**3. Study results**

First, we estimated vaccine effect on distribution pattern of lymphocyte subpopulations in PBMC cultures. Volunteers were divided into three groups according to the baseline antibody (AB) titers against the hemagglutin of the influenza virus A/H1N1, A/H3N2, and B: low AB titers (20–40 U) in the first group, medium AB titers (80–160 U) in the second group, and high AB titers (≥320 U) in the third group. Such differences in AB level indicate that influenza infection in the unvaccinated volunteers could have been masked under the guise of another infection, as all volunteers did not report previous influenza infection.

Immunophenotypic analysis showed changes in the number of T lymphocytes (СD45+/ СD3+), NK cells (CD16+/56+), NKT cells (CD3 + CD16/56+), B lymphocytes (СD45+/CD20+), and activated cells (**Table 1**).

There were statistically significant differences (F = 8.00, p < 0.001, q = 0.001) in T lymphocytes (СD3+) distribution after incubation with different types of vaccines (**Figure 1**). It should be noted that regardless of the AB level vaccines did not have a significant effect on T lymphocyte number except subunit vaccine, which caused a decrease in the percent of T lymphocytes compared to control (PBMC culture without vaccine) while the absolute number did not change. These results may indicate a shift in the number of cells due to an increase in the number of other subpopulations.

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prevention. These vaccines contain hemagglutinin of the influenza virus type A subtypes A/H1N1 и A/H3N2 (15 μg each) and hemagglutinin of the influenza virus type B (15 μg). Grippol plus (LLC "NPO Petrovax Pharm," Russia) – trivalent polymer subunit inactivated influenza vaccine. It contains hemagglutinin of the influenza virus type A subtypes A/H1N1 и A/H3N2 and hemagglutinin of the influenza virus type B (5 μg each), and immunoadjuvant Polyoxidonium (500 μg). All the vaccines contained current influenza virus strains for epide-

Anti-influenza virus A/H1N1/California/07/09, p.149, A/H3N2/Switzerland/9715293/13 (subunit antigen), B/Phuket/3073/13, p. 25 (season 2015–2016); A/H1N1/California/07/09 p.124 till 01.17, A/ H3N2/Hong Kong/4801/14 p.200, and B/Brisbane/60/08 p. 27 (season 2016–2017) **baseline serum antibody levels** were studied in volunteers using the standard method (MU 3.3.2 1758–03) for HAI assay. The 4+ system was applied to HAI assay: an antigen titer, i.e., 1 HAU, was highest antigen dilution giving complete hemagglutination of RBCs (3+ or 4+). In HAI assay the antigen working dose was the antigen dilution containing 4 hemagglutination units (4 HAU) in 0.2 mL.

Cell percentage difference between test groups was measured by a robust dispersion analysis of repeated measures (R Statistical Software, WRS2 package, rmanova function) with subsequent pairwise comparisons (R Statistical Software, WRS2 package, rmmcp function), the obtained significance level was corrected by Holm method [17]. Benjamini-Hoсhberg method was used to account for multiple comparison (false discovery rate control) [18]. The obtained

First, we estimated vaccine effect on distribution pattern of lymphocyte subpopulations in PBMC cultures. Volunteers were divided into three groups according to the baseline antibody (AB) titers against the hemagglutin of the influenza virus A/H1N1, A/H3N2, and B: low AB titers (20–40 U) in the first group, medium AB titers (80–160 U) in the second group, and high AB titers (≥320 U) in the third group. Such differences in AB level indicate that influenza infection in the unvaccinated volunteers could have been masked under the guise of another

Immunophenotypic analysis showed changes in the number of T lymphocytes (СD45+/ СD3+), NK cells (CD16+/56+), NKT cells (CD3 + CD16/56+), B lymphocytes (СD45+/CD20+),

There were statistically significant differences (F = 8.00, p < 0.001, q = 0.001) in T lymphocytes (СD3+) distribution after incubation with different types of vaccines (**Figure 1**). It should be noted that regardless of the AB level vaccines did not have a significant effect on T lymphocyte number except subunit vaccine, which caused a decrease in the percent of T lymphocytes compared to control (PBMC culture without vaccine) while the absolute number did not change. These results may indicate a shift in the number of cells due to an increase in the

miological seasons 2015–2016 and 2016–2017.

data were described with the median and interquartile range.

infection, as all volunteers did not report previous influenza infection.

**2.6. Statistical analysis**

86 Influenza - Therapeutics and Challenges

**3. Study results**

and activated cells (**Table 1**).

number of other subpopulations.

Note. Aliquots of 10 μL vaccines were added to cell suspensions (PBMC, 10<sup>6</sup> cells/mL). Cells were incubated for 72 hours at 37°С in 5% СО<sup>2</sup> . The cells were then washed with RPMI-1640 at 1500 g for 10 min. Monoclonal antibodies against studied cell receptors were added in accordance with the manufacturer's instructions. The number of cells (%) in each sample was determined by flow cytometry.

**Table 1.** Distribution pattern of peripheral blood lymphocyte subpopulations incubated with influenza vaccines.

Analysis revealed significant changes (F = 180.28, p < 0.001, q < 0.001) in percent of natural killer cells (NK, CD16/56+) after incubation with different types of vaccines (**Table 1**, **Figure 1**). Regardless of the AB level there was an increase in number of NK cells from 4.8 (control) to 13.2% (subunit vaccine), 17.2% (adjuvanted vaccine), and 15% (split vaccine). There were statistically significant differences for subunit (13.2 vs. 4.8%, p < 0.001), adjuvanted (17.2 vs. 4.8%, p < 0.001), and split vaccines (15 vs. 4.8%, p < 0.001) compared to control, for subunit vaccine compared to adjuvanted (13.2% vs. 17.2%, p < 0.001) and split vaccines (13.2 vs. 15%, p = 0.003), and for adjuvanted vaccine compared to split vaccine (17.2 vs. 15%, p < 0.001). That means that

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However, the changes in number of NK cells (CD16/56+) after incubation of PBMC with different types of vaccines were observed in all groups of volunteers, regardless of the baseline anti-influenza AB level (F = 48.88, p < 0.001, q < 0.001 – low AB level, F = 103.04, p < 0.001, q < 0.001 – medium AB level, F = 89.09, p < 0.001, q < 0.001 – high AB level) (**Figure 2**).In women with low anti-influenza AB level, percent of NK cells (CD16/56+) was significantly higher after incubation with subunit (11 vs. 5%, p = 0.045), adjuvanted (16.5% vs. 5%, p = 0.001), and split vaccines (14.2 vs. 5%, p = 0.01) compared to control. Immunoadjuvant-containing vaccine had a higher potential for elevation of NK cell number (3.3-fold increase) compared with subunit

In women with medium anti-influenza AB level, percent of NK cells (CD16/56+) was significantly higher after incubation with subunit (12.8 vs. 4.8%, p = 0.001), adjuvanted (17.5 vs. 4.8%, p < 0.001), and split vaccines (15.3 vs. 4.8%, p < 0.001) compared to control. This corresponds to a 2.6- to 3.6-fold increase. Immunoadjuvant-containing vaccine produced more pronounced increase compared to subunit vaccine (17.5 vs. 12.8%, p = 0.029) and split vaccine (17.5 vs. 15.3%, p = 0.011), and number of NK cells was significantly higher after incubation

In women with high anti-influenza AB level, percent of NK cells (CD16/56+) was significantly higher after incubation with subunit (14.8 vs. 4.4%, p = 0.023), adjuvanted (18.2 vs. 4.4%, p = 0.046), and split vaccines (16.1 vs. 4.4%, p = 0.035) compared to control. This corresponds to a 3.3-, 4.1-, and 3.6-fold increase, respectively. There were no statistically significant differ-

For NKT cells (natural killer Т cells, CD3 + CD16/56+), following findings were revealed. Regardless of the AB level there were statistically significant changes (F = 57.52, p < 0.001, q < 0.001) in NKT cells distribution after incubation with different types of vaccines: for subunit (3.6 vs. 1.6%, p = 0.006), adjuvanted (7.5 vs. 1.6%, p < 0.001), and split vaccines (5 vs. 1.6%, p < 0.001) compared to control, for subunit vaccine compared to adjuvanted (3.6 vs. 7.5%, p < 0.001) and split vaccines (3.6 vs. 5%, p = 0.007), and for adjuvanted vaccine compared to split vaccine (7.5 vs. 5%, p = 0.006). Therefore, subunit vaccine caused a 2.2-fold increase in NKT cell number, adjuvanted vaccine caused a 4.6-fold increase, and split vaccine caused a

An increase of NKT cell (CD3 + CD16/56+) number in all cultures was dependent of baseline anti-influenza AB level (F = 22.08, p < 0.001, q < 0.001 – low AB level, F = 20.02, p < 0.001,

q < 0.001 – medium AB level, F = 65.92, p < 0.001, q < 0.001 – high AB level) (**Figure 2**).

with split vaccine compared to subunit vaccine (15.3 vs.12.8%, p = 0.029).

incubation with influenza vaccines increased the number of NK cells in all cultures.

vaccine (2.2-fold increase) (p = 0.017).

ences between various types of vaccines.

3.1-fold increase compared to control (**Table 1**, **Figure 1**).

**Figure 1.** Lymphocyte count in PBMC culture after incubation with influenza vaccines. C = control; Su = inactivated subunit influenza vaccine; A = trivalent inactivated polymer-subunit influenza vaccine; Sp = inactivated split-product influenza vaccine.

The comparison of the T lymphocyte count between vaccines showed a significant decrease in the number of cells after incubation with subunit vaccine only (71.2% vs. 79.8% in control, p = 0.008) (**Figure 1**). However, the changes in the T lymphocyte (СD3+) number after incubation with different types of vaccines were observed only in women with medium AB level (F = 6.40, p = 0.004, q = 0.007). In this group, statistically significant differences were found for subunit vaccine (72 vs. 82.6% in control, p = 0.022) and split-product vaccine (74.8 vs. 82.6% in control, p = 0.022) (**Figure 2**).

**Figure 2.** The impact of influenza vaccines on the lymphocyte count in PBMC cultures from volunteers with different antibody titers against the hemagglutinin of the influenza virus a/H1N1, a/H3N2, and В. Significant differences: \*\*\* p < 0.001, \*\* p < 0.01, \* p < 0.05.

Analysis revealed significant changes (F = 180.28, p < 0.001, q < 0.001) in percent of natural killer cells (NK, CD16/56+) after incubation with different types of vaccines (**Table 1**, **Figure 1**). Regardless of the AB level there was an increase in number of NK cells from 4.8 (control) to 13.2% (subunit vaccine), 17.2% (adjuvanted vaccine), and 15% (split vaccine). There were statistically significant differences for subunit (13.2 vs. 4.8%, p < 0.001), adjuvanted (17.2 vs. 4.8%, p < 0.001), and split vaccines (15 vs. 4.8%, p < 0.001) compared to control, for subunit vaccine compared to adjuvanted (13.2% vs. 17.2%, p < 0.001) and split vaccines (13.2 vs. 15%, p = 0.003), and for adjuvanted vaccine compared to split vaccine (17.2 vs. 15%, p < 0.001). That means that incubation with influenza vaccines increased the number of NK cells in all cultures.

However, the changes in number of NK cells (CD16/56+) after incubation of PBMC with different types of vaccines were observed in all groups of volunteers, regardless of the baseline anti-influenza AB level (F = 48.88, p < 0.001, q < 0.001 – low AB level, F = 103.04, p < 0.001, q < 0.001 – medium AB level, F = 89.09, p < 0.001, q < 0.001 – high AB level) (**Figure 2**).In women with low anti-influenza AB level, percent of NK cells (CD16/56+) was significantly higher after incubation with subunit (11 vs. 5%, p = 0.045), adjuvanted (16.5% vs. 5%, p = 0.001), and split vaccines (14.2 vs. 5%, p = 0.01) compared to control. Immunoadjuvant-containing vaccine had a higher potential for elevation of NK cell number (3.3-fold increase) compared with subunit vaccine (2.2-fold increase) (p = 0.017).

In women with medium anti-influenza AB level, percent of NK cells (CD16/56+) was significantly higher after incubation with subunit (12.8 vs. 4.8%, p = 0.001), adjuvanted (17.5 vs. 4.8%, p < 0.001), and split vaccines (15.3 vs. 4.8%, p < 0.001) compared to control. This corresponds to a 2.6- to 3.6-fold increase. Immunoadjuvant-containing vaccine produced more pronounced increase compared to subunit vaccine (17.5 vs. 12.8%, p = 0.029) and split vaccine (17.5 vs. 15.3%, p = 0.011), and number of NK cells was significantly higher after incubation with split vaccine compared to subunit vaccine (15.3 vs.12.8%, p = 0.029).

The comparison of the T lymphocyte count between vaccines showed a significant decrease in the number of cells after incubation with subunit vaccine only (71.2% vs. 79.8% in control, p = 0.008) (**Figure 1**). However, the changes in the T lymphocyte (СD3+) number after incubation with different types of vaccines were observed only in women with medium AB level (F = 6.40, p = 0.004, q = 0.007). In this group, statistically significant differences were found for subunit vaccine (72 vs. 82.6% in control, p = 0.022) and split-product vaccine (74.8 vs. 82.6% in

**Figure 2.** The impact of influenza vaccines on the lymphocyte count in PBMC cultures from volunteers with different antibody titers against the hemagglutinin of the influenza virus a/H1N1, a/H3N2, and В. Significant differences: \*\*\*

**Figure 1.** Lymphocyte count in PBMC culture after incubation with influenza vaccines. C = control; Su = inactivated subunit influenza vaccine; A = trivalent inactivated polymer-subunit influenza vaccine; Sp = inactivated split-product

control, p = 0.022) (**Figure 2**).

88 Influenza - Therapeutics and Challenges

p < 0.001, \*\* p < 0.01, \* p < 0.05.

influenza vaccine.

In women with high anti-influenza AB level, percent of NK cells (CD16/56+) was significantly higher after incubation with subunit (14.8 vs. 4.4%, p = 0.023), adjuvanted (18.2 vs. 4.4%, p = 0.046), and split vaccines (16.1 vs. 4.4%, p = 0.035) compared to control. This corresponds to a 3.3-, 4.1-, and 3.6-fold increase, respectively. There were no statistically significant differences between various types of vaccines.

For NKT cells (natural killer Т cells, CD3 + CD16/56+), following findings were revealed. Regardless of the AB level there were statistically significant changes (F = 57.52, p < 0.001, q < 0.001) in NKT cells distribution after incubation with different types of vaccines: for subunit (3.6 vs. 1.6%, p = 0.006), adjuvanted (7.5 vs. 1.6%, p < 0.001), and split vaccines (5 vs. 1.6%, p < 0.001) compared to control, for subunit vaccine compared to adjuvanted (3.6 vs. 7.5%, p < 0.001) and split vaccines (3.6 vs. 5%, p = 0.007), and for adjuvanted vaccine compared to split vaccine (7.5 vs. 5%, p = 0.006). Therefore, subunit vaccine caused a 2.2-fold increase in NKT cell number, adjuvanted vaccine caused a 4.6-fold increase, and split vaccine caused a 3.1-fold increase compared to control (**Table 1**, **Figure 1**).

An increase of NKT cell (CD3 + CD16/56+) number in all cultures was dependent of baseline anti-influenza AB level (F = 22.08, p < 0.001, q < 0.001 – low AB level, F = 20.02, p < 0.001, q < 0.001 – medium AB level, F = 65.92, p < 0.001, q < 0.001 – high AB level) (**Figure 2**).

In women with low anti-influenza AB level, NKT cell (CD16/56+) number was significantly higher after *in vitro* incubation with subunit (7 vs. 1.6%, p = 0.033), adjuvanted (8.1 vs. 1.6%, p = 0.007), and split vaccines (5 vs. 1.6%, p = 0.005) compared to control. There were no statistically significant differences between various types of vaccines.

Immunoadjuvant-containing and split vaccines more effectively increased the number of this type of cells. There were statistically significant changes for subunit (1.6 vs. 0.4%, p < 0.001), adjuvanted (1.3 vs. 0.4%, p = 0.050), and split vaccines (1.6 vs. 0.7%, p = 0.002) compared to control, for adjuvanted vaccine compared to subunit vaccine (1.3 vs. 0.7%, respectively, p = 0.046), and for adjuvanted vaccine compared to split vaccine (4.9 vs. 2.6%,

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However, changes in the number of activated cytotoxic T lymphocytes *in vitro* between vaccine types were significant only in women with medium anti-influenza AB level (F = 5.16, p = 0.020, q = 0.035) (**Figure 2**). Incubation with adjuvanted vaccine caused significant increase of the number of activated cytotoxic T lymphocytes compared to control (1.4 vs. 0.4%,

Regardless of the AB level there were significant changes in the number of Т lymphocytes with late activation marker (CD3/HLA-DR+) after incubation with different types of vaccines (F = 8.92, p < 0.001, q = 0.002) (**Table 1**, **Figure 1**). There were statistically significant changes for subunit (2.7 vs. 1%, p < 0.044), adjuvanted (4.9 vs. 1%, p = 0.006), and split vaccines (2.6 vs. 1%, p = 0.010) compared to control, and for adjuvanted vaccine compared to subunit (4.9 vs.

Statistically significant changes in the number of this type of cells were demonstrated only in women with low (F = 30.17, p < 0.001, q < 0.001) and high (F = 12.49, p = 0.001, q = 0.003) anti-influenza AB level (**Figure 2**). In women with low serum AB level, analysis of activated T lymphocytes showed significant activation by adjuvanted vaccine compared to control (3.8 vs. 0.9%, p = 0.047). In women with high serum AB level, the number of activated T lymphocytes was significantly higher after incubation with adjuvanted vaccine compared to split

For lymphocytes with early activation marker (CD45/CD25+), there was statistically significant increase (F = 12.94, p < 0.001, q = 0.001) after incubation of PBMC with different types of vaccines, regardless of the AB level (**Table 1**, **Figure 1**).All types of vaccines increased number of cells with early activation marker. Furthermore, there were statistically significant changes for subunit (3.7 vs. 1.4%, p = 0.007), adjuvanted (4.1 vs. 1.45%, p = 0.049), and split-product vaccines (4.1 vs. 1.4%, p = 0.003) compared to control. There were no statistically significant

Regardless of the AB level there was a significant changes in the number of activated CD45/ CD25+ lymphocytes. It was dependent of the vaccine type in all groups of volunteers (F = 9.96, p = 0.002, q = 0.006 – low AB level, F = 7.92, p = 0.002, q = 0.005 – medium AB level, F = 25.89,

In women with low and medium AB level, percent of T lymphocytes with early activation marker (CD45/CD25+) was significantly increased after incubation of PBMC with subunit vaccine (3.8 and 3.3%, respectively) compared to control (1.1 and 1.2%, respectively) (p = 0.024 and p = 0.036). At the same time, in women with high AB level, the number of these cells was increased after incubation of PBMC with adjuvanted vaccine (11%) compared to control

respectively, p = 0.044).

p = 0.049) and subunit vaccine (1.4 vs. 0.7%, p = 0.047).

2.7%, p = 0.015) and split vaccines (4.9 vs. 2.6%, p = 0.044).

vaccine (8.2 vs. 3.5%, p = 0.027) (**Figure 2**).

differences between various types of vaccines.

p < 0.001, q < 0.001 – high AB level) (**Figure 2**).

(1.5%) (p = 0.009).

In women with medium anti-influenza AB level, NKT cell number in PBMC cultures was significantly higher after incubation with adjuvanted vaccine compared to control (7.4 vs. 1.3%, p < 0.001) and subunit vaccine (7.4 vs. 3%, p < 0.001) (5.7- and 2.48-fold increase, respectively) and after incubation with split vaccine compared to control (4.4 vs. 1.3%, p < 0.001) and subunit vaccine (4.4 vs. 3%, p = 0.009) (3.38- and 1.46-fold increase, respectively).

In women with high anti-influenza AB level, percent of NKT cells (CD3 + CD16/56+) was significantly (4.6-fold) higher after incubation with adjuvanted vaccine compared to control (7.4 vs. 1.6%, p = 0.043).

Analysis also revealed statistically significant differences (F = 167.44, p < 0.001, q < 0.001) in B lymphocytes (CD45/CD20+) distribution after incubation of PBMC with different types of vaccines (regardless of the AB level): for subunit (16.3 vs. 5.1%, 3.1-fold increase, p < 0.001), adjuvanted (21.1 vs. 5.1%, 4.1-fold increase, p < 0.001), and split vaccines (18.1 vs. 5.1%, 3.5 fold increase, p < 0.001) compared to control, and for adjuvanted vaccine compared to subunit (21.1 vs. 16.3%, 1.3-fold increase, p < 0.001) and split vaccines (21.1 vs. 18.1%, 1.1-fold increase, p < 0.001). Therefore, adjuvanted vaccine was the most effective (**Table 1**, **Figure 1**).

Regardless of the AB level there was a significant increase in B lymphocyte number after incubation with different types of vaccines (F = 24.09, p < 0.001, q < 0.001 – low AB level, F = 181.14, p < 0.001, q < 0.001 – medium AB level, F = 150.61, p < 0.001, q < 0.001 – high AB level) (**Figure 2**). In women with low anti-influenza AB level, percent of B lymphocytes (CD20+) was significantly higher after incubation with subunit (15.6 vs. 5%, p = 0.017), adjuvanted (16.3 vs. 5%, p = 0.046), and split vaccines (14.7 vs. 5%, p = 0.014) compared to control. There were no statistically significant differences between various types of vaccines.

In women with medium anti-influenza AB level, percent of B lymphocytes (CD20+) was also significantly higher after incubation with all types of vaccines: subunit (16.2 vs. 5.3%, p < 0.001), adjuvanted (21.6 vs. 5.3%, p < 0.001), and split vaccines (18.1 vs. 5.3%, p < 0.001) compared to control. Immunoadjuvant-containing vaccine had the greatest potential for elevation of B lymphocyte number (21.6%) compared with subunit vaccine (16.2%, 1.3-fold increase) (p < 0.001) and split vaccine (18.1%, 1.2-fold increase) (p = 0.013).

In women with high anti-influenza AB level, there was a significant increase in B lymphocyte number after incubation with subunit (20 vs. 4.7%, p = 0.021), adjuvanted (23.6 vs. 4.7%, p = 0.030), and split vaccines (21.9 vs. 4.7%, p = 0.030) compared to control. Immunoadjuvantcontaining vaccine induced higher (fivefold) increase of B lymphocyte number than split vaccine (4.6-fold, p = 0.011).

Analysis revealed statistically significant differences (F = 13.36, p < 0.001, q < 0.001) in the distribution of activated cytotoxic T lymphocytes (CD8/HLA-DR+) after incubation of PBMC with different types of vaccines (regardless of the AB level) (**Table 1**, **Figure 1**). Immunoadjuvant-containing and split vaccines more effectively increased the number of this type of cells. There were statistically significant changes for subunit (1.6 vs. 0.4%, p < 0.001), adjuvanted (1.3 vs. 0.4%, p = 0.050), and split vaccines (1.6 vs. 0.7%, p = 0.002) compared to control, for adjuvanted vaccine compared to subunit vaccine (1.3 vs. 0.7%, respectively, p = 0.046), and for adjuvanted vaccine compared to split vaccine (4.9 vs. 2.6%, respectively, p = 0.044).

In women with low anti-influenza AB level, NKT cell (CD16/56+) number was significantly higher after *in vitro* incubation with subunit (7 vs. 1.6%, p = 0.033), adjuvanted (8.1 vs. 1.6%, p = 0.007), and split vaccines (5 vs. 1.6%, p = 0.005) compared to control. There were no statisti-

In women with medium anti-influenza AB level, NKT cell number in PBMC cultures was significantly higher after incubation with adjuvanted vaccine compared to control (7.4 vs. 1.3%, p < 0.001) and subunit vaccine (7.4 vs. 3%, p < 0.001) (5.7- and 2.48-fold increase, respectively) and after incubation with split vaccine compared to control (4.4 vs. 1.3%, p < 0.001) and

In women with high anti-influenza AB level, percent of NKT cells (CD3 + CD16/56+) was significantly (4.6-fold) higher after incubation with adjuvanted vaccine compared to control

Analysis also revealed statistically significant differences (F = 167.44, p < 0.001, q < 0.001) in B lymphocytes (CD45/CD20+) distribution after incubation of PBMC with different types of vaccines (regardless of the AB level): for subunit (16.3 vs. 5.1%, 3.1-fold increase, p < 0.001), adjuvanted (21.1 vs. 5.1%, 4.1-fold increase, p < 0.001), and split vaccines (18.1 vs. 5.1%, 3.5 fold increase, p < 0.001) compared to control, and for adjuvanted vaccine compared to subunit (21.1 vs. 16.3%, 1.3-fold increase, p < 0.001) and split vaccines (21.1 vs. 18.1%, 1.1-fold increase,

Regardless of the AB level there was a significant increase in B lymphocyte number after incubation with different types of vaccines (F = 24.09, p < 0.001, q < 0.001 – low AB level, F = 181.14, p < 0.001, q < 0.001 – medium AB level, F = 150.61, p < 0.001, q < 0.001 – high AB level) (**Figure 2**). In women with low anti-influenza AB level, percent of B lymphocytes (CD20+) was significantly higher after incubation with subunit (15.6 vs. 5%, p = 0.017), adjuvanted (16.3 vs. 5%, p = 0.046), and split vaccines (14.7 vs. 5%, p = 0.014) compared to control.

In women with medium anti-influenza AB level, percent of B lymphocytes (CD20+) was also significantly higher after incubation with all types of vaccines: subunit (16.2 vs. 5.3%, p < 0.001), adjuvanted (21.6 vs. 5.3%, p < 0.001), and split vaccines (18.1 vs. 5.3%, p < 0.001) compared to control. Immunoadjuvant-containing vaccine had the greatest potential for elevation of B lymphocyte number (21.6%) compared with subunit vaccine (16.2%, 1.3-fold

In women with high anti-influenza AB level, there was a significant increase in B lymphocyte number after incubation with subunit (20 vs. 4.7%, p = 0.021), adjuvanted (23.6 vs. 4.7%, p = 0.030), and split vaccines (21.9 vs. 4.7%, p = 0.030) compared to control. Immunoadjuvantcontaining vaccine induced higher (fivefold) increase of B lymphocyte number than split vac-

Analysis revealed statistically significant differences (F = 13.36, p < 0.001, q < 0.001) in the distribution of activated cytotoxic T lymphocytes (CD8/HLA-DR+) after incubation of PBMC with different types of vaccines (regardless of the AB level) (**Table 1**, **Figure 1**).

subunit vaccine (4.4 vs. 3%, p = 0.009) (3.38- and 1.46-fold increase, respectively).

p < 0.001). Therefore, adjuvanted vaccine was the most effective (**Table 1**, **Figure 1**).

There were no statistically significant differences between various types of vaccines.

increase) (p < 0.001) and split vaccine (18.1%, 1.2-fold increase) (p = 0.013).

cally significant differences between various types of vaccines.

(7.4 vs. 1.6%, p = 0.043).

90 Influenza - Therapeutics and Challenges

cine (4.6-fold, p = 0.011).

However, changes in the number of activated cytotoxic T lymphocytes *in vitro* between vaccine types were significant only in women with medium anti-influenza AB level (F = 5.16, p = 0.020, q = 0.035) (**Figure 2**). Incubation with adjuvanted vaccine caused significant increase of the number of activated cytotoxic T lymphocytes compared to control (1.4 vs. 0.4%, p = 0.049) and subunit vaccine (1.4 vs. 0.7%, p = 0.047).

Regardless of the AB level there were significant changes in the number of Т lymphocytes with late activation marker (CD3/HLA-DR+) after incubation with different types of vaccines (F = 8.92, p < 0.001, q = 0.002) (**Table 1**, **Figure 1**). There were statistically significant changes for subunit (2.7 vs. 1%, p < 0.044), adjuvanted (4.9 vs. 1%, p = 0.006), and split vaccines (2.6 vs. 1%, p = 0.010) compared to control, and for adjuvanted vaccine compared to subunit (4.9 vs. 2.7%, p = 0.015) and split vaccines (4.9 vs. 2.6%, p = 0.044).

Statistically significant changes in the number of this type of cells were demonstrated only in women with low (F = 30.17, p < 0.001, q < 0.001) and high (F = 12.49, p = 0.001, q = 0.003) anti-influenza AB level (**Figure 2**). In women with low serum AB level, analysis of activated T lymphocytes showed significant activation by adjuvanted vaccine compared to control (3.8 vs. 0.9%, p = 0.047). In women with high serum AB level, the number of activated T lymphocytes was significantly higher after incubation with adjuvanted vaccine compared to split vaccine (8.2 vs. 3.5%, p = 0.027) (**Figure 2**).

For lymphocytes with early activation marker (CD45/CD25+), there was statistically significant increase (F = 12.94, p < 0.001, q = 0.001) after incubation of PBMC with different types of vaccines, regardless of the AB level (**Table 1**, **Figure 1**).All types of vaccines increased number of cells with early activation marker. Furthermore, there were statistically significant changes for subunit (3.7 vs. 1.4%, p = 0.007), adjuvanted (4.1 vs. 1.45%, p = 0.049), and split-product vaccines (4.1 vs. 1.4%, p = 0.003) compared to control. There were no statistically significant differences between various types of vaccines.

Regardless of the AB level there was a significant changes in the number of activated CD45/ CD25+ lymphocytes. It was dependent of the vaccine type in all groups of volunteers (F = 9.96, p = 0.002, q = 0.006 – low AB level, F = 7.92, p = 0.002, q = 0.005 – medium AB level, F = 25.89, p < 0.001, q < 0.001 – high AB level) (**Figure 2**).

In women with low and medium AB level, percent of T lymphocytes with early activation marker (CD45/CD25+) was significantly increased after incubation of PBMC with subunit vaccine (3.8 and 3.3%, respectively) compared to control (1.1 and 1.2%, respectively) (p = 0.024 and p = 0.036). At the same time, in women with high AB level, the number of these cells was increased after incubation of PBMC with adjuvanted vaccine (11%) compared to control (1.5%) (p = 0.009).

Analysis also revealed significant changes (F = 4.27, p = 0.017, q = 0.032) in regulatory T cell (T-regs) number with CD4/CD25/Foxp3+ phenotype after incubation of PBMC with different types of vaccines, regardless of the AB level (**Table 1**, **Figure 1**). Immunoadjuvant-containing vaccine increased T-regs number compared to control (3.7 vs. 2.7%, 1.3-fold increase, p = 0.005). Other types of vaccines did not have a significant effect on these cells.

Significant changes in the number of T-regs between vaccine types were noted only in women with high AB level against influenza viruses A/H1N1, A/H3N2, and В (F = 8.15, p = 0.003, q = 0.006) (**Figure 2**).Incubation of PBMC with adjuvanted vaccine induced significant increase of T-regs count (CD25/CD4/Foxp3+) compared to control (5.5 vs. 2.7%, p = 0.049).

At the next step of the study we evaluated number of TLR-expressing granulocytes in PBMC cultures incubated with influenza vaccines.

All types of vaccines had immunostimulating effect on TLR-expressing cells by increasing the number of granulocytes expressing TLR 2,3,4,6,8, and 9, as shown in **Table 2**.

We found significant differences (F = 270.16, p < 0.001, q < 0.001) in the percent of granulocytes expressing TLR2 (**Table 2**, **Figure 3**) after incubation of PBMC with different types of vaccines, regardless of the AB level against the hemagglutinin of the influenza virus A/H1N1, A/H3N2 and В.. Subunit vaccine increased number of TLR2+ cells in PBMC culture from 16.6 (in control) to 38.2% (p < 0.001), adjuvanted vaccine—to 39.8% (p < 0.001), and split vaccine—to 37.5% (p < 0.001). However, there were no significant differences in TLR2 cell number between vaccine types.

Incubation of cell culture in the presence of influenza vaccines induced an increase in the number of TLR2+ granulocytes regardless of the baseline anti-influenza AB level (F = 53.25, p < 0.001, q < 0.001 – low AB level, F = 169.63, p < 0.001, q < 0.001 – medium AB level, F = 103.89, p < 0.001, q < 0.001 – high AB level) (**Figure 4**). In women with low AB level, the number

of TLR2-expressing granulocytes increased 2.4-fold after incubation with subunit vaccine (ph = 0.019), 2.3-fold after incubation with adjuvanted vaccine (p<sup>h</sup> = 0.019), and 2.2-fold after

**Figure 3.** Number of TLR-expressing granulocytes in PBMC cultures incubated with influenza vaccines. C = control; Su = inactivated subunit influenza vaccine; A = trivalent inactivated polymer-subunit influenza vaccine; Sp = inactivated

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In women with medium AB level, there was similar increase in the number of these cells: 2.3 fold for subunit and split vaccines (ph < 0.001), and 2.4-fold for adjuvanted vaccine (p<sup>h</sup> = 0.001)

In women with high AB level, the number of TLR2-expressing granulocytes increased 2.6-fold after incubation with subunit vaccine (ph = 0.031), 2.8-fold after incubation with adjuvanted vaccine (ph = 0.029), and 2.7-fold after incubation with split vaccine (p<sup>h</sup> = 0.029) compared with

Analysis revealed significant differences (F = 10.62, p < 0.001, q < 0.001) in the percent of granulocytes expressing TLR4 after incubation of PBMC with different types of vaccines, regardless of the AB level against the hemagglutinin of the influenza virus (**Table 2**, **Figure 3**). Subunit vaccine increased number of TLR4+ cells 1.2-fold compared to control (p < 0.001) and

Statistically significant changes in the number of TLR4+ cells (**Figure 4**) between vaccine types were demonstrated only in women with medium AB level (F = 5.24, p = 0.008, q = 0.010): the number of these cells increased 1.1-fold after incubation with subunit vaccine compared to

incubation with split vaccine (ph = 0.003) compared with control.

control (ph = 0.047) and 1.2-fold compared to split vaccine (p = 0.007).

compared with control.

split-product influenza vaccine.

1.1-fold compared to split vaccine (p < 0.001).

control.


**Table 2.** Number of TLR-expressing granulocytes after incubation with influenza vaccines.

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Analysis also revealed significant changes (F = 4.27, p = 0.017, q = 0.032) in regulatory T cell (T-regs) number with CD4/CD25/Foxp3+ phenotype after incubation of PBMC with different types of vaccines, regardless of the AB level (**Table 1**, **Figure 1**). Immunoadjuvant-containing vaccine increased T-regs number compared to control (3.7 vs. 2.7%, 1.3-fold increase,

Significant changes in the number of T-regs between vaccine types were noted only in women with high AB level against influenza viruses A/H1N1, A/H3N2, and В (F = 8.15, p = 0.003, q = 0.006) (**Figure 2**).Incubation of PBMC with adjuvanted vaccine induced significant increase

At the next step of the study we evaluated number of TLR-expressing granulocytes in PBMC

All types of vaccines had immunostimulating effect on TLR-expressing cells by increasing the

We found significant differences (F = 270.16, p < 0.001, q < 0.001) in the percent of granulocytes expressing TLR2 (**Table 2**, **Figure 3**) after incubation of PBMC with different types of vaccines, regardless of the AB level against the hemagglutinin of the influenza virus A/H1N1, A/H3N2 and В.. Subunit vaccine increased number of TLR2+ cells in PBMC culture from 16.6 (in control) to 38.2% (p < 0.001), adjuvanted vaccine—to 39.8% (p < 0.001), and split vaccine—to 37.5% (p < 0.001). However, there were no significant differences in TLR2 cell number

Incubation of cell culture in the presence of influenza vaccines induced an increase in the number of TLR2+ granulocytes regardless of the baseline anti-influenza AB level (F = 53.25, p < 0.001, q < 0.001 – low AB level, F = 169.63, p < 0.001, q < 0.001 – medium AB level, F = 103.89, p < 0.001, q < 0.001 – high AB level) (**Figure 4**). In women with low AB level, the number

21.7 (19.5–23.05) 24.15

25.45 (24–26.32) 26.4

6 23 4.3 (4.05–5.15) 6.5 (5.95–7) 5.7 (5.2–6.9) 6.9 (5.95–7.55) 18.04 <0.001 <0.001

37.5 (35.38–39.27)

23.35 (21.5–25.35)

(21.95–25.95)

(24.48–28.23)

32.7 (30.12–35) 42.5 (37–45.1) 34.4 (29–37) 138.59 <0.001 <0.001

270.16 <0.001 <0.001

10.62 <0.001 <0.001

6.90 <0.001 <0.001

86.57 <0.001 <0.001

**TLR N TLR-expressing granulocytes, %, Me (Q1-Q3) F p q**

39.35 (37.73–42.4)

24.45 (22.15–26.9)

**Table 2.** Number of TLR-expressing granulocytes after incubation with influenza vaccines.

**Control Subunit Adjuvanted Split**

38.2 (36.45–40.05)

26.85 (25.23–29.43)

20 (18.02–24.05)

19.85 (17.95–25.2)

p = 0.005). Other types of vaccines did not have a significant effect on these cells.

of T-regs count (CD25/CD4/Foxp3+) compared to control (5.5 vs. 2.7%, p = 0.049).

number of granulocytes expressing TLR 2,3,4,6,8, and 9, as shown in **Table 2**.

cultures incubated with influenza vaccines.

92 Influenza - Therapeutics and Challenges

between vaccine types.

2 24 16.6

4 24 22.3

3 24 20.2

9 24 11.95

8 24 20.6

(14.2–18.38)

(19.75–25.4)

(18.23–22.95)

(9.825–12.85)

(18.68–22.4)

**Figure 3.** Number of TLR-expressing granulocytes in PBMC cultures incubated with influenza vaccines. C = control; Su = inactivated subunit influenza vaccine; A = trivalent inactivated polymer-subunit influenza vaccine; Sp = inactivated split-product influenza vaccine.

of TLR2-expressing granulocytes increased 2.4-fold after incubation with subunit vaccine (ph = 0.019), 2.3-fold after incubation with adjuvanted vaccine (p<sup>h</sup> = 0.019), and 2.2-fold after incubation with split vaccine (ph = 0.003) compared with control.

In women with medium AB level, there was similar increase in the number of these cells: 2.3 fold for subunit and split vaccines (ph < 0.001), and 2.4-fold for adjuvanted vaccine (p<sup>h</sup> = 0.001) compared with control.

In women with high AB level, the number of TLR2-expressing granulocytes increased 2.6-fold after incubation with subunit vaccine (ph = 0.031), 2.8-fold after incubation with adjuvanted vaccine (ph = 0.029), and 2.7-fold after incubation with split vaccine (p<sup>h</sup> = 0.029) compared with control.

Analysis revealed significant differences (F = 10.62, p < 0.001, q < 0.001) in the percent of granulocytes expressing TLR4 after incubation of PBMC with different types of vaccines, regardless of the AB level against the hemagglutinin of the influenza virus (**Table 2**, **Figure 3**). Subunit vaccine increased number of TLR4+ cells 1.2-fold compared to control (p < 0.001) and 1.1-fold compared to split vaccine (p < 0.001).

Statistically significant changes in the number of TLR4+ cells (**Figure 4**) between vaccine types were demonstrated only in women with medium AB level (F = 5.24, p = 0.008, q = 0.010): the number of these cells increased 1.1-fold after incubation with subunit vaccine compared to control (ph = 0.047) and 1.2-fold compared to split vaccine (p = 0.007).

compared to PBMC culture without stimulation (p<sup>h</sup> = 0.002). However, in women with medium and high AB level, other types of vaccines stimulated TLR9+ granulocytes. There were following differences between vaccine types: subunit vaccine caused 1.6-fold increase (p<sup>h</sup> = 0.017, ph = 0.050), adjuvanted vaccine caused 2- and 1.8-fold increase (p<sup>h</sup> < 0.001, p<sup>h</sup> = 0.040), and split vaccine caused 2.3- and 1.8-fold increase (ph < 0.001, p<sup>h</sup> = 0.050) compared to control,

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Analysis of TLR8-expressing cells showed interesting results (**Table 2**, **Figure 3**). This receptor plays important role in recognition of viral single-stranded RNA.Analysis revealed a significant increase in the number of these cells in PBMC culture dependent on vaccine type (F = 138.59, p < 0.001, q < 0.001). All vaccines induced increase in the number of TLR8-positive granulocytes. This parameter increased 1.6-fold after incubation with subunit vaccine (p < 0.001), twofold after incubation with adjuvanted vaccine (p < 0.001), and 1.7-fold after incubation with split vaccine (p < 0.001) compared to control. Adjuvanted vaccine was 1.3-fold more effective than subunit vaccine (p < 0.001) and 1.2-fold more effective than split vaccine (p < 0.001).

Differences in the number of TLR8+ cells dependent on vaccine type were detected in all groups of volunteers, regardless of the baseline anti-influenza AB level (F = 35.99, p < 0.001, q < 0.001 – low AB level, F = 76.10, p < 0.001, q < 0.001 – medium AB level, F = 116.13, p < 0.001, q < 0.001 – high AB level) (**Figure 4**). In women with low, medium and high serum AB level, subunit vaccine induced 1.7-fold (ph < 0.001), 1.5-fold (p<sup>h</sup> < 0.001), and 1.6-fold (p<sup>h</sup> = 0.002) increase, respectively, adjuvanted vaccine caused 1.9-fold (p<sup>h</sup> < 0.014), twofold (p<sup>h</sup> < 0.001), and 2.1-fold (p<sup>h</sup> = 0.014) increase, respectively, and split vaccine caused 1.8-fold (ph < 0.029), 1.5-fold (p<sup>h</sup> < 0.001), and 1.7-fold (ph = 0.042) increase of TLR8-expressing granulocyte number, respectively, compared to control. In women with medium and high serum AB level, immunoadjuvant-containing vaccine was, respectively, 1.3- and 1.2-fold more effective than split vaccine (p<sup>h</sup> = 0.002 and ph = 0.042), and 1.3-fold more effective than subunit vaccine (p<sup>h</sup> < 0.001 и p<sup>h</sup> = 0.042). In women with medium and high serum AB level, immunoadjuvant-containing vaccine was, respectively, 1.3- and 1.2-fold more effective than split vaccine (p<sup>h</sup> = 0.002 and p<sup>h</sup> = 0.042), and 1.3-fold more

Changes in the distribution of TLR6-expressing granulocytes were similar (**Table 2**, **Figure 3**). Analysis showed significant increase in the number of these cells in PBMC cultures dependent on vaccine type (F = 18.04, p < 0.001, q < 0.001). TLR6-expressing granulocyte number increased 1.5-fold after incubation with subunit vaccine, 1.3-fold after incubation with adjuvanted vaccine, and 1.6-fold after incubation with split vaccine compared to control (p < 0.001). However, there were no statistically significant differences between various types of vaccines. Analysis also showed that effect of different types of vaccines on TLR6-positive cells depended on the baseline AB level (F = 26.38, p < 0.001, q < 0.001 – low AB level; F = 11.71, p < 0.001, q < 0.001 – medium AB level; F = 16.57, p = 0.001, q = 0.001 – high AB level) (**Figure 4**). In women with low and medium serum AB level, subunit vaccine induced 1.6 fold (ph = 0.043) and 1.5-fold (p<sup>h</sup> = 0.004) increase, respectively, adjuvanted vaccine caused 1.2-fold (ph = 0.032) and 1.3-fold (p<sup>h</sup> = 0.004) increase, respectively, and split vaccine caused 1.3-fold (ph = 0.027) and 1.6-fold (p<sup>h</sup> < 0.001) increase of TLR6-expressing granulocyte num-

respectively, in women with medium and high AB level.

effective than subunit vaccine (p<sup>h</sup> < 0.001 и p<sup>h</sup> = 0.042).

ber, respectively, compared to control.

**Figure 4.** The impact of influenza vaccines on TLR-expressing granulocytes in PBMC cultures from volunteers with different AB titers against the hemagglutinin of the influenza virus A/H1N1, A/H3N2 and В.

Analysis of TLR3-expressing granulocytes (**Table 2**, **Figure 3**) revealed significant differences (F = 6.90, p < 0.001, q < 0.001) between groups, meaning that activation of the innate immunity effectors was dependent of the vaccine type, but not baseline AB level. There were significant differences for split vaccines compared to control (1.2-fold increase, p = 0.001) and subunit vaccine (1.2-fold increase, p = 0.008). That means that split vaccine had higher activity.

In women with low and high AB level, there were significant changes in the number of TLR3 expressing cells (**Figure 4**). The significance of differences was (F = 6.05, p = 0.025, q = 0.030) for low AB level and (F = 6.45, p = 0.008, q = 0.010) for high AB level. In women with low and high AB level, percent of TLR3-expressing granulocytes significantly increased after incubation with split vaccine (1.3-fold, ph = 0.042 and p<sup>h</sup> = 0.050, respectively) compared to control.

Analysis also revealed (**Table 2**, **Figure 3**) that different vaccines influenced (F = 86.57, p < 0.001, q < 0.001) the number of TLR9-positive cells regardless of the AB level. All types of vaccines increased the number of TLR9-expressing granulocytes in PBMC culture. Subunit vaccine caused 1.6-fold increase (p < 0.001), adjuvanted vaccine caused 2.1-fold increase (p < 0.001), and split vaccine caused 2.2-fold increase (p < 0.001) compared to control. Subunit vaccine was 1.2-fold less effective than adjuvanted vaccine (p = 0.012) and 1.3-fold less effective than split vaccine (p = 0.003).

Analysis showed that effect of different types of vaccines on TLR9-positive cells depended on the baseline AB level (F = 26.93, p < 0.001, q < 0.001 – low AB level; F = 39.81, p < 0.001, q < 0.001 – medium AB level; F = 29.41, p < 0.001, q < 0.001 – high AB level) (**Figure 4**). In women with low AB level, split vaccine induced threefold increase in the number of TLR9+ granulocytes compared to PBMC culture without stimulation (p<sup>h</sup> = 0.002). However, in women with medium and high AB level, other types of vaccines stimulated TLR9+ granulocytes. There were following differences between vaccine types: subunit vaccine caused 1.6-fold increase (p<sup>h</sup> = 0.017, ph = 0.050), adjuvanted vaccine caused 2- and 1.8-fold increase (p<sup>h</sup> < 0.001, p<sup>h</sup> = 0.040), and split vaccine caused 2.3- and 1.8-fold increase (ph < 0.001, p<sup>h</sup> = 0.050) compared to control, respectively, in women with medium and high AB level.

Analysis of TLR8-expressing cells showed interesting results (**Table 2**, **Figure 3**). This receptor plays important role in recognition of viral single-stranded RNA.Analysis revealed a significant increase in the number of these cells in PBMC culture dependent on vaccine type (F = 138.59, p < 0.001, q < 0.001). All vaccines induced increase in the number of TLR8-positive granulocytes. This parameter increased 1.6-fold after incubation with subunit vaccine (p < 0.001), twofold after incubation with adjuvanted vaccine (p < 0.001), and 1.7-fold after incubation with split vaccine (p < 0.001) compared to control. Adjuvanted vaccine was 1.3-fold more effective than subunit vaccine (p < 0.001) and 1.2-fold more effective than split vaccine (p < 0.001).

Differences in the number of TLR8+ cells dependent on vaccine type were detected in all groups of volunteers, regardless of the baseline anti-influenza AB level (F = 35.99, p < 0.001, q < 0.001 – low AB level, F = 76.10, p < 0.001, q < 0.001 – medium AB level, F = 116.13, p < 0.001, q < 0.001 – high AB level) (**Figure 4**). In women with low, medium and high serum AB level, subunit vaccine induced 1.7-fold (ph < 0.001), 1.5-fold (p<sup>h</sup> < 0.001), and 1.6-fold (p<sup>h</sup> = 0.002) increase, respectively, adjuvanted vaccine caused 1.9-fold (p<sup>h</sup> < 0.014), twofold (p<sup>h</sup> < 0.001), and 2.1-fold (p<sup>h</sup> = 0.014) increase, respectively, and split vaccine caused 1.8-fold (ph < 0.029), 1.5-fold (p<sup>h</sup> < 0.001), and 1.7-fold (ph = 0.042) increase of TLR8-expressing granulocyte number, respectively, compared to control. In women with medium and high serum AB level, immunoadjuvant-containing vaccine was, respectively, 1.3- and 1.2-fold more effective than split vaccine (p<sup>h</sup> = 0.002 and ph = 0.042), and 1.3-fold more effective than subunit vaccine (p<sup>h</sup> < 0.001 и p<sup>h</sup> = 0.042). In women with medium and high serum AB level, immunoadjuvant-containing vaccine was, respectively, 1.3- and 1.2-fold more effective than split vaccine (p<sup>h</sup> = 0.002 and p<sup>h</sup> = 0.042), and 1.3-fold more effective than subunit vaccine (p<sup>h</sup> < 0.001 и p<sup>h</sup> = 0.042).

Analysis of TLR3-expressing granulocytes (**Table 2**, **Figure 3**) revealed significant differences (F = 6.90, p < 0.001, q < 0.001) between groups, meaning that activation of the innate immunity effectors was dependent of the vaccine type, but not baseline AB level. There were significant differences for split vaccines compared to control (1.2-fold increase, p = 0.001) and subunit

**Figure 4.** The impact of influenza vaccines on TLR-expressing granulocytes in PBMC cultures from volunteers with

In women with low and high AB level, there were significant changes in the number of TLR3 expressing cells (**Figure 4**). The significance of differences was (F = 6.05, p = 0.025, q = 0.030) for low AB level and (F = 6.45, p = 0.008, q = 0.010) for high AB level. In women with low and high AB level, percent of TLR3-expressing granulocytes significantly increased after incubation with split vaccine (1.3-fold, ph = 0.042 and p<sup>h</sup> = 0.050, respectively) compared to control. Analysis also revealed (**Table 2**, **Figure 3**) that different vaccines influenced (F = 86.57, p < 0.001, q < 0.001) the number of TLR9-positive cells regardless of the AB level. All types of vaccines increased the number of TLR9-expressing granulocytes in PBMC culture. Subunit vaccine caused 1.6-fold increase (p < 0.001), adjuvanted vaccine caused 2.1-fold increase (p < 0.001), and split vaccine caused 2.2-fold increase (p < 0.001) compared to control. Subunit vaccine was 1.2-fold less effective than adjuvanted vaccine (p = 0.012) and 1.3-fold less effective than split

Analysis showed that effect of different types of vaccines on TLR9-positive cells depended on the baseline AB level (F = 26.93, p < 0.001, q < 0.001 – low AB level; F = 39.81, p < 0.001, q < 0.001 – medium AB level; F = 29.41, p < 0.001, q < 0.001 – high AB level) (**Figure 4**). In women with low AB level, split vaccine induced threefold increase in the number of TLR9+ granulocytes

vaccine (1.2-fold increase, p = 0.008). That means that split vaccine had higher activity.

different AB titers against the hemagglutinin of the influenza virus A/H1N1, A/H3N2 and В.

vaccine (p = 0.003).

94 Influenza - Therapeutics and Challenges

Changes in the distribution of TLR6-expressing granulocytes were similar (**Table 2**, **Figure 3**). Analysis showed significant increase in the number of these cells in PBMC cultures dependent on vaccine type (F = 18.04, p < 0.001, q < 0.001). TLR6-expressing granulocyte number increased 1.5-fold after incubation with subunit vaccine, 1.3-fold after incubation with adjuvanted vaccine, and 1.6-fold after incubation with split vaccine compared to control (p < 0.001). However, there were no statistically significant differences between various types of vaccines.

Analysis also showed that effect of different types of vaccines on TLR6-positive cells depended on the baseline AB level (F = 26.38, p < 0.001, q < 0.001 – low AB level; F = 11.71, p < 0.001, q < 0.001 – medium AB level; F = 16.57, p = 0.001, q = 0.001 – high AB level) (**Figure 4**). In women with low and medium serum AB level, subunit vaccine induced 1.6 fold (ph = 0.043) and 1.5-fold (p<sup>h</sup> = 0.004) increase, respectively, adjuvanted vaccine caused 1.2-fold (ph = 0.032) and 1.3-fold (p<sup>h</sup> = 0.004) increase, respectively, and split vaccine caused 1.3-fold (ph = 0.027) and 1.6-fold (p<sup>h</sup> < 0.001) increase of TLR6-expressing granulocyte number, respectively, compared to control.

In women with high serum AB level, the number of TLR6-expressing granulocytes increased only after incubation with split vaccine (ph = 0.050).

does not adequately reflect the mechanisms of immune response to viruses. Therefore, it is essential to also study the cellular immunity. Immunodominance, which means that the immune system chooses one or more key epitopes for recognition, is an important factor for the development of vaccines stimulating the cellular immune response [28]. Vaccines aimed at producing cytotoxic T lymphocytes specific for an immunodominant epitope can significantly narrow the cross-reactive range of immune response to various virus strains. The role of antigen delivery route and presentation should also be considered when developing such vaccines. To stimulate a strong cytotoxic immune response, an antigen should be processed and presented by dendritic cells and coupled to MHC class I molecules. These may occur either at the moment dendritic cells are being infected or transduced or when dendritic cells engulf apoptotic bodies from other infected cells. Thus, the induction of cytotoxic immune response varies from strong one (with live attenuated vaccines) to a weaker, lower one (with

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B lymphocytes are among the key adaptive immunity effectors in influenza, since they produce anti-hemagglutinin (HA) (mainly against its globular domain) virus-neutralizing antibodies that prevent hemagglutinin from interacting with cellular receptors. Moreover, their Fc portion contributes to virion phagocytosis and to stimulation of antibody-dependent cellular cytotoxicity. HA amino acid sequence homology is about 80% between different strains within one subtype and 40–70% between strains of different subtypes. Besides, anti-neuraminidase antibodies have protective properties. They do not offer virus-neutralizing activity but they can inhibit neuraminidase enzymatic activity, which prevents the virus from spreading. Anti-neuraminidase antibodies also stimulate antibody-dependent cellular cytotoxicity. In addition, anti-neuramini-

Our study showed high stimulating effect of all studied influenza vaccines on B cell counts in PBMC culture. Adjuvanted vaccine was 1.3-fold more effective than subunit vaccine and 1.1-fold more effective than split vaccine. That means that adjuvanted vaccine activated B cell

B cells were found to produce IgA, IgG, and IgM antibody isotypes in primary infection, while no production of IgM antibodies was observed in secondary infection. IgM antibodies are capable of activating the complement cascade as well as of neutralizing the virus [21, 29]. Secretory immunoglobulins A protect respiratory mucosae, through which influenza enters the body, and are indicative of recent virus exposure. Immunoglobulins G ensure the longest

Comparative analysis of the vaccines studied showed that adjuvanted vaccine is more effective in stimulating NK, NKT cells and Tregs, as well. The vaccine was 1.3- and 1.1-fold more effective than subunit and split vaccines in increasing NK cell count, 2.1- and 1.5-fold for NKT cell count, 1.3- and 1.16-fold for B lymphocyte count, and 1.5- and 1.2-fold for Treg count,

Natural thymus-derived regulatory cells (nTreg) of CD4 + CD25+ surface phenotype with constitutive expression of Foxp3 transcription factor responsible for their regulatory activity are one of the best documented cell population. Increased Treg number can possibly be explained by the immunoregulatory effect of PO (adjuvant)-containing vaccine. Immunoregulatory function of

respectively. The studied vaccines were not found to activate other cell types.

dase antibodies have been shown to protect mice from H5N1 influenza virus [29].

proliferation more effectively than the inactivated vaccines studied.

protection against influenza [21, 30].

inactivated whole-virion and subunit vaccines) [21].
