Madin-Darby canine kidney (MDCK) (ATCC CCL-34) cells were cultured in minimum essential medium (MEM) (HyClone) supplemented with 10% fetal bovine serum (HyClone). Mouse macrophage cell line RAW264.7 (ATCC TIB-71) cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (HyClone) supplemented with 10% fetal bovine serum (HyClone).

X-31ca-based CAIVs used in this study are genetic reassortants carrying the surface HA and NA genes of A/Korea/1/09 (H1N1) (GQ131023 and GQ132185), A/New Caledonia/20/99 (H1N1) (CY031336 and CY033624), or A/Indonesia/5/05 (H5N1) (CY116646 and CY116648) virus and the six internal genes from the X-31ca donor backbone strain. The X-31ca was derived from the cold-adaptation of the parent X-31 virus (21, 22), a reassortant carrying the HA and NA genes of A/Hong Kong/1/1968 (H3N2) under A/Puerto Rico/8/34 (H1N1) backbone (18). The GenBank database accession numbers of the six internal genes of the X-31ca are DQ874873 for PB2, DQ874874 for PB1, DQ874875 for PA, DQ874877 for NP, DQ874879 for M, and DQ874880 for NS (22). All the CAIVs have been evaluated for attenuated phenotypes, vaccine efficacy, and safety in animal models in the previous studies (23–25). Challenge viruses included two laboratory strains, PR8 (H1N1) (A/Puerto Rico/8/34) and MA81 (H5N2) (A/aquatic bird/Korea/w81/05), one wild-type virus, Phil82 (H3N2) (A/Philippines/2/82), and one 7:1 genetic reassortant virus, reNet03 (H7N1) (PR8:HA of A/Netherlands/219/03). The mouse lethal dose 50 (MLD₅₀) of each of the virus was determined by preliminary study, 5 × 10³ PFU for PR8 (H1N1), 1 × 10⁴ PFU for MA81 (H5N2), 5 × 10⁴ PFU for Phil82 (H3N2), and 5 × 10³ PFU for reNet03 (H7N1).

The HA proteins expressed in insect cells were purchased from Sino Biological (China). The seven different HA proteins were derived from A/California/6/2009 (H1N1), A/Puerto Rico/8/1934 (H1N1), A/Canada/720/2006 (H2N2), A/Indonesia/5/2005 (H5N1), A/Hong Kong/35820/2009 (H9N2), A/Sydney/5/1997 (H3N2), and A/Anhui/1/2013 (H7N9) influenza viruses. We also expressed the HA proteins using bacterial expression system. The HA without transmembrane domain (HAΔTM) (positions 1−531 in H1 numbering) and the stalk region (positions 345−531 in H1 numbering) in the HA2 domain of PR8 (H1N1) and A/Korea/1/09 (H1N1) viruses were produced in Escherichia coli expression system, as previously described (26). The transmembrane domain (positions 532−566) was excluded from the expression to enable soluble expression of the HAΔTM protein. The expression plasmid (pLysRS-GE) was transformed into E. coli host BL21(DE3)pLysS. After expression, the cell lysates were centrifuged and separated into soluble and pellet fractions and were subjected to SDS-PAGE and visualized by staining with Coomassie brilliant blue R-250. The expressed proteins were purified using nickel affinity chromatography.

All animal studies were carried out in strict accordance with the guidelines of the Ministry of Food and Drug Safety (MFDS) of Korea. The experimental protocols including animal infection with an influenza live virus were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) and the Institutional Biosafety Committee (IBC) of the Yonsei Laboratory Animal Research Center (YLARC) (permit numbers: IACUC-A-201602-138-02, IACUC-A-201605-203-01, and IACUC-A-201605-205-02). Animal infection with CAIV or live virus was carried out in BSL-2 facility in YLARC. For vaccination, 6-week-old balb/c female mice were primed and boosted with 10⁵ PFU of CAIVs with an interval of two weeks. Challenge was done 35 days after the boosting vaccination, and mice that lost weight greater than 25% were considered non-viable and euthanized. Blood was collected from mice through retro-orbital bleeding under anesthesia, and the lungs and the BALF were taken from sacrificed mice.

Microneutralization (MN) assay and HA inhibition (HI) assay were performed to measure neutralizing antibody titers. Before MN assay, sera were pre-treated with a receptor-destroying enzyme at 37°C overnight and then heat-inactivated at 56°C for 30 min. 50 µl of twofold serial dilutions of the sera were incubated with 100 tissue cell infectious dose 50 (TCID₅₀) of viruses, and the mixtures were transferred to MDCK cells grown in a 96-well plate for viral infection. Viral infection was determined by the cytopathic effect (CPE), and the MN antibody titer of sera was calculated as the reciprocal of the highest dilution that completely suppressed the CPE. For HI assay, sera were pre-treated with a receptor-destroying enzyme at 37°C overnight and then heat-inactivated at 56°C for 30 min. 25 µl of twofold serial dilutions of the sera were incubated with the same volume of four hemagglutination units of the virus at 37°C for 1 h. A total of 50 µl of 1% chicken red blood cells were added and incubated at 4°C for 1 h. HI antibody titer was calculated as the reciprocal of the highest dilution that completely inhibited hemagglutination.

Vaccination-induced antibodies specific to the whole virus or the HA proteins were estimated by ELISA. Ninety-six-well plates were coated with 100 µl of 10⁵ PFU of sucrose-gradient purified viruses or 1 µg/ml of the HA proteins overnight. After blocking with 150 µl of 1% BSA in PBS and washing the plates, the wells were incubated with 100 µl of twofold serial dilutions of sera or BALF for 1 h at RT. After washing the plates, the wells were incubated with 100 µl of HRP-conjugated secondary goat anti-mouse IgG antibodies or IgA antibodies for 1 h at RT. The plates were washed and supplemented with 100 µl of TMB solution and incubated for 30 min at RT in the dark. The reaction was stopped by the addition of 50 µl of 2 N H₂SO₄ solution, and OD₄₅₀ was measured on an ELISA reader. Antibody titers were calculated as the endpoint dilution that yielded an OD value greater than the mean + 2 SD of the control group.

To determine the standard virus titer for NA inhibition (NAI) assay, viral NA activity was measured as described elsewhere (27). Ninety-six-well plates were coated overnight with 150 µl of 50 µg/ml fetuin. A total of 100 µl of twofold serial dilutions of influenza viruses dissolved in PBS containing 1% BSA were transferred to the fetuin-coated plates and incubated for 1 h at 37°C. The plates were washed and supplemented with 100 µl of 2.5 µg/ml HRP-conjugated peanut lectin and incubated for 1 h at RT. After washing, 100 µl of TMB solution was added to each well and the reaction was stopped after 5 min by the addition of 50 µl of 2 N H₂SO₄. The NA activity was expressed as OD₄₅₀ measured by an ELISA reader. For NAI assay, twofold serial dilutions of sera were mixed with the predetermined titer of the virus with the NA activity corresponding to OD₄₅₀ of 1 and incubated for 1 h at 37°C. The mixtures were then transferred to fetuin-coated plates and subjected to the subsequent procedures described above to measure the NA activity.

Vaccination-induced sera antibodies were examined for their ability to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) activity against virus-infected MDCK cells. We used primary spleen cells isolated from naive mice instead of natural killer (NK) cells as effector cells in ADCC assay, since pure NK cells isolated from the spleen demonstrated very weak ADCC activity in the preliminary study. Confluent MDCK cells grown in 96-well plates were infected with two multiplicities of infection of the influenza virus and incubated in MEM supplemented with 20 µM of Z-VAD-FMK (Promega), pan-caspase inhibitor, and in the absence of trypsin to minimize cytotoxicity from the multicycle viral infection. Eight hours after the infection, the supernatant was removed and 100 µl of sera (1:10 dilution) was added to the MDCK cells and incubated for 1 h at 37°C. After the incubation, 100 µl of mouse spleen cells (10⁶ cells) pre-treated with RBC lysis buffer were added to each well and incubated for 2 h at 37°C. The cytotoxicity of the target MDCK cells was measured in triplicate by lactate dehydrogenase (LDH) release assay.

To examine whether vaccination-induced sera antibodies promote the viral infection of heterologous influenza viruses, NP-based ELISA method (6) was used with minor modifications. Confluent MDCK or RAW264.7 cells grown in a 96-well plate were infected with 100 TCID₅₀ of the virus. Twenty-four hours later, the infected cells were washed and fixed by an acetone fixative and 100 µl of anti-influenza A/WSN/33 (H1N1) NP antibodies (26) were added to each well and incubated for 1 h at RT. After the binding of secondary antibodies and washings, OD₄₉₀ was measured by an ELISA reader. The virus infectivity was measured in triplicate and expressed as % of control infection with non-vaccination immune sera.

From vaccinated or control mice, blood was taken at 0 (before challenge), 2, 4, and 6 days after challenge with PR8 (H1N1) or Phil82 (H3N2), and the lungs were taken at 6 days after the challenge from the sacrificed mice. To obtain single-cell suspensions, the lungs were homogenized and filtered through 70-µm cell strainers. After centrifugation, the cells were resuspended in fresh MEM and erythrocytes were removed by RBC lysis buffer. The cells were washed with FACS buffer (0.5% FBS, 0.09% NaN3 in PBS) and were blocked with anti-mouse CD16/CD32 (BD Pharmingen) and 5 µg/ml streptavidin (Invitrogen). The cells were then stained with APC-CD8a mAb (clone 53-6.7; Biolegend), FITC-CD44 mAb (clone IM7; Biolegend), and PE-H-2Kd/NP_147–155(TYQRTRALV)-tetramer. After staining, the cells were fixed by 2% paraformaldehyde and analyzed using the FACSCalibur flow cytometer (BD Bioscience) and the Flowjo software (TreeStar Inc.).

For the depletion of CD4+ T cells and CD8+ T cells in vivo, 200 µg of anti-CD8 mAb (clone 2.43; BioXcell) and anti-CD4 mAb (clone GK1.5; BioXcell) were injected intraperitoneally into mice four times at days 1, 3, 5, and 7 before challenge. Non-depleted control mice were given isotype control rat IgG2b antibodies (clone LTF-2, BioXcell). Blood and lungs were taken from the mice 24 h after the last antibody injection and were subjected to flow cytometry to confirm the depletion. For flow cytometric analysis, we used anti-CD8 mAb (clone 53-6.7; Biolegend) and anti-CD4 mAb (clone RM4-5; Biolegend) directed against different epitope of CD4 and CD8 molecules to that of the depleting antibodies. For the depletion of NK cells, 20 µl of anti-asialo GM1 antiserum (Wako Pure Chemical Industries) were injected intraperitoneally into mice four times at days 1, 3, 5, and 7 before challenge. Non-depleted control mice were given normal rabbit serum (Wako Pure Chemical Industries). The spleens were taken from the mice 24 h after the last antibody injection to confirm the depletion by flow cytometry. For flow cytometric analysis, we used anti-CD3 mAb (clone 17A2; Biolegend) and anti-CD49b mAb (clone DX5; Biolegend).

All data were expressed as mean ± SD of each cohort, and the difference comparison between two groups was conducted by the unpaired, two-tailed Student’s t-test. The difference was considered statistically significant when P values were <0.05 (***P < 0.001; **P < 0.01; *P < 0.05).

We previously developed X-31ca as a master strain for CIAVs, by the cold-adaptation of the parent X-31 virus (21, 22). The X-31ca was used as a reliable backbone for CAIVs for the 2009 pandemic A/Korea/1/09 (H1N1) virus (ca-pH1N1), seasonal influenza A/New Caledonia/20/99 (H1N1) virus (ca-NCH1N1), and highly pathogenic H5N1 avian influenza A/Indonesia/5/05 (H5N1) virus (ca-IDH5N1) in subsequent studies (23–25, 28, 29). Using these three CAIVs, four different combinations of prime–boost vaccinations were designed to evaluate and compare induced immune responses and protective efficacy against antigenically distant heterologous influenza viruses in a mouse model. ca-pH1N1 was intranasaly inoculated into the single vaccination group and also used as prime strains in three boosting vaccination groups. Two weeks after the prime vaccination with ca-pH1N1, three different CAIVs were used as boosting strains: ca-pH1N1 for homologous boosting (identical to the prime strain), ca-NCH1N1 for heterologous boosting (different strain of H1N1), and ca-IDH5N1 for heterosubtypic boosting (Figure 1A). Cross-reactive antibody responses elicited by each vaccination were analyzed through various in vitro assays as described later. In addition, to evaluate cross-protection in vivo, the following four heterologous influenza viruses were used as challenge strains: PR8 (H1N1) and MA81 (H5N2) belonging to the HA group 1 and Phil82 (H3N2) and reNet03 (H7N1) belonging to the HA group 2 (Figure 1B). Thus, while three CAIVs had identical six internal gene segments derived from the X-31ca and the surface genes from different viruses belonging to the HA group 1 (H1 and H5), challenge viruses encompass both HA groups. This experimental design allowed us to assess the potency and breadth of immune responses and protective efficacy against diverse influenza viruses covering both HA groups.

Considering the roles of the HA as a major protective antigen in the protection against influenza viral infection, we first examined whether vaccination-induced sera antibodies bind to the HA proteins of heterologous influenza viruses with ELISA using the insect cell-expressed recombinant HA proteins derived from seven different influenza viruses. All the vaccinations elicited high levels of antibodies cross-reactive to antigenically close group 1 HAs including H1, H2, and H5 (Figure 2A). The robustness of the cross-reactivity of the antibody responses extended to antigenically distant H9 HA, although the antibody titers were significantly reduced as compared to those to the closer HAs. Specific antibody binding was observed even in group 2 HAs (H3 and H7), although noticeable antibody binding seen only in sera from heterologous boosting vaccinations (ca-pH1N1 + ca-NCH1N1) (Figure 2A). Next, we estimated vaccination-induced antibody titers directed to the conserved HA stalk domain. For this purpose, we expressed recombinant HA full-length and stalk proteins derived from A/Korea/1/09 (pH1N1) and PR8 (H1N1) viruses, as soluble proteins fused with the E. coli lysyl tRNA synthetase (LysRS) (Figure S1A in Supplementary Material). In ELISA, vaccination-induced antisera strongly bound to the fusion proteins, but not to the LysRS, enabling the measurement of HA-specific antibody titers (Figure S2B in Supplementary Material). The results also suggest that the sequential epitopes present in the HA proteins expressed in E. coli can be recognized by specific antibodies. A single vaccination with ca-pH1N1 resulted in a pH1N1-HA-specific antibody titer of 100, and boosting by ca-pH1N1, ca-NCH1N1, and ca-IDH5N1 increased the antibody titers to 210, 160, and 150, respectively (Figure 2B). The most significant increase in the HA-specific antibodies compared to single vaccination was seen in homologous boosting with ca-pH1N1, while heterologous boosting with ca-NCH1N1 and heterosubtypic boosting with ca-IDH5N1 resulted in only modest increases in the HA-specific antibody titers. Vaccinations also resulted in pH1N1-HA stalk-specific antibodies. A single vaccination yielded the HA stalk-specific antibody titer of 192, and each boosting increased the antibody titers to 250−520. In parallel, the cross-reactivity of the antibodies was examined against the PR8 (H1N1) HA protein. A single vaccination induced the PR8 HA antibody titer of 56, and each boosting resulted in a significant increase in the antibody titers ranging 210−260, an approximate fourfold increase of the single vaccination. The PR8 HA stalk-specific antibody levels were very similar between the single vaccination and homologous boosting groups (mean antibody titer of 384 in both). However, heterologous boosting and heterosubtypic boosting resulted in more than a twofold increase in the HA stalk-specific antibody titer compared to that of homologous boosting. The results suggest that heterologous or heterosubtypic boosting could be more effective way of inducing the HA stalk-specific antibodies than the homologous boosting. We also estimated the NA-specific antibodies induced by the vaccination. All the vaccinations generated high levels of antibodies reactive to closely related N1 NA proteins, and the antibody binding reactivity became weak against heterologous N2, N6, N7, N8, and N9 NA proteins (Figure S2 in Supplementary Material). Additionally, vaccination-induced anti-influenza antibodies were measured using purified whole viruses as coating antigens in ELISA. A single vaccination with ca-pH1N1 induced high levels of serum IgG antibodies against not only homologous pH1N1 but also four heterologous influenza viruses, PR8 (H1N1), MA81 (H5N2), Phil82 (H3N2), and reNet03 (H7N1) (Figure S3A in Supplementary Material). Boosting by homologous, heterologous, or heterosubtypic CAIV induced antibody levels similar to, or greater than, that of the single vaccination. IgA antibody levels in the brochoalveolar lavage fluid (BALF) were also estimated against the same set of viruses. Similar to the results of serum IgG antibodies, BALF IgA antibodies showed strong reactivity against all the viruses tested (Figure S3B in Supplementary Material). These results show that the prime–boost vaccination with CAIVs can induce cross-reactive systemic and mucosal antibodies not only against the HA group 1 (H1 and H5) but also against the antigenically distant HA group 2 (H3 and H7) influenza viruses.

To examine whether the cross-reactivity of vaccination-induced antibodies could translate into viral neutralizing activity, we performed various in vitro assays with the sera or the mucosal samples. MN assay showed that the sera antibodies effectively inhibited the replication of the homologous pH1N1 virus in MDCK cells. Against the homologous pH1N1, a single vaccination with ca-pH1N1 developed the MN antibody titer of 224, and the homologous, heterologous, and heterosubtypic boosting increased MN antibody titers to 570, 350, and 290, respectively (Figure 3A). However, none of vaccination-induced antibodies yielded detectable levels of MN antibodies against the four heterologous influenza viruses. HI assay with the sera showed the same trend as the MN assay results; sera antibodies could inhibit the hemagglutination activity of the pH1N1 but not the heterologous influenza viruses (Figure 3B). Additionally, we performed an NAI assay with the same set of viruses to examine whether the sea antibodies could block the enzyme activity of the NAs of the heterologous influenza viruses. As expected, homologous boosting yielded the highest level of NAI antibodies against the homologous pH1N1, and heterologous or heterosubtypic boosting resulted in the levels of NAI antibodies similar to that of the single vaccination (Figure S4 in Supplementary Material). However, none of the vaccination-induced antibodies demonstrated NAI activities that were greater than 50% against any of the heterologous viral NAs, with only partial inhibition seen against the heterologous viruses at the least diluted sera.

Recent studies have provided considerable evidence that influenza-specific antibodies have Fc-effector functions, such as ADCC, which target the infected cells for killing by immune cells such as NK cells, monocytes, neutrophils, and macrophages (30–32). We examined whether the vaccination-induced antibodies exert such ADCC activity to MDCK cells infected with the heterologous influenza viruses. All sera from the four vaccination groups resulted in a significant increase in the cytotoxicity of PR8 (H1N1)-infected cells, as compared to the non-vaccination control (Figure 3C). Additionally, sera from the three boosting vaccination groups led to the lysis of MA81 (H5N2)-infected or reNet03 (H7N1)-infected cells. Significant lysis of Phil82 (H3N2)-infected cells was seen by sera from heterologous boosting and heterosubtypic boosting vaccination groups. These results suggest that although the antibodies induced by CAIVs could not neutralize heterologous influenza viruses, they exerted a weak but distinct ADCC activity against the heterologous viruses.

There has been substantial experimental and clinical evidence that CAIVs provide varying degrees of cross-protection against heterologous influenza viruses even without detectable antibody-mediated neutralizing activities (28, 33, 34). To evaluate the breadth and potency of this cross-protection in vivo, vaccinated mice were challenged with a lethal dose of each heterologous influenza virus. Non-vaccinated control mice infected with 10 MLD₅₀ of PR8 (H1N1), MA81 (H5N2), Phil82 (H3N2), or reNet03 (H7N1) virus showed rapid weight loss and death upon the challenge (Figure 4A). By contrast, a single vaccination with ca-pH1N1 protected the mice from mortality associated with lethal infections, causing mild weight loss of 5−8% upon challenge with MA81 (H5N2) or reNet03 (H7N1), and less than 1% upon challenge with PR8 (H1N1) or Phil82 (H3N2) (Figure 4B). Homologous boosting also resulted in excellent protection against PR8 (H1N1) and Phil82 (H3N2) challenge and resulted in the weight loss of 8.9% following reNet03 (H7N1) challenge, a comparable protective efficacy to that from a single vaccination (Figure 4C). Interestingly, however, MA81 (H5N2) challenge caused weight loss of 12.9% in the homologous boosting group, which was higher than in the single vaccination. Heterologous boosting with ca-NCH1N1 protected the mice from the morbidity associated with the challenges, except for the temporal weight loss of 5.8% at 2 days post-challenge (dpc) with MA81 (H5N2) (Figure 4D). Remarkably, heterosubtypic boosting with ca-IDH5N1 did not resulted in any weight loss after the challenges, showing the most potent cross-protection efficacy among the four vaccination groups (Figure 4E). The virulence represented by weight loss are summarized in Figure 4F. Evidently, challenge with PR8 (H1N1) or Phil82 (H3N2) caused only mild weight loss of less than 3% in all vaccination groups, making it difficult to discriminate the cross-protective efficacy between prime–boost combinations. On the other hand, challenge with MA81 (H5N2) or reNet03 (H7N1) yielded variable weight loss ranging 0−13% depending on the vaccination combination. Homologous boosting demonstrated seemingly lessened protective potency against MA81 (H5N2) or reNet03 (H7N1) as compared to the single vaccination, but the potency was strengthen by heterologous boosting and became complete by heterosubtypic boosting causing weight loss less than 1% upon the challenge. Protective efficacy was further substantiated by estimating challenge virus titers in the lungs of vaccinated mice. All the challenge viruses replicated to greater than 10⁶ PFU in the lungs of the non-vaccinated control group, whereas vaccinations reduced the viral titers to 10²−10⁴ PFU (Figure 4G), showing potent sterile immunity. As compared to the single vaccination, each boosting vaccination resulted in an approximate 10-fold reduction of the viral titers of PR8 (H1N1) and Phil82 (H3N2). However, the viral titers of MA81 (H5N2) and reNet03 (H7N1) were higher in the homologous boosting group than the single vaccination group. Again, heterologous boosting and heterosubtypic boosting further decreased the viral titers of MA81 (H5N2) and reNet03 (H7N1). Overall, the protective efficacy evaluated by the weight loss was mirrored in the lung viral titer (Figures 4F,G). The results of protection tests in vivo suggest two important points regarding the breadth and potency of our vaccination strategy. First, despite the absence of antibody-mediated viral neutralizing activities, prime–boost vaccination with CAIVs comprising only the HA group 1 strains provided a broad and potent cross-protection covering both HA group 1 and group 2 influenza viruses in vivo. Second, the potency of cross-protection against more virulent strains such as H5 and H7 avian influenza viruses could be enhanced through the heterologous or heterosubtypic boosting.

To further define immunological correlates responsible for the cross-protection, we focused on T cells which have been considered as the key factor for cross-protection. We first examined whether CD8+ cytotoxic T lymphocytes (CTLs) directed to the conserved NP_147−155 epitope could be recalled in vaccinated mice upon a heterologous challenge. For this purpose, separate groups of mice were primed and boosted with CAIVs and then challenged with PR8 (H1N1) or Phil82 (H3N2), and the specific CTLs in the blood and lungs were analyzed by flow cytometry (Figure 5A). In the peripheral blood, a variable but slightly increased recall of NP_147−155+ CTLs was observed through six days after the challenge with PR8 (H1N1) or Phil82 (H3N2) (Figures 5B,C). In the lungs, a much more significant increase in the NP_147−155+ CTLs were observed in the vaccinated groups upon PR8 (H1N1) challenge, in which NP_147−155+ CTLs accounted for 2.7−3.7% of the whole CD8+ T-cell population, corresponding to a 7.3−9.8-fold increase relative to that of the non-vaccinated control (Figure 5D; Figure S5A in Supplementary Material). Phil82 (H3N2) challenge also recalled the NP_147−155+ CTLs in the lungs of vaccinated mice, albeit not significantly (Figure 5E; Figure S5B in Supplementary Material). However, IFN-γ producing CTLs were significantly increased upon Phil82 (H3N2) challenge in all three boosting vaccination groups (Figure 5F; Figure S5C in Supplementary Material), suggesting that the boosting vaccination generated CTLs with a specific memory, which then can be re-stimulated to produce IFN-γ cytokine upon the heterologous challenge.

To further determine cell-mediated immunity for cross-protection in vivo, a particular subset of the T cells or NK cells were depleted by injecting anti-CD4, anti-CD8, or anti-asialo GM1 antibodies into mice (Figure 6A). Injection of the antibodies to vaccinated mice resulted in the depletion of >98% of CD4+ T cells and CD8+ T cells in the peripheral blood and the lung, and the depletion of 70−80% of NK cells in the spleen (Figures S6A,B in Supplementary Material). First, the lethality of 10 MLD₅₀ of PR8 (H1N1) and Phil82 (H3N2) was confirmed in non-vaccinated mice (Figure 6B). Next, separate mice were vaccinated with ca-pH1N1 (single vaccination) or ca-pH1N1 + ca-NCH1N1 (heterologous boosting) and their T cells or NK cells were depleted before challenge with either PR8 (H1N1) or Phil82 (H3N2). Challenge with PR8 (H1N1) caused little morbidity in the single vaccination group that was given isotype control antibodies (Figure 6C). Depletion of CD8+ T cells resulted in a weight loss of ~11% upon the challenge without death. When both CD8+ T cells and CD4+ T cells were depleted, PR8 (H1N1) challenge caused a weight loss of ~13% and death in one of the five mice, and the recovery of the survived mice was significantly delayed (Figure 6C). In a single vaccinated mice, NK cell-depleted group and CD8+ T-cell-depleted group demonstrated similar levels of weight loss upon PR8 (H1N1) challenge, and depletion of both T-cell populations as well as NK cells led to a substantial weight loss of ~20% and the death of three mice (Figure 6C). In heterologous boosting group (ca-pH1N1 + ca-NCH1N1), depletion of both T-cell populations and NK cells resulted in a weight loss of ~10% and the death of one mouse (Figure 6D). Phil82 (H3N2) was also used as a challenge strain. In the single vaccination group, Phil82 (H3N2) challenge caused a weight loss of ~10% and the death of one mouse when CD8+ T cells, CD4+ T cells, and NK cells were depleted (Figure 6E). Heterologous boosting vaccination group did not develop any morbidity upon the Phil82 (H3N2) challenge even in the absence of both T cells and NK cells (Figure 6F). Of note, depletion of CD4+ T cells, CD8+ T cells, and NK cells did not completely abolish cross-protection against the heterologous challenges. These results suggest not only that T cells and NK cells contribute significantly to the cross-protection, but also that there remained other protection mechanisms operating robustly.

As described earlier, VAERD has been reported in the cases of influenza virus vaccines, in which non-neutralizing HA stalk antibodies enhanced viral infectivity and aggravated diseases upon heterologous infection (6), raising a concern on the HA stalk-based approaches for cross-protection. Considering that prime–boost vaccinations with CAIVs induced high levels of HA stalk antibodies (Figure 2B), we examined whether the antibodies caused enhanced viral infectivity of the heterologous viruses. Each virus was pre-incubated with the serial dilutions of sera for binding and absorbed into MDCK or RAW264.7 cells, and the viral replication was monitored by NP-based ELISA (6). In both cell lines, vaccination-induced sera antibodies effectively inhibited the replication of the homologous pH1N1 virus, as compared to the non-vaccinated control sera (Figures 7A,B). The sera antibodies showed partial inhibition of the replication of the four heterologous influenza viruses, resulting in the infectivity ranging 50−85%, as compared to the control. However, none of the sera antibodies promoted the viral infectivity of heterologous influenza viruses. The results, along with in vivo protection tests described above, suggest that our strategy provides a safe cross-protection against heterologous influenza viruses without causing VAERD by non-neutralizing antibodies. Another potential safety issue of prime–boost vaccination with CAIVs is related to the phenomenon of original antigenic sin. The original antigenic sin theory, first described in 1953 by Thomas Francis (19), refers the phenomenon in which sequential exposure to antigenically different virus strains results in preferential antibody responses to the first strain and impaired immune responses to the second strain. In line with this, recent studies have demonstrated that sequential infections of mice with two different influenza virus strains resulted in almost an exclusive neutralizing antibody response to the first strain and a severely impaired protective immunity to the second strain (20, 35). Thus, we examined if a similar phenomenon occurred in the prime–boost vaccination strategy, in which antigenically heterologous CAIVs were sequentially given to mice. ca-NCH1N1 and ca-IDH5N1 were used as the second vaccine strain in heterologous boosting (ca-pH1N1 + ca-NCH1N1) and heterosubtypic boosting (ca-pH1N1 + ca-IDH5N1) vaccinations, respectively. Neutralizing antibodies to each homologous wild-type virus were estimated by MN and HI assays and were compared to those induced by a single vaccination with ca-NCH1N1 or ca-IDH5N1. A single vaccination with ca-NCH1N1 and heterologous boosting vaccination with ca-pH1N1 + ca-NCH1N1 yielded very similar levels of MN antibody titers (1,330 and 1,270) against the antigenically matched A/NC/20/99 wild-type virus (Figure 7C). Consistently, these two vaccination groups produced similar HI antibody titers (341 and 363) against the same virus. In parallel, a single vaccination with ca-IDH5N1 induced the mean HI and MN antibody titers of 128 and 310, respectively, which were similar to those developed by heterosubtypic boosting vaccination with ca-pH1N1 + ca-IDH5N1 (Figure 7C). The results show that prime vaccination did not prevent the generation of neutralizing antibody responses to the second antigenically heterologous vaccination. Taken together, our data demonstrate that prime–boost vaccination with CAIVs does not accompany adverse effects from heterogeneity not only between the vaccine strains (VAERD) but also between the vaccine and challenge virus (antigenic sin-like phenomenon), supporting the safety of our vaccination strategy.