphere 2014 season of whom 49.6% were seronegative. After vaccination with two doses of IIV4 (day 56), for each strain, <2% of participants remained seronegative. Post-vaccination HAI GMTs ranged from 445 to 819 for A(H1N1), 517 to 1901 for A(H3N2), 416 to 1183 for B/Victoria, and 783 to 1610 for B/Yamagata. Geometric mean post-/prevaccination ratios of HAI titres ranged from 34.9 to 61.3 for A (H1N1), 32.7 to 63.4 for A(H3N2), 70.4 to 122 for B/Victoria, and 68.2 to 275 for B/Yamagata. Rates of seroconversion or significant increase in titre were 87% for all strains. Irrespective of the serological status at baseline, HAI antibody titres increased markedly after two injections of IIV4, although HAI GMTs were higher in participants with detectable titres (10) at baseline than in participants with undetectable titres (<10) (2149 vs. 408 for A(H1N1), 3632 vs. 585 for A(H3N2), 3986 vs. 562 for B/Victoria, and 1698 vs. 870 for B/Yamagata) (Table S8). Non-inferiority of post-vaccination HAI titres for IIV4 was demonstrated for both A strains when compared to the pooled IIV3s (Table 3). Non-inferiority was also demonstrated for B/Yamagata when compared to the IIV3 containing the same B strain (IIV3-2), although not for B/Victoria when compared to the IIV3 containing the same B strain (IIV3-1). However, for both B strains, superiority was demonstrated for IIV4 when compared to IIV3 conTable 1 Participant demographics and baseline characteristics. Characteristic IIV4 Placebo IIV3-1 IIV3-2 N = 2721 N = 2715 N = 183 N = 186 Sex, n (%) Male 1388 (51.0) 1425 (52.5) 94 (51.4) 98 (52.7) Female 1333 (49.0) 1209 (47.5) 89 (48.6) 88 (47.3) Age in months, mean ± SD 19.7 ± 8.4 19.8 ± 8.4 19.7 ± 8.4 19.3 ± 8.1 At risk for influenza-related complications a , n (%) 30 (1.1) 30 (1.1) 3 (1.6) 3 (1.6) Ethnicity, n (%) White 505 (18.6) 507 (18.7) 76 (41.5) 76 (40.9) Asian 1504 (55.3) 1504 (55.4) 0 (0.0) 3 (1.6) Black or African American 261 (9.6) 252 (9.3) 3 (1.6) 2 (1.1) American Indian b or Alaska Native 207 (7.6) 205 (7.6) 102 (55.7) 102 (54.8) Native Hawaiian or Pacific Islander 0 (0.0) 1 (<0.1) 0 (0.0) 0 (0.0) Region, n (%) Africa 251 (9.2) 249 (9.2) – – Asia 1498 (55.1) 1501 (55.3) – – Europe 528 (19.4) 523 (19.3) 81 (44.3) 84 (45.2) Latin America 444 (16.3) 442 (16.3) 102 (55.7) 102 (54.8) Values are for all randomised participants. Abbreviations: IIV3-1, investigational trivalent split-virion inactivated vaccine containing the B lineage (Victoria) not recommended by the World Health Organization; IIV3-2, licensed trivalent split-virion inactivated vaccine containing the B lineage (Yamagata) recommended by the World Health Organization; IIV4, quadrivalent split-virion inactivated influenza vaccine; SD, standard deviation. a Participants were considered to be at risk for influenza-related complications if they had chronic respiratory, heart, renal, metabolic, or haematological disorders. b Latin American participants were classified as American Indian. Table 2 Efficacy of the quadrivalent influenza vaccine by laboratory confirmation method and strain. IIV4 Placebo n (%) n (%) Outcome N = 2489 N = 2491 Vaccine efficacy Primary outcome % (97% CI) Laboratory-confirmed influenza illness caused by Any influenza A or B type 120 (4.82) 245 (9.84) 50.98 (37.36; 61.86) Vaccine-similar strains 24 (0.96) 76 (3.05) 68.40 (47.07; 81.92) Secondary outcomes % (95% CI) RT-PCR-confirmed influenza illness caused by Any influenza A or B type 118 (4.74) 243 (9.76) 51.40 (39.20, 61.33) Vaccine-similar strains 24 (0.96) 76 (3.05) 68.40 (49.42, 80.91) Culture-confirmed influenza illness Caused by any influenza A or B type 91 (3.66) 214 (8.59) 57.44 (45.36, 67.07) Caused by vaccine-similar strains 22 (0.88) 74 (2.97) 70.25 (51.56, 82.40) Laboratory-confirmed influenza illness by strain Any influenza A 65 (2.61) 147 (5.90) 55.75 (40.35, 67.47) A(H1N1) 11 (0.44) 44 (1.77) 74.98 (50.77, 88.35) A(H3N2) 53 (2.13) 103 (4.13) 48.50 (27.59, 63.75) Any influenza B 58 (2.33) 106 (4.26) 45.24 (23.88, 60.94) B/Victoria lineage 12 (0.48) 20 (0.80) 39.95 (28.98, 73.24) B/Yamagata lineage 26 (1.04) 63 (2.53) 58.70 (33.81, 74.90) Values are for participants with efficacy data completing the efficacy evaluation period according to protocol. Abbreviations: CI, confidence interval; IIV4, quadrivalent splitvirion inactivated influenza vaccine; RT-PCR, reverse transcription-polymerase chain reaction. S. Pepin et al./Vaccine 37 (2019) 1876–1884 1881 taining the alternate lineage (Table 3). Results were similar when the non-inferiority and superiority analyses were repeated in alternate data sets (Table S9). During the revaccination period, HAI GMTs at baseline (day 365) were higher in participants who had been vaccinated with IIV4 than in those who had been vaccinated with placebo (Fig. S1). After one dose of vaccine (day 393), HAI GMTs for each strain were at least four-fold higher in participants who had previously received IIV4 than in those who had previously received placebo. 3.4. Safety and reactogenicity Except for a higher proportion of participants reporting solicited injection-site reactions in the IIV4 group (39.9% [95% CI, 37.5– 42.4%]) than in the placebo group (31.9% [95% CI, 29.6–34.2%]), proportions reporting solicited reactions and adverse events were similar for the IIV4, IIV3, and placebo groups (Tables 4,S10, and S11). A single vaccine-related serious adverse event (benign febrile seizure) was reported for a participant vaccinated with IIV4. The event was secondary to an upper respiratory tract infection, did not lead to study discontinuation, and the participant recovered. 4. Discussion This randomised, placebo-controlled study confirmed the clinical efficacy of two full doses of IIV4 (15 mg HA/strain) for preventing influenza in children aged 6–35 months. Efficacy was demonstrated against vaccine-similar strains as well as against any strain, even though only about one-quarter of confirmed infections were due to vaccine-similar strains. The level of efficacy observed here is similar to that reported for another full-dose inactivated quadrivalent influenza vaccine in this same age group [24]. Together, the results provide convincing evidence that inactivated quadrivalent influenza vaccines are effective at preventing influenza in children aged 6–35 months. This study also showed that a full dose of IIV4 could be safely administered to young children. With the exception of more frequent injection-site reactions with IIV4 than with placebo, the overall safety profile for IIV4 appeared similar to placebo and IIV3. This agrees with other studies showing that a full dose of antigen can be used to increase the immune response to influenza vaccines in young children without adversely affecting safety [19– 21,24,25]. Furthermore, adding a fourth strain and using a full dose of antigen (15 mg HA/strain) appeared acceptable for this age group. Table 3 Non-inferiority and superiority of antibody responses induced by the quadrivalent influenza vaccine vs. trivalent comparators. Analysis/strain IIV4 IIV3 Ratio of GMTs (IIV4/IIV3) Non-inferior/ superior N Day 56 HAI GMT (95% CI) N Day 56 HAI GMT (95% CI) Non-inferiority a A(H1N1) 300 650 (549, 769) 320 c 629 (530, 746) 1.03 (0.81, 1.31) Yes A(H3N2) 300 1075 (917, 1261) 320 c 989 (845, 1158) 1.09 (0.87, 1.36) Yes B/Victoria lineage 300 593 (519, 678) 152 d 806 (657, 988) 0.74 (0.58, 0.93) No B/Yamagata lineage 300 997 (863, 1153) 168 e 983 (824, 1172) 1.01 (0.80, 1.28) Yes Superiority b B/Victoria lineage 341 623 (550, 706) 179 e 10.0 (8.26, 12.1) 62.33 (50.04, 77.64) Yes B/Yamagata lineage 341 1010 (885, 1153) 171 d 39.9 (31.2, 51.0) 25.3 (19.63, 32.62) Yes Non-inferiority was demonstrated if the two-sided 95% CI of the ratio of the HAI GMT between IIV4 and that of the comparator IIV3, calculated using a normal approximation of log-transformed titres, was >2/3. Superiority was demonstrated if the two-sided 95% CI of the ratio of the HAI GMT between IIV4 and that of the comparator IIV3, calculated using a normal approximation of log-transformed titres, was >1. Abbreviations: CI, confidence interval, GMT, geometric mean titre; HAI, hemagglutination inhibition, IIV3, trivalent inactivated split-virion influenza vaccine; IIV4, quadrivalent inactivated split-virion influenza vaccine. a The primary analysis for non-inferiority was performed in all randomised participants who completed the vaccination schedule, had blood sample drawn after the last dose, and completed the efficacy evaluation period according to protocol. b The primary analysis of superiority was performed in all randomised participants who received at least one dose of vaccine and had a blood sample drawn after the last dose. c Comparator was pooled IIV3s (IIV3-1 and IIV3-2). d Comparator was IIV3-1 (investigational IIV3 containing the B/Victoria lineage). e Comparator was IIV3-2 (licensed IIV3 containing the B/Yamagata lineage). Table 4 Adverse events and solicited reactions. Event IIV4 Placebo Pooled IIV3 n/N % (95% CI) n/N % (95% CI) n/N % (95% CI) Immediate unsolicited adverse event (<30 min) 1/1614 <0.1 (0.0, 0.3) 2/1612 0.1 (0.0, 0.4) 1/367 0.3 (0.0, 1.5) Vaccine-related 1/1614 <0.1 (0.0, 0.3) 1/1612 <0.1 (0.0, 0.3) 0/367 0.0 (0.0, 1.0) Solicited reaction 7 days after vaccination 1017/1592 63.9 (61.5, 66.2) 921/1595 57.7 (55.3, 60.2) 216/362 59.7 (54.4, 64.8) Injection-site 635/1591 39.9 (37.5, 42.4) 508/1593 31.9 (29.6, 34.2) 124/361 34.3 (29.5, 39.5) Systemic 772/1592 48.5 (46.0, 51.0) 741/1595 46.5 (44.0, 48.9) 180/362 49.7 (44.5, 55.0) Unsolicited adverse event 28 days after vaccination 1044/1614 64.7 (62.3, 67.0) 1079/1612 66.9 (64.6, 69.2) 261/367 71.1 (66.2, 75.7) Vaccine-related 91/1614 5.6 (4.6, 6.9) 96/1612 6.0 (4.9, 7.2) 7/367 1.9 (0.8, 3.9) Leading to study discontinuation 3/1614 0.2 (0.0, 0.5) 0/1612 0.0 (0.0, 0.2) 0/367 0.0 (0.0, 1.0) Serious adverse event 180 days after vaccination 68/1614 4.2 (3.3, 5.3) 78/1612 4.8 (3.8, 6.0) 14/367 3.8 (2.1, 6.3) Death a 4/1614 0.2 (0.1, 0.6) 1/1612 <0.1 (0.0, 0.3) 0/367 0.0 (0.0, 1.0) Adverse event of special interest b 29/2718 1.1 (0.7, 1.5) 31/2711 1.1 (0.8, 1.6) 1/367 0.3 (0.0, 1.5) Values are for all participants who received at least one dose of vaccine or placebo. Abbreviations: CI, confidence interval, IIV3, trivalent inactivated split-virion influenza vaccine, IIV4, quadrivalent inactivated split-virion influenza vaccine. a None of the deaths were considered vaccine-related. b Included anaphylaxis, Guillain-Barre syndrome, encephalitis/myelitis, neuritis, febrile convulsions, non-febrile convulsions, thrombocytopenia, and vasculitis. 1882 S. Pepin et al./Vaccine 37 (2019) 1876–1884 Immunogenicity is used as a correlate of protection, but identifying a clear threshold for protection has been difficult, especially for young children [26,27]. Because of this and to help strengthen the evidence base supporting efficacy of inactivated influenza vaccines in very young children, the study focused on demonstrating efficacy, with immunogenicity as a secondary and supporting endpoint. The study demonstrated that IIV4 induced high HAI antibody titres in this population and that it provided superior titres for the added B-lineage strain and non-inferior titres vs. IIV3 for all shared strains, except for B/Victoria. This lack of noninferiority for the B/Victoria strain does not appear to be a general feature of IIV4 because non-inferiority against all vaccine strains has been demonstrated for individuals aged 3 years [22,28]. Whether this unexpected finding will prove to be common for very young children will require further study. This study was designed to include unprimed young children. According to parents and guardians, the participants had not been previously vaccinated for influenza or infected with influenza virus. Nevertheless, depending on the region and season, up to half of the participants had detectable baseline HAI titres for each strain, suggesting that they had, in fact, been exposed to influenza viruses. As expected, post-vaccination titres were higher in these participants, although the IIV4 was highly immunogenic even in participants without detectable baseline antibodies. The revaccination portion of the study revealed that the antibody titres induced by a single dose of IIV4 increased when the participants had received IIV4 the previous season. This indicates that IIV4 could adequately prime this population. It also suggests that the two-dose schedule currently recommended for vaccinating unprimed children aged <9 years [29] should be continued for the full dose of IIV4. A major strength of this study was its representativeness. It was a large study conducted over a wide geographical area in both hemispheres and over several influenza seasons. The study was, however, limited by the vaccine strains that circulated. During the four seasons included, influenza A(H3N2) and B/Yamagata strains dominated. Thus, efficacy could be demonstrated against the A/H1N1, A/H3N2, and B/Yamagata strains but not against the B/Victoria strain. Efficacy of IIV4 against the B/Victoria strain will have to be established in further studies. In conclusion, this study showed that IIV4 safely protected children aged 6–35 months against influenza illness. By including a second B-lineage strain, IIV4 should provide additional protection beyond IIV3, irrespective of which B lineage circulates during a given season or region. As IIV4 gradually replaces IIV3 globally, it may help further reduce influenza-associated morbidity and mortality in young children. Acknowledgments The study was funded by Sanofi Pasteur. Medical writing was provided by Dr. Phillip Leventhal (4Clinics, Paris, France) and paid for by Sanofi Pasteur. Employees of Sanofi Pasteur participated in conceiving and designing the study, drafting and editing the article, deciding to submit the article for publication, and approving the final, submitted version. S.P. conceived and designed the study and helped draft the article; J.D.D., C.D.G., and F.M.T. participated in data acquisition. S.P., M.D., I.D.B., S.G., M.P.K., C.F.C., M.M., L.B., J.D.C., D.M.R.M., C.C., and M.A. participated in data analysis and interpretation. All authors helped critically revise the manuscript, approved the final version, and agree to be accountable for the accuracy and integrity of its content. The members of the GQM05 study group included: Karina Abalos (Sanofi Pasteur, Taguig City, Philippines), Helena Aurell (Sanofi Pasteur, Marcy-l’Étoile, France), José Maria Baldó (Centro de Salud Quart de Poblet, Valencia, Spain), Gianni Bona (Universita del Piemonte Orientale, Novara, Italy), Miguel Angel Cabanero (Centro de Salud Illes Columbretes), Josefina Cadorna-Carlos (University of the East Ramon Magsaysay), Marcela Cangrejo (Sanofi Pasteur, Bogota, Columbia), Brindusa Ruxandra Capilna (Ultratest S.R.L., Târgu Mureș, Romania), Alexandra Carmen Cara (private practice, Ca ˘la ˘r ași, Romania), Alfonso Carmona Martinez (Instituto Hispalense de Pediatria de Sevilla, Sevilla, Spain), Frédéric Chemin (private practice, Ecully, France), Ricardo Closa (Universitat Rovira i Virgili, Tarragona, Spain), Manuel Baca Cots (Hospital quirón salud), Florence Coux (Sanofi Pasteur, Marcy-l’Étoile, France), Javier Diez-Domingo (Centro Superior de Investigación en Salud Pública), Laura Larisa Dracea (private practice, Brasov, Romania), Maria Emporiadou (Aristotle University of Thessaloniki), María Espiau (University Hospital Vall d’Hebron, Barcelona, Spain), Susanna Esposito (Universita degli Studi, Genoa, Italy), Oana Asso Falup Pecurariu (Transylvania University, Brasov, Romania), Maria GarcésSanchez (Centro Superior de Investigación en Salud Pública, Valencia, Spain), Sanjay Garg (Sanofi Pasteur, Marcy-l’Étoile, France), Amparo Gil (Centro de Salud Malvarrosa), Laurie Gonzales (Sanofi Pasteur, Marcy-l’Étoile, France), Ronan Guevel (Sanofi Pasteur, Marcy-l’Étoile, France), Sara Guillen (Hospital Universitario de Getafe, Madrid, Spain), Giancarlo Icardi (Universita degli Studi, Genoa, Italy), Thelma Laot (Sanofi Pasteur, Manilla, Philippines), Isabelle Lacroix (Sanofi Pasteur, Marcy-l’Étoile, France), Josep Marès (Institut Pediàtric Marès-Riera, Girona, Spain), Manuel Martinez Pons (CS Republica Argentina), Catherine Moreau (Sanofi Pasteur, Marcy-l’Étoile, France), Mihai Leonida Neamtu (Lucian Blaga University of Sibiu, Sibiu, Romania), Andrea Elena Neculau (private practice, Brasov, Romania), Joyce Ojeda (Sanofi Pasteur, Mexico City, Mexico), Vana Papaevangelou (University of Athens School of Medicine, Athens, Greece), Maria Gabriella Penon (ULSS179 Presidio Ospedaliero di Este Dipartimento (PD)), Celine Petit (Sanofi Pasteur, Marcy-l’Étoile, France), Marie Philibert (Sanofi Pasteur, Marcy-l’Étoile, France), Victoria Planelles Cantarino (Centro Superior de Investigación en Salud Pública, Valencia, Spain), MarieLaure Py (Sanofi Pasteur, Marcy-l’Étoile, France), José T. Ramos (Hospital Clínico San Carlos), Enrique Rivas (Sanofi Pasteur, Mexico City, Mexico), Emmanouel Roilides (Aristotle University, Thessaloniki, Greece), Angels Rosich (Centro de Salud Malvarrosa, Valencia, Spain), Camille Salamand (Sanofi Pasteur, Marcy-l’Étoile, France), Eva Suarez (Centro de Salud Burriana 2), Gabriel Doru Surdu (private practice, Ploiești, Romania), Teresa Cerdán Vera (private practice, Valencia, Spain), Mariza Tsolia (University of Athens School of Medicine, Athens, Greece), Mira Vicedo (Centro de Salud Petrer 1), and Anne Woods (Sanofi Pasteur, Marcy-l’Étoile, France). Conflict of interest C.C. reports grants from Sanofi Pasteur during the conduct of the study. J.D.D. reports other support from Sanofi Pasteur during the conduct of the study and personal fees from Sanofi Pasteur outside the submitted work. M.E.M. reports personal fees from Sanofi Pasteur and GlaxoSmithKline outside the submitted work. F.M.T. reports other support from Ablynx, Janssen, GlaxoSmithKline, Regeneron, Medimmune, Pfizer, MSD, and Sanofi Pasteur outside the submitted work; and personal fees from Pfizer, MSD, and Sanofi Pasteur outside the submitted work. D.M.R.M. reports grants from Sanofi Pasteur during the conduct of the study and outside the submitted work. S.P, M.D., M.P.K.D., S.G., and I.D.B. are employees of Sanofi Pasteur. All others declare no conflicts of interest. S. Pepin et al./Vaccine 37 (2019) 1876–1884 1883 Author contributions All authors attest they meet the ICMJE criteria for authorship. Appendix A. Supplementary material Supplementary data to this article can be found online at https://doi.org/10.1016/j.vaccine.2018.11.074. References [1] Fraaij PL, Heikkinen T. Seasonal influenza: the burden of disease in children. Vaccine 2011;29:7524–8. [2] Iskander M, Booy R, Lambert S. The burden of influenza in children. Curr Opin Infect Dis 2007;20:259–63. 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