Comparison of the Immunogenicity of Cell Culture-Based and Recombinant Quadrivalent Influenza Vaccines to Conventional Egg-Based Quadrivalent Influenza Vaccines Among Healthcare Personnel Aged 18–64 Years: A Randomized Open-Label Trial

Abstract Background RIV4 and cell-culture based inactivated influenza vaccine (ccIIV4) have not been compared to egg-based IIV4 in healthcare personnel, a population with frequent influenza vaccination that may blunt vaccine immune responses over time. We conducted a randomized trial among healthcare personnel (HCP) aged 18–64 years to compare humoral immune responses to ccIIV4 and RIV4 to IIV4. Methods During the 2018–2019 season, participants were randomized to receive ccIIV4, RIV4, or IIV4 and had serum samples collected prevaccination, 1 and 6 months postvaccination. Serum samples were tested by hemagglutination inhibition (HI) for influenza A/H1N1, B/Yamagata, and B/Victoria and microneutralization (MN) for A/H3N2 against cell-grown vaccine reference viruses. Primary outcomes at 1 month were seroconversion rate (SCR), geometric mean titers (GMT), GMT ratio, and mean fold rise (MFR) in the intention-to-treat population. Results In total, 727 participants were included (283 ccIIV4, 202 RIV4, and 242 IIV4). At 1 month, responses to ccIIV4 were similar to IIV4 by SCR, GMT, GMT ratio, and MFR. RIV4 induced higher SCRs, GMTs, and MFRs than IIV4 against A/H1N1, A/H3N2, and B/Yamagata. The GMT ratio of RIV4 to egg-based vaccines was 1.5 (95% confidence interval [CI] 1.2–1.9) for A/H1N1, 3.0 (95% CI: 2.4–3.7) for A/H3N2, 1.1 (95% CI: .9–1.4) for B/Yamagata, and 1.1 (95% CI: .9–1.3) for B/Victoria. At 6 months, ccIIV4 recipients had similar GMTs to IIV4, whereas RIV4 recipients had higher GMTs against A/H3N2 and B/Yamagata. Conclusions RIV4 resulted in improved antibody responses by HI and MN compared to egg-based vaccines against 3 of 4 cell-grown vaccine strains 1 month postvaccination, suggesting a possible additional benefit from RIV4.

Influenza is estimated to result in 9-45 million illnesses, 140 000-810 000 hospitalizations, and 12 000-61 000 deaths each season in the United States [1]. Observed influenza vaccine effectiveness has been lower against A/H3N2 viruses than A/H1N1 viruses during recent seasons in the United States [2][3][4] which is concerning because influenza A/H3N2 viruses have been associated with higher influenza-associated hospitalization and mortality rates among older adults [5,6].
Mutations incurred during egg-based vaccine strain production may reduce vaccine effectiveness against influenza A/ H3N2 viruses in some seasons [7][8][9]. The conventional method of inactivated influenza vaccine (IIV) production relies on propagation in embryonated chicken eggs of a vaccine seed strain derived from a circulating influenza virus. Serial passage of influenza viruses in chicken eggs can result in mutations that cause important antigenic differences between the vaccine strain and circulating wild-type strains [7,[10][11][12]. Historically, the immunogenicity of influenza vaccines was assessed by measuring antibody responses to egg-grown influenza viruses, which may be a suboptimal measure of efficacy if egg-grown viruses differ antigenically from circulating wild-type viruses.
Vaccine strains that do not rely on egg-based production may induce higher immune responses to circulating influenza strains than egg-based vaccines [7]. During the past decade, a cell culture-based influenza vaccine (Flucelvax Quadrivalent™ by Seqirus, Inc., ccIIV4) and a recombinant influenza vaccine (Flublok Quadrivalent ® by Sanofi Pasteur, RIV4) were licensed for use in the United States. RIV4 has a higher hemagglutinin (HA) content (45 µg of HA per strain) than standard-dose IIV4 and ccIIV4 (both 15 µg of hemagglutinin [HA] per strain) but does not contain any neuraminidase (NA) antigen. In contrast, both ccIIV4 and IIV4 contain varying amounts of NA. Prelicensure trials evaluating these vaccines measured antibody responses to a variety of targets including egg-grown viruses, cell-grown viruses, and baculovirus expression vector systems (BEVS)-derived antigen [13][14][15][16]. Although several recent trials have documented improved humoral immune responses to RIV4 compared to IIV4 in adults 18-64 years [17] and ≥65 years of age [18,19], RIV4 and ccIIV4 have not been evaluated against IIV4 in highly influenza-vaccinated working-age adult populations in whom immune responses to influenza vaccination may be blunted over time [20]. To date, there are few data directly comparing the immunogenicity of cell-based and recombinant influenza vaccines to egg-based vaccines using the same immunogenicity outcome measures against the same antigenic targets.
This randomized, open-label trial assessed humoral immune responses to ccIIV4 and RIV4 compared to egg-based standard dose IIVs (Fluarix Quadrivalent™, GlaxoSmithKline; and Fluzone Quadrivalent™, Sanofi Pasteur) among United States (US) healthcare personnel (HCP) aged 18-64 years using cellgrown vaccine reference viruses. Because multiple egg-based IIVs with varying non-HA components such as NA and preservatives are available in the United States, 2 egg-based standard dose IIVs were combined as a single comparator group to improve generalizability of results. The primary study hypothesis was that a single dose of ccIIV4 or RIV4 would induce comparable or higher antibody titers against cell-grown vaccine viruses than a single dose of egg-based influenza vaccine in HCP with frequent prior influenza vaccination.

Randomization and Blinding
Both participants and study investigators were aware of study arm assignments. Laboratory investigators were blinded to assignment until testing was completed. Enrolled HCP stratified by age groups (18-44 years and 45-64 years) were assigned to receive ccIIV4, RIV4, Fluzone IIV4, or Fluarix IIV4 using a site-stratified REDCap-based randomization system (see Supplementary Methods for details).

Intervention
At enrollment, randomized HCP received a 0.5-mL dose of study vaccine via intramuscular injection into the deltoid muscle of the upper arm. During the 2018-2019 influenza season, egg-based IIV4 were produced from egg-derived seed viruses (or viral isolates), ccIIV4 contained cell culture derived H3N2 and B seed viruses and egg-derived H1N1 seed virus, and RIV4 contained recombinant HA proteins based on cell-culture derived seed viruses.

Study Procedures
At enrollment, eligible and consented HCP had 20 mL of venous blood drawn for serologic assays. HCP also completed online enrollment surveys and were asked to come back at approximately 1 and 6 months postvaccination for collection of 20 mL of venous blood at each visit. During the period of influenza circulation, sites conducted active surveillance for influenzalike illness (ILI) with mid-turbinate nasal swab collection and testing for influenza viruses. Surveillance was conducted to identify vaccine failures and not to assess clinical efficacy end points. See Supplementary Methods for additional details about ILI surveillance.

Outcomes Measures
The coprimary outcomes were serologic responses to cell-grown vaccine reference viruses by hemagglutination inhibition (HI) for influenza A/H1N1, influenza B/Yamagata, and influenza B/Victoria vaccine and by microneutralization (MN) assay to influenza A/H3N2 at approximately 1 month postvaccination using the following measures: seroconversion rate (SCR), geometric mean titers (GMT), mean fold rise (MFR), and geometric mean titer ratio. SCR was defined as the proportion of participants with either a prevaccination titer of <1:10 and 1 month postvaccination titer ≥1:40 or a prevaccination titer ≥1:10 and a ≥ 4-fold rise between pre-and postvaccination titers. MFR was defined as the geometric mean of the ratio of postvaccination titer and prevaccination titer for each subject. GMT ratio was defined as the ratio of postvaccination GMTs between either ccIIV4 or RIV4 compared to the egg-based vaccine group. Secondary outcomes were titers ≥1:40, 1:80, and 1:160 against cell-grown vaccine reference viruses by HI or MN at approximately 1 month postvaccination.
Subgroup analyses to evaluate for heterogeneity of effects among HCP stratified by number of influenza vaccines received during the preceding 5 years were prespecified in the study protocol.
All viruses used in the study were sequenced and confirmed with no additional mutations compared to seed strains. A/H1N1 and A/H3N2 antigens were cultivated at the Centers for Disease Control and Prevention (CDC), and the B antigens were provided by Seqirus and then further ether treated at CDC. See Supplementary Methods for details about blood specimen collection and processing.

Sample Size
Assuming a Type 1 error of 5% and a Type 2 error of 20%, a minimum sample size of at least 696 with at least 203 participants in the ccIIV4 and RIV4 arms and 145 participants in each of the Fluzone IIV4 and Fluarix IIV4 arms was anticipated to provide adequate statistical power to detect a difference in postvaccination GMT of ≥2-fold between study arms if postvaccination GMT was ≥20 in the combined IIV4 arms and a relative difference in postvaccination SCR of 30% if the postvaccination SCR was ≥50% in the combined egg-based IIV4 arms.

Data Analysis
The full analytic intention-to-treat (ITT) population comprised randomized HCP meeting eligibility criteria regardless of vaccine receipt. The 1 month and 6 month per protocol populations comprised randomized HCP who received study vaccine and had serum samples drawn and tested at 1 month or 1 and 6 months postvaccination, respectively, within the protocol-specified acceptable time periods. Primary analyses for outcomes at 1 month postvaccination were ITT. Secondary analyses for outcomes at 6 months postvaccination were per protocol. To address missing data, a "worst-case scenario" analytic approach was used for ITT analyses in which a titer of 1:5 (ie, undetectable) was assigned for all missing data.
Participants in the Fluzone IIV4 and Fluarix IIV4 groups were initially evaluated separately for the primary endpoints of SCR and GMT at 1 month postvaccination using prespecified criteria to determine whether the 2 groups would be collapsed into a single comparator group (combined egg-based IIV4). The prespecified criteria were based on effect sizes for which there would be adequate statistical power to detect differences based on the goal sample size. Comparison of Fluzone IIV4 and Fluarix IIV4 participants met the prespecified criteria of <15% absolute difference in SCR and a ≤2-fold difference in postvaccination GMT between participants in the 2 groups (Supplementary Tables 1a, 1b, 2a, and 2b). Therefore, participants in both groups were combined into a single egg-based vaccine comparator group for subsequent analyses evaluating ccIIV4 and RIV4 recipients.
Frequencies of seroconversion and postvaccination HI and MN titers greater than prespecified cutoffs were compared between vaccine arms using χ 2 test. GMTs, GMT ratios, and MFR were compared using Student t test. All tests were 2-tailed with a level of significance of .05. See Supplementary Methods for details about prespecified subgroup analyses and post hoc analyses. Analyses were performed with SAS (Version 9.3) (SAS Institute, Cary, North Carolina, USA).

Ethical Review
The study protocol was reviewed and approved by the institutional review boards (IRBs) of the 2 study sites and Abt Associates, which provided site oversight and data management support. The IRB of the CDC relied upon the single IRB review of the BSWH IRB. This study is registered in ClinicalTrials.gov, number NCT03722589. Study findings are reported in accordance with CONSORT (Consolidated Standards of Reporting Trials) statement guidelines.

Study Enrollment and Participant Baseline Characteristics
Overall, 952 HCP were assessed for eligibility, of whom 225 (24%) were excluded (Figure 1). The remaining 727 HCP were enrolled, randomized and included in the ITT population. Of these, all participants allocated to the Fluzone IIV4, Fluarix IIV4, and ccIIV4 arms received study vaccine, and 98% (198/202) allocated to the RIV4 arm received study vaccine. One and 6 month per protocol retention rates by vaccine arm were 90% and 70% for Fluzone IIV4, 98% and 81% for Fluarix IIV4, 99% and 86% for ccIIV4, and 97% and 82% for RIV4.
Among the ITT population, participants in each vaccine arm were similar with respect to age, sex, race, ethnicity, body mass index (BMI), and mean subjective health status score (Table 1). Participants in all study arms reported receiving an average of 5 influenza vaccines during the preceding 5 seasons; only 1-2% in each vaccine arm reported having never received an influenza vaccine during the preceding 5 seasons. Baseline geometric mean HI or MN titers were similar against vaccine reference viruses among participants in the combined IIV4, ccIIV4, and RIV4 arms ( Table 2, Supplementary Table 3).

Antibody Responses at One Month Postvaccination
At 1 month postvaccination, there were no consistent differences in antibody responses against HA between participants in the  12 participants were withdrawn from the study and did not have serum samples tested after they received vaccine that was left out at room temperature for an extended period; in addition, 1 participant completed the 1 month visit outside the per protocol time window; an additional 1 participant completed the 6 month visit per protocol but did not have a serum sample tested, 10 participants did not complete the 6 month visit, and 12 completed it outside the per protocol time window. For Fluarix IIV4, 1 participant did not complete the 1 month visit, 1 participant completed it outside the per protocol time window, and 1 completed it per protocol but did not have serum samples tested; an additional 11 participants did not complete the 6 month visit, and 9 participants completed it outside the per protocol time window. For ccIIV4, 2 participants did not complete the 1 month visit, 1 participant completed it outside the per protocol time window, and 1 completed it per protocol but did not have serum samples tested; an additional 14 participants did not complete the 6 month visit, and 21 participants completed it outside the per protocol time window. For RIV4, 2 participants did not complete the 1 month visit, and 1 participant completed it outside the per protocol time window; an additional 19 participants did not complete the 6 month visit, and 11 participants completed it outside the per protocol time window. Abbreviations: ccIIV4, cell-culture based IIV4 represented by Flucelvax Quadrivalent™ by Seqirus; IIV4, quadrivalent inactivated influenza vaccine represented by Fluzone by Sanofi Pasteur and Fluarix by GSK Biologicals; RIV4, recombinant IIV4 represented by Flublok by Sanofi Pasteur.
The small numbers of participants who received <5 influenza vaccines during the preceding 5 years precluded subgroup analyses to assess the interaction between number of prior influenza vaccinations during the preceding 5 years and vaccine type on seroconversion rates.

Antibody Responses at Six Months Postvaccination
At 6 months postvaccination, GMTs and GMT ratios did not differ between participants in the ccIIV4 arm compared to the combined egg-based IIV4 arm for any vaccine reference virus. Participants in the RIV4 arm had higher HI or MN GMTs compared to combined egg-based IIV4 recipients against the A/H3N2 and influenza B/Yamagata vaccine reference viruses ( Figure 2). As a post hoc analysis, GMTs were analyzed by vaccine arm after excluding participants with reverse transcription polymerase chain reaction (RT-PCR)-confirmed ILI (5 in the combined egg-based IIV4 arm, 14 in the ccIIV4 arm, and 7 in the RIV4 arm, Supplementary Table 4) between the 1 and 6 month postvaccination visits. Findings were consistent with the per protocol analysis. DISCUSSION We evaluated the immunogenicity of quadrivalent cellculture based and recombinant influenza vaccines compared to standard-dose egg-based vaccines among HCP aged 18-64 years using the same set of cell-grown influenza vaccine reference viruses for all vaccine types. Despite a history of frequent influenza vaccination among participants, egg-based IIV4, ccIIV4, and RIV4 all induced increases in Original answer choice converted to numeric scale where 5 = excellent and 1 = poor. c Based on report of vaccination by participant interview or electronic medical record extraction Table 2  The intention-to-treat population comprised randomized participants meeting eligibility criteria regardless of vaccine receipt. To address missing data, a "worst-case" analysis approach was used in which a titer of 1:5 (ie, undetectable) was assigned for all missing data. c P-value based on t test for postvaccination geometric mean titers and mean fold rises and χ 2 test for seroconversion rate and postvaccination titers ≥1:40, 1:80, 1:160 comparing either ccIIV4 or RIV4 recipients to combined egg-based IIV4 recipients.

. Antibody Responses Prior to Vaccination and at One Month Postvaccination by Hemagglutination Inhibition or Microneutralization Against Cell-Grown Vaccine Reference Viruses
postvaccination antibody titers. RIV4 induced more robust antibody responses against HA than standard dose egg-based vaccines against the A/H1N1, A/H3N2, and B/Yamagata influenza vaccine strains at 1 month postvaccination, but response to the B/Victoria reference virus was similar. RIV4 recipients also had higher GMTs at 6 months postvaccination against 2 of the 4 vaccine strains (A/H3N2 and B/Yamagata), but GMT ratios comparing RIV4 recipients to egg-based vaccine recipients at 6 months were only significant for the A/ H3N2 strain. In contrast, ccIIV4 induced similar responses against HA to all vaccine reference viruses at 1 and 6 months postvaccination compared to the egg-based vaccines. Our findings expand on those from previous studies that suggest that RIV4 may induce higher antibody responses against HA  The intention-to-treat population comprised randomized participants meeting eligibility criteria regardless of vaccine receipt. To address missing data, a "worst-case" analysis approach was used in which a titer of 1:5 (ie, undetectable) was assigned for all missing data.