ISSN 2500-2236
DOI-prefix: 10.18527/2500-2236

Enhanced CD8+ T-cell response in mice immunized with NS1-truncated influenza virus


Influenza viruses with truncated NS1 protein stimulate more intensive innate immune response compared to their wild type counterparts. Here we investigate whether the shortening of the NS1 protein enhances the immunogenicity of conserved T-cellular epitopes of influenza virus. Using flow cytometry, we showed that intraperitoneal (i.p.) immunization of mice with influenza virus encoding 124 N-terminal amino acid residues of the NS1 protein (A/PR8/NS124) induced increased quantities of CD8+ T-cells recognizing immunodominant (NP366-374) and sub-immunodominant (NP161-175, NP196-210, HA323-337, HA474-483, NA427-433) epitopes compared to the virus expressing full-length NS1 (A/PR8/NSfull). It is important to note that the response to the immunodominant influenza epitope NP366-374 was achieved with the lower immunization dose of A/PR8/NS124 virus comparing to the reference wild type strain. Despite the polyfunctional CD8+ effector memory T-lymphocytes simultaneously producing two (IFNγ and TNFα) or three (IFNγ, IL2 and TNFα) cytokines prevailed in the immune response to both viruses, the relative number of such T-cells was higher in A/PR8/NS124-immunized mice. Furthermore, we found that polyfunctional populations of lymphocytes generated upon immunization with the mutant virus demonstrated increased capacity to produce IFNγ compared to corresponding populations derived from the A/PR8/NSfull-immunized mice. Thus, attenuated influenza viruses encoding truncated NS1 protein ensures generating more potent CD8+ T-cell immune response.


According to the meta-analysis the effectiveness of modern influenza vaccines in people aged 18-65 years is about 60% [1]. Inactivated vaccines primarily induce a humoral immune response and effective protection only when the antigenic structure of surface influenza proteins in vaccine strains coincides with the antigenic structure of surface proteins of circulating viruses [2]. Live attenuated influenza vaccines (LAIV) induce not only humoral but also mucosal and T-cellular immunity. The capacity of LAIV strains to replicate in the upper respiratory tract provides a possibility to trigger the MHC-I/II-dependent presentation of conserved epitopes to CD8+ and CD4+ T-cells. It has been shown in recent studies that circulating influenza viruses of types A, B and C share common CD8+ T-cellular epitopes which could induce the cross-protective immune response [3]. Memory CD8+ T cells directed against these universal epitopes could be found in blood and lungs of healthy humans [3]. Despite the existence of such highly conserved cross-protective influenza epitopes, the T-cellular immune response induced by LAIVs is insufficient to provide a broad cross-protection [4]. It is noteworthy that all licensed LAIVs contain an active NS1 protein which inhibits the host immune response by interacting with components of the IFN signaling system [5, 6]. The immunosuppressive mechanism of LAIVs can prevent the formation of the immune response to weakly immunogenic conserved epitopes of influenza proteins and suppress the ability of vaccine strain to provide cross-protection against a wide range of influenza viruses. The development of approaches to increase the immunogenicity of conserved influenza epitopes can be a promising strategy for creating a new generation of live influenza vaccines with enhanced cross-protection activity. It is known that abrogation of the immunosuppressive activity of NS1 protein by the truncation of its effector domain increases innate cytokine immune response to influenza virus and reduces its reproductive activity in respiratory tract of the host organism [7]. The immunization with live influenza virus with modified NS gene protects mice from challenge with heterologous strains of the same subtype [8, 9].

Previously we showed that the influenza virus encoding NS1 protein shortened to 124 aa induced higher proinflammatory cytokine production and enhanced innate immune cell stimulation compared to influenza A/PR8/34 wild type virus after the intraperitoneal immunization [10]. Here we investigate whether the augmented ability of the influenza virus with truncated NS1 protein to activate the innate immune system could be translated into a more efficient adaptive T-cellular immune response against immunodominant and sub-immunodominant influenza epitopes. Given the known data on the importance of polyfunctional T-lymphocytes in mediating the protective immune response against different bacteria and viruses [11–15], we analyzed the level and functional activity of T-cells producing IFNγ, IL2 and TNFα in different combinations. To equalise the antigenic load of two viruses with different reproduction activity in the respiratory tract of immunized mice we used the intraperitoneal route of immunization, as it was shown that influenza virus does not replicate in the peritoneal cavity but retains the ability to induce humoral and T-cellular immune response [16]. 

Materials and Methods


Two strains based on the influenza A/Puerto Rico/8/1934 (H1N1) virus were used for immunization of mice: (1) the virus, encoding full-length NS1 protein (A/PR8/NSfull), (2) A/PR8/NS124 strain, encoding shortened to 124 a.a. NS1 protein. The strains were obtained by the method of “reverse genetics” [17] accumulated in developing chicken embryos (RCE) and purified by fractionation in a sucrose density gradient.

Laboratory animals

The study was performed on C57BL/6 female mice obtained from the Biomedical Science Center (Stolbovaya, Russia). All of the experiments in this research study were conducted according to the guidelines for care and work with laboratory animals [18].


The A/PR8/NSfull and A/PR8/NS124 viruses were titrated on Vero cells to equalize the immunizing dose. A limiting dilution assay was performed in 96-well culture plates (Nunc, Denmark) by the addition of 100 μl of prepared dilutions of the virus-containing material into the wells, and subsequent incubation for 5 days at 34°C and 5% CO2 in OptiPRO SFM (Invitrogen) with 2% L-glutamine (Invitrogen) and 5 mkg/ml of trypsin (Sigma-Aldrich). The results were evaluated visually by cytopathic effect. Hemagglutination assay (using a 0.5% suspension of chicken red blood cells) was used as the control. The calculation of virus 50% tissue culture infectious dose (TCID50) was performed using the Reed and Munch method [19], and the viral titer was expressed as log10TCID50/ml. To assess the immunogenicity of strains, mice were immunized intraperitoneally with 4 or 7 log10 TCID50/ml of each virus (500 μl of a virus suspension in sodium phosphate buffer (PBS, Biolot). The control group received PBS in an equivalent volume. Each group included 5 mice. In total, 45 experimental animals were used in present work.

Flow cytometry

T-cellular immune response was evaluated in splenocytes 8- and 21-days post-immunization. The spleens were manually homogenized using pestle homogenizers (Eppendorf, Germany). Red blood cells were lysed using the RBC Lysis Buffer reagent (Biolegend, USA). Cells were cultivated in RPMI1640 media (Gibco), containing 10% of fetal bovine serum (FBS, Gibco) and 1% of penicillin/streptomycin solution (Gibco). To stimulate cytokine production splenocytes were incubated with NP366-374, NP161-175, NP196-210, HA474-483, HA323-337 or NA427-433 peptides and brefeldin A (Biolegend) for 6 h at 37º С and 5% CO2. Epitopes were selected using the IEDB database ( Peptides were synthesized by Verta Ltd. (Saint‐Petersburg, Russia). Quantities of 10 mg were dissolved in DPBS (Biolot, Russia) at 10 mg/ml and stored in small aliquots at −20 °C. The purity of the peptides was >90% as it was determined by high‐performance liquid chromatography. After the stimulation, cells were stained with CD8-PE/Cy7, CD4-PerCP-Cy5.5, CD44-BV510, CD62L-APC/Cy7, IFNγ-FITC, TNFα-BV421 and IL2-PE antibodies using the Fixation and Permeabilization Solution reagent kit (BD Biosciences, USA) according to the manufacturer’s instructions. Zombie Red viability marker (BioLegend, USA) was used to identify the dead cells. True Stain reagent, containing antibodies to CD16/CD32, was used to block non-specific antibody binding (BioLegend, USA). Data were collected on a BD FACSCanto II flow cytometer (BD Biosciences, USA). The results were analyzed using the Kaluza Analysis 1.5a program (Beckman Coulter, USA). To estimate the increase in the cytokine production levels upon the peptide stimulation, the background values obtained from the nonstimulated cells were subtracted from the corresponding values of stimulated samples before the statistical analysis. 

Statistical analysis

RStudio Desktop 1.0.153 (RStudio Inc, USA) was used for statistical data analysis. The Dunnet test was used to compare several experimental groups with one control group. A comparison of two experimental groups was carried out using Student’s t-test. Multiple comparisons of several groups were performed using univariate analysis of variance (ANOVA) followed by pairwise comparison of groups using the Tukey criterion. Cellular polyfunctionality index (PI) was calculated using the formula   (where n is the number of analyzed functions (n = 3 for IFNγ, IL-2, and TNFα), F is the percentage of cells performing i functions) as it was described previously [14, 20]. Integrated mean fluorescence intensity (iMFI) was calculated by multiplying the frequency of the particular population by the MFI of IFNγ, IL-2, or TNFα of this population.

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Selection of epitopes for T-cell immune response analysis

The data on the epitopes selected for the study are shown in table 1. The set of peptides used in this work allowed us to evaluate the effect of NS1 protein modification on the immunogenicity of the immunodominant and sub-immunodominant epitopes from the internal (NP) and surface (HA, NA) proteins of influenza virus.






1.8% (63 / 3448)



98.9% (3411 / 3448)



67.28% (2320 / 3448)



41.9% (4174 / 9949)



47.7% (4750 / 9949)



42.2% (3288 / 7776)

Table 1. T-cell epitopes of influenza virus

The conservancy of each epitope was determined by calculating the relative number of unique sequences of the corresponding protein of influenza A virus containing a given epitope. The sequences presented in the Influenza Research Database were used for the conservancy analysis. Data on the immunogenicity of epitopes were obtained from published works that compare the immune response to different epitopes of the influenza virus [21–26].

A/PR8/NS124 mutant virus induces CD8+ response at lower dose of immunization

To determine the optimal immunizing dose C57BL/6 mice were intraperitoneally injected with A/PR8/NSfull and A/PR8/NS124 influenza viruses at a dose of 4 and 7 log10TCID50/mouse. The antigen-specific T-cellular response to immunodominant NP366-374 CD8+ epitope was evaluated in spleens 8 days after immunization. Cells were stimulated in vitro with the peptide for 6 hours and levels of IFNγ, IL-2 and TNFα-producing CD8+CD44+CD62L- effector memory T-cells (CD8+ EM) cells were estimated by flow cytometry.

The immune response to both strains was dose-dependent. After the immunization with 4 log10TCID50/mouse 2.88 ± 2.01% of CD8+ effector T-lymphocytes produced cytokines in response to the in vitro stimulation in the NS124 group, while in the A/PR8/NSfull and control groups the number of antigen specific cells was 0.34 ± 0.10 and 0.26 ± 0.16%, respectively. This result shows that mutant virus was capable of inducing CD8+ T-cell response to NP366-374 epitope at lower dose than influenza strain with the full-length NS1-protein. After immunization with 7 log10TCID50 of each virus the level of antigen-specific CD8+ EM T-lymphocytes was 15.07 ± 2.77% of total CD8+ EM T-cells in the NSfull group and 26.86 ± 1.72% in the NS124 group (p = 0.013, Fig.1). Thus, A/PR8/NS124 virus was also more immunogenic at a dose that induces the substantial immune response to both tested viruses.

The shortening of NS1 protein enhances immunogenicity of sub-immunodominant CD8+ T-cell epitopes

Next, we assessed the immunogenicity of sub-immunodominant T-cell epitopes using the highest immunizing dose of 7 log10 TCID50/mouse. According to Cox M.A. et al. the peak of CD8+ T cell response occurs 8–10 days after the immunization followed by decrease in the number of antigen-specific T-cells [15]. At the same time the spectrum of epitopes inducing adaptive immune response shrinks on the later stages after immunization [27]. Considering these data, we estimated the immune response to influenza epitopes on day 8 and 21 after the intraperitoneal immunization.

Fig. 2 represents levels of cytokine-producing EM (CD44+CD62L-) CD8+ T-cells. Eight days after immunization A/PR8/NS124 virus induced higher CD8+ T cell immune response to NP, HA. NA proteins epitopes compared to A/PR8/NSfull (Fig. 2A). The most pronounced differences between NSfull and NS124 groups in a total amount of cytokine-producing cells were obtained upon stimulation with immunodominant NP366-374 epitope. However, stimulation with HA474-483 NA427-433 and NP196-210 peptides representing sub-immunodominant epitopes also showed significant strengthening of A/PR8/NS124 virus as immunogen: for NA427-433 10.7 ± 0.8% of CD8+ EM T-cells in NS124 group versus 8.4 ± 1.9%  in NSfull group (p = 0.05);  for HA474-483 9.9 ± 0.9% of CD8+ EM T-cells in NS124 group versus 7.2 ± 1.8%  in NSfull group (p = 0.03); and for  NP196-210 11.6 ± 1.7% in  NS124 group versus 8.3 ± 2.0%  in NSfull group (p = 0.05) (Fig. 2A). Neither of the studied epitopes induced the significant CD4+ T-cellular response after the in vitro stimulation (data not shown).

On 21 d.p.i. only two epitopes (NP366-374 and NA427-433) retained the ability to induce cytokine production in CD8+ EM T-cells upon in vitro stimulation in both A/PR8/NSfull and A/PR8/NS124-immunized groups (Fig.2B). The proportion of cytokine-producing T-cells was 9.4 ± 1.5% and 14.0 ± 1.3% for NP366-374 and 7.0 ± 0.8% and 11.1 ± 2.4% for NA427-433 in NSfull and NS124 groups, respectively (p = 0.003, p = 0.01). Thus, the total CD8+ EM T-cellular immune response was higher in the NS124 compared to A/PR8/NSfull groups 21 d.p.i.

The percentage of distinct cytokine-producing populations from the total amount of CD8+ EM T-cells or from the total number of cytokine-producing CD8+ EM T-cells 8 and 21 d.p.i. in NSfull and NS124 groups is shown on Fig.3. Upon the stimulation with NP366-374 peptide the majority of antigen-specific T-cells in both NSfull and NS124 groups were polyfunctional CD8+ T-cells (IFNγ+IL2+TNFα+). Double cytokine-producers (IFNγ+IL2-TNFα+) represented the second dominant population of antigen-specific T-lymphocytes. The rest part (about 25% of total cytokine-producing T-cells in both groups) included the minor populations of single IFNγ-, IL2- or TNFα-producers or double-positive T-cells (IFNγ-IL2+TNFα+ and IFNγ+IL2+TNFα-). As well as NP366-374 epitope the peptides corresponding to the sub-immunodominant influenza epitopes induced a higher level of polyfunctional IFNγ+IL2-TNFα+ and IFNγ+IL2+TNFα+ CD8+ T-lymphocytes in the NS124 group compared to the NSfull group 8 d.p.i. In contrast to NP366-374 specific response all evaluated sub-immunodominant epitopes induced an increased level of IFNγ+IL2-TNFα- single-producers in the NS124 group. At the same time A/PR8/NSfull strain induced a higher proportion of IFNγ-IL2-TNFα+ T-cells. The stimulation with HA474-483 and NP161-175 revealed the significant differences between the experimental groups in the amount of TNFα single-producers (NSfull: 0.8 ± 0.2%, NS124:  0.4 ± 0.3%, p = 0.04 after HA474-483 stimulation; 1.1 ± 0.4%, 0.7 ± 0.3%, p = 0.05 after NP161-175 stimulation).  

The contribution of polyfunctional T-lymphocytes (IFNγ+IL2-TNFα+ and IFNγ+IL2+TNFα+) to the cumulative cytokine response has increased on 21 d.p.i. Only a minor part of the total influenza-specific cells was represented by IFNγ single producers in both NSfull and NS124 groups. The IFNγ+IL2+TNFα- CD8+ EM T-cells were observed only in A/PR8/NS124-immunized animals. Statistically significant difference was found between the NSfull and NS124 groups in the proportion of IFNγ+IL2+TNFα+ T cells after stimulation with NP366-374 peptide (p = 0.0003). The immune response to NA427-433 epitope was characterized by the formation of the higher amount of IFNγ+IL2-TNFα+ and IFNγ+IL2+TNFα+ T-lymphocytes in the NS124 group (p = 0.02, p = 0.01).

Thus, immune response to sub-immunodominant epitopes of A/PR8/NS124 influenza strain was characterized by the higher level of total cytokine-producing T-cells and an increase of the proportion of minor IFNγ+IL2+TNFα- population of CD8+ EM T-lymphocytes compared to A/PR8/NSfull virus 8 and 21 d.p.i. However, the number of epitopes inducing the CD8+ T-cell response simultaneously decreased in both groups at 21 d.p.i.

The A/PR8/NS124 mutant virus promotes appearance of polyfunctionl CD8+ lymphocytes with enhanced IFNγ expression

To estimate the difference in the functional activity of cytokine-producing T-cell populations upon the immunization with A/PR8/NSfull or A/PR8/NS124 viruses we analyzed the expression levels (mean fluorescence intensity, MFI) of IFNγ, IL2 and TNFα in different populations of cytokine-producing T-cells 8 and 21 d.p.i. Fig.4A represents the distribution of CD8+ T-cells from A/PR8/NS124-immunized mice (8 d.p.i.) by their fluorescence intensity  of IFNγ, IL2 and TNFα which corresponds to the expression level. The polyfunctional IFNγ+IL2-TNFα+ and IFNγ+IL2+TNFα+ subpopulations were characterized by the highest cytokine-producing activity among all antigen-specific T-lymphocytes. In particular IFNγ+IL2+TNFα+ triple-producers secreted more IFNγ, IL2 and TNFα than their bifunctional or monofunctional analogs. IFNγ+IL2-TNFα+ T-cells produced higher levels of IFNγ and TNFα than the single-producers expressing these cytokines (Fig.4A). The similar fluorescence intensity (FI) distributions were obtained for A/PR8/NSfull-immunized animals (data not shown). These results underline the dominance of polyfunctional T-cells in the mediation of CD8+ T-cell cytokine response after i.p. immunization regardless of NS1 functionality.

Nevertheless, the comparison of the NSfull and NS124 groups showed that A/PR8/NS124 virus induced higher levels of the IFNγ MFI in IFNγ+IL2-TNFα+ T-cells upon the stimulation with NP366-374 peptide (Fig.4B). Similar, NP161-175, NP196-210, HA323-337 and HA474-483 peptides induced the higher values of IFNγ MFI in IFNγ+IL2+TNFα+ CD8+ EM T-cells in NS124 group compared to NSfull group (p < 0.05). No statistically-significant differences between the experimental groups were found in the MFI expression levels of IL2 and TNFα (data not shown). The observed differences in the expression level of IFNγ in the polyfunctional T-lymphocytes remained during  21 days after the immunization. The IFNγ MFI of IFNγ+IL2-TNFα+ was higher in the NS124 group compared to the NSfull group after the stimulation with NP366-374 (p < 0.001). The NA427-433 peptide induced the enhanced level of the IFNγ MFI in the IFNγ+IL2+TNFα+ T-cells in the A/PR8/NS124-immunized animals (p = 0.02).

It can be concluded that intraperitoneal administration of influenza A/PR8/NS124 virus not only stimulates a greater amount of antigen-specific CD8+ T lymphocytes than the A/PR8/NSfull strain but also ensures formation of polyfunctional T cells with the enhanced IFNγ -producing activity.


CD8 T-cell immune response to conserved antigens is essential for cross-protection against broad variety of influenza viruses [3, 28]. T-lymphocytes recognize linear (8-24 a.k.) epitopes of the internal influenza antigens [29, 30]. Unfortunately, the majority of the conserved influenza epitopes maintains low immunogenicity during the course of natural influenza infection or vaccination. Thus, the development of vaccine approaches promoting cell-mediating immune responses is of great importance.   

Adaptive T-cell immune response is strongly dependent on cytokine milieu and activated antigen-presenting cells generated during the innate immune response. Previously we showed that A/PR8/NS124 influenza virus injected intraperitoneally induces 300 times higher IFNβ production in the peritoneal washes and promotes increased expression of co-stimulatory CD86-molecule participating in the T-cell differentiation process compared to the virus expressing full-length NS1 protein [10]. In this study we estimated the effect of the modification of the NS1 protein on the immunogenicity of immunodominant and sub-immunodominant epitopes of internal and surface proteins of the influenza virus. It was shown that A/PR8/NS124 required the lower dose for triggering CD8+ response comparing to NS1 competent virus.  In addition, the NS1 mutant virus induced the formation of an increased number of antigen-specific effector CD8+ T-lymphocytes compared to the A/PR8/NSfull strain at the highest immunization dose. The shortening of the NS1 protein resulted in simultaneous enhancement of the immunogenicity of all studied epitopes. This was reflected in the increased formation of polyfunctional CD8+ IFNγ+IL2-TNFα+ and IFNγ+IL2+TNFα+ T-lymphocytes and monofunctional IFNγ+IL2-TNFα- T-cells in mice immunized with A/PR8/NS124. Antigen-specific polyfunctional T-cells play a key role in providing protection against reinfection with different microbial and viral pathogens [11, 12]. It is known that polyfunctional T-lymphocytes have a prolonged life cycle compared to monofunctional subpopulations and serve as a source for the formation of long-lived memory cells [33]. Polyfunctional T-lymphocytes also provide a significant contribution to the formation of the immune response to conserved influenza antigens  and could be considered as effectors for cross- protection against antigenically divergent influenza viruses [35]. In the present work we analyzed only the cytokine-producing function of T-lymphocytes, however, from previous researches it is known that the polyfunctional T-cells demonstrate more efficient killing capacities compared to their monofunctional analogues [36–39]. Thus, we can speculate that influenza virus-specific polyfunctional T-cells generated in response to the A/PR8/NS124 immunization may be characterized by higher cytotoxic activity compared to the T-lymphocytes formed after the immunization with A/PR8/NSfull strain. This hypothesis deserves further evaluation.   

The explanation of an increased potency of A/PR8/NS124 virus in the induction of polyfunctional T-cells could be obtained from the known data about the role of TLR7/8 and RIG-I signaling pathways in the dendritic cells activation and the induction of T-lymphocytes maturation. Mice with impaired RIG-I expression are characterized by the formation of the reduced number of polyfunctional T-lymphocytes in response to the immunization and weakened protection against heterologous strains of the influenza virus [40]. Considering that NS1-protein mediates its immunosuppressive function through direct interaction with RIG-I it should be expected that immunization of mice with influenza virus lacking its effector domain of NS1 protein would lead to the enhanced production of the polyfunctional T-lymphocytes. We found that IFNγ+IL2-TNFα+ and IFNγ+IL2+TNFα+ T-cells generated after immunization with A/PR8/NS124 strain produced higher amount of IFNγ compared to the corresponding populations of the A/PR8/NSfull-immunized mice. To compare the cumulative functional properties of influenza-specific T cell immune response to A/PR8/NSfull and A/PR8/NS124 strains after the intraperitoneal immunization we calculated an integrated MFI (iMFI) of IFNγ in predominant polyfunctional populations and cellular polyfunctionality index (PI) for each observation as it was described previously [14, 20]. The results are presented in supplementary table 2 and 3. iMFI and PI reflect both the magnitude and quality of immune response. The higher values of these two metrics in the NS124 group compared to NSfull group after the stimulation with HA474-483, HA323-337, NP366-374 and NP196-210 8 d.p.i. and NP366-374, NA427-433 21 d.p.i.  showed that shortening of NS1 protein leads to the increase in both magnitude of T-cell immune response to influenza virus and functional activity of antigen-specific T-lymphocytes. Since the aim of the present work was to compare the immunogenicity of two viruses with different reproduction activity in the respiratory system, we analyzed only systemic immune response to A/PR8/NSfull and A/PR8/NS124 viruses after the intraperitoneal immunization. However, the growing literature evidence underlines the importance of the local immune response to the influenza virus in the lung tissue for the mediation of heterologous protection. It was shown that the populations of CD69+CD103+ tissue resident memory CD4+ and CD8+ T-cells (Trm) play a critical role in local immune protection through the direct killing of infected cells [41, 42] and cytokine release [43], promoting the recruitment of the immune cells from the circulation and mounting the non-permissive state in the surrounding cells [44]. Trms are indispensable for providing optimal heterosubtypic immunity [45, 46] and vaccination strategies that induce the formation of influenza virus-specific Trm cells in the lungs provide superior protection against heterologous influenza strains [47, 48]. Moreover, as it was shown by Zhao et al., Trm generated in lungs after intranasal vaccine administration were more protective against challenge with pathogenic human coronaviruses than those generated after systemic vaccination [49]. Based on the obtained data we could expect that live attenuated vaccines, based on the viruses with shortened NS1 protein will have higher potential in the induction of cross-reactive Trm compared to the existing LAIVs.

It should be noted that the inhibition of the immunosuppressive function of the NS1 protein did not prevent the loss of immunogenicity of several sub-immunodominant epitopes of influenza virus 21 d.p.i. Given the known data about the importance of the conserved low immunogenic epitopes for the formation of a cross-protective immune response [3], the influenza viruses with the shortened NS1 protein could be considered as a tool for the creation of influenza virus vectors overexpressing important cross-protective epitopes sequences integrated into various genomic fragments. Such vaccine strategy may possibly solve the problem of the loss of immunogenicity of conserved subdominant epitopes at the later stages after immunization. 

Conflict of interest

The authors declare no commercial or financial conflict of interest.


Vasilyev KA, Shurygina A-PS, Stukova MA, Egorov AY. Enhanced CD8+ T-cell response in mice immunized with NS1-truncated influenza virus. MIR J 2020; 7(1), XX-XX. doi: 10.18527/2500-2236-2020-7-1-XX-XX.

© 2020 Vasilyev et al. This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International Public License (CC BY-NC-SA), which permits unrestricted use, distribution, and reproduction in any medium, as long as the material is not used for commercial purposes, provided the original author and source are cited.












11.0 ± 0.1

5.6 ± 1.4

6.1 ± 1.6

5.3 ± 1.4

5.0 ± 1.2

6.3 ± 1.7


19.6 ± 4.1

7.4 ± 1.1

8.4 ± 1.2

7.5 ± 0.6

6.2 ± 0.2

8.0 ± 0.5









iMFI (x1000)


17.9 ± 3.3

6.3 ± 1.5

6.2 ± 2.3

4.5 ± 0.8

5.8 ± 2.4

5.8 ± 1.9


32.5 ± 10.6

6.6 ± 1.6

8.3 ± 1.9

7.3 ± 1.7

9.0 ± 2.9

8.6 ± 2.8









iMFI (x1000)


42.8 ± 3.5

13.9 ± 5.2

17.9 ± 5.9

15.0 ± 5.8

14.7 ± 5.2

20.9 ± 7.7


79.8 ± 13.9

24.5 ± 5.2

27.0 ± 3.6

20.6 ± 1.9

26.6 ± 2.9

26.9 ± 2.9








P-value was determined in Student’s t-test

Table 2. Cellular polyfunctionality index and the integrated IFNγ MFI of influenza-specific CD8+ EM T‑lymphocytes 8d after the intraperitoneal immunization with А/PR8/NSfull and А/PR8/NS124 strains







7.7 ± 1.2

5.4 ± 0.5


11.6 ± 1.1

8.8 ± 1.8





iMFI (x1000)


25.5 ± 15.8

19.6 ± 8.7


40.6 ± 14.0

35.7 ± 4.8





iMFI (x1000)


39.6 ± 10.6

22.4 ± 3.8


66.2 ± 10.5

45.4 ± 16




P-value was determined in Student’s t-test

Table 3. Cellular polyfunctionality index and the integrated IFNγ MFI of influenza-specific CD8+ EM T‑lymphocytes 21d after the intraperitoneal immunization with А/PR8/NSfull and А/PR8/NS124 strains


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Fig. 1. CD8+ T-cellular immune response to different doses of A/PR8/NSfull or A/PR8/NS124 influenza strains. Total levels of cytokine-producing effector memory CD8+CD44+CD62L- T-lymphocytes after 6h of in vitro stimulation of splenocytes of C57BL/6 mice with the NP366-374 peptide. Groups were compared using Student’s t-test (*: p < 0.05, n = 5).
Fig. 2. T-cellular immune response to different epitopes of influenza virus in spleens of C57BL/6 mice 8 and 21 days after intraperitoneal immunization with А/PR8/NSfull and А/PR8/NS124 influenza strains. Box-plots represent total levels of cytokine-producing CD8+ EM T-lymphocytes after 6h of in vitro stimulation of splenocytes with peptides corresponding to sub-immunodominant conservative influenza epitopes. Groups were compared using ANOVA followed by Tukey’s post-hoc comparison. Significant differences between groups (p < 0.05, n = 5) are marked by *.
Fig. 3. Relative content of different cytokine-producing populations of CD8+ EM T-lymphocytes 8 and 21 days after the intraperitoneal immunization with А/PR8/NSfull and А/PR8/NS124 influenza strains. Radar charts represent the differences in the mean values of epitope-specific immune response of each population of cytokine-producing CD8+ EM T-cells of immunized mice. Each point is located in the range from 0 to maximal mean value of corresponding population in NSfull or NS124 group. The pie charts represent the percentage of cells, producing any combination of IFNγ, IL2 or TNFα cytokines in the total cytokine-producing CD8+ EM T-cell subset. (*: p < 0.05, Student’s t-test, n = 5).
Fig. 4. IFNγ expression level in IFNγ+IL2-TNFα+ and IFNγ+IL2+TNFα+ cytokine-producing populations of CD8+ EM T-lymphocytes 8 and 21 days after the intraperitoneal immunization with А/PR8/NSfull and А/PR8/NS124 influenza strains. A. The distribution of fluorescence intensity of cytokine-producing cells from mice immunized with 7.0 log10TCID50 of A/PR8/NS124 influenza strain. The density plots represent the differences in the cytokine expression level between the subsets of cytokine-producing cells. B. IFNγ mean fluorescence intensity (MFI) values of the polyfunctional subpopulations after peptide stimulation were compared using Student’s t-test (*: p < 0.05, n = 5).