**4. Discussion**

Considering that inactivated influenza vaccines have a number of drawbacks (lack of efficacy in certain patients [1–7], no protection against drift influenza viruses [8–11]), there is a need for next generation vaccines to be developed. Besides, the effect many influenza vaccines have on the cellular and molecular immunologic mechanisms remains poorly studied.

The effects of inactivated influenza vaccines on key effectors of innate and acquired immunity are being investigated at the Mechnikov Research Institute of Vaccines and Sera (Moscow). Various types of influenza vaccines were selected for the study. First, their effect on distribution pattern of lymphocyte subpopulations was estimated *in vitro*.

Analysis of the vaccine effect on the immunophenotype of lymphocytes cultured for 72 hours, showed activation of the innate and acquired immunity effectors: NK cells (CD16/56), NKT cells (CD3/CD16/56), В lymphocytes (CD45/CD20), cells with early activation marker (CD45/ CD25), Т lymphocytes with late activation marker (CD3/HLA-DR), and regulatory Т cells (Tregs, CD4/CD25/Foxp3). In view of this, below are characteristics of the cells that most actively responded to influenza vaccines added to PBMC culture.

Natural killer cells are essential to the innate immunity in influenza. Their function is to lyze tumor and virus-infected cells and to regulate innate and adaptive immune responses [19, 20]. Natural killer cells have been reported to identify influenza-infected cells through the NKp44 and NKp46 receptors that bind influenza hemagglutinin. Natural killer cells have also been reported to stimulate cellular immune response, regulate eosinophil maturation, and protect respiratory epithelium [21]. When interacting with peripheral mononuclear cells, PO, a component of the adjuvanted vaccine, significantly increases NK cells' cytotoxic effect on target cells. The phenomenon was observed almost in all donors examined, with the increased effect being especially pronounced in patients with the baseline activity of NK cells at the lower normal limit or decreased [22].

Being phenotypically heterogenous, NKT cells duplicate the functions of NK cells and link innate and acquired immunity [23].

Cytotoxic T lymphocytes identify and kill virus-infected cells. Infected cells present virus core antigens coupled to MHC class I molecules, which ensures their identification and subsequent killing by cytotoxic T lymphocytes [24, 25].

Specific cytotoxic lymphocytes cannot prevent cells from being initially infected with the virus, but they can restrict virus reproduction and enhance virus elimination out of the body. In unvaccinated adults, cytotoxic lymphocytes are crucial for clearing the body from influenza. They release perforin and stimulate apoptosis of virus-infected cells [26, 27].

Efficacy of influenza vaccines is currently assessed from their ability to activate the humoral immune response, as recommended in WHO guidelines. We think that this assessment 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 inactivated whole-virion and subunit vaccines) [21].

In women with high serum AB level, the number of TLR6-expressing granulocytes increased

Considering that inactivated influenza vaccines have a number of drawbacks (lack of efficacy in certain patients [1–7], no protection against drift influenza viruses [8–11]), there is a need for next generation vaccines to be developed. Besides, the effect many influenza vaccines have

The effects of inactivated influenza vaccines on key effectors of innate and acquired immunity are being investigated at the Mechnikov Research Institute of Vaccines and Sera (Moscow). Various types of influenza vaccines were selected for the study. First, their effect on distribu-

Analysis of the vaccine effect on the immunophenotype of lymphocytes cultured for 72 hours, showed activation of the innate and acquired immunity effectors: NK cells (CD16/56), NKT cells (CD3/CD16/56), В lymphocytes (CD45/CD20), cells with early activation marker (CD45/ CD25), Т lymphocytes with late activation marker (CD3/HLA-DR), and regulatory Т cells (Tregs, CD4/CD25/Foxp3). In view of this, below are characteristics of the cells that most

Natural killer cells are essential to the innate immunity in influenza. Their function is to lyze tumor and virus-infected cells and to regulate innate and adaptive immune responses [19, 20]. Natural killer cells have been reported to identify influenza-infected cells through the NKp44 and NKp46 receptors that bind influenza hemagglutinin. Natural killer cells have also been reported to stimulate cellular immune response, regulate eosinophil maturation, and protect respiratory epithelium [21]. When interacting with peripheral mononuclear cells, PO, a component of the adjuvanted vaccine, significantly increases NK cells' cytotoxic effect on target cells. The phenomenon was observed almost in all donors examined, with the increased effect being especially pronounced in patients with the baseline activity of NK cells at the lower

Being phenotypically heterogenous, NKT cells duplicate the functions of NK cells and link

Cytotoxic T lymphocytes identify and kill virus-infected cells. Infected cells present virus core antigens coupled to MHC class I molecules, which ensures their identification and sub-

Specific cytotoxic lymphocytes cannot prevent cells from being initially infected with the virus, but they can restrict virus reproduction and enhance virus elimination out of the body. In unvaccinated adults, cytotoxic lymphocytes are crucial for clearing the body from influ-

Efficacy of influenza vaccines is currently assessed from their ability to activate the humoral immune response, as recommended in WHO guidelines. We think that this assessment

enza. They release perforin and stimulate apoptosis of virus-infected cells [26, 27].

on the cellular and molecular immunologic mechanisms remains poorly studied.

tion pattern of lymphocyte subpopulations was estimated *in vitro*.

actively responded to influenza vaccines added to PBMC culture.

only after incubation with split vaccine (ph = 0.050).

**4. Discussion**

96 Influenza - Therapeutics and Challenges

normal limit or decreased [22].

innate and acquired immunity [23].

sequent killing by cytotoxic T lymphocytes [24, 25].

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

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 proliferation more effectively than the inactivated vaccines studied.

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 protection against influenza [21, 30].

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

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 nTreg is implemented both through cytokine secretion, such as TGF-β and IL-10, and through contact interaction with the effector T lymphocytes and antigen-presenting cells [31, 32].

Influenza vaccines have been reported to activate innate effectors—the first line of defense to infection—dendritic cells, both myeloid and lymphoid lineages [48]. TLR3 plays an important part in cross-priming of naive CD8 T cells that differentiate to cytotoxic T cells [49, 50]. They are key to killing virus-infected cells. TLR3 expressed on dendritic cells is also essential for

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*Adjuvanted vaccine* showed high induction potential with respect to TLR9- and TLR8-expressing cells, compared to subunit vaccine (p = 0.012 and p < 0.001, respectively) and split vaccine (p = 0.003 and p < 0.001, respectively). TLR8 has been found to recognize viral single-stranded RNA and to be a specific receptor responsible for influenza virus recognition [45, 52]. The increased TLR8-positive cell count in this study can be attributed to the co-stimulating effect

TLR9 along with TLR2 and TLR4 are involved in the regulation of B lymphocyte activation, proliferation, differentiation, and survival (this is considered an alternative pathway of B lymphocyte activation) [53]. TLR9 is also supposed to be a PRR key to influenza identification and binding, while recognition of influenza virions by TLR7/8 is significant for the induction of

Two different intracellular signaling systems are generally recognized at the moment. One of them involves TLR2, TLR4, TLR5, TLR7, TLR9 and intracellular molecules MyD88, IRAK, TRAF, NFkB. This intracellular signaling system usually activates an early pro-inflammatory response. The other intracellular system involves TLR3, TLR4, (might involve TLR7 and TLR8), adaptor protein TRIF and intracellular proteins TRAM, TBK1, and IRF3. This signaling system ensures the activation of anti-virus response. TLR3 is the key component of this signaling pathway, since it interacts with double-stranded viral RNA. TLR4 is equally effective in the activation of both intracellular signaling systems. Thus, there are two important types of innate immune responses. The first type activates antibacterial protection along with the tissue inflammation. The second type provides type I interferon-mediated antiviral response,

Thus, the studies have shown that influenza vaccines activate cellular immunity effectors as well as induce humoral immune response. PO-containing adjuvanted vaccine showed the strongest capability of inducing the cellular response, among the three vaccines studied.

Influenza vaccines *in vitro* induced an increase in the number of the innate and acquired immunity effectors: NK cells, NKT cells, В lymphocytes, cells with early activation marker, Т

Despite the fact that influenza vaccines must activate endosomal receptors, they cause nonspecific activation of the surface TLRs. This might be due to the influence exerted by antigen complexes contained in influenza vaccines of various types and due to the presence of an adjuvant in one of the vaccines studied. These vaccines activate TLR signaling cascade and, thus, can probably stimulate key effectors of the innate (DC, NK, and NKT cells) and adaptive

protective immune response to main antigens (hemagglutinin) [54].

with interferon being the primary antiviral mediator in innate immunity [55].

lymphocytes with late activation marker, and regulatory Т cells.

NK cell activation via INAM molecule [51].

of the adjuvant in the adjuvanted vaccine.

**5. Conclusion**

Innate immune mechanisms are key to protection against pathogens, since they ensure prompt inflammatory reactions including detection of highly conservative structures, which are common to many microorganisms, through special receptors of broad specificity. These are signal PRRs, and TLRs are the most important of them [33–36].

Having recognized a specific pattern, PRRs initiate a series of signal cascades, which make the first line of defense against microorganisms. Besides, these signals initiate maturation of dendritic cells, which prepare the second line of immune response to the infection, known as acquired immunity. Thus, TLRs contribute to the regulation of innate and acquired immunity. Currently known are 11 types of TLRs in humans and 13 types in mice [37, 38]. Four of them (TLR3, TLR7, TLR8, and TLR9) recognize virus RNA and DNA. TLRs have an established role in physiological regulation of pro-inflammatory cytokine production, which are required for immune response to infections caused by bacteria, fungi, and viruses [39]. Inflammation is known to be directly associated primarily with neutrophils, which express almost all identified TLRs, as it has been shown recently. This explains the importance of TLRs in neutrophil activity regulation: LPS-induced TLR4 activation induces the production of pro-inflammatory cytokines and chemokines (IL-1β, IL8, and TNFα); TLR2, TLR4, and TLR9 stimulation is accompanied by respiratory burst and changed expression of adhesion molecules [40, 41].

The study of the effect influenza vaccine has on TLR-positive cell (granulocyte) expression gave the following results.

Patients with initially different anti-influenza AT titers *in vitro* showed statistically significant differences in TLR3, TLR8, and TLR9-expressing cell counts, depending on the type of influenza vaccine added to leukocyte culture.

All the influenza vaccines studied, caused a statistically significant (p < 0.05) increase in TLR2-, TLR6-, TLR8-, and TLR9-positive granulocyte counts in PBMC culture, compared to non-stimulated cells.

*Subunit vaccine* showed statistically significant (p < 0.001) stimulating effect on the expression of TLR4-positive granulocytes, compared to control group and split vaccine. TLR4 is known to be an important regulator of neutrophil survival [40–42].

*Split vaccine* provided better increase in TLR3- (p = 0.008) and TLR9- (p = 0.001) positive cell counts, compared to subunit vaccine. Both vaccines had similar effect on TLR8+ granulocyte proliferation. TLR3 is an important receptor in recognition of viral double-stranded RNA generated during replication [43]. TLR3 expression by CD4+ и CD8+ lymphocytes is known to be accompanied by their activation, which allows them to get directly involved in various types of immune response [44].

Dendritic cell activation has been reported to occur predominantly with TLR2, TLR3, TLR4, TLR7, and TLR9. TLRs are effective contributors to APC activation, not only because they induce pro-inflammatory cytokine production, but also because they enhance expression of various co-stimulating molecules required for effective antibody recognition [45, 46]. Moreover, TLRs control dendritic cell maturation and antigen-presenting function [47]. Influenza vaccines have been reported to activate innate effectors—the first line of defense to infection—dendritic cells, both myeloid and lymphoid lineages [48]. TLR3 plays an important part in cross-priming of naive CD8 T cells that differentiate to cytotoxic T cells [49, 50]. They are key to killing virus-infected cells. TLR3 expressed on dendritic cells is also essential for NK cell activation via INAM molecule [51].

*Adjuvanted vaccine* showed high induction potential with respect to TLR9- and TLR8-expressing cells, compared to subunit vaccine (p = 0.012 and p < 0.001, respectively) and split vaccine (p = 0.003 and p < 0.001, respectively). TLR8 has been found to recognize viral single-stranded RNA and to be a specific receptor responsible for influenza virus recognition [45, 52]. The increased TLR8-positive cell count in this study can be attributed to the co-stimulating effect of the adjuvant in the adjuvanted vaccine.

TLR9 along with TLR2 and TLR4 are involved in the regulation of B lymphocyte activation, proliferation, differentiation, and survival (this is considered an alternative pathway of B lymphocyte activation) [53]. TLR9 is also supposed to be a PRR key to influenza identification and binding, while recognition of influenza virions by TLR7/8 is significant for the induction of protective immune response to main antigens (hemagglutinin) [54].

Two different intracellular signaling systems are generally recognized at the moment. One of them involves TLR2, TLR4, TLR5, TLR7, TLR9 and intracellular molecules MyD88, IRAK, TRAF, NFkB. This intracellular signaling system usually activates an early pro-inflammatory response. The other intracellular system involves TLR3, TLR4, (might involve TLR7 and TLR8), adaptor protein TRIF and intracellular proteins TRAM, TBK1, and IRF3. This signaling system ensures the activation of anti-virus response. TLR3 is the key component of this signaling pathway, since it interacts with double-stranded viral RNA. TLR4 is equally effective in the activation of both intracellular signaling systems. Thus, there are two important types of innate immune responses. The first type activates antibacterial protection along with the tissue inflammation. The second type provides type I interferon-mediated antiviral response, with interferon being the primary antiviral mediator in innate immunity [55].
