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. 2015 Aug;96(Pt 8):2061–2073. doi: 10.1099/vir.0.000156

Influenza B virus-specific CD8+ T-lymphocytes strongly cross-react with viruses of the opposing influenza B lineage

Carolien E van de Sandt 1, YingYing Dou 1,†, Stella E Vogelzang-van Trierum 1, Kim B Westgeest 1, Mark R Pronk 1, Albert D M E Osterhaus 1,2, Ron A M Fouchier 1, Guus F Rimmelzwaan 1,2,✉, Marine L B Hillaire 1
PMCID: PMC4681061  PMID: 25900135

Abstract

Influenza B viruses fall in two antigenically distinct lineages (B/Victoria/2/1987 and B/Yamagata/16/1988 lineage) that co-circulate with influenza A viruses of the H3N2 and H1N1 subtypes during seasonal epidemics. Infections with influenza B viruses contribute considerably to morbidity and mortality in the human population. Influenza B virus neutralizing antibodies, elicited by natural infections or vaccination, poorly cross-react with viruses of the opposing influenza B lineage. Therefore, there is an increased interest in identifying other correlates of protection which could aid the development of broadly protective vaccines. blast analysis revealed high sequence identity of all viral proteins. With two online epitope prediction algorithms, putative conserved epitopes relevant for study subjects used in the present study were predicted. The cross-reactivity of influenza B virus-specific polyclonal CD8+ cytotoxic T-lymphocyte (CTL) populations obtained from HLA-typed healthy study subjects, with intra-lineage drift variants and viruses of the opposing lineage, was determined by assessing their in vitro IFN-γ response and lytic activity. Here, we show for the first time, to the best of our knowledge, that CTLs directed to viruses of the B/Victoria/2/1987 lineage cross-react with viruses of the B/Yamagata/16/1988 lineage and vice versa.

Introduction

Influenza A viruses of the H1N1 and H3N2 subtypes and influenza B viruses cause annual outbreaks of respiratory tract disease in humans (WHO, 2014b). Seasonal recurrence of these viruses is a result of selection of variants that evade recognition by virus neutralizing antibodies induced by previous infections or vaccination (antigenic drift) (Chen & Holmes, 2008; Koel et al., 2013; Smith et al., 2004). In contrast to influenza A viruses, influenza B viruses are not further subdivided in antigenically distinct subtypes. In addition, the host range of influenza B viruses is more limited (Bodewes et al., 2013) than that of influenza A viruses, which infect a wide range of animal species and for which aquatic birds constitute a major reservoir of most subtypes (Boyce et al., 2009). As animal influenza A viruses of various subtypes have been shown to be able to cross the species barrier and can cause pandemic outbreaks, they continue to pose a threat for public health (Gao et al., 2013; van de Sandt et al., 2012).

Although no subtypes of influenza B viruses have been identified, two antigenically distinct lineages are distinguished based on their haemagglutinin (HA): the B/Victoria/2/1987 and B/Yamagata/16/1988 lineages (Rota et al., 1990). The B/Yamagata/16/1988 lineage was the dominant lineage until the mid-1980s when B/Victoria/2/1987 made a global appearance after its earlier detection in the 1970s in China (Chen et al., 2007). Viruses of both influenza B lineages co-circulated with influenza A/H3N2 and A/H1N1 viruses during various seasonal epidemics (Ambrose & Levin, 2012; CDC, 2009; McCullers et al., 2004). However, the relative contribution of these types and subtypes of influenza virus to the respective epidemics varied over the years. Influenza B viruses can be the major cause of seasonal epidemics or be almost completely absent (Ambrose & Levin, 2012; Heikkinen et al., 2014; Rota et al., 1990; Thompson et al., 2003).

Overall, influenza A/H3N2 virus infections are associated with the highest disease severity; however, infections with influenza B virus contribute considerably to morbidity and mortality in the human population (Ambrose & Levin, 2012; Feng et al., 2012; Simonsen et al., 1997; Thompson et al., 2003, 2004). Although influenza B viruses cause disease in all age groups, the burden of influenza B virus infections is highest amongst children and young adults (Ambrose & Levin, 2012; Heikkinen et al., 2014; Olson et al., 2007). To prevent severe disease and mortality, annual vaccination of individuals at high risk for influenza is recommended (WHO, 2014b). For many years trivalent seasonal influenza vaccines have been used. These vaccines contain components of three strains that match circulating influenza viruses antigenically: influenza A/H3N2 and A/H1N1, and one influenza B strain of either the B/Yamagata/16/1988 or B/Victoria/2/1987 lineage (WHO, 2014a). These vaccines aim at eliciting virus neutralizing strain-specific antibody responses. Unfortunately, antibodies directed against a virus of one lineage of influenza B poorly cross-react with viruses of the opposing influenza B lineage (Belshe et al., 2010; Shaw et al., 2002). The time-consuming process of vaccine production requires recommendation of vaccine strains months in advance of the upcoming influenza season (Russell et al., 2008). As only one strain of influenza B virus is included in most current trivalent seasonal influenza vaccines, vaccine effectiveness is reduced when the epidemic strain is of the opposing lineage (Belshe et al., 2010; Beran et al., 2009; Heinonen et al., 2011). The increased co-circulation of both influenza B lineages in the last decade has led to more frequent mismatches between the vaccine strain and the most dominant circulating influenza B lineage (Ambrose & Levin, 2012; Belshe, 2010; Belshe et al., 2010; CDC, 2014; Heikkinen et al., 2014). To address this problem, quadrivalent influenza vaccines, containing components of both influenza B lineages, have become available in some countries (CDC, 2013; FDA, 2012; WHO, 2014a). However, vaccine effectiveness may still be reduced in the case of unforeseen antigenic drift within either influenza B lineage (Belshe et al., 2010). This spurred an increased interest in identifying other correlates of protection, which could be relevant for future developments of broadly protective vaccines (van de Sandt et al., 2012). Of interest, antibodies cross-reactive with viruses of both the B/Yamagata/16/1988 and B/Victoria/2/1987 lineages have been demonstrated, but they contributed only to a limited extent to the overall antibody repertoire (Dreyfus et al., 2012). Here, we investigated the cross-reactivity of influenza B virus-specific CD8+ cytotoxic T-lymphocytes (CTLs). The main function of CTLs is to detect and eliminate virus-infected cells, thereby restricting viral replication and accelerating viral clearance (Sridhar et al., 2013; van de Sandt et al., 2012). Numerous studies have demonstrated that influenza A virus-specific CTLs contribute to heterosubtypic immunity against antigenically distinct influenza A virus strains. Influenza A virus-specific CTLs are predominantly directed to more conserved internal proteins (Assarsson et al., 2008; Hillaire et al., 2013; Jameson et al., 1998; Kreijtz et al., 2008; Lee et al., 2008; Quinones-Parra et al., 2014; van de Sandt et al., 2014a; Yewdell et al., 1985) and their contribution to cross-protective immunity has been demonstrated in various animal models (Flynn et al., 1998; Hillaire et al., 2011; Kreijtz et al., 2007, 2009; O'Neill et al., 2000; Weinfurter et al., 2011). Although in vivo evidence for the role of CTLs in protective heterosubtypic immunity in humans is limited (Epstein, 2006; McMichael et al., 1983; Slepushkin, 1959; Sridhar et al., 2013), several in vitro studies have demonstrated that human CTLs directed to seasonal influenza A viruses cross-react with possible pandemic influenza A viruses, including avian influenza viruses of the H5N1 and H7N9 subtype and swine origin vH3N2 viruses (Hillaire et al., 2013; Jameson et al., 1999; Kreijtz et al., 2008; Lee et al., 2008; Quinones-Parra et al., 2014; van de Sandt et al., 2014a). Virus-specific CTLs are also induced after influenza B virus infections (Robbins et al., 1989, 1995, 1997), but it is unknown to what extent human CTLs directed to an influenza B virus of one lineage can cross-react with viruses of the opposing lineage. Here, we show for the first time, to the best of our knowledge, that polyclonal CD8+T-cells directed to influenza B viruses of the B/Victoria/2/1987 lineage can cross-react with viruses of the B/Yamagata/16/1988 lineage and vice versa, although the antigen specificity of these cross-reactive CD8+T-cells was not defined. Furthermore, by using the prototypic viruses of both lineages (B/Victoria/2/1987 and B/Yamagata/16/1988) and more recent descendants (B/Netherlands/455/2011 and B/Netherlands/712/2011, respectively) we showed that these CD8+T-cells also recognize intra-lineage drift variants.

Results

Virus characterization

Phylogenetic analysis was performed to confirm the lineage of the influenza B viruses used in the present study. A dataset comprising the HA nucleotide sequences of 51 influenza B viruses isolated between 1987 and 2013 was used to determine the lineage of influenza viruses B/Netherlands/455/2011 and B/Netherlands/712/2011. A maximum-likelihood (ML) phylogenetic tree was reconstructed to study the nucleotide evolution of the HA gene segment of the influenza B/Yamagata/16/1988 and B/Victoria/2/1987 lineage. As expected, both prototypic strains B/Victoria/2/1987 and B/Yamagata/16/1988 were located near the base of the respective lineage. Based on this ML phylogenetic tree, it was concluded that the B/Netherlands/455/2011 virus belonged to the B/Victoria/2/1987 lineage, whilst B/Netherlands/712/2011 belonged to the B/Yamagata/16/1988 lineage (Fig. 1).

Fig. 1. Phylogenetic ML tree of the HA gene segment of human influenza B viruses. The phylogenetic ML tree was inferred from 51 HA nucleotide sequences of both influenza B virus lineages. Bootstrap values of 1000 replicates of ML trees are shown as percentages (values < 70 % are omitted). Bar roughly represents 1 % of nucleotide substitutions between close relatives. Bold type underlined sequences were used in the present study.

Fig. 1.

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High amino acid sequence identity between both influenza B lineages

The percentage amino acid sequence identity of influenza A viral proteins has proven to be a good predictor of cross-reactivity of virus-specific T-cells with influenza A virus of various subtypes (van de Sandt et al., 2014a). Therefore, we wished to compare the overall amino acid sequence identity between the influenza B viruses used in the present study. blast analysis revealed that the sequence identity of all viral proteins was remarkably high (86–100 %) (Table 1).

Table 1. Percentage amino acid sequence identity between internal proteins of the influenza B viruses used in this study.

Virus Gene segment Identity (%)
B/Victoria/02/1987 B/Netherlands/712/2011 B/Netherlands/455/2011
B/Yamagata/16/1988 PB2 99 99 99
PB1 99 99 99
PA 98 99 98
HA 95 96 95
NP 98 99 99
NA 97 95 94
NB 90 91 90
M1 100 99 100
BM2 93 98 96
NS1 99 94 93
NS2 100 98 98
B/Victoria/02/1987 PB2 99 99
PB1 99 99
PA 98 98
HA 94 97
NP 98 98
NA 93 93
NB 87 86
M1 99 100
BM2 91 89
NS1 94 93
NS2 98 98
B/Netherlands/712/2011 PB2 99
PB1 99
PA 99
HA 93
NP 99
NA 94
NB 93
M1 99
BM2 96
NS1 97
NS2 98
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Prediction of conserved CD8+ T-cell epitopes

CD8+T-cell epitope prediction tools Syfpeithi and Immuneepitope were used to predict the presence of putative conserved CD8+T-cell epitopes in influenza B viral proteins. As the amino acid sequence identity between the respective strains was very high, this analysis was performed with the prototypic strains B/Victoria/2/1987 and B/Yamagata/16/1988 only. Epitope predictions were performed for the HLA alleles that corresponded with those of the study subjects (Table 2). Syfpeithi and Immuneepitope predicted a large number of putative epitopes in viral internal proteins of both B/Victoria/2/1987 and B/Yamagata/16/1988 (Fig. S1, available in the online Supplementary Material). Due to the large difference in the number of epitopes predicted by both programs, we considered an epitope a putative epitope when predicted by both programs. In addition, to be a putative conserved epitope, the epitope needed to be present in both viruses (Fig. 2). As shown in Fig. 2, most of the epitopes predicted by both programs were present in both virus strains. Only a small proportion of the predicted epitopes was unique for one of the two viruses. The data suggested that the majority of CTL epitopes were conserved between the two lineages of influenza B.

Table 2. HLA-A and HLA-B haplotypes of the study subjects.

Group Donor HLA-A and HLA-B haplotypes
I 8904 HLA-A*0101, A*0201, B*0801, B*3501
6888
1578
II 7482 HLA-A*0101, A*0201, B*0801, B*2705
2501
8801
III 6877 HLA-A*0101, A*0301, B*0801, B*3501
9465
5891
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Fig. 2. Epitopes predicted by Syfpeithi and Immuneepitope. Only epitopes predicted by both algorithms were considered to increase the fidelity of the prediction. Bars represent the total number of putative epitopes for B/Victoria/2/1987 (V) or B/Yamagata/16/1988 (Y); back bars represent the number of putative conserved epitopes present in both viruses, whilst white bars represent the number of putative epitopes present in one virus only.

Fig. 2.

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To test the robustness of these prediction algorithms, we wished to establish the in vitro reactivity of influenza B virus-specific CD8+T-cells with the predicted epitopes. HLA-B*0801-restricted putative epitopes were selected as HLA-B*0801 was previously shown to be dominant in stimulating influenza B virus-specific CTLs (Boon et al., 2004) and the HLA-B*0801 allele was present in all the study subjects (Table 2). The HLA-B*0801 allele also gave us the opportunity to use three previously described peptides, i.e. NP263–271(ADRGLLRDI), NP413–421(ALKCKGFHV) and NP30–38(RPIIRPATL) (Robbins et al., 1997), of which only the NP30–38 epitope was predicted by our prediction algorithms. One study subject of each HLA group (Table 2; 6888, 8801 and 6877) was selected from which in vitro expanded polyclonal CD8+T-cells specific for B/Yamagata/16/1988 were tested for their reactivity with the predicted HLA-B*0801-restricted epitopes using peptide-loaded HLA class I-matched B-lymphoblastoid cell lines (BLCLs). To this end, we determined the IFN-γ production of the polyclonal CD8+T-cells in an ELISpot assay. Although donor 6877 responded to NP30–38 and M145–52, we did not observe a significant response to any of the other predicted or previously identified epitopes by donors 6877, 6888 and 8801, whilst all donors had a high response to BLCLs infected with the homologous virus (data not shown). These results clearly indicated that the prediction algorithms were not very reliable and therefore putative epitopes with other HLA restrictions were not tested.

Cross-reactivity of influenza B virus-specific CD8+ T-cells assessed by ELISpot

Next, we determined the extent of cross-reactivity of influenza B virus-specific CD8+T-cells with intra-lineage drifted variants and viruses of the opposing lineage. To this end, polyclonal CD8+T-cells derived from B/Victoria/2/1987 or B/Yamagata/16/1988 virus-stimulated peripheral blood mononuclear cell (PBMC) cultures were restimulated with HLA class I-matched BLCLs infected with the prototypic viruses (B/Victoria/2/1987 and B/Yamagata/16/1988) and the more recent viruses (B/Netherlands/455/2011 and B/Netherlands/712/2011, respectively). Activation of the polyclonal CD8+T-cells was assessed by measuring the number of IFN-γ-producing cells per 10 000 CD8+T-cells with the ELISpot assay (Fig. 3).

Fig. 3. Cross-reactivity of virus-specific polyclonal CD8+T-cells assessed by IFN-γ ELISpot. (a) B/Victoria/2/1987 virus-specific polyclonal CD8+T-cells or (b) B/Yamagata/16/1988 virus-specific polyclonal CD8+T-cells of nine study subjects were co-cultured with HLA-matched BLCLs infected with B/Victoria/2/1987, B/Netherlands/455/2011, B/Yamagata/16/1988 or B/Netherlands/712/2011.The number of IFN-γ-producing cells per 10 000 polyclonal CD8+T-cells was determined by ELISpot assay. Experiments were performed in triplicate; bar, sd. Uninfected BLCLs were used as negative controls. Identification number of the respective study subject is indicated in the left upper corner of each graph.

Fig. 3.

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The reactivity of B/Victoria/2/1987 virus-specific CD8+T-cells is shown in Fig. 3(a). These cells of all study subjects responded to reactivation with the homologous B/Victoria/2/1987 virus, although two study subjects (7482 and 2501) were low responders. A similar response was observed after restimulation with the intra-lineage drift variant B/Netherlands/455/2011. In addition, after stimulation with a virus of the opposing lineage B/Yamagata/16/1988 or B/Netherlands/712/2011, a cross-reactive response was observed that did not substantially differ in magnitude from the response to viruses of the B/Victoria/2/1987 lineage, in most subjects. Fig. 3(b) shows the response of B/Yamagata/16/1988 virus-specific CD8+T-cells. Again, all study subjects responded to the restimulation with HLA-matched BLCLs infected with the homologous virus strain and also after stimulation with the intra-lineage drift variant B/Netherlands/712/1988. In addition, a cross-reactive response was observed after stimulation with HLA-matched BLCLs infected with both viruses of the opposing B/Victoria/2/1987 lineage (B/Victoria/2/1987 and B/Netherlands/712/2011), which was similar in magnitude as compared with the response to viruses of the B/Yamagata/16/1988 lineage.

Cross-reactivity of influenza B virus-specific CD8+ T-cells assessed by lytic activity

Next, we wished to assess the cross-reactive lytic capacity of these polyclonal CD8+T-cell populations. Based on the IFN-γ ELISpot results, we selected a strong responder from each HLA group of study subjects (6888, 8801 and 9465) to test the lytic capacity of the CD8+T-cells. To this end, in vitro expanded B/Victoria/2/1987 or B/Yamagata/16/1988 virus-specific polyclonal CD8+T-cells were incubated with carboxyfluorescein succinimidyl ester (CFSE)-labelled HLA-matched BLCLs infected with B/Victoria/2/1987, B/Yamagata/16/1988, B/Netherlands/455/2011 or B/Netherlands/712/2011. The gating strategy used for flow cytometry to detect lytic activity of the CD8+T-cells is shown in Fig. 4(a, b).

Fig. 4. Cross-reactivity of virus-specific polyclonal CD8+ T cells assessed by lytic activity.To assess the degree of lytic activity a gating strategy was used to determine the number of viable CFSE positive target cells in the (a) absence and (b) presence of virus-specific T cells using FlowJo software. (c) B/Victoria/2/1987 and (d) B/Yamagata/16/1988 virus-specific polyclonal CD8+ T cells from study subjects 6888, 8801 and 9465 were isolated after stimulation of PBMCs with the respective virus. Lytic activity against HLA-matched CFSE-labeled BLCLs infected with B/Victoria/2/1987, B/Netherlands/455/201, B/Yamagata/16/1988 and B/Netherlands/712/2011 was assessed. Uninfected BLCLs were used as negative controls. Experiments were performed in triplicate; bar, sd.

Fig. 4.

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B/Victoria/2/1987 virus-specific CD8+T-cells displayed lytic capacity against cells infected with the homologous virus and the B/Netherlands/455/2011 virus of the same lineage in a effector-to-target cell (E : T) ratio-dependent fashion. In addition, cells infected with the heterologous viruses B/Yamagata/16/1988 and B/Netherlands/712/2011 of the opposing lineage were also lysed (Fig. 4c). A similar pattern of reciprocal lytic activity was observed for the B/Yamagata/16/1988 virus-specific CD8+T-cells (Fig. 4d). These results confirmed the cross-reactive nature of influenza B virus-specific CD8+T-cells observed in the ELISPOT assay.

Discussion

Influenza B viruses display less antigenic drift than influenza A viruses (Chen & Holmes, 2008; Lindstrom et al., 1999; Rota et al., 1992), yet they efficiently evade recognition by virus neutralizing antibodies present in the human population. This necessitates regular updates of the influenza B component of seasonal influenza vaccines. The circulation of influenza B viruses belonging to two antigenically distinct lineages further complicates the production of efficacious influenza vaccines. Inactivated influenza vaccines typically aim at the induction of virus neutralizing antibodies directed to the variable globular head region of the HA molecules of the respective influenza viruses. Consequently, there is interest in protective immune responses directed to more conserved proteins or regions thereof. Understanding humoral and cell-mediated immune responses to these conserved proteins may aid the development of more universal vaccines. Here, we assessed the cross-reactivity of influenza B virus-specific CD8+T-cells with viruses of the opposing lineage. It was concluded that influenza B virus-specific CD8+T-cells display a high degree of cross-reactivity with intra-lineage drift variants and viruses of the opposing lineage.

Although it was beyond the scope of this study to identify novel influenza B epitopes, we were interested whether predicted putative CTL epitopes were conserved between both influenza B lineages. The amino acid sequence identity of all viral proteins was very high between both lineages, which already suggested the existence of cross-reactive T-cell epitopes, as was also demonstrated for influenza A viruses (van de Sandt et al., 2014a). For the in silico prediction of epitopes, we excluded proteins encoded by the HA and NA gene segments, as these proteins undergo antigenic drift after positive selection by antibodies. Furthermore, it has been shown for influenza A viruses that these envelope proteins are minor targets for CTL responses (Lee et al., 2008). Two epitope prediction algorithms that are publicly available, i.e. Syfpeithi and Immuneepitope, were used in order to predict putative epitopes with the highest possible fidelity. A large number of putative epitopes was predicted by both programs, of which most were present in viruses of both lineages. However, the total number of epitopes predicted by both programs varied widely. The performance of these algorithms is not completely clear, and most likely false positives have also been predicted and false negatives omitted (Lundegaard et al., 2006; Roider et al., 2014). Indeed, out of six in vitro confirmed influenza B epitopes described previously (Robbins et al., 1989, 1995, 1997) that corresponded to the HLA alleles investigated in our study, only the NP30–38 RPIIRPATL epitope (HLA-B*0801 restricted) was predicted by both prediction programs. Other epitopes were solely predicted by Immuneepitope [NP85–94 KLGEFYNQMM and NP85–93 KLGEFYNQM (HLA-A*0201 restricted)] or Syfpeithi [NP413–421 ALKCKGFHV (HLA-B*0801 restricted)], and two epitopes [NP263–271 ADRGLLRDI (HLA-B*0801 restricted) and NP82–94 MVVKLGEFYNQMM (HLA-A*0201 restricted)] were not predicted at all. As these programs predict epitopes based on binding affinity of the epitope with the selected HLA allele, they do not take into account other possible factors that might play a role, such as the dissociation rate of the epitope (van der Burg et al., 1996), folding of the MHC class I molecules (Silver et al., 1991) or antigen processing (van de Sandt et al., 2012). To further test the robustness of the prediction algorithms, we determined the reactivity of polyclonal influenza B virus-specific polyclonal CD8+T-cells of three study subjects with the HLA-B*0801-restricted putative epitopes. We were unable to confirm any of the predicted epitopes, which included three previously described HLA-B*0801-restricted epitopes (NP30–38, NP263–271 and NP413–422). The lack of response to these previously described epitopes may be explained by mis-match of the HLA-C alleles of our study subjects. As the HLA-C alleles of our study subjects were not defined and Robbins et al. (1997) did not exclude the HLA-Cw7 allele as the presenting MHC class I molecule, it is possible that these epitopes are restricted by HLA-Cw7 instead of HLA-B*0801. This might also explain why only one study subject responded to the NP30–38 epitope (data not shown). Alternatively, differences in HLA makeup may have influenced immunodominance patterns (Boon et al., 2002). Thus, results obtained with epitope prediction algorithms should be interpreted with caution, which is in agreement with previous studies (Roider et al., 2014). Therefore, viral vectors expressing a single influenza B viral protein and/or overlapping peptide pools are considered more useful than in silico predictions for the identification of CD8+T-cell epitopes and establishing their immunodominance patterns.

The extent of cross-reactivity of influenza B virus-specific polyclonal CD8+T-cells was tested with two independent assays, i.e. IFN-γ ELISpot and CTL assays. In both assays, B/Victoria/2/1987 and B/Yamagata/16/1988 virus-specific CD8+T-cell populations displayed a high degree of cross-reactivity with drifted intra-lineage variants and across lineages, confirming that influenza B viruses indeed contain cross-reactive CTL epitopes. Differences in magnitude of the influenza B virus-specific CD8+T-cell responses between study subjects may reflect differences in HLA class I makeup (Boon et al., 2002) and/or differences in the history of influenza infections.

Most likely, subjects with alternative HLA alleles will also mount cross-reactive CD8+T-cell responses, considering the high level of sequence identity. Thus, in theory, infection with any influenza B virus will induce a cross-reactive influenza B virus-specific CD8+T-cell response. These cross-reactive CD8+T-cells may afford some degree of protection against a subsequent infection with an antigenically distinct influenza B virus, to which antibodies induced by previous infection will not be protective. Indeed, it was demonstrated during the pandemic of 2009 that, in the absence of virus neutralizing antibodies, the frequency of influenza A virus-specific CD8+T-cells correlated with a favourable disease outcome (Sridhar et al., 2013).

To address the problem of two co-circulating antigenically distinct influenza B lineages, quadrivalent seasonal influenza vaccines have become available that contain components of both influenza B lineages (CDC, 2013; FDA, 2012; WHO, 2014a). As quadrivalent vaccines elicit antibody responses against viruses of both lineages (Domachowske et al., 2013; Kieninger et al., 2013; Langley et al., 2013; Tinoco et al., 2014), they eliminate the risk that the incorrect B lineage is selected for inclusion in the vaccine. However, unforeseen antigenic drift within either influenza B lineage may affect vaccine effectiveness, although not as dramatically as a lineage mismatch (Belshe et al., 2010).

Vaccines that would induce cross-reactive T-cell-mediated immunity may offer another layer of protective immunity that is less sensitive to antigenic drift or circulation of an opposing lineage of influenza B virus. In particular, the use of live attenuated influenza vaccines has been shown to induce virus-specific CD8+T-cells, in contrast to inactivated vaccines (He et al., 2006; Hoft et al., 2011). Of interest, the viral proteins of live attenuated vaccines currently in use also display a high degree of sequence identity with recent circulating strains (93–100 % for B/Ann Arbor/1/66; data not shown), which supports the notion that these vaccines may also induce CD8+T-cell responses that cross-react with epidemic strains of the opposing lineage.

In conclusion, the present study shows for the first time, to the best of our knowledge, that human influenza B virus-specific CD8+T-cells are highly cross-reactive with influenza B viruses of the opposing lineage. Although quadrivalent influenza vaccines will be more commonly used in the near future, the induction of cross-reactive virus-specific T-cell responses may be a promising approach to broaden the protective efficacy of influenza vaccines, against both influenza A and B viruses. The induction of virus-specific CTL responses may be achieved with live attenuated influenza vaccines, especially in children (He et al., 2006). However, the administration of live attenuated influenza vaccines is restricted for certain high-risk groups (CDC, 2013; Fiore et al., 2010). The induction of virus-specific CD8+T-cell responses may also be achieved, e.g. by the use of specific adjuvants (Antrobus et al., 2014; Pérez-Girón et al., 2014; van de Sandt et al., 2014b) or novel antigen delivery systems (Altenburg et al., 2014; Berthoud et al., 2011; Daemen et al., 2005; Rimmelzwaan et al., 2000; Ulmer, 2002).

Methods

Cells

PBMCs were obtained from nine HLA-typed healthy blood donors (18–64 years of age) (Sanquin Bloodbank) by means of Lymphoprep (Axis-Shield) gradient centrifugation and were subsequently cryopreserved at − 135 °C. Study subjects were divided into three groups based on their HLA class I alleles (Table 2). The use of PBMCs for scientific research was approved by the Sanquin Bloodbank after informed consent was obtained from the blood donors.

Viruses

Prototypic influenza viruses B/Victoria/2/1987 (kindly provided by Vicki Gregory, WHO Collaborating Centre for Influenza, National Institute for Medical Research, London, UK) and B/Yamagata/16/1988 as well as the two more recent viruses, B/Netherlands/455/2011 and B/Netherlands/712/2011 belonging to each lineage, respectively, were propagated in Madin–Darby canine kidney cells at 37 °C. Culture supernatants were clarified by low-speed centrifugation and subsequently concentrated by ultracentrifugation, after which their infectious virus titres were determined as described previously (Rimmelzwaan et al., 1998).

Sequence analysis

Sequences of the eight gene segments of the above-described viruses were obtained as described previously (Westgeest et al., 2012) using segment-specific primers. Nucleotide sequences of the HA gene segments were used for phylogeny, and amino acid sequences of all gene segments were used to determine the amino acid sequence identity and for epitope prediction analysis, as described below.

Phylogeny

In addition to the above-mentioned viruses, an additional 47 human influenza B viruses, either used as vaccine strains or for which the lineage was previously confirmed by means of haemagglutination inhibition assay (Belshe, 2010; Belshe et al., 2010; Paiva et al., 2013; Rimmelzwaan et al., 2003; WHO, 2014a) and for which the complete HA nucleotide sequence was available from the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/genomes/FLU) or the Global Initiative on Sharing Avian Influenza Data (http://gisaid.org) influenza database, were selected for phylogenetic analysis. Previously published HA GenBank accession numbers are provided in Table S1. Nucleotide sequences of the HA gene segment of all 51 human influenza B viruses were aligned using the clustal w program running within BioEdit version 7.2.5 (Hall, 1999) and manually edited to maintain the correct reading frame. Nucleotides before the start codon and after the stop codon were removed. The nucleotide sequence alignment was used to determine the best-fit models of nucleotide substitution by jModelTest version 2.1.4 (Darriba et al., 2012; Guindon & Gascuel, 2003). The preferred ML-optimized model of nucleotide substitution, based on the Akaike information criterion, was TPM1uf+I+Γ4: Kimura three-parameter model (K81) (Kimura, 1981) with unequal base frequencies (uf), the proportion of invariant sites (I) and the Γ distribution of among-site rate variation with four categories estimated from the empirical data (Γ4). ML phylogenetic trees were reconstructed using the selected model of nucleotide substitution and PhyML version 3.1 (Guindon et al., 2010), performing a full heuristic search and subtree pruning and regrafting searches. The reliability of all phylogenetic groupings of each tree was determined through a non-parametric bootstrap resampling analysis with PhyML: 1000 replicates of ML trees were analysed by applying the TPM1uf+I+Γ4 model of nucleotide substitution. A detailed HA tree, including bootstrap values, is shown in Fig. 1. Trees were visualized through FigTree version 1.4.0 (http://tree.bio.ed.ac.uk/software/figtree/).

Amino acid sequence identity

blast analysis (http://blast.ncbi.nlm.nih.gov) was used to determine the amino acid sequence identity of all viral proteins of influenza B viruses used in this study, i.e. B/Victoria/2/1987, B/Yamagata/16/1988, B/Netherlands/455/2011 and B/Netherlands/712/2011 (Table 1).

Immunoinformatic analysis

Epitopes restricted for the HLA alleles of the study subjects were predicted based on the amino acid sequence of all internal proteins for B/Victoria/2/1987 and B/Yamagata/16/1988. For robustness, we used two online programs, i.e. Syfpeithi (http://www.syfpeithi.de) and Immuneepitope (http://tools.immuneepitope.org/mhci). In Syfpeithi, the MHC (HLA) type of the study subjects was selected in combination with prediction of epitopes of all possible lengths (all ‘mers’). The program indicated that putative epitopes were amongst the top 2 % of all top-scoring possibilities. An additional cut-off was set at a score of ≥ 19 as all known influenza A epitopes for these HLA alleles scored ≥ 19. In Immuneepitope, we also selected the HLA alleles of the study subjects in combination with all possible epitope lengths. The program indicated that most putative epitopes had an artificial neural network IC50 (nM) score of ≤ 500, so this was used as a cut-off value. Predicted amino acid sequences of putative epitopes are available on request.

Peptides

The HLA-B*0801-restricted putative epitopes predicted by Immuneepitope and Syfpeithi, and present in viruses of both lineages, were ordered as synthetic immunograde peptides (>85 % purity) (Eurogentec), in addition to previously in vitro confirmed HLA-B*0801-restricted influenza B epitopes NP263–271 ADRGLLRDI and NP413–421 ALKCKGFHV (Robbins et al., 1997).

In vitro expansion of influenza B virus-specific CD8+ T-cells

PBMCs obtained from HLA-typed healthy blood donors were stimulated with B/Victoria/2/1987 or B/Yamagata/16/1988 at m.o.i. 3, as described previously (Boon et al., 2002). Polyclonal CD8+T-cells were isolated from the expanded PBMC cultures 8–9 days after stimulation by means of CD8+ magnetically activated cell sorting according to the manufacturer's instructions (Miltenyi Biotec). These polyclonal CD8+T-cells were used as effector cells in IFN-γ ELISpot and lytic assays (see below).

Target cells

HLA-matched BLCLs were prepared as described previously (Rimmelzwaan et al., 2000). The cells (106) were incubated with or without 10 μM peptide for 1 h at 37 °C, and subsequently washed and resuspended in RPMI 1640 medium (Lonza) containing antibiotics and 10 % FBS (Sigma-Aldrich; R10F medium). Virus-infected target cells were prepared by inoculating BLCLs at m.o.i. 3 with B/Victoria/2/1987, B/Yamagata/16/1988, B/Netherlands/455/2011 or B/Netherlands/712/2011, or left untreated (negative control). After 1 h, cells were washed and resuspended in R10F medium, and then cultured for 16–18 h at 37 °C before being used for the stimulation of T-cells in IFN-γ ELISpot assays or as target cells in the CTL assays (see below).

IFN-γ ELISpot assay

The IFN-γ responses of in vitro expanded polyclonal CD8+T-cells were determined by ELISPOT assays, which were performed according to the manafacturer's recommendations (Mabtech). In brief, 10 000 in vitro expanded polyclonal CD8+T-cells were used as effector cells and incubated overnight with 30 000 peptide-loaded, virus-infected or untreated HLA class I-matched target cells, in triplicate. The mean number of spots was determined using an ELISpot reader and image analysis software (Aelvis).

CTL assay

To examine the lytic capacity of the in vitro expanded polyclonal CD8+T-cells, a CTL assay was used with CFSE-labelled target cells. In brief, 5 × 106 HLA class I-matched BLCLs were incubated with 50 μM CFSE (Sigma-Aldrich) for 5 min at 37 °C. These cells were subsequently inoculated with B/Victoria/2/1987, B/Yamagata/16/1988, B/Netherlands/455/2011 or B/Netherlands/712/2011 at m.o.i. 3 for 16–18 h. The infected and CFSE-labelled BLCLs were used as target cells and co-cultured with the in vitro expanded polyclonal CD8+ effector T-cells at E : T ratios of 5, 2.5 and 1.25. After a 3 h incubation period, dead cells were stained with ToPro3 (Invitrogen) for 10 min at 37 °C. Lysis in the target cell population was determined by flow cytometry using BD FACSDiva software (Becton Dickinson). Experiments were performed in triplicate. Percentage lysis was calculated by the formula: 100–[100 × (viable target cells in sample in presence of effector cells/viable target cells in absence of effector cells)].

Acknowledgements

R. A. M. F. received funding from the NIAID/NIH (HHSN272201400008C). G. F. R. received funding from EU grant FLUNIVAC (project ID 602604). A. D. M. E. O. was supported by the European Commission ERC project FLUPLAN (project ID 101920).

Supplementary Data

Supplementary Data

Click here for additional data file. (169.5KB, pdf)

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