Evaluation of Immune Responses to Seasonal Influenza Vaccination in Healthy Volunteers in South Apulia, Italy: A Pilot Study

Background The aim of our pilot study is to investigate different components of the immune response to influenza vaccination in a group of healthy volunteers. We evaluated the cellular immune response (CD4+ T lymphocytes) by flow cytometry. The humoral immune response was assessed by measuring the serum haemagglutination inhibition antibody response. Methods Healthy adult donors (n = 18), were vaccinated with a commercially influenza virus vaccine (FLUARIX® GlaxoSmithKline S.p.a. Verona, Italy), peripheral blood was drawn the same day as influenza virus vaccination and one month later in order to enumerate the antigen-specific CD4+ T lymphocytes. Hemagglutination inhibition assay was performed to enumerate the titer of neutralizing antibodies. Samples of nasal-pharyngeal secretions were taken by swabbing, from ILI (Influenza like Illness) subjects among the studied group, in order to verify influenza infections and eventually identify viruses using Real Time PCR. Results and Conclusions Parenteral influenza vaccination results in significant increase in the CD4+ Th cell population after vaccination. The number of pre-vaccination CD4+ T cells was 0.018 [the results are presented as number of percent fluorescent cells per 10 000 lymphocytes (fixed cells)], while there was a significantly higher number of CD4+ Th cells one month after vaccination (statistical significance was set at the level of α = 0.01). Twenty-two percent of patients demonstrated protective antibody levels to influenza A H1/N1 serotype. None was diagnosed with influenza type A or B. Keywords T cells; Flow cytometry; Influenza virus; Influenza vaccine


Introduction
Infl uenza virus is a common respiratory pathogen that causes substantial morbidity, hospitalization and mortality worldwide every year especially in the case of infants, the elderly and immuno-compromised patients [1,2]. The incidence of the illness depends on the immunity acquired by previous exposure (infection or vaccination) to the circulating strain. Each year, 10% to 20% of the population is infected. In clusters (schools, healthcare facilities, etc.), incidence can reach 40 -50%. At the present, the infection mainly affects the young population ≤ 14 years and adults in the 15 -64 years old age group, while infl uenza-related complications occur most frequently in the elderly [3,6]. Resistance to infl uenza virus infection and disease is mediated by an intricate pattern of innate and acquired immunity with both the local (mucosal) and the systemic arms involving a variety of immuncompetent cells including B cells (humoral immunity), T cells (cellular immunity), antigen presenting cells and matrix cells. Antibodies secreted locally, particularly secretory IgA (S-IgA), in the upper respiratory tract are a major factor in resistance to natural infection [7], whereas serum IgG plays an important role in protection of the lower respiratory tract. Cell-mediated immunity is important in recovery from infl uenza infection and may also prevent infl uenzaassociated complications, but it does not seem to contribute signifi cantly to prevention of infection. Infl uenza infection induces a strong CD4 + T-helper (Th) response, which plays an important role in stimulating antibody production against the virus [8].
Infl uenza causes a broad spectrum of illness in humans, Despite the increasing availability of antivirals, vaccination is still the most cost-effective prevention alternative.
Vaccinaton is the principal way of preventing infl uenza and its complications, but it is less effective in immunocompromised patients, compared with healthy individuals [9,11,12].
The inactivated infl uenza virus vaccine, used since 1945, has been generally well tolerated and has been reported to induce substantial levels of protection, in the range of 70% to 90% when the vaccine and circulating wild-type strains are antigenically similar [13].
At present, the trivalent inactivated infl uenza virus vaccine (TIV), produced by several manufacturers, is licensed worldwide and recommended for many populations, including children 6 months to 5 years, adults over the age of 50, people with a variety of chronic illnesses, and health care workers.
Improvements could be made in the current vaccines effectiveness, duration of response, ease of administration and compliance. Thus, it is important to have a detailed knowledge of the protective immunity and immune processes occurring in the upper respiratory tract in man to allow development of new vaccines and vaccination strategies [14].
The present preliminary study was carried out to investigate different components of the immune response to infl uenza vaccination in a group of healthy volunteers. We evaluated the cellular immune response (CD4 + T lymphocytes) by fl ow cytometry. The humoral immune response was assessed by measuring the serum haemagglutination inhibition antibody response.

Study population
Eighteen healthy volunteers [7 men and 11 women, the median age 36.8 (27-55) years] were recruited by General Practitioners. Ten controls, unvaccinated healthy adults (median age, 35 years; range, 27 -45 years) tested before and 4 weeks after infl uenza vaccination were included in the study.

Analysis of lymphocyte populations by fl ow cytometry
Peripheral blood was drawn in EDTA-containing tubes the same day as infl uenza virus vaccination and one month later. Phenotypic detection of T cells was performed by three-color fl ow cytometry; to stain cell surface molecules, 100 μL of anti-coagulated blood were incubated for 2 h at room temperature (RT) in the dark with R-PE-labeled DRB1*0101(PKYVKQNTLKLAT)-restricted MHC Class II Ultimers TM containing peptide derived from Infl uenza hemaglutinin (DR4/HA 306-318) that allowed the enumeration of antigen-specifi c CD4 + T lymphocytes (Proimmune Ltd., Oxford, UK) and CD4 FICT monoclonal antibody.
The assay was performed according to the manufacturers' specifi cations. First, the erythrocytes were lysed with Lysing buffer 1X (BD Pharm Lyse TM ) and the leukocytes fi xed by incubating for 10 minutes at RT with 300 μL of Fix solution (1% fetal calf serum, 2.5% formaldehyde in PBS).
For the analysis of T cell subpopulation, multiparametric fl ow cytometry was performed by using a FACSCanto fl ow cytometer (Becton Dickinson, California). A total of 10 000 live events were acquired, gated on small viable lymphocytes and analyzed with BDFacsDIVA software (version 4.1.2) (Becton Dickinson, California). The instrument was routinely calibrated according to the manufacturer's instructions.
The results are presented as number of percent fl uorescent cells per 10 000 lymphocytes (fi xed cells).

Serum antibody titers
The hemagglutination inhibition assay was performed on serum samples using a single stock source for each of the HA antigens representing the strains of virus contained in the vaccine [11]. The antibodies titers were determined before and 4 weeks after vaccination. Immunity to infl uenza was defi ned as a HI titer more than or equal to 40.

Controls
Whole blood from unvaccinated patients were treated in the same manner as the samples from vaccinated patients.

Analysis of infl uenza viruses by Real Time PCR
Between December 2007 and March 2008, samples of nasalpharyngeal secretions were taken, by swabbing, from ILI (Infl uenza like Illness) subjects among the studied group, in order to verify infl uenza infections and eventually identify viruses using Real Time PCR.
During 2007-2008 infl uenza season a real-time RT-PCR reaction was performed on the studied samples; both the re-verse transcription and PCR steps were reacted in the same tube; primers and probes for infl uenza viruses A and B were based on genomic regions highly conserved in various subtypes and genotypes of infl uenza virus A matrix protein gene) and infl uenza virus B (haemagglutinin gene segment) (Fast set InfA/InfB-Arrow Diagnostics S.r.l., Genova, Italy) [15].

Statistical analysis
Pre-post-vaccine comparisons were done using ANOVA test. Statistical signifi cance was set at the level of α = 0.01.

Analysis of lymphocyte populations by fl ow cytometry
CD4 + Th cells are pivotal cells in the immune system, secreting cytokines to control and regulate the immune system. Parenteral infl uenza vaccination results in signifi cant in-crease in the antigen-specifi c CD4 + Th cell population after vaccination.
The number of antigen-specifi c CD4 + T cells lymphocytes in the peripheral blood was determined by fl ow cytometry. As shown in Figure 1, the number of pre-vaccination CD4 + T cells was 0.018 (range, 0 -0.03%) (Median = 0.02%, StDev = 0.01). This value is consistent with the hypothesis that most if not all of the donors had been exposed to infl uenza during their lifetime. There was a signifi cantly higher number of antigen-specifi c CD4 + Th cells one month after vaccination; the mean of percentage of number of post-vaccination CD4 + T cells was 1.95 (range, 0 -4.2%) (Median = 1.5%, StDev = 1.26) and ANOVA test presents a P = 0 (P < 0.01); when T0 and T30 data were compared, no signifi cant differences in the peripheral blood count of antigen-specifi c CD4 + T cells lymphocytes in control subjects were detected.

Serology
Serum antibody titers were measured pre-and postvacci-

Analysis of infl uenza viruses by Real Time PCR
Of the 18 healthy donors and of the 10 controls, none was diagnosed with infl uenza type A or B.

Discussion
Infl uenza viruses are unique in their capacity to cause recurrent illnesses, yearly epidemics and more extended pandemics that spread quickly and can have an effect on all population groups. Currently, there are two options to control infl uenza-vaccination and treatment with antiviral drugs. During the past decade there have been new developments in improving the effi cacy of infl uenza vaccination, including the construction of live attenuated, recombinant or nucleicacid vaccines [2]. The effectiveness of protective antibodies specifi c for HA for both treatment and prophylaxis of infl uenza A infection has been shown in animals models [16][17][18], but use of animal-derived antibodies in humans is limited because of severe anaphylactic reactions [19]. Thus, vaccination with inactivated vaccine remains the main strategy for infl uenza prevention but it might fall short in immunocompromised individuals. Therefore, monitoring the state of the immune system is a vital element in our understanding of disease progression and pathology.
CD4 cells are an important component of the anti-viral response to local and systemic infections.
More recent studies have determined that CD4 cells were necessary for long lasting, effective CD8 memory.
CD4 effector cells can also promote survival to a lethal dose of infl uenza infection and may contribute to immunemediated pathology; CD4 effector T cells and memory contribute to immunity to infl uenza via multiple mechanism [20].
Recent studies have recognized intrinsic limitations of the serological methods currently used as a sole measure to evaluate infl uenza virus vaccine effi cacy, and it has been suggested that evaluation of the cellular immune response could provide additional information to enable better estimation of protection against the disease [21,22]. We monitored the precursor frequency of the proliferating CD4 + T cells in response to specifi c antigen stimulations by using the fl ow dye dilution assay in 18 healthy donors, 0-30 days post vaccination with inactivated infl uenza virus vaccine.
This test, compared to traditional methods for assaying antigen-specifi c proliferation, offers the additional ability to evaluate the phenotype of the responding cells and is able to determine their rate of proliferation. The sensitivity of the cytometric assay is likely the result of a combination of factors, including (a) the high sensitivity of fl uorescence detection by modern fl ow cytometers; (b) the highly effi cient capture of produced cytokine within the cytoplasm of the sectretion-inhibited responding cell; (c) the independence of culture conditions and the single cell signal detection strategy, allowing these conditions to be set up with optimization of response as the only concern; and (d) the relatively short stimulation period (4 h) [23].
All of the adults and elderly enrolled in our pilot study demonstrated that vaccination against seasonal infl uenza induce cellular immunity against infl uenza viruses. We do not found signifi cant differences in the number of CD4+ T cells lymphocytes responses elicited after immunization between subjects among the studied group.
Among control subjects, the frequency of proliferating antigen-specifi c CD4 + T cells was essentially the same at T0 and T30; in contrast, among healthy donors, a statistically signifi cant increase in the frequency of proliferating T cells was already detected at day 30 post-vaccination.
Based on this body evidence, we conclude that MHC Class II tetramers can be used to reliably detect CD4 + T cells specifi c for prevalent pathogens in normal donors. Regards serology, in our study with only 22% of patients developing a titer to H1N1 generally accepted as being protective, although no patient developed protective titers to either H3N2 or infl uenza B. The low response could be due to the fact that these are previously unvaccinated, and therefore would need a second dose of vaccine, on the contrary, as evidenced by our data, a single dose of vaccine would be effective anyway to make the priming of CD4 + T lymphocytes in the majority of vaccine subjects.
Cell-mediated immunity may also play a role in competition among infl uenza strains. Althoug T lymphocytes do not confer clinically signifi cant protection against infection in humans, they can mediate cross-reactive and heterotypic protection in responce to conserved viral proteins in mouse models, and reduced viral shedding has been seen in the absence of antibodies against HA and NA. Cytotoxic T lymphocytes that are generated by seasonal infl uenza viruses against conserved epitopes might provide heterotypic immune responses that could dampen transmission, even in the absence of measurable antibody protection [24].
In conclusion, our study shows that cell-mediated immunity is important in recovery from infl uenza infection and may also prevent infl uenza-associated complications. Vaccination remains a priority, improved vaccines are needed, especially for vulnerable patients who are at increased risk of hospitalisation such as infants, the immunosuppressed and the elderly.
However, to establish if cellular immune response is able to provide an adequate level of protection against infl uenza would require further studies involving a larger number of subjects and for a longer time.