Immunogenicity and safety of high-dose quadrivalent influenza vaccine in Japanese adults ≥65 years of age: a randomized controlled clinical trial.

ABSTRACT A trivalent high-dose inactivated influenza vaccine has been licensed in healthy adults ≥65 years of age and provides better protection against influenza infection and related complications than trivalent standard-dose vaccine. This phase I/II clinical trial (NCT03233217), conducted at two sites in Japan, examined the safety and immunogenicity of a quadrivalent formulation of the high-dose inactivated influenza vaccine (IIV4-HD). Healthy adults ≥65 years of age were randomized to receive IIV4-HD by intramuscular injection (n = 60), IIV4-HD by subcutaneous injection (n = 60), or a quadrivalent standard-dose inactivated influenza vaccine (IIV4-SD) by subcutaneous injection (n = 55). Irrespective of administration route, post-vaccination (day 28–35) hemagglutination inhibition geometric mean titers and seroconversion rates were higher for IIV4-HD than for IIV4-SD. Hemagglutination inhibition geometric mean titers and seroconversion rates were also higher for intramuscular than subcutaneous administration of IIV4-HD. Solicited reactions were more common in participants who received IIV4-HD administered subcutaneously than in those who received IIV4-HD administered intramuscularly or IIV4-SD administered subcutaneously. Unsolicited adverse events were similar between the vaccine groups, and no safety signals were detected. This study showed that IIV4-HD administered by either intramuscular or subcutaneous injection was well tolerated and highly immunogenic in healthy Japanese adults ≥65 years of age. Although this study was descriptive, the results add to the evidence that high-dose inactivated influenza vaccines are more immunogenic than standard-dose vaccines in this age group and that intramuscular administration provides greater immunogenicity and lower reactogenicity than subcutaneous administration.


Introduction
Most influenza-related hospitalizations and deaths occur in adults ≥65 years of age. 1 This appears to be due to increasing comorbidities and waning immune responses associated with aging. 2,3 Because of the increased risk, the World Health Organization and most national health authorities recommend that, along with young children, pregnant women, and individuals with certain underlying conditions, adults ≥65 years of age should be prioritized for influenza vaccination. 4 Influenza vaccination is becoming increasingly important in Japan because it has one of the oldest and most rapidly aging populations globally. 5 Routine vaccination against influenza was instituted in Japan in 2001 for adults ≥65 years of age and adults 60-64 years of age with respiratory, cardiac, or renal disease or infection with human immunodeficiency virus. 6,7 Since the 2015/16 season, quadrivalent influenza vaccines have been used to vaccinate eligible individuals in Japan. 8 Quadrivalent influenza vaccines contain antigen from two influenza A strains (A/H1N1 and A/H3N2) and both influenza B-strain lineages (Victoria and Yamagata), whereas trivalent vaccines contain the two A strains and a single B-lineage strain. Quadrivalent influenza vaccines were developed to avoid mismatches between the B-strain lineage in trivalent vaccines and the predominant circulating B lineage, 9 which occurred in about one-quarter of influenza seasons between 2000 and 2013. 10 Switching from trivalent to quadrivalent influenza vaccines in Japan has prevented an estimated 2030 hospitalizations and 98 deaths each year and saved an estimated 10.75 million US dollars from a societal perspective. 11 To provide improved protection against influenza infection, a trivalent high-dose, split-virion inactivated influenza vaccine (IIV3-HD; Fluzone® High-Dose, Sanofi Pasteur) 12 has been licensed in adults ≥65 years of age in the US since 2009, Canada since 2015, Australia since 2017, Brazil since 2018, and the United Kingdom since 2019. This vaccine contains 60 µg hemagglutinin per influenza strain, which is four times the antigen content of standard-dose influenza vaccines. A multicenter phase III trial in the US and Canada showed that, in adults ≥65 years of age, IIV3-HD was 24.2% more effective than a standard-dose trivalent influenza vaccine (IIV3-SD) in preventing laboratory-confirmed influenza caused by any strain and 35.4% more effective than IIV3-SD at preventing laboratory-confirmed influenza caused by vaccine-similar strains. 13 IIV3-HD is well tolerated in adults ≥65 years of age, although as expected with the increased antigen dose, local reactions are more common with IIV3-HD. 14 These findings have been supported by post-marketing studies, which have shown that IIV3-HD provides improved protection against influenza, serious pneumonia, postinfluenza death, and all-cause, influenza-related, and cardiorespiratory-associated hospitalization. [15][16][17][18][19] To further improve protection against influenza in older adults, a quadrivalent formulation of the high-dose inactivated influenza vaccine (IIV4-HD) is being developed. A recent phase III trial in the US showed that, in healthy adults ≥65 years of age, IIV4-HD was well tolerated and induced immune responses that were non-inferior to responses induced by IIV3-HD for the shared strains and superior responses for the additional B-lineage strain. 20 The current study, performed in Japan during the 2017-2018 influenza season, compared the safety and immunogenicity of IIV4-HD and standard-dose quadrivalent inactivated influenza vaccine (IIV4-SD) in adults ≥65 years of age. In Japan, the standard of care for influenza vaccination is administration by subcutaneous (SC) injection, whereas the safety and efficacy of high-dose vaccines have so far been demonstrated following intramuscular (IM) injection. In this study, IIV4-HD was therefore administered by both IM and SC injection, whereas IIV4-SD was administered by SC injection.
Strains included in influenza vaccines are selected from the list of World Health Organization-recommended strains for each season, although the specific strains are selected by the Vaccines and Related Biological Products Advisory Committee for US vaccines and the National Institute of Infectious Diseases for Japanese vaccines. During the 2017-2018 influenza, three of four strains differed slightly between the US IIV4-HD and Japanese licensed IIV4-SD. This study therefore measured immunogenicity against all seven strains included in the two vaccines.

Study design
This was a phase I/II, randomized, parallel, modified double-blind, multi-center study in adults ≥65 years of age (NCT03233217) conducted between September and November 2017 at two clinics in Japan (ToCROM Clinic, Tokyo, Japan and OCROM Clinic, Osaka, Japan). The study was approved by the independent ethics committee or institutional review board for each institution and conducted in compliance with the standards established by the Declaration of Helsinki, the International Conference for Harmonization guidelines for Good Clinical Practice, and all local and national regulations and directives. All participants provided signed, informed consent before taking part in the study. An abbreviated version of the study protocol is available at https://clinicaltrials.gov/ct2/show/NCT03233217. No amendments were made to the protocol.

Participants
The study included adults ≥65 years of age not vaccinated against influenza in the preceding 6 months. Potential participants were excluded if they had any condition or were receiving any treatment that, in the opinion of the investigator, could interfere with the trial conduct, completion, or assessments. Other exclusions are listed in Supplementary table S1.  Table 1. Hemagglutinin content in the vaccines was measured by single radial immunodiffusion assay.

Randomization
A computer-generated randomization list was generated and provided by the sponsor and used for labeling and packaging. In a first cohort, 10 participants were enrolled (cohort 1) and assigned in a 1:1 ratio by block randomization to receive IIV4-HD by IM or SC injection. After review of the local and systemic adverse events (AEs) occurring within 7 days postvaccination by the sponsor's safety review team, 165 participants were enrolled in a second cohort (cohort 2) and assigned in a 1:1:1 ratio by block randomization to receive IIV4-HD by IM injection, IIV4-HD by SC injection, or IIV4-SD by SC injection, with stratification by age (<75, ≥75 years), sex, and site. Site staff were provided with vaccine dose numbers via interactive response technology. Randomization codes were kept securely in the interactive response technology and an internal system.

Blinding
An unblinded administrator at each site administered the vaccine. The vaccine administrator was not involved in any of the blinded study assessments. Investigators or delegates in charge of safety assessments, the trial staff who collected the safety data, and the laboratory personnel who analyzed the blood samples did not know which product was administered. Participants were blindfolded during vaccination to prevent them from identifying which vaccine was administered.

Safety assessments
Unsolicited AEs, serious adverse events (SAEs), and solicited reactions were defined as described in the International Unsolicited systemic AEs within the first 30 min after vaccination were recorded as immediate unsolicited systemic AEs. SAEs included any medical occurrence resulting in death, was life-threatening, required inpatient hospitalization or prolongation of existing hospitalization, resulted in persistent or significant disability/incapacity, was a congenital anomaly/birth defect, or was an important medical event according to the investigator's judgment. AEs of special interest included new onset of Guillain-Barré syndrome, encephalitis/myelitis (including transverse myelitis), Bell's palsy, optic neuritis, and brachial neuritis. For all AEs and SAEs, investigators recorded duration and the potential relationship to vaccination.
An early safety data review was performed by the sponsor after 7 days of post-vaccination safety data were available for cohort 1. The review included immediate reactions, solicited reactions, unsolicited AEs, SAEs, and AEs of special interest occurring within 7 days after vaccination. The study could have been put on hold or terminated if any of the following were detected during the review: ≥1 death or other SAE considered related to the vaccination; ≥40% of participants experiencing a grade 3 solicited reaction persisting ≥48 h; ≥20% of participants experiencing a grade 3 unsolicited, nonserious reaction persisting for ≥48 h and reported as related by the investigator.

Hemagglutination inhibition antibody titers
All subjects provided a pre-vaccination (baseline) blood sample at day 0 and a post-vaccination blood sample on day 28-35 for HAI testing against US-or Japanese-recommended strains (Table 1) , and B/Texas/2/2013. As described previously, 22 anti-influenza antibodies were measured using a hemagglutination inhibition (HAI) assay. Briefly, test and control sera were incubated with Type III neuraminidase to eliminate nonspecific inhibitors and then with a red blood cell suspension to adsorb spontaneous antispecies agglutinins. Ten two-fold dilutions of each sera were mixed and incubated with 4 hemagglutination units/25 µL of influenza virus. A red blood cell suspension was added to the mixture and, following incubation, titers were recorded as the highest serum dilution in which complete inhibition of hemagglutination occurred. If the lowest/first serum dilution used in the assay (1:10) did not result in complete inhibition of hemagglutination, the serum HAI titer was reported as < 10. If the highest/last serum dilution used in the assay (1:10,240) exhibited complete inhibition of hemagglutination, the serum HAI titer was reported as ≥ 10,240. The primary endpoints for the evaluation of immunogenicity were HAI antibody titers obtained on day 28 and seroconversion, defined as either (i) a HAI titer < 10 (1/dilution) at day 0 and a post-vaccination (day 28-35) HAI titer ≥ 40 or (ii) a HAI titer ≥ 10 at day 0 and a ≥ 4-fold increase in HAI titer between day 0 and postvaccination.

Statistical analysis
Because only descriptive analyses were performed for this study, no sample size calculation was made. Safety events were analyzed in the safety analysis set, defined as all participants who received a study vaccine. Immunogenicity was analyzed in the immunogenicity analysis set, defined as all participants in cohort 2 completing the study according to protocol. The 95% confidence intervals (CIs) for the geometric mean titers (GMTs) were calculated using a normal approximation of log-transformed titers. The 95% CIs of GMT ratios of each IIV4-HD group (IM or SC) compared to the IIV4-SD group (SC) were calculated using normal approximation of log-transformed titers. The 95% CIs for the proportions were calculated using the Clopper-Pearson method. Differences in the seroconversion rates between each IIV4-HD group (IM or SC) and the IIV4-SD group (SC) were computed along with the 2-sided 95% CIs by the Newcombe-Wilson method without continuity correction.
Statistical analyses were performed using SAS version 9.4 or later (SAS Institute, Cary, NC, USA). No search for outliers and no imputations were performed, although unsolicited AEs were considered as related if the investigator's assessment of the relationship was missing.

Participants
As planned, the study recruited 175 adults ≥65 years of age ( Figure 1) between September 15 and November 28, 2017 in two cohorts. A total of 60 participants were randomized to IIV4-HD by IM injection (IIV4-HD IM), 60 to IIV4-HD by SC injection (IIV4-HD SC), and 55 to IIV4-SD by SC injection (IIV4-SD SC). All participants were vaccinated as randomized and completed the study according to protocol, although one participant who received IIV4-SD by subcutaneous injection was excluded from the immunogenicity analysis set because of taking a prohibited medication before day 28-35 to treat a SAE.
All participants in this study were Asian, and age, sex, and body mass indices were similar in the three vaccine groups ( Table 2). Proportions of participants vaccinated for influenza the previous year were similar in the IIV4-HD SC (31.7%) and IIV4-SD SC (30.9%) groups but lower in the IIV4-HD IM group (16.7%).

Solicited reactions
The most frequently reported solicited injection-site reaction was pain, followed by erythema and swelling ( Figure 2). The most frequently reported solicited systemic reaction was myalgia, followed by headache and malaise. Most solicited reactions were of grade 1 or 2 intensity, started within the first 3 days after vaccination (Supplementary table S2), and resolved spontaneously within 7 days of onset (Supplementary table S3). Except for shivering, which was reported by few participants, and injection-site bruising, which was not reported, proportions reporting each solicited reaction were highest for the IIV4-HD SC group. Fever was reported in only one subject in the IIV4-HD SC group.

Unsolicited adverse events
Treatment-related unsolicited AEs, mostly injection-site pruritus, were reported more often in participants vaccinated SC than IM, although in fewer than 10% of the participants in each group (Table 3). No immediate unsolicited AEs, AEs of special interest, or deaths were reported. A single SAE of sudden hearing loss 3 days after vaccination was reported for a participant in the IIV4-SD SC group. The event was not considered related to the vaccination.

HAI antibody response
Before vaccination (day 0), most subjects were seropositive (HAI titer ≥10), although HAI GMTs were similar between IIV4-HD and IIV4-SD for all strains (Supplementary table S4). Between  Figure 1. Study design and participant disposition. The first 10 participants enrolled (cohort 1) were assigned in a 1:1 ratio to receive IIV4-HD by IM or SC injection. After review of the local and systemic adverse events (AEs) occurring within 7 days post-vaccination by the sponsor's safety review team, the remaining 165 participants enrolled (cohort 2) were assigned in a 1:1:1 ratio to receive IIV4-HD by IM injection, IIV4-HD by SC injection, or IIV4-SD by SC injection. Safety analysis was conducted for all participants who received a study vaccine (safety analysis set). Immunogenicity analysis was conducted for all participants in cohort 2 completing the study according to protocol (immunogenicity analysis set). Abbreviations: IIV4-HD, high-dose quadrivalent inactivated influenza vaccine; IIV4-SD, standard-dose quadrivalent inactivated influenza vaccine; IM, intramuscular; SC, subcutaneous.

IIV4-SD SC
baseline and day 28-35 post-vaccination, HAI titers increased for all strains in all vaccine groups by at least 2-fold and in most cases by at least 4-fold ( Table 4). The highest geometric mean postvaccination/pre-vaccination HAI titer ratios were for strains A/ H1N1 (16.00 for IIV4-HD IM, 9.25 for IIV4-HD SC, and 6.56 for IIV4-SD SC) and A/H3N2 (16.93 for IIV4-HD IM, 8.31 for IIV4-HD SC, and 4.85 for IIV4-SD SC). HAI GMTs were similar between male and female participants (data not shown).

Comparison of IIV4-HD and IIV4-SD
Post-vaccination HAI GMTs were higher for IIV4-HD than for IIV4-SD, irrespective of administration route (IM or SC) or whether the US-or Japanese-recommended strains were tested (Figure 3a). Ratios of post-vaccination HAI GMTs between IIV4-HD and IIV4-SD ranged from 1.98 to 2.89 for the IIV4-HD IM group, and from 1.65 to 2.70 for the IIV4-HD SC group (Supplementary table S5). Similarly, for all strains, point estimates for seroconversion rates for both the IIV4-HD IM and IIV4-HD SC groups were higher than for the IIV4-SD SC group, irrespective of whether the US or Japanese strains were tested. (Figure 3b).
Similarly, for all strains, point estimates for seroconversion rates were also higher for the IIV4-HD IM group than for the IIV4-HD SC group (Figure 3b).
Influence of strains tested HAI GMTs for A/H1N1 and B Victoria-lineage strains (Figure 3a) tended to be higher when immunogenicity was measured using the US-recommended than using the Japanese-recommended strains, although seroconversion rates were similar (Figure 3b).

Discussion
This study showed that IIV4-HD was well tolerated and highly immunogenic in healthy adults in Japan ≥65 years of age. No important safety events were detected for IIV4-HD. Although this study was not designed or powered to make statistical comparisons between IIV4-HD and IIV4-SD, the results are similar to those from studies comparing IIV3-HD and IIV3-SD in this population. 14,23,24 Because influenza vaccines are typically administered by SC injection in Japan and IM injection in the US and many other countries, this study assessed the immunogenicity and a b 100 1000 10000 IIV4-HD IM  IIV4-HD SC  IIV4-SD SC  IIV4-HD IM  IIV4-HD SC  IIV4-SD SC  IIV4-HD IM  IIV4-HD SC  IIV4-SD SC  IIV4-HD IM  IIV4-HD SC  IIV4-SD SC  IIV4-HD IM  IIV4-HD SC  IIV4-SD SC  IIV4-HD IM  IIV4-HD SC  IIV4-SD SC  IIV4-HD IM  IIV4-HD SC  IIV4-SD -HD IM  IIV4-HD SC  IIV4-SD SC  IIV4-HD IM  IIV4-HD SC  IIV4-SD SC  IIV4-HD IM  IIV4-HD SC  IIV4-SD SC  IIV4-HD IM  IIV4-HD SC  IIV4-SD SC  IIV4-HD IM  IIV4-HD SC  IIV4-SD SC  IIV4-HD IM  IIV4-HD SC  IIV4-SD SC  IIV4-HD IM  IIV4-HD SC  IIV4-SD   Values are for the immunogenicity analysis set. Abbreviations: CI, confidence interval; GMT, geometric mean titer; HAI, hemagglutination inhibition; IIV4-HD, highdose quadrivalent inactivated influenza vaccine; IIV4-SD, standard-dose quadrivalent inactivated influenza vaccine; IM, intramuscular; SC, subcutaneous safety of IIV4-HD administered by both routes. HAI antibody responses appeared higher and reactogenicity lower by IM administration than by SC administration. Thus, despite a higher antigen dose, the reactogenicity of IIV4-HD administered by IM injection was similar to that of IIV4-SD administered SC. Few other studies have directly compared SC and IM administration of influenza vaccines. A study in 120 adults 20 to 40 years of age in Japan found higher HAI antibody responses and fewer local adverse events following IM than SC administration of an adjuvanted influenza vaccine. 25 Also, a study in 720 Australian adults ≥65 years found that a standard-dose split-virion trivalent inactivated influenza vaccine induced higher HAI antibody titers and fewer local reactions when administered by IM injection than SC injection. 26 Although the immunogenicity differences in the Australian study were limited to women, sex did not appear to affect HAI antibody responses in the current study. The greater reactogenicity by SC injection is thought to be due to slower absorption of substances from the injection site, resulting in greater antigen processing by the antigenpresenting cells in the SC subcutaneous tissue and therefore greater inflammation. 27,28 Despite some differences in reactogenicity and immunogenicity, IIV4-HD was well tolerated and highly immunogenic when administered by SC or IM injection. IIV4-HD included the strains recommended by the US Vaccines and Related Biological Products Advisory Committee, whereas IIV4-SD included the strains recommended by the National Institute of Infectious Diseases of Japan. Although both the US and Japan used the WHOrecommended list of "-like" strains for influenza vaccines and the strains were from the same clades, three of four strains were not identical between the two vaccines. For all vaccine groups, HAI antibody titers were therefore measured for all seven strains. Irrespective of which strains were used for immunogenicity testing, conclusions were the same, with only small differences in HAI antibody titers.
In this study, 26.3% of the participants were vaccinated for influenza the previous year. By comparison, estimated influenza vaccination rates for Japanese adults ≥65 years of age were 50% in 2014 and 51% in 2015. 29 The lower rate in the current study might be because of the limited sample and the inclusion of only healthy, community-dwelling individuals. Regardless, coverage in Japan appears to be below the World Health Organization target of 75% for this age group. 30 An important limitation of this study is that it was not designed to make statistical comparisons between groups on immunogenicity or safety, and it was too small to detect lowfrequency safety events. Rather, the study was primarily designed to confirm tolerability and immunogenicity and to provide data for designing future clinical trials. Additional studies in larger populations will be conducted to confirm these findings. Nonetheless, the current immunogenicity findings, coupled with the results of other studies comparing the efficacy and effectiveness of high-and standard-dose inactivated influenza vaccines, 13,[17][18][19]31 suggest that IIV4-HD can further improve protection against influenza infection in adults ≥65 years of age. Finally, this study included only healthy, community-dwelling older adults. Further study will be required to determine whether the findings apply to frail or institutionalized individuals.
In conclusion, this study showed that IIV4-HD can be safely administered by either IM or SC injection and that it is highly immunogenic against both the US and Japanese strains in adults ≥65 years of age. The results also add to the evidence that high-dose influenza vaccines are more immunogenic than standard-dose vaccines in this age group. 24 Accordingly, IIV4-HD is predicted to provide better protection than IIV4-SD against influenza infection in adults ≥65 years of age.
Disclosure of potential conflicts of interest TI, TN, and KK (Sanofi) and LS, YM, AP, and LC (Sanofi Pasteur) are employees of the study sponsor. OM and SI declare no conflicts of interest related to the study reported in this manuscript.

Funding
This work was supported by Sanofi Pasteur. Sanofi Pasteur participated in study conception and design, writing and revision of the manuscript, final approval, and the decision to publish the findings.

Data sharing statement
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