Antibody responses to intradermal or intramuscular MF59-adjuvanted influenza vaccines as evaluated in elderly institutionalized volunteers during a season of partial mismatching between vaccine and circulating A(H3N2) strains

The study included a total of 80 elderly subjects living in two nursing homes located in Umbria, a region of central Italy, (42 at the “Opera Pia Bartolomei-Castori” and 38 at the “Casa Serena” nursing homes). Forty volunteers were vaccinated with IM-MF59 and 40 with ID influenza vaccine commercially available for the 2011–2012 Winter season.

As reported in Table 1, the baseline characteristics of the two groups were similar and for this reason the results obtained with the two vaccines could be compared.

Vaccine immunogenicity was evaluated by comparing HI titers in blood samples collected before and 30 days after vaccination and persistence of immune response by considering titers at 6 months of vaccination. The results are reported as protection rate (numbers of volunteers showing HI titers ≥40, considered to be associated with protection from influenza infection [15]), geometric mean titers (GMT), mean fold increase (MFI) of GMT (ratio of post-immunization to pre-immunization titers), seroconversion rate (subjects with a fourfold or greater increase in titer in pre-vaccination seropositive subjects or from <10 to ≥40 in seronegative volunteers).

As reported in Table 2, the pre-vaccination seroprotection rate and the values of GMT were similar in the IM-MF59 and ID groups and a significant increase in these values was observed 1 month after vaccination in both vaccine groups against the A(H3N2) and A(H1N1) vaccine components. The increases against the B vaccine antigen were not significant, except for GMT values in the ID group. HI titers at 6 months of vaccination decreased, as compared with those found at 1 month in both groups.

No significant differences were observed across vaccine groups against the A(H3N2) and A(H1N1) vaccine antigens 1 and 6 months after vaccination. On the contrary, serocoversion rates against the B antigen were higher, both at 1 (40.0% vs. 10.0%, p < 0.01) and 6 (17.5% vs. 0.0%, p < 0.01) months, in the ID group as compared with the IM-MF59 group.

The serological results observed 1 month post vaccination were also evaluated according to the Committee for Medicinal Products for Human Use (CHMP) criteria for approval of influenza vaccines in the elderly, which require that at least one of the following criteria must be met, protection rate ≥60%, MFI of GMT ≥2 and seroconversion rate ≥30% [16]. Although the pre-requisite of at least 50 persons per group was not met (the two groups examined were of only 40 people) and although there are some controversies on the identification of a single threshold (HI titer ≥40) for defining protection [17], all three CHMP criteria were met by both vaccines against A(H3N2) and A(H1N1) viruses and by ID vaccine against the B virus. IM-MF59 vaccine failed MFI and seroconversion criteria against B virus.

In order to have more comparable data, post-vaccination GMT values were adjusted for pre-vaccination status (Figure 1), as suggested by Beyer et al. [18]. One month after vaccination not only, as previously found, the responses against B virus (3.0 vs. 0.8, p < 0.01), but also against A(H3N2) virus (3.2 vs. 2.4, p < 0.05) were significantly higher in the ID group as compared with the IM-MF59 group. Six months after vaccination statistically higher values were found in the ID group against A(H1N1) (1.3 vs. 0.7, p < 0.01) and B (1.9 vs. 0.0, p < 0.01) viruses.

During the 2011–2012 Winter season in our laboratory, the regional reference laboratory for Umbria of INFLUNET (Italian Surveillance Influenza Network), a total of 61 influenza viruses were identified on examining 91 throat swabs by cultivation in Madin-Darby canine kidney (MDCK) cells and/or Real-Time Reverse-Transcriptase-Polymerase-Chain-Reaction (RT-PCR). Swabs were collected from people with influenza-like illness (ILI) living in the area where the two nursing homes were located. Most of the viruses isolated not only in Umbria (53/61, i.e. 87%), but also in other parts of Italy, were A(H3N2) viruses, presenting antigenic and genetic patterns different from the A(H3N2) component of the 2011–2012 influenza vaccine [19]. The results obtained analyzing the nucleotide sequence of the HA1 domain of the hemagglutinin (HA) gene of a selected number of these A(H3N2) viruses, encompassing four viruses isolated in Umbria, are reported in Figure 2. The data show a high genetic affinity of these four viruses with the recent circulating viruses belonging in the A/Victoria/208/2009 clade, different from the clade of the 2011–2012 A(H3N2) vaccine component (A/Perth/16/2009 clade). Because of the importance of documenting the ability of the influenza vaccine to induce heterologous immune responses, we examined the induction of HI antibody responses against those four drifted A(H3N2) viruses following immunization with the two different enhanced 2011–2012 influenza vaccines. The results obtained, as reported in Table 3, show that, in most instances, pre-vaccination titers were comparable in the two vaccinated groups (except for seroprotection rate in the ID group, higher than in the IM-MF59 group against A/Perugia/44/12, p < 0.05) and in the same range as those found against the vaccine A(H3N2) virus (Table 2). Seroprotection rates ranged between 25.0% and 65.0%, as compared to 50.0% and 50.0% and GMT values ranged, indeed, between 15.4 and 33.7, as compared with 25.5 and 28.3 against A(H3N2) vaccine antigen. One month after vaccination, significant increases were observed both in seroprotection and in GMT values, with the exception of seroprotection rate against the A/Perugia/06/12 virus. On comparing the two vaccinated groups, significantly higher values were found in the ID group as compared with the IM-MF59 group against A/Perugia/20/12 and A/Perugia/44/12 viruses if considering the values of seroprotection (72.5% vs. 47.5%, p < 0.05, 87.5% vs. 67.5% p < 0.05, respectively, Table 3) and against A/Perugia/06/12 on considering the values of GMT corrected for pre-vaccination status (2.5 vs. 1.9, p < 0.01, Table 3). Moreover, the post-vaccination values tended to be lower as compared to those observed against the A(H3N2) vaccine component. On comparing circulating (Table 3) with vaccine A(H3N2) (Table 2) viruses, the GMT values ranged, respectively, from 35.4 to 75.9 and from 83.7 to 131.3, the MFI of GMT from 2.0 to 2.5 and from 3.3 to 4.6, and the percentage of seroconversions from 17.5% to 37.5% and from 47.5% to 60.0%.

As reported in Table 3, of the three CHMP criteria, the seroprotection rate and the MFI of GMT were always met, with the exception of the seroprotection rate against A/Perugia/20/12 antigen (47.5%) after IM-MF59. The seroconversion rate was in most instances lower than 30.0%, except for IM-MF59 group against A/Perugia/44/12 (37.5%) and for ID group against A/Perugia/06/12 (35.0%).

The study included a total of 80 elderly people living in two nursing homes located in Umbria (Italy) immunized with a single dose of trivalent influenza vaccine in November 2011. Two commercialized vaccines were freely offered for the Winter season 2011–2012 by the Public Health Authorities of Umbria (Italy) to the high risk group of elderly people: intramuscular MF59-adjuvanted (Fluad®, Novartis Vaccines, Italy) (IM-MF59) and intradermal (Intanza® 15 mcg, Sanofi-Pasteur MSD, France) (ID) influenza vaccine. The two vaccines contained 15 mcg of A/Perth/16/09 (H3N2), A/California/7/09 (H1N1) and B/Brisbane/60/08, respectively in 0.5 ml (IM-MF59) or 0.1 ml (ID). After obtaining informed consent, subjects were randomly assigned to receive in the deltoid region one dose of intradermal Intanza® 15 mcg (ID) or of intramuscular Fluad® (IM-MF59). Forty volunteers were immunized with IM-MF59 and 40 with ID influenza vaccine. Serum samples were examined for each volunteer before and approximately 1 and 6 months after vaccination. The study was conducted in accordance with all relevant regulations and with ethical standards set out in the Helsinki Declaration and Good Practice Guidelines and since both vaccines were assigned to the two nursing homes for the vaccination of elderly residents within the annual influenza vaccination campaign and sera were leftover sera from samples collected for clinical routine controls, the study did not need to be registered as a formal trial. Demographic, current medical conditions, prescribed medications, and previous influenza vaccination data were obtained from each subject at the time of vaccination.

Serum samples taken from the same subject and frozen at −20°C were tested simultaneously for HI antibodies titers against different influenza antigens. HI titers were determined by a standard microtiter method using 0.5% turkey erythrocytes. All sera were treated with receptor-destroying enzyme and heat-inactivated at 56°C for 30 min to remove non-specific inhibitors [25]. To eliminate any subjective bias, HI titers determinations were determined in a blind fashion, i.e. with the tester unaware of which treatment the donor had received.

The antibody responses were evaluated against the three egg-grown vaccine strains and against four circulating A(H3N2) field viruses (A/Perugia/06/12, A/Perugia/20/12, A/Perugia/44/12 and A/Perugia/50/12) isolated examining throat swabs (by culturing in MDCK cells and by RT-PCR) from people with ILI living in Umbria, Italy. Viruses were genetically characterized by sequencing the complete HA1 domain of the HA gene with specific primers (deposited in Gisaid; A/Perugia/06/12 [EPI:438358], A/Perugia/20/12 [EPI:438360], A/Perugia/44/12 [EPI:392313] and A/Perugia/50/12 [EPI:392315]). PCR products were amplified as previously reported [26] and purified using QIAquick PCR purification kit (Qiagen). Nucleotide sequences were obtained with the Big Dye Terminator Cycle Sequencing v1.1 ready Reaction kit using an ABI PRISM 310 sequencer (Applied Biosystems) and were aligned by using ClustalW 105 program (EMBL-EBI, European Bioinformatics Institute). Phylogenetic analysis was performed by using version 3.1 of the MEGA software package [27]. The Kimura-2-distance method and the Neighbor-Joining algorithm were used for the phylogenetic tree reconstruction.

Differences between groups and results obtained using vaccine and epidemic antigens were analyzed by Student’s t test for continuous statistics (GMT), and by chi-square test for qualitative statistics (protection, seroconversion rate and clinical and demographic characteristics). HI titers were also transformed into binary logarithms, corrected for pre-vaccination status as described by Beyer et al. [18] and expressed as median titers, with the corresponding 25–75° inter-quartile range. Comparisons of corrected post-vaccination titers were analyzed by Wilcoxon test.