**2. Materials and methods**

**1. Introduction**

84 Influenza - Therapeutics and Challenges

against drift variants of influenza virus [8–11].

ratory infections in the postvaccinal period [13–15].

21–28 days after vaccination)—at least 70% and

differ from that of non-adjuvanted vaccines.

criteria:

cination)—at least 40%;

Vaccination is the most effective means of preventing influenza and consequently reducing incidence and severity of complications. Modern influenza vaccines include a live attenuated, inactivated (whole-virion, split-virion and subunit) vaccines. Currently, inactivated split and subunit vaccines are used for influenza prevention as the safest ones and stimulating the production of a protective level of strain-specific virus-neutralizing antibodies to the globular domain of hemagglutinin protein and neuraminidase protein of contemporary serotypes of the influenza virus. These vaccines protect against infection with the appropriate antigenic variants of influenza virus. Not all inactivated vaccines have been reported to be effective enough for certain categories of vaccinated people [1–7]. Some of them are not able to protect

Due to the continuous antigenic drift of influenza viruses and the emergence of pandemic influenza viruses, the study of influenza vaccines causing broader protective immunity is of great interest [12]. In this regard, before influenza pandemic of 2009–2010 vaccination with adjuvanted vaccines began, aiming to enhance the synthesis of specific antibodies. In addition, given poor population health in the modern era, there is the need to enhance the efficacy of vaccines meant to activate all the components of the immune system. According to the literature data, adjuvanted vaccines seem to have such effect. However, a small number of human studies to investigate, how adjuvanted vaccine influence cellular immunity and activate of not only adaptive, but also innate immunity, have been conducted. In addition, unlike foreign adjuvanted influenza vaccines developed in 2009–2010, the National Immunization Calendar of the Russian Federation for more than 20 years applies polymer-subunit influenza vaccine containing immunomodulator PO as the adjuvant. Furthermore, immunomodulators have long been used in vaccination practice for immunocompromised patients in the Russian Federation. Immunomodulator use to support the vaccination was shown to promptly enhance the synthesis of specific antibodies and significantly decrease the incidence of respi-

To date, the vaccine immunogenicity is assessed according to the requirements of the European Committee for influenza vaccines [16], and must meet at least one of the three

• seroconversion (percentage of subjects with a fourfold increase in antibody titers after vac-

• seroprotection (percentage of subjects with a protective antibody titers before and

Taking into account a new type of vaccine (adjuvanted), not only humoral, but also cellular immune response is important for the evaluation of immunological efficacy. The activation of cellular immunity parameters, important to the formation of immunological memory, may

• multiplicity factor for the increase of antibody titers compared to baseline—at least 2.5.

#### **2.1. Clinical characteristics of patients**

An open-label non-randomized monocenter study enrolled 27 healthy women of childbearing potential (aged 18–40 years) without co-morbidities who were not influenza-vaccinated within the previous 3 years and acquired no influenza or influenza-like illnesses within the previous 6 months.

#### **2.2. Legal basis of the study**

Once the signed informed consent for study participation was obtained, venous blood samples were drawn from volunteers with all applied aseptic and antiseptic techniques met and in accordance with the Study Protocol approved in 2015 by the Ethics Committee at the Mechnikov Research Institute of Vaccines and Sera. The study was conducted at the certified laboratory of the Mechnikov Research Institute of Vaccines and Sera (Moscow) using modern reagents and equipment.

#### **2.3. Distribution pattern of lymphocyte subpopulations**

The distribution pattern of peripheral blood lymphocyte subpopulations *in vitro* in healthy women exposed to influenza vaccine was tested by flow cytometer FC-500 (Beckman Coulter, USA), using anti-CD45/CD3, anti-CD45/CD3/CD4, anti-CD45/CD3/CD8, anti-CD16/56, anti-CD3/ CD16/56, anti-CD45/CD20, anti-CD8/HLA-DR, anti-CD3/HLA-DR, anti-CD45/CD25, and anti-СD4/ CD25/Foxp3 FITC- and PE-labeled monoclonal antibodies mAbs (Beckman Coulter, USA).

#### **2.4. Toll-like receptors**

The concentration of granulocytes with TLR expression was evaluated by flow cytometer FC-500 (Beckman Coulter, USA) using anti-TLR2, anti-TLR 3, anti-TLR4, anti-TLR6, anti-TLR8, and anti-TLR9 mAbs (eBioscience, USA).

Mononuclear WBCs were isolated from the whole blood using Ficoll-Urografin density gradients. We incubated 10<sup>6</sup> cells/mL in RPMI-1640 complete growth medium (PanEco, Russia) with 10% FBS (PanEco, Russia) and antibiotic (streptomycin) in the presence of 10 μL of a corresponding vaccine for 72 hours.

#### **2.5. Study vaccines**

Influvac ("Abbott biologicals" B.V., Netherlands) – inactivated subunit influenza vaccine, Vaxigrip ("Sanofi Pasteur", France)– inactivated split-virion influenza vaccine for influenza 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 epidemiological seasons 2015–2016 and 2016–2017.

**Lymphocyte subpopulations**

T lymphocytes (CD45/СD3+)

Helper T cells (CD45/CD3/СD4+)

Cytotoxic

Natural killer cells, NK cells (CD16/56+)

Natural killer T cells, NKT (CD3/CD16/56+)

B lymphocytes (CD45/CD20+)

Activated cytotoxic T lymphocytes, CTL(CD8/HLA-DR+)

Activated T lymphocytes (CD3/HLA-DR+)

Activated lymphocytes (CD45/CD25+)

Tregs

IRI (CD4/CD8)

Regulatory T cells,

(CD4/CD25/Foxp3+)

at 37°С in 5% СО<sup>2</sup>

sample was determined by flow cytometry.

T lymphocytes, CTL (CD45/CD3/СD8+)

**N % in comparison groups – Me(Q1–Q3) F p q**

The Impact of Adjuvanted and Non-Adjuvanted Influenza Vaccines on the Innate and Adaptive…

**Splitproduct V**

73.91 (66.92– 78.22)

41.9 (35.6–47.7)

21.5 (18.4–5.8)

15

5

18.1 (15.88– 19.62)

1.35 (0.4875– 1.975)

2.6 (1.9–3.575)

4.15 (3.075– 5.275)

4.2 (2.2–4.5)

1.65 (1.475–.525)

. The cells were then washed with RPMI-1640 at 1500 g for 10 min. Monoclonal antibodies against

(13.8–16.25)

(4.625–6.75)

8.00 <0.001 0.001

http://dx.doi.org/10.5772/intechopen.77006

87

2.50 0.071 0.107

0.64 0.533 0.601

180.28 <0.001 <0.001

57.52 <0.001 0.00001

167.44 <0.001 <0.001

13.36 <0.001 <0.001

8.92 <0.001 0.002

12.94 <0.001 0.001

4.27 0.017 0.032

1.26 0.300 0.389

cells/mL). Cells were incubated for 72 hours

**Adjuvanted** 

(66.38–79.17)

**V**

74.6

40.2 (31.8–46.5)

22.5 (16.9–26.9)

17.25 (15.93–18.25)

7.5

21.15 (18.93–22.9)

1.6 (1.2–2.4)

4.95 (3.775–7.1)

4.15 (3.2–9.075)

3.7 (3.2–5.5)

1.85 (1.4–2.5)

studied cell receptors were added in accordance with the manufacturer's instructions. The number of cells (%) in each

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

(6.675–8.225)

**Control Subunit V**

> 71.25 (64.7– 79.75)

> 37.5 (32.7– 43.8)

> 21.2 (17.4– 23.6)

13.2 (11.15– 14.85)

3.6 (2.775– 5.825)

16.36 (15.47– 17.7)

0.7 (0.3–1.2)

2.7 (1.875– 3.375)

3.7 (2.6–4.85)

3.5 (3.2–4.9)

1.85 (1.45– 2.325)

18 79.85

21 43.5

21 23.5

24 4.85

24 1.6

24 5.15

20 0.4

12 1.05

16 1.45

13 2.7

20 1.825

(74.17– 83.35)

(41–49.8)

(17.3–24.7)

(4.175–5.9)

(1.3–2.25)

(4.475– 6.725)

(0.275–0.5)

(0.65–1.65)

(1–1.775)

(1.7–2.9)

(1.5–3.275)

Note. Aliquots of 10 μL vaccines were added to cell suspensions (PBMC, 10<sup>6</sup>

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.

#### **2.6. Statistical analysis**

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 data were described with the median and interquartile range.
