Quadrivalent Influenza Vaccine-Induced Antibody Response and Influencing Determinants in Patients ≥ 55 Years of Age in the 2018/2019 Season.

The effects of immunization with subunit inactivated quadrivalent influenza vaccine (QIV) are not generally well assessed in the elderly Polish population. Therefore, this study evaluated vaccine-induced antibody response and its determinants. Methods: Consecutive patients ≥ 55 years old, attending a Primary Care Clinic in Gryfino, Poland, received QIV (A/Michigan/ 45/2015(H1N1)pdm09, A/Singapore/INFIMH-16-0019/2016 (H3N2), B/Colorado/06/2017, B/Phuket/ 3073/2013) between October-December 2018. Hemagglutination inhibition assays measured antibody response to vaccine strains from pre/postvaccination serum samples. Geometric mean titer ratio (GMTR), protection rate (PR) and seroconversion rate (SR) were also calculated. Results: For 108 patients (54.6% males, mean age: 66.7 years) the highest GMTR (61.5-fold) was observed for A/H3N2/, then B/Colorado/06/2017 (10.3-fold), A/H1N1/pdm09 (8.4-fold) and B/Phuket/ 3073/2013 (3.0-fold). Most patients had post-vaccination protection for A/H3N2/ and B/Phuket/3073/ 2013 (64.8% and 70.4%, respectively); lower PRs were observed for A/H1N1/pdm09 (41.8%) and B/Colorado/06/ 2017 (57.4%). The SRs for A/H3N2/, A/H1N1/pdm09, B Victoria and B Yamagata were 64.8%, 38.0%, 46.8%, and 48.2%, respectively. Patients who received QIV vaccination in the previous season presented lower (p < 0.001 and p = 0.03, respectively) response to B Victoria and B Yamagata. Conclusions: QIV was immunogenic against the additional B lineage strain (B Victoria) without significantly compromising the immunogenicity of the other three vaccine strains, therefore, adding a second B lineage strain in QIV could broaden protection against influenza B infection in this age group. As the QIV immunogenicity differed regarding the four antigens, formulation adjustments to increase the antigen concentration of the serotypes that have lower immunogenicity could increase effectiveness. Prior season vaccination was associated with lower antibody response to a new vaccine, although not consistent through the vaccine strains.


Introduction
Influenza is a contagious, acute respiratory disease, usually caused by Influenza A or B viruses, with seasonal infections that can lead to numerous complications, hospitalization and even death.
(TIV), composed of the three seasonal influenza virus strains currently circulating: Two influenza A virus types (H3N2 and H1N1), but only one B lineage (either B/Victoria or B/Yamagata). However, the B-lineage strain in TIVs and the dominant circulating B-lineage strain have differed in about 25% of the influenza season [16]. To reduce the chance of vaccine mismatch, a quadrivalent influenza vaccine (OIV) has been recently approved that includes an additional type B strain to represent both antigenic lineages [13,17,18]. Its immunogenicity and a safety profile, comparable to those of TIV, have the potential to overcome the drawbacks of erroneously predicting which B lineage will predominate in a given year [19][20][21]. Quadrivalent influenza vaccine (QIV) is expected to provide significant public health and economic benefit, as shown in recent studies [21].
The  [22]. Influenza vaccination with QIV was recommended for 2018/2019 season in the Polish National Immunization Program for all citizens aged >55 years [23].
One commonly accepted approach is the measurement of influenza-specific antibody titers as a correlate of protection. Titers are traditionally measured using a hemagglutination inhibition (HAI) assay, which quantifies the ability of hemagglutinin (HA)-specific antibodies to block the N-acetyl-neuraminic acid-mediated viral agglutination of red blood cells [24,25]. Using the set guidelines of this assay, vaccine immunogenicity can be measured based on HAI antibody titers obtained on day ≥28. Parameters commonly used to expresses seroresponse to influenza vaccination are mean fold increase, seroprotection and seroconversion rates [26][27][28]. While discrepancies can be found in surveys focusing on antibody response to influenza vaccine in the elderly, a quantitative review concluded that host-related factors, such as gender, BMI, preexisting immunity, genetic polymorphisms, and the presence of chronic underlying conditions could compromise influenza vaccine responsiveness [29,30]. HAI antibodies are significantly lower in older adults who were vaccinated, than compared to younger adults [30]. Although limited data exist, gender-differences have also been reported in response to diverse influenza vaccines with females having greater antibody responses than males following vaccination [29]. Obesity has also been associated with a decreased immune response to influenza vaccination. In addition, several studies document the influence of host genetic background on the immune response to influenza vaccination [29]. There is also a correlation between health status in older adults and HAI titers, i.e., healthy individuals having significantly higher levels of titers than those with chronic diseases [29,31].
There are methodological discrepancies among the meta-analyses of seasonal influenza vaccines efficacy and effectiveness for the elderly [32]. Although most vaccines show statistically significant efficacy, this is within a highly variable range [32,33]. The results of the measurement of influenza-specific antibody titers have been described previously; however, these mainly referred to older adults from the US and Western Europe [16,19,20]. Polish data on QIV immunogenicity in this age group are lacking. Therefore, the objective of this study was to evaluate the immunogenicity of a subunit inactivated QIV vaccine in Polish adults ≥55 years of age.

Setting, Study Population, and Sampling
The study was conducted among consecutive patients reporting to the primary care clinic (PCC) in Gryfino, Poland, in the vaccination season between October 2018-January 2019. The study group consisted of consecutive patients vaccinated with a QIV recommended by the WHO for that season. Inclusion criteria: Age ≥ 55 years, lack of co-existing diseases that could affect the cognitive functions of the subject, lack of contraindications to vaccination and informed written consent. Participation was voluntary.

Vaccine and Vaccination
Subunit, inactivated QIV (Abbott Biologicals, Olst, The Netherlands) was provided in prefilled syringes and administered by injection intramuscularly, using a 19 mm needle into the deltoid muscle to all subjects during the 2018-2019 influenza season. The cold chain was preserved, and the vaccines were stored at 2-8°C. A standard dose of QIV (0.

Serological Testing
Blood samples were collected twice, once before vaccination and four weeks after. Samples (1 mL) were centrifugated (15 min/4500 r.p.m.), stored at ≤−20 • C and then transported to the laboratory at the Department of Influenza Research (National Influenza Center, the NIPH-NIH) in Warsaw where they were tested. Briefly, sera were inactivated to remove non-specific hemagglutination inhibitors, which may affect a false positive result in an HAI test. Therefore, sera were treated with Receptor Destroying Enzyme (RDE), obtained from the Vibrio cholerae cell filtrate, and incubated at +37 • C. The test consisted of determining antibody titers (anti-HA) in serum by means of an HAI using a 0.75% solution of turkey red blood cells and reference strains of influenza virus, multiplied in chicken embryos, according to WHO recommendations [34].
Each study participant was given a code number, also placed both on the questionnaire and on a test tube. On 31 January 2019, participants were able to obtain information about their before/after vaccination serological tests results.

Vaccination Immunogenicity Assessment
On the basis of the results obtained after sero-testing, relevant parameters were calculated to assess the immunogenicity of a QIV. The current study assessed QIV-induced HAI antibody geometric mean titers (GMTs), seroconversion and seroprotection rates. Humoral responses were assessed on the basis of guidelines developed by the Committee for Proprietary Medicinal Products (CPMP) and the European Agency for the Evaluation of Medicinal Products (EMEA, now the European Medicine Agency, EMA) [1,26]. The following parameters were evaluated: • GMT (geometric mean titers) calculated at baseline (day 0) and 28-36 days after vaccination, • Average increase in antibody titers: GMTR (geometric mean titers ratio)-geometric mean of the individual post-vaccination/pre-vaccination titer ratios, • PR (protection rate)-the proportion of subjects with an HAI antibody titer ≥ 1:40, For the purpose of this study, the widely accepted HAI antibody titer of at least 1:40 was used to define seroprotection [16,35,36]. However, there is an ongoing debate whether this definition and serum antibody titers, in general, are valid correlates of protection [37][38][39][40][41]. According to Greenberg [36] and Chang [16] who evaluated the immunogenicity of a QIV in independent RCTs, as well as to current US guidelines [42], the HAI antibody titer remains an acceptable surrogate marker that is likely to predict clinical benefit.

Ethical Approval
The project received consent from the Bioethical Committee of Pomeranian Medical University in Szczecin (KB-0012/109/18).

Statistical Analysis
Data were analyzed using a customized program STATISTI-CA PL, Version 12.5 (StatSoft, Kraków, Poland). Categorical data were presented as frequencies with percentages and continuous data as means. The primary endpoints, HAI antibody titer, GMTR, seroconversion and seroprotection rate, were analyzed. HAI antibody titers were analyzed using geometric mean titer (GMT) and geometric standard deviations. GMTR, seroprotection and seroconversion were defined as in 2.4 sub-section. Protective HAI antibody titers before vaccination in the 2018/2019 season by the previous season vaccination were compared using the Fisher exact test.
Regarding determinants influencing seroconversion categorical (binary) variables (such as age: Up to 67/ ≤ 67 years; gender: Male/female; BMI: < 25/ ≥ 25 kg/m 2 ; smoking: Yes/no; alcohol consumption: Yes/no; chronic diseases: Yes/no; the self-reported occurrence of symptoms of upper respiratory tract infection in the current epidemic season: Yes/no; previous influenza vaccinations: Yes/no; vaccination in the previous season: Yes/no) groups were compared using the Fisher exact test.
A p-value was statistically significant if ≤0.05.

Influenza Vaccination
About two-thirds of the participants (63.0%) had never been vaccinated against influenza, 13.9% were vaccinated only once in their lifetime, 23.2%-more than once. Only 15.7% of respondents reported being vaccinated in the previous season; their vaccination records showed they were vaccinated with QIV.

Serologic Antibody Response after Influenza Vaccination
Paired pre-and ≥28 days (28-36 days) post-vaccination sera were available from 108 vaccinated patients. Serologic antibody response after QIV vaccination in terms of GMT, GMTR, PR and SR are presented in Table 2.
HAI antibody titers at baseline were the highest for B Yamagata lineage strain and similar regarding the rest studied strains (Table 2). Immunization with a QIV increased HAI antibody titers by 62-fold against the A/H3N2/ strain and by 3.0 to 10.3-fold against the B strains. Post-vaccination PRs were 42-65% against the A strains and 57% to 70% against the A/H1N1/pdm09 and B strains, and SRs occurred in 38-65% of participants for the A strains and in 47% to 48% for the B strains.
In detail, no protection against A/H3N2/ was observed in the study group before vaccination (PR 0.0%), however, the percentage of participants with antibody titers ≥ 1:40 increased significantly (PR 64.8%) after immunization and the proportion of participants with seroconversion also equaled 64.8%; the GMTR was 61.5 (Table 2). Regarding protection against A/H1N1/pdm09, before vaccination it was observed in only 5.6% of patients, however, the percentage of patients with protective anti-HAI titer increased significantly after immunization (to 41.8%) with the SR 38%; the GMTR ratio in this group was 8.35. More than 46% of vaccinated subjects seroconverted following vaccination regarding B Victoria lineage strain and the proportion of subjects with anti-HAI titer ≥ 1:40 was 57.4%; the mean fold increase was 10.3. A moderate response was observed for the B Yamagata lineage strain, i.e., seroconversion regarding vaccination was 48%; 17.6% of patients had protective anti-HAI titer before vaccination, this increased significantly (70.4%) after immunization. However, the mean fold increase was low (3.0).  (2) an HAI titer ≥ 10 at day 0 and a ≥4-fold increase in HAI titer between day 0 and post-vaccination; * Pre-vaccination; ** Post-vaccination.

Determinants of Seroconversion
Determinants of seroconversion after QIV vaccination by viral strain are presented in Table 3. Significant between-group differences were found with regards to seroconversion for B/Phuket/ 3073/2013 and BMI (p = 0.02), and influenza vaccination in the previous season/vaccination in life time (p = 0.03; p = 0.046). Similarly, for B/Colorado/06/2017 statistically significant differences were found in relation to proportions of patients who seroconverted and influenza vaccination in the previous season, as well as vaccination in life time (both: p < 0.0001).
No significant between-group differences were found for any of 4 strains regarding sero-conversion after QIV vaccination and gender (p > 0.12), age (p > 0.33), alcohol uptake (p > 0.26), comorbidities (p > 0.23) and self-reported respiratory symptoms in the current season (p = 1.00).

Results Overview
The results of this study showed a remarkably high GMTR after vaccination (61.5-fold) in the case of the A/H3N2/ strain. A much lower GMTR (3 to 10-fold) was observed regarding A/H1N1/ pdm09, B Victoria, and B Yamagata strains. About two-thirds of patients had post-QIV immunization protection for A/H3N2/ and B Yamagata vaccine strains; the lower rates (about 50%) were observed for A/H1N1/pdm09 and B Victoria. The SR was high for A/H3N2/ (64.8%) and relatively lower for B Yamagata (48.2%), B Victoria (46.8%) and A/H1N1/pdm09 (38%). Vaccination in the previous season significantly impaired the SR regarding both B strains.

Serologic Antibody Response after Influenza Vaccination
In this study anti-HAI titers against A (A/H1N1/pdm09 and A/H3N2/), and B (Victoria and Yamagata) influenza viruses were low among unvaccinated individuals (GMT: 1.7, 1.0, 3.1, and 14.2, respectively). However, despite the weakening of numerous components of the immune system in the study group, due to the natural aging process [21,43], substantial antibody response following vaccination was observed. This referred to all four vaccine antigens, particularly to A/H3N2. Thus, adding a second B strain to a subunit, QIV did not compromise the immunogenicity induced by the other three strains. This outcome corresponds to the results of previous studies in elderly patients [29,30], including randomized controlled trials (RCTs) [16,36].
As an example, the results of phase III, randomized, double-blind, active-controlled, multi-center trial performed during the 2010/2011 influenza season in the US showed that -in adults ≥ 65 years of age -QIV induced non-inferior antibody titers compared with control TIVs for all four vaccine strains [36]. This finding is in line with the more current, similar randomized, multicenter trial conducted in the US in the same group of age, in the 2017 /2018 season [16]. The results showed that a quadrivalent high-dose (HD) vaccine-induced HAI antibody responses that were non-inferior to responses induced by a trivalent-HD vaccine for the three shared strains and superior HAI antibody titers for the additional B-lineage strain.
In this study, a moderate response was observed for the B Yamagata lineage strain. The relatively low average post-vaccination increase in antibody titers (3.0) and seroconversion rate (47%) might be partly influenced by the fact that the same lineage strain was used in a QIV for the 2017/2018 season. Of note, about 16% of the study participants reported being vaccinated in the previous season with a QIV, which reflects the generally low uptake of influenza vaccines in Poland, especially in the elderly [11]. Previous exposure to influenza vaccine could have an impact on reduced antibody titers and SRs. Almost one in six patients showed protective antibody titers before vaccination, which increased significantly (70.4%) after immunization; QIV induced superior PR for the B Yamagata-lineage strain when compared with the other strains.
The relative proportion of circulating influenza A/H1N1/pdm09 and influenza A/H3N2/ viruses in the European region varied by country in the 2018/2019 season. The proportion of influenza A viruses subtyped in patients from EU PCCs was ≥ 95%; about 60% were influenza A/H1N1/pdm09 viruses; however, this proportion was > 80% in Denmark, the UK and Poland [44].
Based on this mix of circulating influenza subtypes and variation within the antigenic likeness of circulating viruses with the egg-propagated vaccine component, vaccine effectiveness might vary across Europe [44]. Although, according to current research, the vaccine was less effective against A/H3N2/ influenza viruses in recent years [45], in the 2018/2019 season, the protection rate against the A/H3N2/ strain among elderly Polish patients from a PCC was higher than A/H1N1/pdm09 and influenza B Victoria. The same was noted in the A/H3N2/ strain and GMTR, showing over a sixty-fold increase after vaccination.
In the current study, protection after QIV immunization, with an HAI antibody titer of ≥ 40, was found to be acceptable, particularly regarding A/H3N2/ and B viral strains; however, it was much lower for A/H1N1/pdm09. Interestingly, the high post-vaccination protection rate against A/H3N2/ was not the result of a high pre-protection rate, as none of the patients had protection before vaccination. Some previous observations also found that the A/H3N2/ vaccine antigen was able to induce a satisfactory immune response [49]. The reason for potential differences observed between studies in PRs and SRs could be related to the vaccine, the viruses or population exposure history [50].
Remarkably, the vast majority of the studied patients who had a QIV consisting of the same A/H1N1/pdm09 and B Yamagata antigens as in the previous season did not have protective antibody titers. This was also observed by Loebermann et al., who evaluated the immunogenicity of aTIV produced in mammalian cell culture administered to elderly adults. This may suggest that either antibody titers decline rapidly or that individuals did not develop a protective antibody titer earlier.
Due to the fact that protective antibody titers from the QIV received the previous season could only be detected in a minority of immunized elderly patients, to recommend annual vaccination in this cohort, even if the antigen composition did not change from the previous season would be of value [51].
In the 2018/2019 influenza season, the B Yamagata vaccine strain had not been changed [22]. Therefore, one of the reasons for the relatively high pre-protection rate against B Yamagata (Table 4) might have been the long-term vaccine antigen stimuli. Another cause could be a pre-existing immunity derived from previous natural infection. However, the relatively low percentage of participants having protective HAI titers before vaccination in the 2018/2019 season in the group which had not been vaccinated previously indicates the first scenario is more likely.
The response rate to QIV antigens, measured by the percentage of participants showing at least a 4-fold increase in the HAI antibody titer after vaccination, as well as an average increase in antibody levels, was excellent regarding A/H3N2/, and relatively lower in the case of the other antigens. Therefore, formulation adjustments to increase the antigen concentration of the serotypes that have lower immunogenicity could increase effectiveness. It has been shown in the case of the elderly that for both, TIV and QIV, higher doses of antigens are associated with higher antibody responses to a vaccine [16,29].

Determinants of Serologic Antibody Response
Repeated vaccination, as well as obesity, are well-known factors affecting the immune response after influenza vaccination [52][53][54].
In the case of influenza B, vaccination in the previous season, as well as vaccinations in the earlier seasons, had a negative impact on vaccine response among our participants; this was also observed by others [55][56][57]. As an example, Nebeshima et al. found that the HAI antibody titers to both influenza B strains in the repeated vaccination group of hospital workers were significantly lower than in the single vaccination group. This phenomenon had no relation to the pre-vaccination HAI titer, which suggested that the decreased HAI response to repeated influenza vaccination was mainly affected by the previous vaccination per se, rather than by the pre-existing antibody titer [57]. Similarly, to our findings, Sasaki et al. demonstrated that prior year vaccination was associated with sustained high HAI antibody titer one year on, but lower antibody response to the new vaccination [58]. In the current study vaccine responses were also impaired by pre-existing HAI titers regarding influenza B strains: 29.4% of patients vaccinated with a QIV in the previous season presented protective HAI-antibody titers for B Yamagata vs 15.4% not vaccinated patients; however, only 23.5% previously vaccinated patients seroconverted vs 55.9% in the not vaccinated group.
Human studies regarding obesity and its association with a decreased immune response to influenza vaccination have presented conflicting results [29]. The findings of the current study show that for B Yamagata strain significantly more normal weight patients seroconverted when compared to overweight and obese patients. This was also observed recently by Frasca et al. [59] who found reduced antibody responses to influenza vaccination in both young and elderly obese individuals.
A correlation between health status in the elderly and HAI titers, with healthy older adults having significantly higher HAI titers after influenza vaccination than those with comorbidities was reported by other authors [29,43,60,61]. However, this was not observed in this study, possibly due to the relatively small sample size. Additional strategies that provide better protection of at-risk populations will be required to reinforce the efficacy of QIV in chronically ill elderly patients [29]. The immunization of family members, including children, and the vaccination of medical personnel, should be highly advocated.

Limitations
Several limitations exist in this study. Firstly, the sample size was relatively small, and power was limited. Therefore, the results obtained, particularly those from subgroup analyses, should be interpreted with caution. Secondly, information about the vaccination/infection history in the current season was obtained through a questionnaire, not from vaccination or medical records. Therefore, undocumented influenza exposure was likely, particularly among patients unvaccinated in the previous season, who presented a high pre-vaccination titer [60]. In addition, information reported as grams of pure alcohol consumption per week would be more instructive than the frequency of alcohol intake. However, to minimize potential measurement errors regarding cumulative measures of alcohol consumption, especially in the older age group, participants were queried on the frequency of alcohol intake. Other (unmeasured) factors could have also affected serologic response to QIV. Further studies on larger populations are needed to assess the response and its determinants better. Finally, only HAI titers were used to assess the immune response to vaccination. This might be challenging for influenza A/H3N2/, due to their fluctuating capacity to agglutinate red blood cells [34]. Consequently, to assess other measures of the immune response, such as anti-neuraminidase antibody levels or cell-mediated the immunity would be of great value.

Conclusions
Even though the influenza vaccination is reported to be less effective in the elderly compared to young individuals, partly due to decreased generation of specific serum antibodies and switched memory B cells [21,46], the QIV-induced antibody response in the study cohort was satisfactory. While the QIV vaccine had a tendency to work better against A/H3N2/ and influenza B viruses than A/H1N1/pdm09 in the elderly Polish population, this introductory vaccine immunogenicity study supports its use.
The results show that a subunit QIV was immunogenic against the additional B lineage strain (B Victoria) without significantly compromising the immunogenicity of the other three vaccine strains. Adding a second B lineage strain in QIV could, therefore, provide broader protection against influenza B infection in this age group. As the QIV immunogenicity differed regarding the four antigens, formulation adjustments to increase the antigen concentration of the serotypes that have lower immunogenicity could increase effectiveness.
The study adds important data on the immunogenicity of influenza vaccines in Poland which have been lacking, even though QIVs have been available on the Polish market since the 2017/2019 season. These results should help encourage the switch to subunit QIV or other QIVs to protect Polish elderly patients against influenza.
The reduced response to immunization with influenza B strains in patients who had previously had influenza vaccinations, not consistent through the vaccine strains, needs further research to better understand influencing factors.