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Effectiveness, immunogenicity, and safety of influenza vaccines with MF59 adjuvant in healthy people of different age groups

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Introduction
Influenza vaccines were first developed to prevent the attack of influenza virus in 1938, and then recommended to be inoculated annually based on the results from randomized clinical trials (RCTs) and laboratory detection showing effectiveness and safety. [1] A previous meta-analysis of RCTs showed no evidence for decreasing protection with annually repeated influenza vaccination. [2] Inoculation of influenza vaccine should result in effective and safe protection. Present efforts mainly focus on the prevention of seasonal and pandemic influenza. However, it is urgent to enhance the efficacy of influenza vaccines to decrease flu-related morbidity and mortality in all age groups. [3] The antibody titer levels would decrease to levels without protection no more than a year after the inoculation. Moreover, age-associated weakening of innate and adaptive immune responses would lead to impairment of influenza vaccine responses. [4][5][6][7] Lower T-cell responses are companied with a decrease in the antibody titers postvaccination with aging, and the level of cytolytic effector CD8+ T-cells is a vital factor for the clinical protection against influenza. [8] The degradation of immunity related with aging limits vaccine effects (VE) in healthy older people. These obstacles emphasize the importance to develop adjuvants to distinctly improve the efficacy of conventional influenza vaccines.
Addition of the adjuvants is a powerful approach to enhance the immunogenicity of influenza vaccines and decrease the content of hemagglutinin protein. In addition, the adjuvants would balance humoral immunity and cellular immunity, which may trigger the quicker and durable immune response. [9] Microfluidized emulsion 59 (MF59) is a squalene-based adjuvant, which is an oil-in-water emulsion. MF59 recruits the antigen present cells (APCs) at the injected site, subsequently transmits immune activation signals to lymphocyte T cells and B cells. Ansaldi et al concluded that seasonal trivalent influenza vaccines adjuvanted with MF59 provided a greater advantage compared to nonadjuvanted vaccines in protecting against a broader range of virus strains during the influenza season. [10] The effectiveness and safety of influenza vaccines adjuvanted with MF59 had been evaluated in several systematic analysis. [11][12][13][14] One study showed that influenza vaccines adjuvanted with MF59 were no better than conventional vaccines in some people. [15] In addition, RCTs comparing the effectiveness of MF59-adjuvanted influenza vaccines with conventional non-adjuvanted vaccines reported inconsistent consequences. [16] Therefore, the priority of MF59 adjuvant did not reach a consensus for extensive application.
Up to now, no meta-analysis and systematic review has systematically inspected the relation between the addition of MF59 adjuvant and immunogenicity outcomes in all age groups inoculated with adjuvanted vaccines. Therefore, we performed a systematic review and meta-analysis to evaluate the effect of MF59 adjuvant on humoral immune responses among healthy people over 6 months old inoculated with influenza vaccines.

Methods
Our systematic review and meta-analysis was performed following the Preferred Reporting Items for Systematic review and Meta-Analysis Protocols (PRISMA-P) guidelines (Table S1, http://links.lww.com/MD/D755).
We retrieved the following databases up to March 2019: PubMed, Embase, Medline, OVID, and Cochrane Library Trials (CENTRAL) using medical subject heading (MeSH) terms and free words of the keywords "influenza vaccine", "MF59" and "adjuvant". The search strategy was shown in Table S2, http:// links.lww.com/MD/D756. Only articles with English language were retrieved, and the reference lists from all retrieved studies and the most recent review articles were reviewed to collect additional undetected published studies.
The protocol of this study was registered: PROSPERO (Prospective register of systematic reviews) registration No. CRD42019129579. No ethics approval was necessary because this was a meta-analysis.

Study selection and methodological quality assessment
The retrieved articles would be identified based on the inclusion criteria: 1. Healthy people over 6 months old, and the exclusion criteria: 1. animal studies, 2. people allergic to eggs and other vaccines, 3. people vaccinated with adjuvant influenza vaccine 6 months before enrollment, 4. people with immunodeficiency, 5. people infected with influenza virus within 12 months, 6. participated in any other clinical studies, 7. pregnant and lactating women, and 8. people with acute, chronic or serious diseases.
The quality assessment of the included studies was conducted independently by 2 researchers employing the Cochrane Review Risk of Bias Assessment Tool. The methodological heterogeneity was evaluated by the strategies of randomization, allocation concealment, and blinding. Subsequently, the data integrity was assessed by dropout rate, complete outcome data, selective data reporting. Risk of bias of every portion was classified into low, high, or unclear.

Data extraction and analysis
The data were extracted from the included studies by 2 investigators (Jing Yang and Jiayou Zhang), respectively, including first author's name, time of publication, country of experimental locations, number of subjects, age of subjects, sex ratio of subjects, measures of intervention, information of vaccine, type of comparison vaccine, local and systemic adverse events, clinical and laboratorial outcome measures. The immunogenicity of influenza vaccine with MF59 adjuvant was evaluated for Geometric Mean Titer (GMT), and then the seroconversion and seroprotection rates were extracted from the studies. HI antibody titer was the maximum dilution capable of inhibiting the agglutination of red blood cells with the influenza viruses under standardized conditions. Seroconversion rate was defined as the percentage of subjects per group achieving at least a 4-fold increase in HI titer from a seropositive prevaccination titer (>10) or a rise from <10 to 40 in those who were originally seronegative. Seroprotection rate was defined as the proportion of subjects achieving an influenza antibody titer greater than or equal to 40 in HI laboratory detection.
Licensure criteria of influenza vaccines for adults were slightly different between Center for Biologics Evaluation and Research (CBER) and Committee for Medicinal Products for Human Use (CHMP) guidelines ( Table 1). Immunogenicity of the influenza vaccine in our studies should at least satisfy the requirements of the CBER guidelines, which set valid levels of vaccine immunogenicity for a population over the age of 65 years as at least a 30% seroconversion rate, at least a 60% subjects achieving antibody titer ≥40. As for the population less than 65 years, the rate of seroconversion should be no less than 40%, or at least a 70% subjects achieving antibody titer ≥40. Each vaccine antigen should meet at least 1 of the following criteria in the CBER guidelines. Meta-analysis was implemented as the trials had acceptable clinical homogeneity and statistical heterogeneity. A randomeffect model was employed on account of the significant heterogeneity expected among the studies. Dichotomous data were analyzed using a Risk Ratio (RR) with 95% confidence intervals (CI) for identifying the differences in effectiveness and immunogenicity between the intervention and the control groups. Heterogeneity was quantified using the Cochrane Q TEST and I 2 statistics. Potential publication bias was assessed by observing the symmetry of funnel plots and by using Egger test. Meta-analysis was performed using Review Manager (RevMan) program Version 5.3.
Vaccination strategies ranged from 1 dose immunization to 1 or 2 booster immunization for better immune responses. Among the 8932 participants included, 14 individuals had severe adverse effects related to vaccination.

Subgroup analysis of immunogenicity of influenza vaccines with MF59 adjuvant in different age groups
A subgroup meta-analysis was conducted based on the different age groups of the healthy subjects. The participants included were divided into the following 3 age groups: healthy young children (6 to 72 months old), healthy adults (7 to 64 years old), and healthy older adults (over 64 years old). The heterogeneity of the subgroups seroconversion rate was low in 2 extreme age groups (Fig. 5). We used the outcome measure of seroconversion rate to assess the effects of MF59 adjuvant on influenza vaccine strains, participants from the second age group with the RR for seroconversion rate was 2.10, with a 95% CI of 1.28 to 3.44, I 2 = 94%, P = .003, which benefited the most from inoculation of MF59-adjuvanted influenza vaccine followed by the healthy older adults, but the young children (36-72 months old) group had no significant differences in immune responses between MF59-adjuvant and Non-adjuvant influenza vaccine.

Subgroup analysis of immunogenicity differences of influenza vaccines with MF59 adjuvant between the monovalent prepandemic vaccine and trivalent vaccine
Because of high heterogeneity of seroconversion rates in adult group, another subgroup analysis was conducted in accordance Table 3 Vaccine information of included studies.  [15] A monovalent inactivated subunit influenza vaccine: A/Duck/Singapore/ 97 (H5N3) strain 0.5 ml/HA (7.5, 15, or 30 mg)

Subgroup analysis of immunogenicity of influenza vaccines with MF59 adjuvant in different content of HA antigen
Furthermore, to investigate appropriate content of HA antigen with standard dose MF59 adjuvant and the necessity of 2-dose schedule vaccination, we performed a subgroup analysis of the content of HA antigen, and compared the seroconversion rate between second dose vaccination and first dose vaccination with same vaccine formulation that contained 7.5 mg HA antigen with standard dose MF59 adjuvant. Both 15 mg HA antigen group and 7.5 mg HA antigen group showed high homogeneity after dividing participants into MF59adjuvanted influenza vaccines containing 15 mg HA antigen and MF59-adjuvanted influenza vaccines containing 7.5 mg HA antigen groups. Based on the seroconversion rate of MF59adjuvanted influenza vaccine, we found more favorable effect in the 7.5 mg HA antigen group (RR = 1.30, 95% CI = 1.15-1.47, I 2 = 54%, P < .0001) (Fig. 7a).

Discussion
To our knowledge, this is the first systematic review and metaanalyzed on the efficacy of MF59 adjuvant in different types of influenza vaccines among various age groups. We found that MF59 adjuvant enhanced the immunogenicity against specific influenza viral strains H1N1, H3N2, B strains, H9N2, and H5N1, but not the H7N9 strain. [15] The seroconversion rates from included RCTs showed better immune protective effect in the participants inoculated with MF59 adjuvanted influenza vaccine against vaccine homologous H1N1 and B strain, and the seroprotection rate showed significantly enhanced efficacy of MF59-adjuvanted influenza H1N1 vaccine.
In this study, the subjects inoculated with MF59-adjuvanted influenza vaccine met at least 1 of the CBER license criteria for the influenza vaccine strains (seroprotection rates and seroconversion rates against the specific influenza viral strains), and had superior seroconversion rates against the B strain, H1N1, H9N2 and H5N1 strains compared to non-adjuvanted groups. In this meta-analysis, only1 study [17] might have slightly overestimated the seroconversion rate against H9N2, as the HI titer ≥ 32, and a >4 fold increase was used as the standard, which would not affect the final results by the sensitivity analysis.
The efficacy of MF59 adjuvant for enhancing the effectiveness of influenza vaccines did not reach a consistent conclusion in previous studies. Our results show that the majority of the 17 enrolled RCTs reached a consistent conclusion, and only 1 trial [1] showed opposite results. The inconsistency might be attributed to study design, dose-schedule, immune procedure, as well as the demographic characteristics of the subjects enrolled such as health status and age. We attempted to analyze the potential mixed effect of these variables on MF59-adjuvanted influenza vaccine. The age and influenza vaccine type had significant effects on the participants' immune responses to MF59-adjuvanted influenza vaccines. Subjects from adult group were more sensitive    practices to make appropriate recommendations for different age groups to strengthen influenza vaccine efficacy. Although compliance of the participants might be a confounding factor in RCTs. In this study, the compliance was recorded in all RCTs and most of the studies showed good compliance, and 7 studies further confirmed the immune persistence of MF59adjuvanted influenza vaccines half or 1 year after inoculation. Only 1 study declared no good compliance, and the dropout rate was over 30%. [16] Furthermore, a subgroup meta-analysis was performed on different types of influenza vaccine in adult group, we found that MF59-adjuvanted monovalent inactivated influenza vaccines had distinguish positive effects on immunogenicity changes against all kinds of influenza vaccine strains except H7N9 strain. On the contrary, MF59 adjuvant showed no significant difference in the efficacy in young children group. Further investigation is needed to identify the influences of lower MF59 adjuvant content in young children.
The potential mechanisms of MF59 adjuvant to enhance immune efficacy of influenza vaccine strains remain to be elucidated. In cellular immunity, MF59 adjuvant can recruit neutrophil, phagocyte, and NK cells [30] and promote IgA secretion to the injection site to enhance the vaccine effects. [31] In addition, Th1 cells produce interferon g, which has antiviral effects.
No previous meta-analysis has compared the efficacy of MF59 adjuvant in various age groups in enhancing the immunogenicity of various types of influenza vaccines. In this meta-analysis, subgroup analysis revealed that monovalent MF59 adjuvanted influenza vaccine in adult group achieved more immunogenicity changes. Consequently, the addition of MF59 adjuvant might be a significant approach to increase the immune responses to influenza vaccines.
Low heterogeneity of the pooled analysis is the advantage of this study. Furthermore, the RCTs included in this analysis were retrieved from various databases and included publications in English from different perspectives and cultural viewpoints. However, our meta-analysis has several limitations. First, we only used the rates of seroprotection and seroconversion as outcome measures, and did not use the changes in antibody geometric mean titer (GMT) because none of the included studies recorded GMT with a standard deviation before and after vaccination. Second, only 1 trial was included for subgroup analysis of different MF59 adjuvant content, therefore, the comparison of different MF59 adjuvant content was limited. Third, due to the limited number of included RCTs and the lack of sufficient data on basic immune status and original antibody titers against influenza, it was impossible to conduct more subgroup analysis to identify the effects of these variables. Finally, the medications used by the subjects might be confound factors. However, no medication records had been reported in the included studies.

Conclusions
Our meta-analysis showed that MF59 adjuvant can improve the immunogenicity of influenza vaccines in healthy adults and the aged. We propose that MF59-adjuvanted influenza vaccines can be widely inoculated both in healthy adults and the aged, especially monovalent pre-pandemic influenza vaccines. Further large RCTs focusing on the optimal dose, inoculation interval, and the antagonism effect of a combination of MF59 adjuvant and trivalent seasonal influenza vaccine are required to testify these findings.