ABSTRACT
It has been reported that the high‐growth reassortant (HGR) A(H3N2) influenza viruses used for split influenza vaccine (SV) production have some amino acid substitutions in hemagglutinin due to egg adaptation during virus propagation, causing antigenic differences between HGR and epidemic viruses. To clarify whether inactivated whole‐virus vaccine (WV) derived from the A(H3N2) HGR virus possessing egg adaptation could induce cross‐protective immune responses against epidemic A(H3N2) viruses, the efficacy of WV was compared with that of SV in a ferret model. When the ferrets immunized with WV or SV derived from HGR A/Victoria/361/2011 (IVR‐165) virus were challenged with the homologous virus A/Victoria/361/2011 (IVR‐165) or its original cell‐propagated A/Victoria/361/2011 virus, respectively, WV successfully shortened the duration of virus shedding of both challenge viruses, whereas SV shortened only that of the homologous virus, A/Victoria/361/2011 (IVR‐165). When WV‐immunized ferrets were challenged with A/Fukushima/69/2015 virus, which is an epidemic virus antigenically different from the A/Victoria/361/2011 virus, WV could shorten the duration of shedding of this virus. In addition, we found that early induction of nasal IgG and IgA antibodies by vaccines helped shorten the virus‐shedding period, although this was dependent on the degree of difference in antigenicity of the challenge virus. These results indicate that vaccination with WV, not with SV, would be a solution to avoid decreased vaccine effectiveness due to the antigenic change of HGR virus by egg adaptation during virus propagation.
Keywords: ferret, H3N2, human sera, IgA, IgG, influenza, nasal wash, vaccine
Abbreviations
- ADCC
antibody‐dependent cytotoxicity
- CDC
complement‐dependent cytotoxicity
- CTLs
cytotoxic T lymphocytes
- FBS
fetal bovine serum
- Fuk69
A/Fukushima/69/2015
- GMT
geometric mean titer
- HA
hemagglutinin
- HGR
high‐growth reassortant
- HI
hemagglutination‐inhibition
- IVR165
A/Victoria/361/2011 (IVR‐165)
- MDCK
Madin‐Darby canine kidney
- NA
neuraminidase
- NT
neutralization
- SRD
single radial immunodiffusion
- SV
split influenza vaccine
- TCID
tissue culture infectious doses
- Vic361
A/Victoria/361/2011
- WV
whole‐virus vaccine
1. Introduction
Protection against infection with influenza virus is largely mediated by antibodies directed against the major viral surface glycoproteins, hemagglutinin (HA), and neuraminidase (NA) [1, 2]. However, the antigenicity of HA and NA is frequently altered due to antigenic drift and infrequent antigenic shifts. Therefore, to prepare effective vaccines, it is necessary that the vaccine antigenically matches circulating viruses as closely as possible.
Embryonated hen eggs (hereafter referred to as eggs) are useful substrates for the propagation of viruses used in influenza vaccine production. Therefore, the high growth ability of vaccine viruses in eggs and the yields of viral HA and NA antigens are important issues. However, during virus propagation in eggs, the high‐growth reassortant viruses used by manufacturers in vaccine production acquire mutations in viral genes, especially HA, due to egg adaptation. These mutations frequently lead to amino acid substitutions around the receptor‐binding site in HA, causing antigenic changes [3, 4]. Consequently, the antigenic change in vaccine viruses by egg adaptation is a major obstacle in the production of effective influenza vaccines, even if the prototype of vaccine viruses has been properly selected by the World Health Organization on the basis of antigenic and genetic characteristics of viruses circulating globally. It has been previously demonstrated that the 2011/12 season vaccine strain of A/Victoria/210/2009 (X‐187) (H3N2) used for vaccine production was antigenically distinguishable from its prototype virus (A/Victoria/210/2009) using hemagglutination‐inhibition (HI) tests with post‐infection ferret antisera and the cross‐reactivity of vaccinated human sera to the epidemic circulating viruses was low [3].
In the 2012/13 season in Japan, inactivated trivalent ether‐split influenza vaccine (SV) consisting of three viruses, A/California/7/2009 (X‐179A) (H1N1)pdm09 and A/Victoria/361/2011 (IVR‐165) (H3N2) subtypes and B/Wisconsin/01/2010 (BX‐41A), was manufactured and used. Skowronski et al. suggested that the low influenza vaccine effectiveness of A/Victoria/361/2011 (IVR‐165) of the H3N2 subtype in the 2012/13 season was attributable to mutations at antigenic sites B (H156Q and G186V substitutions) and D (an S219Y substitution) in the HA of the egg‐adapted H3N2 vaccine strain A/Victoria/361/2011 (IVR‐165) compared with that of the prototype virus A/Victoria/361/2011 [4]. In Japan, SV has been used since 1972 because of the reactogenicity of the inactivated whole‐virus vaccine (WV), which had been used previously. However, it has been shown that WV has superior immunogenicity compared to SV in unprimed persons, especially after one time vaccination [5, 6, 7, 8, 9, 10], and it effectively induces memory cross‐protective cytotoxic T lymphocytes (CTLs), thus inducing (heterosubtypic) cross‐protective CTLs in humans [11].
Both secretory immunoglobulin A (S‐IgA) and IgG antibodies contribute to protection against influenza virus in the respiratory tract [12, 13]. Intranasal vaccination, a vaccination mode that mimics natural infection, can induce virus‐specific S‐IgA antibodies in the upper respiratory mucosa as well as IgG antibodies in serum. Intramuscular administration of inactivated trivalent virus vaccines has been shown to elicit IgA responses in human tonsils and saliva [14, 15].
Therefore, we evaluated the protective efficacy of the WV of A/Victoria/361/2011 (IVR‐165) possessing egg adaptation and compared it with that of its SV in ferrets, a standard model for human influenza vaccine efficacy studies, by examining virus and antibody titers in nasal wash samples and changes in the body weight of the ferrets.
2. Materials and Methods
2.1. Viruses and Cells
A/California/7/2009 (X‐179A) (H1N1)pdm09, A/Victoria/361/2011 (IVR‐165) (H3N2) (hereafter IVR165), and B/Wisconsin/01/2010 (BX‐41A) were propagated in the allantoic cavities of 10‐day‐old embryonated chicken eggs at 35°C for 48 h. A/Victoria/361/2011 (hereafter Vic361) and A/Fukushima/69/2015 (hereafter Fuk69) cells were propagated in confluent Madin‐Darby canine kidney (MDCK) and MDCK‐SIAT1 [16] cells, respectively, at 34°C for 72 h. The Fuk69 virus of the epidemic strain of the 2015/16 influenza season belonged to genetic clade 3C.2a and was antigenically different from Vic361 and IVR165, which belonged to genetic clade 3C.1 (Table 1). The MDCK and the MDCK‐SIAT1 cells were maintained in Dulbecco's modified Eagle medium (DMEM; Gibco, Shanghai, China) supplemented with 10 or 5% fetal bovine serum (FBS), 100 U/mL penicillin, 100 µg/mL streptomycin, and 0.5 µg/mL fungizone at 37°C in 5% CO2. The MDCK‐SIAT1 cells were further supplemented with 2 mg/mL G418.
Table 1.
Antigenic difference in high‐growth reassortant virus for vaccine and circulating viruses.
| HI titer of ferret antisera against | |||||
|---|---|---|---|---|---|
| Virus | IVR165 | Vic361 | Fuk69 | Genetic clade | Amino acid differences in antigenic sites from Vic361 |
| IVR165 | 2560 | 640 | 320 | 3C.1 | H156Q, G186V, S219Y |
| Vic361 | 320 | 640 | 320 | 3C.1 | |
| Fuk69 | 160 | 160 | 1280 | 3C.2a | N144S, N145S, F159Y, K160T |
Note: A 1% guinea pig red blood cell solution was used for the hemagglutination‐inhibition assay, which was conducted in the presence of 20 nM oseltamivir.
2.2. Pre‐ and Post‐Vaccination Human Sera and Ethical Statement
For H3 vaccine evaluation, pre‐ and post‐vaccinated human sera of recipients who received 2012/13 season vaccine [A/California/7/2009 (X‐179A) (H1N1)pdm09, A/Victoria/361/2011 (IVR‐165) (H3N2), and B/Wisconsin/01/2010 (BX‐41A)] were collected from an adult group (n = 30; age range, 20–39 y; mean = 28.9 y), a middle‐aged group (n = 30; age range, 41–58 y; mean = 47.9 y), and an elderly group (n = 30; age range, 62–102 y; mean = 83.5 y).
After obtaining written informed consent, all individuals were vaccinated with one dose of a standard commercial trivalent influenza SV, containing 15 μg each of the hemagglutinin proteins of the vaccine viruses. The sera were collected twice at the time of vaccination and 3–4 weeks after the vaccination from the participants. The study using these sera was reviewed and approved by the Ethics Committee of the Medical School of Niigata University (No. 2300) and the National Institute of Infectious Diseases (No. 747).
2.3. HI Assay
Serum samples were treated with RDEII (Denka Co. Ltd., Japan) to remove non‐specific inhibitors. A 1:10 dilution of the treated sera was prepared with phosphate‐buffered saline. Two‐fold serial dilutions of sera were mixed with four hemagglutination units of antigen virus per well and pre‐incubated for 60 min in 96‐well plates at room temperature. The HI titers of sera were determined as the reciprocal of the highest dilution that did not display hemagglutinating activity. The HI assay for antigenic characterization of H3 viruses was performed with 1% guinea pig red blood cells in the presence of 20 nM oseltamivir to avoid the NA‐mediated binding of Fuk69 to the red blood cells [17]. The HI assay for cross‐reactivity of human sera was performed with 0.5% turkey red blood cells, and the plate was incubated for 45 min at room temperature.
2.4. Neutralization (NT) Assay
Human and ferret serum samples were treated with RDEII to remove nonspecific inhibitors of influenza virus replication. One hundred, 50% tissue culture infectious doses (TCID50) of H3N2 viruses were pre‐incubated with two‐fold serial dilutions of treated sera for 30 min and then inoculated into MDCK or MDCK‐SIATI cells. The hemagglutination activity of the supernatant of these cells was measured on day four to determine the neutralizing activity of the test sera. The NT titers of sera were determined as the reciprocal of the highest dilution that did not show cytopathic effect.
2.5. Vaccine Preparation
SV and WV contained 2012/13 seasonal influenza vaccine strains [A/California/7/2009 (X‐179A) (H1N1)pdm09, A/Victoria/361/2011 (IVR‐165) (H3N2), and B/Wisconsin/01/2010 (BX‐41A)]. The immunization of ferrets with SV, a commercial 2012/13 seasonal influenza SV (Kitasato Daiichi Sankyo Vaccine Co. Ltd., Japan), was conducted in the early phase of 2013. Each virus was inactivated with β‐propiolactone and purified by sucrose density gradient centrifugation methods. WV was prepared by mixing 15 μg/0.5 mL of HA from each of the purified viruses. The HA content in the purified virus was determined by using the single radical immunodiffusion (SRD) test [18].
2.6. Immunization and Infection of Ferrets
Healthy, young, adult, outbred female ferrets between 6 and 12 months of age were used in the experiment. These ferrets were confirmed to be seronegative for antibodies against circulating influenza A(H1N1)pdm09, A(H3N2), B Yamagata, and B Victoria viruses using the HI assay prior to the experiment.
Twenty‐two ferrets were immunized intramuscularly five times with an adult human dose (0.5 mL) of SV at intervals of 3 weeks. HI antibodies (HI titers against IVR165 ≥ 10) were detected in eight out of 22 immunized ferrets. These eight ferrets were subsequently divided into two groups of four animals each (groups 1 and 2). For immunization with WV, 12 ferrets were immunized twice with WV (0.5 mL of WV at an interval of 3 weeks). HI antibodies (HI titers against IVR165 ≥ 40) were detected in all ferrets immunized with WV, and these ferrets were divided into three groups of four animals each (groups 3, 4, and 5). Three weeks after the final immunization, the ferrets of groups 1 and 2 were inoculated intranasally with 106 TCID50 of IVR165 virus and prototype Vic361 virus, respectively, and the ferrets of groups 3, 4, and 5 were inoculated intranasally with 106 TCID50 of IVR165 and Vic361, and 105.5 TCID50 of Fuk69 viruses, respectively, in a total volume of 0.5 mL. During the infection experiment, body weights were monitored and nasal washes were collected every day for a week. Viral titers in nasal washes were determined by using the method of Reed and Muench [19]. Serum samples were collected prior to vaccination and viral challenge. All immunizations and sampling were performed under anesthesia.
2.7. Enzyme‐Linked Immunosorbent Assay
Titers of IgG and IgA antibodies specific for IVR165, Vic361, or Fuk69 in nasal wash samples collected after virus challenge were determined by use of an ELISA. Half‐area flat‐bottomed microtiter plates (Costar, Corning, NY, USA) were coated with each ether‐split antigen of purified virus (32 HA/50 µL) [20], followed by blocking with 0.1% BSA in phosphate‐buffered saline (PBS) (pH 7.4). Two‐fold serial dilutions of nasal wash samples were then added to each well of the plates. IgG or IgA antibodies were detected by using biotin‐conjugated affinity purified anti‐ferret IgG (Brookwood Biomedical, Jemison, AL, USA) or anti‐ferret IgA (Rockland, Philadelphia, PA, USA) antibody, respectively, followed by alkaline phosphatase‐conjugated streptavidin (Southern Biotech, Birmingham, AL, USA). The enzymatic reaction was initiated by the addition of p‐nitrophenylphosphate substrate (Sigma Chemical Co., St. Louis, MO, USA). Before each step, plates were washed three times with PBS containing 0.05% Tween 20. The absorbance at 405 nm was measured in a SpectraMax Plus 384 (Molecular Devices, San Jose, CA, USA). Each antigen‐specific antibody titer was calculated as the reciprocal of the highest dilution of the test sample that gave an absorbance greater than the cut‐off value, equal to the mean absorbance of naïve ferret nasal wash plus fifth‐fold SDs [21].
2.8. Statistical Analysis
Data analysis and visualization were performed using GraphPad Prism 8.4.3 software (GraphPad Software Inc., San Diego, CA). Differences in HI and NT titers between IVR165 and Vic361 or Fuk69 were assessed by using the Mann–Whitney U test. Comparisons of body weight, nasal viral titer, and nasal antibody titer were performed by using two‐way repeated measures analysis of variance followed by Bonferroni's multiple comparison tests.
3. Results
3.1. Antigenicity of A/Victoria/361/2011 (IVR‐165)
To confirm that the antigenicity of IVR165 differs from that of the original prototype Vic361, HI tests with post‐infection ferret antiserum raised against IVR165 or prototype Vic361 were performed (Table 1). IVR165 reacted well with the antiserum raised against prototype Vic361, with the same HI titer as the homologous titers of Vic361. However, the antiserum against IVR165 poorly inhibited Vic361, with an HI titer eight‐fold lower than the homologous titer of IVR165. These results indicate that the antigenicity of IVR165 differs from that of the cell‐propagated prototype, Vic361. Our results were consistent with the data of Skowronski et al. in which a 16‐fold reduction in HI titer using antiserum raised against the egg‐passaged virus was detected when the MDCK‐cell‐passaged prototype virus was examined [4]. In addition, the antiserum against IVR165 (genetic clade 3C.1) poorly inhibited Fuk69, which has many amino acid differences (genetic clade 3C.2a), with an HI titer 16‐fold lower than the homologous titer of IVR165 (Table 1).
3.2. Cross‐Reactivity of Human Serum Antibodies Elicited by SV Containing A/Victoria/361/2011 (IVR‐165)
To assess the cross‐reactivity of human serum antibodies elicited by the 2012/13 season influenza vaccine containing IVR165, the geometric mean titer (GMT) against vaccine virus IVR165 and its prototype virus, Vic361, was compared using HI and NT tests (Figure 1a,b). In HI tests (Figure 1a), the GMT of post‐vaccination serum antibodies of the adult group to the vaccine virus, IVR165, was 1:98.5, indicating that the vaccine induced a sufficient level of immunogenicity, whereas the GMT to the prototype virus, Vic361, was below 1:40. In the middle‐aged and elderly groups, serum samples also showed high GMT against the IVR165 vaccine virus, whereas the GMT against the prototype virus, Vic361, was significantly lower than that against the IVR165 vaccine virus. These results suggest that IVR165 may not induce sufficient reactive serum HI antibody against circulating H3N2 strains.
Figure 1.
Cross‐reactivity of post‐vaccination human serum antibodies elicited by the 2012/13 season vaccine against A/Victoria/361/2011 (IVR‐165) (IVR165) and A/Victoria/361/2011 (Vic361). Human serum samples were collected from an adult group (n = 30, age range: 20–39 y, mean = 28.9 y), a middle‐aged group (n = 30; age range, 41–58 y; mean = 47.9 y), and an elderly group (n = 30; age range, 62–102 y; mean = 83.5 y) and analyzed using hemagglutination‐inhibition (HI) (a) and neutralization (b) tests; 0.5% turkey red blood cells were used in the HI test. Results are shown as geometric mean ± geometric standard deviations. The p‐values were calculated using the Mann–Whitney test (*p < 0.05).
Similar results were obtained in the NT tests (Figure 1b). In all groups, serum samples showed extremely high GMT against the IVR165 vaccine virus, whereas GMT against prototype Vic361 was fairly low compared with that against the IVR165 vaccine virus.
Taken together, these results suggest that human serum antibodies induced by IVR165 in SV did not cross‐react with epidemic viruses because of the different antigenicity of IVR165 from that of the corresponding prototype virus, likely resulting in insufficient vaccine efficacy.
3.3. Immunogenicity of SV and WV in Ferrets
To examine the immunogenicity of SV and WV in ferrets, we immunized ferrets with either SV or WV. HI antibodies against IVR165 in 8 out of 22 immunized ferrets were detected after five immunizations with SV, and yet HI antibody titers of six out of those eight ferrets in which antibody titers were detected, were low (1:10 or 1:20, blue in Figures 2a and 3a). The remaining 14 ferrets did not develop anti‐HI antibodies (data not shown). Although we initially assumed that two immunizations were sufficient to raise HI titers, five immunizations were required for detection of HI titer. In contrast, HI antibodies against IVR165 in all eight immunized ferrets were detected after two immunizations with WV, and HI antibody titers of all ferrets were equal to or greater than 1:40 (pink in Figures 2a, 3a, and 4a). Similar trends were observed in NT antibody titers against IVR165 elicited by both SV and WV. Notably, the HI titers against the prototype virus Vic361 in the ferrets immunized with SV or WV (shown in Figure 2a), and the HI and NT titers against Vic361 in the ferrets immunized with WV (shown in Figure 3a,b), were statistically significantly lower than those against IVR165. However, only two doses of WV induced similar or even higher HI and NT antibody titers against both IVR165 and Vic361 than SV, although there was no statistically significant difference between these groups (Figures 2a,b, and 3a,b). These results suggest that the immunogenicity of WV is higher than that of SV in serologically naïve ferrets.
Figure 2.
Antibody titers against homologous virus A/Victoria/361/2011 (IVR‐165) (IVR165) in ferrets immunized with inactivated whole‐virus (WV) or split influenza (SV) vaccines. Before virus challenge, sera were collected and analyzed for hemagglutination‐inhibition (HI) (a) and neutralization (b) titers. In the HI test, 0.5% turkey red blood cells were used. The statistical analysis of differences between IVR165 and Vic361 was performed by using the Mann–Whitney test. Body weight change (c), IVR165‐specific nasal IgG (d), and IgA (e) titers were evaluated after the homologous virus challenge with IVR165. The statistical analysis was performed by using two‐way repeated measures ANOVA followed by Bonferroni's multiple comparison tests. *significant difference between non‐treated and WV‐immunized groups (p < 0.05). In each panel, the gray, blue, and pink colors indicate data from non‐treated ferrets, SV‐ immunized ferrets, and WV‐immunized ferrets, respectively. Results are shown as the geometric mean ± geometric standard deviations, except the body weight change, which is shown as the mean ± standard deviations.
Figure 3.
Antibody titers against heterologous virus A/Victoria/361/2011 (Vic361) in ferrets immunized with inactivated whole‐virus (WV) or split influenza (SV) vaccines. Before virus challenge, sera were collected and analyzed for hemagglutination‐inhibition (HI) (a) and neutralization (b) titers. In the HI test, 0.5% turkey red blood cells were used. Statistical analysis of differences between IVR165 and Vic361 was performed by using the Mann–Whitney test. Body weight change (c), Vic361‐specific nasal IgG (d), and IgA (e) titers were evaluated after the heterologous virus challenge with Vic361. Statistical analysis was performed by using two‐way repeated measures ANOVA followed by Bonferroni's multiple comparison tests. Significant differences (p < 0.05) between non‐treated and WV‐immunized, and between SV‐immunized and WV‐immunized groups are indicated by * and §, respectively. In each panel, the gray, blue, and pink colors indicate data from non‐treated ferrets, SV‐immunized ferrets, and WV‐immunized ferrets, respectively. Results are shown as the geometric mean ± geometric standard deviations, except for the body weight change, which is shown as the mean ± standard deviations.
Figure 4.
Antibody titers against heterologous virus A/Fukushima/69/2015 (Fuk69) in ferrets immunized with inactivated whole‐virus vaccine (WV). Before virus challenge, sera were collected and analyzed for hemagglutination‐inhibition (HI) (a) and neutralization (b) titers. In the HI test, 0.5% turkey red blood cells were used. Statistical analysis of differences between IVR165 and Vic361 or Fuk69 was performed by using the Mann–Whitney test. Body weight change (c), Fuk69‐specific nasal IgG (d), and IgA (e) titers were evaluated after the heterologous virus challenge with Fuk69. Statistical analysis was performed by use of two‐way repeated measures ANOVA followed by Bonferroni's multiple comparison tests. *Significant difference between the non‐treated and WV‐immunized groups (p < 0.05). Gray and pink colors in each panel indicate data from non‐treated and WV‐immunized ferrets, respectively. Results are shown as the geometric mean ± geometric standard deviations, except body weight change, which is shown as the mean ± standard deviations.
3.4. Inoculation of A(H3N2) Viruses Into the Ferrets Immunized With SV and WV From the 2012/13 Season
To examine the protective efficacy of SV and WV, immunized ferrets were infected with either IVR165, a component of SV and WV, or the prototype, Vic361. Nasal washes were collected daily until 7 days post‐infection and viral titers in nasal washes were determined. Viruses were detected in non‐treated ferrets until 5‐ or 6‐days post‐challenge with IVR165 (Table 2). Viruses were detected in ferrets immunized with both WV and SV until 2‐ and 3‐days post‐challenge with IVR165, respectively (Table 2). Both immunized groups shortened the duration of shedding of the homologous challenge virus. At 2‐days post‐challenge, there was a statistically significant difference in virus titers between the non‐treated and WV‐immunized groups (p < 0.05) (Table 2). However, there were no significant differences between the non‐treated and SV‐immunized groups or between the SV‐ and WV‐immunized groups. Although all groups showed mild body weight loss and there was no statistical significance among groups, the non‐treated group showed the greatest weight loss (6%), followed by the SV and WV groups (4% and 2%, respectively) (Figure 2c).
Table 2.
Viral shedding after challenge with A/Victoria/361/2011 (IVR‐165) (IVR165) in ferrets immunized with split influenza vaccine (SV) or inactivated whole‐virus vaccine (WV).
| Nasal wash | ||||||||
|---|---|---|---|---|---|---|---|---|
| Virus titer (log10 TCID50/100 μL) | ||||||||
| 1 dpi | 2 dpi | 3 dpi | 4 dpi | 5 dpi | 6 dpi | 7 dpi | ||
| Non‐treated | 1 | 4.3 | 4.7 | 3.5 | 2.0 | 4.5 | 3.7 | — |
| 2 | 1.7 | 4.5 | 2.3 | 2.5 | 3.5 | — | — | |
| 3 | 5.0 | 4.7 | 2.5 | — | 3.7 | 2.5 | — | |
| SV | 4 | 3.7 | 3.3 | — | — | — | — | — |
| 5 | 3.7 | 3.7 | — | — | — | — | — | |
| 6 | 3.0 | 2.5 | — | — | — | — | — | |
| 7 | 3.0 | 3.0 | 3.0 | — | — | — | — | |
| WV | 8 | 2.5 | — | — | — | — | — | — |
| 9 | 5.0 | 4.0 | — | — | — | — | — | |
| 10 | 3.0 | — | — | — | — | — | — | |
| 11 | 2.0 | — | — | — | — | — | — | |
Note: Ferrets were inoculated intranasally with 106 TCID50/mL of each virus. Viral titers were measured in nasal washes collected daily for 1 week following serial titration in Madin‐Darby canine kidney (MDCK) cells. The limit of virus detection was 100.7 TCID50/100 μL; a 1% guinea pig red blood cell solution was used in the hemagglutination‐inhibition assay.
When non‐treated ferrets were infected with prototype Vic361, viruses were detected until 5‐ or 6‐days post‐challenge, as was observed in ferrets challenged with IVR165 (Table 3). At 1‐ and 2‐days post‐challenge, there was a statistically significant difference in virus titers only between the non‐treated and the SV‐immunized groups (p < 0.05) (Table 3). Importantly, similar to non‐treated ferrets, in ferrets immunized with SV, viruses were detected until 5‐ or 6‐days post‐challenge, showing that SV did not shorten the duration of shedding of Vic361, unlike when SV‐immunized ferrets were challenged with IVR165. Viruses were detected only until 2‐ or 3‐days post‐challenge in ferrets immunized with WV, unlike in non‐treated and SV‐immunized ferrets. All groups showed a similar course of weight loss until 3‐days post‐challenge. However, after that, the non‐treated and SV‐immunized groups showed slight but further weight loss, whereas weight loss stopped in the WV‐immunized group. In the case of Vic361 infection, significant differences in body weight were observed between the WV‐ and SV‐immunized groups on days 6 and 7 post‐infection (Figure 3c). These results indicate that WV induced much better protective immunity than SV when the antigenicity of the challenge virus (which was circulating in nature) was different from that of the virus contained in the vaccine, although both SV and WV induced good protective immunity when ferrets were infected with the same virus as a component in the vaccine.
Table 3.
Viral shedding after challenge with A/Victoria/361/2011 (Vic361) in ferrets immunized with split influenza vaccine (SV) or inactivated whole‐virus vaccine (WV).
| Nasal wash | ||||||||
|---|---|---|---|---|---|---|---|---|
| Virus titer (log10 TCID50/100 μL) | ||||||||
| 1 dpi | 2 dpi | 3 dpi | 4 dpi | 5 dpi | 6 dpi | 7 dpi | ||
| Non‐treated | 1 | 3.7 | 5.3 | 2.0 | 2.7 | 3.0 | — | — |
| 2 | 3.7 | 5.3 | 0.5 | — | 3.3 | 2.5 | — | |
| 3 | 3.5 | 5.3 | 2.3 | — | 2.7 | — | — | |
| SV | 4 | 2.7 | 4.7 | 1.7 | 2.5 | 1.0 | 3.3 | — |
| 5 | 2.7 | 3.7 | 2.7 | 3.3 | 3.0 | — | — | |
| 6 | 2.7 | 4.7 | 1.7 | 3.5 | 3.7 | 2.5 | — | |
| 7 | 3.5 | 4.0 | 3.5 | 4.0 | 3.0 | 4.0 | — | |
| WV | 8 | 4.5 | 5.7 | 2.5 | — | — | — | — |
| 9 | 4.3 | 4.3 | 1.7 | — | — | — | — | |
| 10 | 3.5 | 5.5 | 0.7 | — | — | — | — | |
| 11 | 4.5 | 4.3 | — | — | — | — | — | |
Note: Ferrets were inoculated intranasally with 106 TCID50/mL of each virus. Viral titers were measured in nasal washes collected daily for 1 week following serial titration in Madin‐Darby canine kidney (MDCK) cells. The limit of virus detection was 100.7 TCID50/100 μL; a 1% guinea pig red blood cell solution was used for the hemagglutination‐inhibition assay. TCID50: 50% tissue culture infectious doses.
Given these results, whether WV conferred protective immunity against Fuk69, which showed an antigenic difference from Vic361, was investigated. Fuk69 virus belongs to genetic clade 3C2a, which has different antigenicity to 3C.1 [22], and thus is indeed antigenically different from IVR165 and Vic361 belonging to 3C.1, showing 16‐ and 4‐fold reductions, respectively, in HI titers compared with the corresponding homologous titers (Table 1). We only investigated efficacy of WV against Fuk69 because SV was not effective even against prototype Vic361. HI and NT antibodies against IVR165 in all four immunized ferrets were detected after two immunizations with WV, and the HI and NT antibody titers of all ferrets were equal to or greater than 1:40 and 1:160, respectively (Figure 4a,b). In contrast, HI and NT antibody titers against Fuk69 were statistically significantly low compared with IVR165, and two of the four animals had an HI value of < 10. Notably, HI and NT titers against Fuk69 in the sera of ferrets immunized with WV were lower than even against the prototype Vic361 (Figure 4a,b). This is again because the antigenicity of Fuk69 differs from that of IVR165 and Vic361 (Table 1). Following inoculation with Fuk69, viruses were recovered from non‐treated ferrets until 6 days post‐challenge, whereas in WV‐immunized ferrets, viruses were recovered until 4 days post‐challenge (Table 4), showing that WV shortened the duration of virus shedding by 2 days. At 4‐days post‐challenge, there was a statistically significant difference in virus titers only between the non‐treated and WV‐immunized groups (p < 0.05) (Table 4). In addition, WV‐immunized ferrets gained weight from 3 days post‐challenge, whereas non‐treated ferrets showed modest weight loss until 4 days post‐challenge, although there was no statistically significant difference between these groups (Figure 4c). These results indicate that WV confers much better protective immunity against challenge viruses, even when they are antigenically different from a virus in the vaccine.
Table 4.
Viral shedding after challenge with A/Fukushima/69/2015 (Fuk69) in ferrets immunized with inactivated whole‐virus vaccine (WV).
| Nasal wash | ||||||||
|---|---|---|---|---|---|---|---|---|
| Virus titer (log10 TCID50/100 μL) | ||||||||
| 1 dpi | 2 dpi | 3 dpi | 4 dpi | 5 dpi | 6 dpi | 7 dpi | ||
| Non‐treated | 1 | 3.5 | 4.5 | 4.5 | 4.0 | 4.0 | 1.0 | — |
| 2 | 4.0 | 4.0 | 3.0 | 4.0 | 3.5 | 2.5 | — | |
| 3 | 4.5 | 4.0 | 2.5 | 4.0 | 4.0 | 1.0 | — | |
| WV | 4 | 3.5 | 2.5 | — | 1.0 | — | — | — |
| 5 | 4.0 | 3.0 | 3.5 | 2.0 | — | — | — | |
| 6 | 4.5 | 3.5 | 4.5 | 3.5 | — | — | — | |
| 7 | 4.0 | 3.5 | 2.5 | 3.0 | — | — | — | |
Note: Ferrets were inoculated intranasally with 105.5 TCID50/mL of each virus. Viral titers were measured in nasal washes collected daily for 1 week following serial titration in Madin‐Darby canine kidney (MDCK)‐SIAT1 cells. The limit of virus detection was 100.5 TCID50/100 μL; a 1% guinea pig red blood cell solution was used in the hemagglutination‐inhibition assay. TCID50: 50% tissue culture infectious doses.
3.5. Virus‐Specific IgG and IgA Antibody Responses in the Nasal Wash
To examine the relationship between the antibody responses and the virus‐shedding period in the upper respiratory tract, titers of IgG and IgA antibodies specific to IVR165, Vic361, and Fuk69 in nasal wash samples collected after virus challenge were assessed by use of an ELISA. WV and SV groups infected with IVR165 showed higher titers of IgG and IgA than the non‐treated group from Day 4 after challenge (Figure 2d,e). A significant difference between the WV‐immunized and non‐treated groups was found only on Day 5. In ferrets infected with Vic361, the WV‐immunized group showed great induction of IgG and IgA titers from Day 5 after the challenge. By contrast, the SV‐immunized group showed delayed induction of IgG and IgA antibodies (Figure 3d,e). The non‐treated group showed weaker and similar IgG and IgA titers, respectively, compared with the SV‐immunized group (Figure 3d,e). There was a significant difference in IgA titers between the WV‐ and SV‐immunized groups and between the non‐treated and WV‐immunized groups on Day 7 (Figure 3e). By contrast, there was no apparent rapid induction of nasal IgG and IgA antibodies against heterologous virus Fuk69 in the WV‐immunized group compared with the non‐treated group (Figure 4d,e), although the nasal washes of the WV‐immunized group showed significantly higher IgG titers compared with the non‐treated group in Days 6 and 7 after Fuk69‐challenge (Figure 4d). These results indicate that intramuscular administration of WV or SV induces rapid nasal antibody responses to infection with homologous virus. Furthermore, WV shows a similar effect after infection with antigenically distinct heterologous viruses, although the effect depends on the degree of difference in antigenicity.
4. Discussion
Skowronski et al. demonstrated, using ferret antisera, that the egg‐adapted H3N2 vaccine strain, IVR165, was antigenically different not only from the prototype strain, Vic361 but also from circulating viruses because IVR165 acquired mutations at antigenic sites during egg passages, suggesting an association with low 2012/13 influenza vaccine effectiveness [4]. The current study shows similar results for antigenic analysis using ferret antisera (Table 1). In addition, we demonstrated that the GMTs of sera from humans who were vaccinated with the 2012/13 influenza (split) vaccine containing IVR165 were much lower against the prototype virus (Vic361) than against those of the homologous virus (IVR165) (Figure 1), supporting the low effectiveness of the 2012/13 influenza vaccine described by Skowronski et al. [4]. The current study also demonstrated, using naïve ferrets, that WV was more immunogenic, elicited broader cross‐reactive immunity, and conferred better protection than SV, even if the vaccine virus possessed amino acid substitutions acquired through egg adaptation. The results suggest a solution for low vaccine effectiveness by using WV even if WV contained a virus antigenically different from the prototype and circulating viruses. Although the difference in antigenicity between the vaccine and prototype viruses of A(H1N1)pdm09 and B is not as great as that of A(H3N2), A(H1N1)pdm09 and B vaccine viruses also undergo antigenic changes due to egg adaptation [3, 4]. Therefore, the use of WV could improve the efficacy of not only A(H3N2) influenza vaccines but also A(H1N1)pdm09 and B influenza vaccines.
In our study, SV showed extremely low immunogenicity in ferrets. It has been reported that SV likely has a small priming effect in naïve mouse models [23] and that HI antibody titers are less likely to be induced in infants [24]. In our study, we used naïve ferrets, and HI titers generally remained low even in ferrets that showed detectable titers. Thus, the phenomenon observed in previous studies might also apply to ferrets.
We demonstrated that intramuscular administration of SV or WV‐induced nasal IgG and IgA antibodies earlier against homologous virus (IVR165) in immunized ferrets after challenge compared with non‐treated ferrets (Figure 2d,e). During this early period, although low antibody titers were observed in both SV‐ and WV‐immunized ferrets, IVR165 was clearly eliminated (Table 2). As antibody levels subsequently increased, it is likely that significant antibodies involved in neutralization were raised during this phase. Therefore, we think that IgG and IgA contributed significantly to the elimination of IVR165 in both SV‐ and WV‐immunized ferrets because SV is known to induce little cellular immunity in naïve individuals [23]. A similar process likely occurred against Vic361 in WV‐immunized ferrets (Figure 3d,e and Table 3). In the case of Fuk69 infection, although antibodies were raised in both the non‐treated and WV‐immunized groups (Figure 4d,e), we think that the proportion of neutralizing antibodies may be higher in the WV‐immunized group than in the non‐treated group due to immunization, resulting in the earlier elimination of the virus in the WV‐treated group (Table 4).
Previous studies have shown that HI titers of 30–40 are associated with a 50% reduction in the risk of influenza infection [25, 26], but no such cut off value has been shown for NT. Therefore, the HI titer of immunized ferret sera is considered to be an important indicator for evaluating the efficacy of vaccines. Even low HI and NT titers (HI titers: 10–20, NT titers: 20–80) against the homologous virus IVR165 in SV‐immunized ferrets shortened the viral shedding period (Figure 2a,b and Table 2). However, when SV‐immunized ferrets were challenged with the antigenically different virus Vic361, no reduction of the viral shedding period was observed, although the sera of SV‐immunized ferrets exhibited 20 HI and 40 NT titers against Vic361 (Figure 3a,b and Table 3). This finding suggests that the efficacy is different in the case of similar HI and NT titers against other challenge viruses (i.e., IVR165 vs. Vic361). Consequently, given our finding that nasal IgG and IgA antibodies are linked to the suppression of virus shedding, it is difficult to evaluate the efficacy of the vaccine using only the antibody HI titer of serum.
The ferrets used in this study were serologically naïve, which may represent individuals who have not previously been exposed to influenza antigens, such as children. Upon single‐dose administration to unprimed children, WV showed superior immunogenicity to that of SV even with a smaller dose of antigen in WV than in SV [5, 6, 8, 9, 10]. Similar results in ferret experiments have shown that fewer immunizations of WV conferred similar or higher antibody titers than SV. The higher immunogenicity of WV compared to SV is likely because endogenous viral single‐strand RNAs in WV stimulate Toll‐like receptor 7 as a built‐in adjuvant [23, 27]. Therefore, WV may induce antibodies even in naïve individuals. Considering the high immune induction ability of WV, it may sufficiently induce immunity in naïve ferrets with a single dose, although ferrets were immunized with two doses of WV in our study because children under the age of 13 receive two doses of SV in Japan. SV has little to no effect in infants as it does not prime naïve children [24, 28, 29]. Therefore, WV, which has a high ability to induce nasal IgG and IgA antibodies early after infection and protect against influenza virus in the upper respiratory tract even in naïve individuals, is particularly beneficial for naïve infants who belong to the high‐risk group [30]. In addition, children play a key role in spreading influenza viruses in the community; therefore, WV helps reduce the spread of the disease to the broader population. Purification methods have been developed to reduce adverse effects [31], and a reduced dose for children could also help reduce the incidence of adverse reactions [32].
The administration of WV shortened the duration of virus shedding after challenge with Fuk69 virus, which is antigenically distinct from IVR165 (Table 1 and Figure 4a,b), compared to that of non‐treatment (Table 4). WV, but not SV, effectively induces memory CTLs, and thus induces (heterosubtypic) cross‐protective CTLs in mice and humans [11, 33]. In the present study, although the challenge virus (Fuk69) was homosubtypic, not heterosubtypic, to a vaccine virus (IVR165), serum antibodies and nasal IgG and IgA antibodies against Fuk69 were not efficiently elicited compared to those against IVR165 and Vic361 (Figure 4d,e); this is likely because the virus was antigenically distinct from IVR165 (Table 1 and Figure 4a,b). Based on our results, CTLs might be activated by vaccination with WV and play a role in virus clearance. In addition, the method of inactivation of the virus of WV likely contributed to CTL activation. Budimir et al. demonstrated that fusion‐active WV treated with β‐propiolactone more efficiently primed naïve CTLs than fusion‐inactive WV treated with formalin, although both WVs reactivated memory CTLs [34]. In the current study, we used WV treated with β‐propiolactone; therefore, our WV likely induced immunity in naïve ferrets. The lack of CTL analyses is a limitation of our study, as there were difficulties in analyzing CTLs in ferrets because of the lack of reagents when our animal study was conducted. However, we plan to analyze CTLs in ferrets in the future by using the method described by DiPiazza et al. [35].
Another limitation of our study is the lack of assessment of the effect of NA on immunity elicited by WV. Chen et al. demonstrated that WV could induce NA antibodies in humans and mice, but SV could not [36]. NA antibodies inhibit viral release from the infected cell surface [37]. Moreover, NA antibodies bound to NA at the surface of infected cells might aid in viral clearance through antibody‐dependent cytotoxicity (ADCC) and complement‐dependent cytotoxicity (CDC) [38, 39]. Therefore, it is also possible that NA antibodies induced by WV contribute to reducing the duration of viral shedding. Our results suggest that vaccination with WV is a solution for low vaccine effectiveness due to antigenic changes in HGR virus by egg adaptation during virus propagation.
Author Contributions
Conceptualization: Noriko Kishida, Masaki Imai, and Shinji Watanabe. Methodology: Noriko Kishida, Masaki Imai, Akira Ainai, Hideki Asanuma, Seiichiro Fujisaki, and Masayuki Shirakura. Investigation: Noriko Kishida, Masaki Imai, and Akira Ainai. Resources: Reiko Saito, Hideki Asanuma, Kazuya Nakamura, Tomoko Kuwahara, and Emi Takashita. Data curation: Noriko Kishida. Writing–original draft preparation: Noriko Kishida. Writing–review and editing: Noriko Kishida, Masaki Imai, and Shinji Watanabe. Visualization: Noriko Kishida and Akira Ainai. Supervision: Masato Tashiro, Takato Odagiri, and Shinji Watanabe. Project administration: Takato Odagiri and Shinji Watanabe. Funding acquisition: Takato Odagiri and Masato Tashiro. All authors have read and agreed to the published version of the manuscript.
Disclosure
The authors declare no conflicts of interest.
Ethics Statement
The study using human sera was reviewed and approved by the Ethics Committee of the Medical School of Niigata University (No. 2300) and the National Institute of Infectious Diseases (No. 747). The animal study protocol was approved by the Institutional Review Board of the National Institute of Infectious Diseases (NIID) [protocol code (date of approval): no. 112080 (2012. 5. 9), 113080 (2013. 5. 23), 213113‐2 (2013. 6.19), and 115111 (2015. 8. 14)]. Informed consent was obtained from all subjects involved in the study.
Acknowledgments
We would like to thank Fukushima Prefectural Institute of Public Health, Fukushima, Japan who provided A/Fukushima/69/2015 (H3N2) isolate. We would like to thank Dr. Y. Ami and Dr. Y. Suzaki, Division of Experimental Animal Research, National Institute of Infectious Diseases, for their excellent support in the ferret experiment; Dr. N. Shimasaki for her excellent support for SRD; and H. Sugawara, A. Sato, M. Akimoto, H. Miura, E. Nishiyoshi, and Y. Koike for their technical assistance. We would like to thank Editage (www.editage.com) and Susan Watson for English language editing. This study was supported by Grants‐in‐Aid for Emerging and Reemerging Infectious Diseases (Grant No. 10110400) from the Ministry of Health, Labor, and Welfare of Japan. We declare no potential conflict of interest relevant to this article.
Data Availability Statement
All data associated with this study are present in the paper.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Data Availability Statement
All data associated with this study are present in the paper.