The Effect of Influenza Vaccines on Maturation of Dendritic Cells Generated from Bone Marrow

Research Article

Austin J Vaccines & Immunother. 2021; 5(1): 1012.

The Effect of Influenza Vaccines on Maturation of Dendritic Cells Generated from Bone Marrow

Kostinova AM1*, Yukhacheva DV2, Akhmatova EA3, Akhmatova NK3,4, Kostinov MP3,5, Stolpnikova VN3, Bisheva IA3 and Mitrofanova NN4

1National Research Center-Institute of Immunology Federal Medical-Biological Agency of Russia, Moscow, Russia

2Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology Ministry of health of Russia, Moscow, Russia

3Federal State Budgetary Scientific Institution «I.I. Mechnikov Research Institute of Vaccines and Sera», Moscow, Russia

4Federal State Budgetary Educational Institution of Higher Education «Penza State University» Мedical Institute, Penza, Russia

5Federal State Autonomous Educational Institution of Higher Education I.M. Sechenov First Moscow State Medical University of the Ministry of Health of the Russian Federation (Sechenov University), Moscow, Russia

*Corresponding author: Kostinova Aristitsa Mikhailovna, National Research Center-Institute of Immunology Federal Medical-Biological Agency of Russia, Moscow, Russia

Received: September 27, 2021; Accepted: October 23, 2021; Published: October 30, 2021


Background: Possibility to control immune system by regulating the activity of Dendritic Cells (DC) with the help of vaccines or other immunobiological drugs opens great prospects for infectious, oncological and autoimmune control. The aim of this study was to evaluate in vitro the effect of adjuvant subunit and non-adjuvant split influenza vaccines on maturation of DCs from human bone marrow.

Methods: From bone marrow cells of healthy volunteers, DCs were obtained using rGM-CSF and IL-4. On the 8th day of cultivation, 10μl of vaccines against influenza were introduced into the culture of Immature DCs (i-DCs): a non-adjuvant split vaccine (Vaxigripp, Sanofi Pasteur) and an immunoadjuvant subunit vaccine (Grippol plus, Petrovax), as well as immunomodulator Polyoxidonium.

Results: Insertion of influenza vaccines into i-DC culture induced the acquisition by DCs typical morphological signs of maturation. DCs became large with eccentrically located of irregular shape nucleus, densified cytoplasm, numerous processes. By immunophenotypic examination decrease in monocyte/macrophage pool, cells with expression of CD34 immaturity marker, increase in expressing CD11c/CD86 costimulatory molecules and CD83 terminal differentiation molecules were observed. Although Polyoxidonium caused a decrease in number of CD11c/CD14 cells (18, 5%), but compared to vaccines, its activity was lower (p<0, 05). Grippol plus more actively induced differentiation of TLR2 and TLR8 expressing cells, whereas Vaxigripp-expression of TLR4 and TLR8 on DCs.

Conclusion: The possibility of using in vitro model of DCs obtained from human bone marrow cells by cytokine stimulation for examination of the ability of influenza vaccines to induce DC maturation processes has been demonstrated.

Keywords: Dendritic cells; Maturation; Morphology; Immunophenotype; Influenza vaccines


DC: Dendritic Cells; FCS: Fetal Calf Serum; i-DC: Immature Dendritic Cells; PBS: Phosphate-Buffered Saline; PO: Polyoxidonium; RBC: Red Blood Cell Lysis Buffer; rGM-CSF: Recombinant Granulocyte-Macrophage Colony-Stimulating Factor; rmIL-4: Recombinant Murine Interleukin-4; TLR: Toll-Like Receptor.


Dendritic Cells (DCs) are the most potent specialized antigenpresenting cells in the body. They initiate immune responses due to their ability to activate naive T-cells [1]. DCs also play an important role in antitumor and post-vaccination immunity, as they can stimulate antigen-specific immune response. DCs can be cultured in vitro and then in immuno-therapy in vivo, and they can also be amplified in vivo using immunobiological drugs-inductors of cytokines or other factors such as Flt-3 [2,3].

DC-based antitumor vaccines as an approach to immunotherapy have attracted much attention in biomedical research and are recognized as one of the most promising approaches for targeted antitumor therapy [4-9].

However, studies aimed at the ability to control the immune system by managing the activation of DCs with the help of vaccines or other immunobiological drugs are also important [10-12]. Of no less importance is the interest in studying the mechanisms of action of various vaccines on the immune response effectors, including innate immunity, and the possibility of activating DCs with their subsequent maturation.

DCs are dispensated throughout the body, accounting for less than 1% of peripheral leukocytes in animals and humans. In order to obtain a sufficient amount of DCs for research in this work, mononuclear cells of healthy volunteers were isolated from the bone marrow and their differentiation in DCs was induced in vitro using cytokines. Subsequently, DCs were activated by various influenza vaccines, evaluating the processes of subsequent stages of differentiation, up to the terminal phenotypically manifested increase in expression of the surface CD83 molecule [13].

This technology can help to understand the mechanisms of action of various drugs on the immune system. We investigated morphology and immunophenotype of the DCs. This study can be the basis for further studies of antitumor vaccines and drugs aimed at activating of innate immunity, as well as provide a model for assessing the immunogenicity of vaccines and other immunobiological drugs.

The aim of the study was to evaluate in vitro the effect of adjuvant subunit and non-adjuvant split influenza vaccines on the maturation of dendritic cells generated from human bone marrow.

Materials and Methods

Volunteer group characteristics

In the single-center, open-label, non-randomized study, participated 5 healthy volunteers - 3 men and 2 women (18-40 years old) without concomitant pathology; they have never been vaccinated against influenza and had not have the symptoms of influenza during last 6 months before.

Legal framework of the study

After signing the informed consent for participation in the study, according to the protocol No.3, approved by the ethics committee in Dmitry Rogachev National Medical Research Center of Pediatric Hematology, Oncology and Immunology and I.I. Mechnikov Research Institute for Vaccines and Sera in 2018, bone marrow was taken from volunteers in compliance with the rules of asepsis and antiseptics. The study was conducted in an accredited laboratory of the I.I. Mechnikov Research Institute for Vaccines and Sera (Moscow) using modern reagents and equipment.

Studied influenza vaccines

Vaxigrip (Sanofi Pasteur, France) is a trivalent inactivated split influenza vaccine containing 15μg of hemagglutinin of two type A subtypes (A/H1N1 and A/H3N2) and 15 μg of type B (Victoria lineage) influenza viruses.

Grippol plus (NPO Petrovax Pharm LLC, Russia) is a trivalent inactivated subunit influenza vaccine containing 5μg of hemagglutinin of two type A subtypes (A/H1N1 and A/H3N2) and 5μg of type B (Victoria lineage) influenza viruses and also 500 μg of the immunoadjuvant Polyoxidonium (Azoximer bromide).

All vaccines contained actual influenza virus strains of the season 2019-2020:

- A / Brisbane / 02/2018 (H1N1) pdm09-like virus;

- A / Kansas / 14/2017 (H3N2) -like virus;

- B / Colorado / 06/2017-like virus (lineage B / Victoria / 2/87);

Characteristics of the adjuvant used in the vaccine

According its chemical structure, Polyoxidonium (PO) is the copolymer of N-oxide of 1, 4-ethylene piperazine and (N-carboxymethyl)-1, 4-ethylene piperazinium bromide with molecular mass about 80kDa (INN: Azoximer bromide). This high molecular compound is analogue of natural wide-spread physiologically active N-oxide polymers having strong physiological and pharmacological properties including immune tropic potency.

PO possesses expressed immune modulating effects acting first of all on autarcesis factors such as monocytic-macrophagal system cells, neutrophils and NK-cells and inducing them activation under initially reduced functions. Flow cytochemistry data showed that PO does interact with three lymphocyte subclasses, predominantly binds with monocytes and neutrophils and to a lesser extent with lymphocytes, enhancing intracellular H2O2 production. Hydrogen peroxide being the secondary messenger activates the transcriptional NF-kB factor that is the participant of the cytokines synthesis regulation. The enhancement of the pro-inflammatory cytokines IL-1β, IL-6, TNF-α synthesis takes place. Activation by PO cells of monocyticmacrophagal cluster and natural killers promotes mobilization of both cellular and humoral immunity. Finally all immunity starts up for adequate response development similarly to that as it occurs in natural way [14].

Besides its own clinical application as independent drug, Polyoxidonium is used as immunoadjuvant in new generation vaccines and is a compound in subunit adjuvanted Grippol family vaccines since 1997 when first Grippol® vaccine was registered in Russian market. Due to Polyoxidonium, all Grippol family vaccines contain 3-times lower antigen content in one immunizing dose - 5 mcg per strain, in comparison to 15mcg per strain in other subunit and split influenza vaccines. This provides Grippol family vaccines with higher safety profile [15-27]. These recommendations were made based on relevant clinical trials results followed by many years practical mass vaccine application experience [28-29]. PO is registered in Russian Federation, CIS countries, Slovakia, Cuba

DC cultivation

DCs were obtained from bone marrow cells of 5 healthy volunteers. Bone marrow was homogenized in RPMI-1640 (Sigma, USA), sedimented three times by centrifugation (250g x 5min), then the cell pellet was resuspended using Tris-NH4Cl Red Blood Cell Lysis Buffer (RBC) and the cells were pelleted again and washed with RBS. Then the cells were transferred to an enriched culturing medium (106cells in 1ml of RPMI-1640 medium supplemented with 10μg / ml gentamicin sulfate (AppliChem, Germany) and 10% thermally inactivated fetal calf serum - FCS (Thermo Fisher Scientific, USA) containing 20ng/ml each of the recombinant GM-CSF and IL-4 (Biosource, USA).

After 12 hours the medium was changed to remove loose cells and cell debris. On the 8-th day 10μl/ml of influenza vaccines were added to the Immature-DC (i-DC) culture. Polyoxidonium was added at the rate of 10μg/ml of culture medium. Commercial TNF-α (20ng/ml, Biosource, USA) was used as a classical maturation inducer (positive control).

Morphological characteristics of cells

Morphological changes in cells were observed every day with the use of inverted optical phase contrast microscope. Cells were harvested on the 8-th and 11-th days, washed with PBS and centrifuged at 900rpm during 5minutes. Cells were fixed with 2% glutaraldehyde for 2 hours at 4oC and washed twice with PBS. Then were stained with eosin azure according to Romanovsky-Giemsa. Light and phase contrast microscopy and cell photography were performed using the AxioVision 4 system (Carl Zeiss, Germany).

Determination of DC immunophenotype

The immunophenotype of DC was evaluated using flow cytometry on a Cytomix FC-500 (Beckman Coulter, USA) and Monoclonal

Antibodies (mAbs) (eBiosciences, USA) marked with fluorochrome to a detectable molecules: CD11c-PE (3.9, Cat # 12-0116 -42), CD11c- PerCP (3.9, Cat # 46-0116-42), CD34-FITC (4H11, Cat # 11-0349- 42), CD14-FITC (61D3, Cat # 11-0149-42), CD86 FITC (BU63, Cat # MHCD8601), CD83-FITC (HB-15e, Cat # 11-0839-42), HLA-DRFITC (LN3, Cat # 11-9956-42) (eBioscience, USA).

Cells were harvested after 8 and 11 days, washed with PBS and divided into several fractions of 5×105cells/100μl. Each sample was measured three times. PE and FITC-tagged antibodies were added to the suspension to a final concentration of 5μg/ml and incubated during 30 minutes at 4oC in the dark. Cells were washed twice with PBS and analyzed on a flow cytometer. FITC- tagged IgG isotypes were used as a control. Population of DCs and their subtypes was isolated using negative selection for linear markers CD3, CD14, CD19 and positive selection for markers MHCII, CD11c. Maturity of DC subtypes was evaluated by expression of surface markers on them - CD34, CD83, CD86, MHCII.

Description of surface and endocytotic toll-like receptors (TLRs) of immunocompetent cells

The content of TLRs-expressing DC was studied in vitro by flow cytometry with the use of mAbs: TLR2-FITC (CD282, TL2.1, Cat # 11-9922-42, e-bioscience, USA), TLR3-PE (CD283, TLR3.7, Cat # 12-9039- 82, e-bioscience, USA), TLR4-FITC (CD284, HTA, Cat # MA1-22766, e-bioscience, USA), TLR8-FITC (CD288, Cat # 395508, BioLegend, USA), TLR9-PE (CD289, Cat # HM2087F-100UG, HycultBiotech, USA). Then they were detected on the Cytomix FC-500 flow cytometer (Beckman Coulter, USA) according to the procedure, described in the manufacturer’s instructions.

Statistical analysis

Statistical analysis of data was carried out using the program “Statistica 10”. The significance of differences between the compared values was determined using nonparametric basic statistics using the Mann-Whitney U-test. The differences were considered significant when p ≤ 0.05.


Morphological study

After 3-4 hours bone marrow precursors of DCs adhered to coverslips placed in the wells of the culture plate. When cells were cultured during 24 hours in the presence of Recombinant Murine Granulocyte Macrophage Colony Stimulating Factor (rmGM-CSF) and interleukin-4 (rmIL-4), in addition to adherent cells, suspended cells were detected by phase contrast microscopy. After 72 hours the number and volume of adhered cells was increasing, and the formation of cell colonies was beginning.

On the 7-th day floating cells with dendritic protrusions appeared. On the 8-th day the suspended cells formed conglomerates, and the dendrites lengthened. After 72-hour incubation (11-th day of DC culture) with studied preparations, the colonies scattered, the cells acquired numerous heterogeneous processes and were evenly distributed in the medium.

Analysis of the DC phenotype using flow cytometry

On the 8-th day of incubation a moderate amount of CD11c+ cells (75.8%) was detected in the cell culture from human bone marrow. The proportion of cells with expression of CD34 molecule, which is a marker of DC Immaturity (i-DC), was 31%. The cell culture also contained a pool of monocytes expressing the CD11c/CD14+ marker (36.9%). The content of cells with CD11c/MHC-II antigenic presentation molecules and CD11c/CD86 costimulatory molecules was low (13.36 and 6.2%, respectively). In i-DC culture mature DCs - CD11c/CD83 - were found in trace amounts (5.5%), among CD83/ CD86 double positive cells only 2.32% of them were found (Table 1).

Citation: Kostinova AM, Yukhacheva DV, Akhmatova EA, Akhmatova NK, Kostinov MP, Stolpnikova VN, et al. The Effect of Influenza Vaccines on Maturation of Dendritic Cells Generated from Bone Marrow. Austin J Vaccines & Immunother. 2021; 5(1): 1012.