Clade 2.3.4.4 H5 chimeric cold-adapted attenuated influenza vaccines induced cross-reactive protection in mice and ferrets

Weiyang Sun; Jiaqi Xu; Zhenfei Wang; Dongxu Li; Yue Sun; Menghan Zhu; Xiawei Liu; Yuanguo Li; Fangxu Li; Tiecheng Wang; Na Feng; Zhendong Guo; Xianzhu Xia; Yuwei Gao

doi:10.1128/jvi.01101-23

. 2023 Nov 2;97(11):e01101-23. doi: 10.1128/jvi.01101-23

Clade 2.3.4.4 H5 chimeric cold-adapted attenuated influenza vaccines induced cross-reactive protection in mice and ferrets

Weiyang Sun ¹, Jiaqi Xu ^1,², Zhenfei Wang ^1,³, Dongxu Li ^1,⁴, Yue Sun ^1,⁵, Menghan Zhu ^1,⁶, Xiawei Liu ^1,⁶, Yuanguo Li ¹, Fangxu Li ^1,², Tiecheng Wang ¹, Na Feng ¹, Zhendong Guo ¹, Xianzhu Xia ^1,^7,^✉, Yuwei Gao ^1,^7,^✉

Editor: Kanta Subbarao⁸

PMCID: PMC10688331 PMID: 37916835

ABSTRACT

Clade 2.3.4.4 H5Nx subtype avian influenza viruses (AIVs) have circulated in poultry and wild birds worldwide. Recently, an increasing number of H5Nx human infection cases have been reported occurred. Live attenuated influenza vaccines demonstrate more advantages than other types of vaccines, such as ease of administration, elicitation of systemic immune responses, and the ability to generate breadth protection. However, the attenuated viruses also bear the risk of reassortment with the wild-type influenza A virus. To overcome this reassortment problem, we designed and constructed Clade 2.3.4.4 H5 chimeric cold-adapted attenuated influenza vaccines (CAIVs) by introducing the hemagglutinin ectodomains of H5N6 into a cold-adapted attenuated master donor of an influenza B virus. These H5 CAIVs induce humoral antibody response, mucosal immune response, and cellular immune response in mice models. After two doses were administered in mice, H5 CAIVs provided cross-protection responses with 100% survival against wild-type Clade 2.3.4.4 H5 subtype AIVs. The immunized mice exhibited more significant reductions of lung viral titers or lung pathology than those in the mock group mice. In ferret models, Clade 2.3.4.4b and 2.3.4.4h H5 CAIVs produced a cross-protective efficacy against wild-type Clade 2.3.4.4b and Clade 2.3.4.4h H5 AIVs. The findings of the current study indicate that our H5 CAIVs may have the potential to prevent and control H5Nx influenza viruses in humans.

IMPORTANCE

Clade 2.3.4.4 H5Nx avian influenza viruses (AIVs) have circulated globally and caused substantial economic loss. Increasing numbers of humans have been infected with Clade 2.3.4.4 H5N6 AIVs in recent years. Only a few human influenza vaccines have been licensed to date. However, the licensed live attenuated influenza virus vaccine exhibited the potential of being recombinant with the wild-type influenza A virus (IAV). Therefore, we developed a chimeric cold-adapted attenuated influenza vaccine based on the Clade 2.3.4.4 H5 AIVs. These H5 vaccines demonstrate the advantage of being non-recombinant with circulated IAVs in the future influenza vaccine study. The findings of our current study reveal that these H5 vaccines can induce cross-reactive protective efficacy in mice and ferrets. Our H5 vaccines may provide a novel option for developing human-infected Clade 2.3.4.4 H5 AIV vaccines.

KEYWORDS: H5Nx influenza viruses, cold-adapted attenuated influenza vaccine, mice, ferrets, immunity

INTRODUCTION

The influenza virus threatens human health every year, posing a heavy economic burden on the global health budget every year. Seasonal influenza viruses cause 3–5 million severe cases and 300,000–650,000 deaths annually. The avian influenza viruses (AIVs) can infect several hosts, such as humans, pigs, wild birds, and poultry (1). AIVs are classified into two types based on their ability to produce diseases in chickens: highly pathogenic avian influenza virus (HPAI) and low pathogenic avian influenza virus. H5 and H7 influenza viruses belong to the HPAI subtype. The H5 influenza virus has evolved into 10 clades and continues to affect poultry and public health (2). Clade 2.3.4.4 H5Nx influenza viruses have circulated worldwide and evolved into eight subclades (3). In 2014, China reported the first case of human-infected Clade 2.3.4.4 H5N6 in the Sichuan province (4). Further, Clade 2.3.4.4b H5N8 influenza is lethal to poultry and wild birds, and Russia reported the first human case of infection in 2020 (5, 6). Over time, further instances of human infection with H5N6 influenza viruses were reported (7 – 9). The World Health Organization (WHO) had reported 84 human cases globally, including 33 deaths until 5 May 2023 (10). Most cases were identified in China. H5N1 influenza virus vaccines were manufactured as National Stockpile that were recommended by WHO. However, only a few Clade 2.3.4.4 H5Nx influenza vaccines were licensed in humans to date.

The live influenza vaccine has been developed and presents broader immune protection over the past years (11 – 13). The live influenza vaccine was administered intranasally but not intramuscularly and could induce mucosal immunity in the nasal cavity. Live influenza vaccine has been used in America and Russia for decades (14, 15). The licensed live influenza vaccines were cold-adapted, safe, and attenuated in populations (16). In 2012, the newest quadrivalent cold-adapted attenuated influenza vaccine (CAIV) was approved and contained four components, including H1N1, H3N2, and two lineages of influenza B viruses (IBVs) (17). Therefore, it could provide more broader protection against seasonal influenza viruses. However, the cold-adapted live influenza vaccine can reassort or mutate with wild-type influenza viruses, which might be detrimental to the individuals treated with the vaccine (18). The influenza A virus (IAV) contains two major surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA). Over 16 HA and 9 NA glycoproteins have been found in many hosts. When humans are infected with two or more different subtypes of IAVs, these viruses may reassort and form a new subtype (19 – 21). To alleviate these potential risks, we designed a chimeric A/B influenza vaccine based on a cold-adapted attenuated master donor of the IBV. No reassortment has been identified between the IAV and IBV to date because of the differences in their genome packaging signals (22). However, scientists have studied the packaging signals and succeeded in exchanging HA and NA genes between the IAV and IBV in the laboratory (23, 24). Although some chimeric influenza virus has been developed, most of them utilize the backbone of IAV (23). IAV backbone virus has been on the market for several years and is safe in humans.

The influenza virus can be engineered to serve as viral vectors expressing sequences encoding antigens of interest, including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), HIV, West Nile virus, and bacteria (25 – 28). Some attenuated modified strategies of the influenza virus were used to develop these vaccines, such as NS1-deletion or NS1-truncation. However, most influenza virus-based vector vaccines were the master donors of the IAV. IBV-infected a lesser number of individuals compared IAV, and the disease is milder compared to that caused by the IAV. In addition, some cold-adapted attenuated master donors of the IBV have been developed, such as B/Ann Arbor/1/66, B/USSR/60/69, and B/Leningrad/14/17/55 (29 – 32). The live attenuated influenza virus vaccine offers more advantages, such as the induction of mucosal antibodies, cell-mediated immunity, and ease of administration. To overcome potential risks and develop novel live influenza virus vaccines, we chose an A/B chimeric HA strategy and a master donor of a cold-adapted attenuated IBV. In the current study, a master donor of B/Vienna/1/99 was chosen, previously used for live attenuated influenza vaccine (33).

Herein, we designed and constructed four Clade 2.3.4.4 H5 chimeric influenza virus vaccines (mainly circulated in China) on the backbone of a cold-adapted IBV (B/Vienna/1/99 strain). The systemic immune responses were elicited and measured in mice and ferret models. Notably, these chimeric vaccines in the current study could induce cross-protection to Clade 2.3.4.4 wild-type H5Nx AIVs. Therefore, our new vaccine design strategy can potentially be used in the clinical development of the H5Nx influenza virus vaccines.

RESULTS

Construction and biological characteristics of Clade 2.3.4.4 H5 CAIVs

Herein, we designed a novel chimeric A/B live influenza vaccine. The four Clade 3.4.4.4 H5 CAIVs were constructed using the eight-plasmid reverse genetic systems of the influenza virus. These H5 CAIVs were chosen for study based on the WHO recommendation for H5 preparedness vaccines (34). Four H5 HA sequences were downloaded from the Global Initiative on Sharing Avian Influenza Data Epiflu online database as shown in Table S3. The chimeric A/B H5 CAIVs were based on a cold-adapted master donor of the IBV with the ectodomain in the HA of H5N6 wild-type influenza viruses (Fig. 1A). The signal peptide, transmembrane domain, and cytoplasmic domain were from the IBV as reported previously (33). To attenuate the pathogenicity of the H5 influenza virus, the polybasic cleavage site in the HA was changed from PLREKRRKRGLF to PLRETRGLF. We retained the last five amino acids (LDNHT) of the transmembrane domain of HA in IBV to improve the growth efficiency of the rescued viruses. Then, the chimeric A/B HA plasmid and other seven plasmids of BV99 were cocultured with 293T and Madin-Darby canine kidney (MDCK) cells (Fig. 1B). The four H5 influenza chimeric vaccines were constructed successfully and named rA/B-SC14 ca (Clade 2.3.4.4a), rA/B-FS17 ca (Clade 2.3.4.4b), rA/B-HB16 ca (Clade 2.3.4.4d), and rA/B-GD18 ca (Clade 2.3.4.4h).

Fig 1 — Study design and identification of Clade 2.3.4.4 H5 CAIVs. (A) Chimeric HA design of H5 CAIVs. (B) Flow chart of protocols in reverse genetic method of H5 CAIVs. The figure was "Created with MedPeer (www.medpeer.cn)." (C) Growth curves of H5 CAIVs in MDCK cells.

The cold-adapted temperature sensitivity characteristics of the constructed H5 vaccines were evaluated in 8-day-old embryonated chicken eggs. All four H5 CAIVs exhibited cold-adapted and temperature-sensitivity phenotypes (see Table 1). The four H5N6 CAIVs grew well at 27°C, but no virus titers were detected at 39°C. The four H5 candidates showed no more than 1,000 times virus titers at 27°C than that at 33°C. In addition, these H5 candidate vaccines showed more than 1,000 times virus titers at 33°C than those at 39°C. These results indicate that our four H5 vaccines were cold-adapted and temperature-sensitive. Additionally, we evaluated the growth curve of the four H5 candidates in MDCK cells. The results showed that the four H5 CAIVs could replicate well in MDCK cells (Fig. 1C). These four H5 candidate vaccines reached high titers in 36–48 h.

TABLE 1.

The cold adaptivity and temperature sensitivity of H5N6 candidate vaccines ^a

Virus	Virus titer [log₁₀(EID₅₀ mL⁻¹)]			RCT₃₉	RCT₂₇	Phenotype
	33°C	39°C	27°C
rA/B-SC14 ca	7.4 ± 0.1	–	5.9 ± 0.3	7.4	1.5	ts, ca
rA/B-FS17 ca	7.3 ± 0.3	–	6.2 ± 0.1	7.3	1.1	ts, ca
rA/B-HB16 ca	6.8 ± 0.1	–	5.1 ± 0.5	6.8	1.7	ts, ca
rA/B-GD18 ca	7.3 ± 0.3	–	6.1 ± 0.1	7.3	1.2	ts, ca

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^{^a}

RCT, the reproduction capacity at the restrictive temperature. RCT₃₉[log₁₀(EID₅₀)] = [log₁₀(EID₅₀ mL^-1)] at 33℃ - [log₁₀(EID₅₀ mL^-1)] at 39℃. RCT₂₇[log₁₀(EID₅₀ mL^-1)] = [log₁₀(EID₅₀ mL^-1)] at 33℃ - [log₁₀(EID₅₀ mL^-1)] at 27℃. ts, temperature-sensitive; ca, cold-adapted.

To confirm the attenuation and safety of the four H5 CAIVs, we used the BALB/c mice as a standard animal model. The findings demonstrated no severe body weight loss or death in the immunized and mock groups (Fig. 2A and B). Low virus titers were detected in the lungs on day 3, and no virus titers were detected in the lungs on day 6 (Fig. 2C). Furthermore, we detected low virus titers in the nasal turbinates on day 3. However, no viruses were replicated in the other tissues, such as the brain, heart, liver, spleen, kidney, and intestine (Fig. 2D).

Fig 2 — Safety evaluation of H5 CAIVs in mice. The mice were divided into the infected and mock groups. The lungs, nasal turbinates, and other tissues were collected, and virus titers were measured by EID₅₀. (A) Body weight changes in mice (n = 5). (B) Survivals in mice (n = 5). (C) Virus titers of lungs in mice (n = 3). (D) Virus titers of tissues in mice (n = 3). Data are shown as the mean ± SD. The limit of detection was 0.75 log₁₀ EID₅₀/g.

Vaccination with H5 CAIVs induces a cross-reactive antibody response in mice

Mice in the one-dose group (Fig. 3A) and two-dose group (Fig. 3B) were immunized with the four Clade 2.3.4.4 H5 CAIVs. We evaluated the humoral antibody responses in the mice serum and mucosal antibody responses in the lung lavage fluids. Serum was collected from the one- or two-dose group 3 weeks after inoculation with the H5 CAIVs.

When using RE-11, SC18, L1P5, XY165, and LY1 as test antigens, the results showed that we could detect different hemagglutinin inhibition (HAI) antibody titer levels in the one-dose group (Fig. 4A) and two-dose group (Fig. 4B). The HAI antibody titers in the two-dose group were higher than in the one-dose group.

The results of the MN assay showed different MN titers in the one-dose group (Fig. 4C) and two-dose group (Fig. 4D). The MN levels in the two-dose group were higher than those in the one-dose group.

We used the purified HA proteins to detect the IgG antibody titers using enzyme-linked immunosorbent assay (ELISA). When using the four HA proteins as the coating antigens, the IgG antibody titers were detected in the one-dose group (Fig. 4E) and the two-dose group (Fig. 4F). Notably, the IgG antibody titers in the one-dose group were higher than those in the two-dose group.

Furthermore, we measured the IgG antibody titers against more H5 HA proteins in the serum using protein microarrays. The protein microarray results showed that the serum from immunized mice had a cross-reactivity response against different H5 HA proteins belonging to varied clades (Fig. S1). The serum from the two-dose group demonstrated a greater response than the one-dose group. These results suggest that the H5 CAIVs developed in the current study might induce a broad cross-reactivity against different clades of H5 HA antigens.

Mucosal immunity is an advantage of live attenuated influenza vaccines. The lung lavage fluids from the immunized mice were collected 3 weeks after one- or two-dose inoculations. The findings revealed that the IgA antibody titers in the two-dose group (Fig. 4H) were higher than that in the one-dose group (Fig. 4G).

These results indicated that the H5 CAIVs induce humoral and mucosal immune responses in mice models and produce cross-reaction antibody responses against other clades of H5 influenza viruses.

Vaccination with H5 CAIVs induces cellular immune responses in mice

Cellular immunity produces long-lasting antibody responses (11). The splenocytes were isolated from the mice in all groups and then stimulated with Clade 2.3.4.4 H5 HA proteins. The enzyme-linked immunospot (ELISpot) assay was performed to determine the levels of IFN-γ or IL-4 cytokine-secreting splenocytes. The results showed that the immunized mice in the one- or two-dose groups produced significantly higher IFN-γ-secreting cells than those in the mock group (Fig. 5A and B). Similarly, more IL-4-secreting cells were observed in the immunized group than in the mock group (Fig. 5C and D). In addition, our results showed that the population of the IFN-γ-secreting cells was higher than that of the IL-4-secreting cells.

Fig 5 — Cellular immunity responses in the mice. Splenocytes were isolated from the immunized mice. The splenocytes were cultured and stimulated with the purified HA proteins of Clade 2.3.4.4 H5 candidate vaccines. ELISpot assays measured the secreted IFN-γ and IL-4 cytokines. (A) IFN-γ level in the one-dose group. (B) IFN-γ level in the two-dose group. (C) IL-4 level in the one-dose group. (D) IL-4 level in the two-dose group. ICS assays for IFN-γ⁺CD8⁺ T cells (E), IL-2⁺CD8⁺ T cells (F), and TNF-α⁺CD8⁺ T cells (G). ICS assays for IFN-γ⁺CD4⁺ T cells (H), IL-2⁺CD4⁺ T cells (I), and TNF-α⁺CD4⁺ T cells (J). All data are shown as mean ± SD. * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and **** indicates P < 0.0001.

We also measured multiple cytokine levels in the supernatants of the cultured splenocytes, which were stimulated with H5N6 HA proteins at a concentration of 10 µg/mL. The results showed an increase in the cytokine expression in the one- and two-dose groups compared to the mock group, including IFN-γ, TNF-α, IL-2, IL-4, IL-5, IL-6, and IL-13 cytokines (Fig. S2A and B). The IFN-γ, TNF-α, and IL-2 were assumed to be derived from T helper 1 (Th1) cells, while the IL-4, IL-5, IL-6, and IL-13 were likely from T helper 2 (Th2) cells.

To investigate T cell immunity, the levels of the cytokines secreted from antigen-specific T cells were measured. The cultured splenocytes were stimulated with purified H5 HA proteins. The results showed a significant difference between the IFN-γ⁺CD8⁺ T cells (Fig. 5E) and IFN-γ⁺CD4⁺ T cells (Fig. 5H). Other secreted cytokines such as IL-2⁺CD8⁺ T cells (Fig. 5F), TNF-α⁺CD8⁺ T cells (Fig. 5G), IL-2⁺CD4⁺ T cells (Fig. 5I), and TNF-α⁺CD4⁺ T cells (Fig. 5J) showed no significant difference in the splenocytes.

In summary, all the aforementioned results indicated that H5 CAIVs induce a wide cross-reactive cellular immune response.

Vaccination with H5 CAIVs reduces body weight and virus titers of the tissues after challenge in mice

To evaluate the cross-protection response of the H5 CAIVs, we challenged the mice belonging to different groups with one dose or two doses of inoculation against Clade 2.3.4.4 H5 subtype AIVs (Fig. 3A and B). When the body weights of the mice decreased over 25% in the challenge study, the mice were humanely euthanized.

When the one-dose group was challenged with SC18 (Clade 2.3.4.4a), the immunized mice showed slight weight loss in 14 days. By contrast, the mock mice showed severe body weight loss until they died (Fig. 6A). Notably, all mock mice died within 10 days. The survival rate of the rA/B-HB16 ca group mice was 80% (Fig. 6B). The two-dose group showed little body weight loss, but the mock group mice eventually showed severe body weight loss and died (Fig. 6C). The immunized mice group showed a 100% survival rate, but all mock mice died (Fig. 6D).

Fig 6 — Cross-protection of H5 CAIVs against wild-type H5Nx influenza viruses in mice. The immunized mice were divided into a one- and two-dose group. All the groups were challenged with Clade 2.3.4.4 H5Nx wild-type influenza viruses containing SC18, L1P5, XY165, and LY1. The body weight changes and survivals were observed for 14 days (n = 5). The body weight changes and survivals of mice intranasally challenged with 10 MLD₅₀ against SC18 in the one-dose group (**A and B**) and two-dose group (**C and D**). The body weight changes and survivals of mice intranasally challenged with 10 MLD₅₀ against L1P5 in the one-dose group (**E and F**) and two-dose group (**G and H**). The body weight changes and survivals of mice intranasally challenged with 10^7.5 EID₅₀ against XY165 in the one-dose group (**I and J**) and two-dose group (**K and L**). The body weight changes and survivals of mice intranasally challenged with 10^7.5 EID₅₀ against LY1 in the one-dose group (**M and N**) and two-dose group (**O and P**).

The immunized mice showed a slight body weight loss when challenged with L1P5 (Clade 2.3.4.4b). Similar to other investigations, the mice in the mock group died (Fig. 6E). The survival rate of the mock group mice was 0%, while that of the immunized group was 100%, except for the rA/B-HB16 ca group (80%, Fig. 6F). Similarly, the immunized mice showed a light body weight loss in the two-dose group, while all the mock group mice died (Fig. 6G). The mock group mice had a 0% survival rate, while that of the immunized mice was 100% (Fig. 6H).

When challenged with XY165 (Clade 2.3.4.4d), the immunized group mice showed moderate body weight loss, whereas the mock group mice showed severe body weight loss until they died (Fig. 6I). The survival rate of the mock group was 0%, while that of the immunized group was 100% (rA/B-HB16 ca and rA/B-GD18 ca groups), 80% (rA/B-FS17 ca group), and 60% (rA/B-SC14 ca group) (Fig. 6J). The immunized mice in the two-dose group showed slight body weight loss. However, all the mock group mice died, similar to that observed in the other investigations (Fig. 6K). The mock group mice showed a 0% survival rate, while the immunized mice showed a 100% survival rate (Fig. 6L).

When challenged with LY1 (Clade 2.3.4.4h), the immunized mice showed moderate body weight loss, but the mock group mice showed severe body weight loss until they died (Fig. 6M). The survival rate of the mock group was 0%, while that of the immunized mice was 100% (rA/B-HB16 ca, rA/B-FS17 ca, and rA/B-GD18 ca groups) or 60% (rA/B-SC14 ca group) (Fig. 6N). The immunized mice in the two-dose group showed slight body weight loss, while all the mock group mice died (Fig. 6O). The survival rate of the mock group mice was 0%, while the immunized group mice were 100% (Fig. 6P).

After challenging, on days 3 and 6, the mock group mice exhibited high virus titers in the lungs and nasal turbinates against SC18 (Fig. 7A through D), L1P5 (Fig. 7E through H), XY165 (Fig. 7I through L), and LY1 (Fig. 7M through P). However, the immunized mice group (one- and two-dose) exhibited zero or lesser virus titers in lungs and nasal turbinates.

On day 3, the lungs of the mice from all groups were collected, and a histological lesion observation was conducted. The hematoxylin and eosin (HE) results showed large areas of lung consolidation with hemorrhage and infiltration of inflammatory cells in the mock mice. Conversely, only bronchiolar and perivascular inflammatory cell infiltration was observed in the immunized mice (Fig. S4). The immunohistochemistry (IHC) results showed a low number of positive cells stained to a lesser extent in the immunized mice, while the number of positive cells was higher, and the degree of staining was moderate to severe in the mock group (Fig. S5).

These results showed that the four H5 CAIVs developed in this study offered a cross-protection in immunized mice. Moreover, H5 CAIVs can provide 100% protection against Clade 2.3.4.4 H5Nx AIVs in the two-dose group.

Vaccination with H5 CAIVs induces humoral and cellular immune responses in ferrets

Two groups of the ferrets were immunized with Clade 2.3.4.4b (rA/B-FS17 ca) and Clade 2.3.4.4h (rA/B-GD18 ca) H5 CAIVs, respectively. All the ferrets were inoculated with the H5 candidate vaccines on day 0 and day 21. The mock group ferrets were inoculated with PBS. We evaluated the humoral antibody responses in the serum and cellular antibody responses in the splenocytes. Serum was collected on days 7, 14, 21, and 42 after inoculation with the H5 CAIVs.

The serum from ferrets was used to measure humoral antibody responses. The MN assay results showed moderate antibody titers in the ferret serum (Fig. 8A through D). Using SC18 or L1P5 as test antigens, the rA/B-FS17 ca group ferrets showed the MN titers on days 21 and 42 (Fig. 8A and B). When using XY165 as test antigens, the rA/B-GD18 ca group ferrets showed the MN titers on day 42 (Fig. 8C). When using LY1 as test antigens, the rA/B-GD18 ca group ferrets produced the MN titers on days 21 and 42 (Fig. 8D).

Fig 8 — Humoral antibody responses in the serum of ferrets. The ferrets were immunized with 10⁵ EID₅₀ of rA/B-FS17 ca, rA/B-GD18 ca, or PBS. On days 7, 14, 21, and 42, the serum in the ferrets was collected and used to detect the HAI antibody, MN, and IgG ELISA titers. MN titers in the ferrets against SC18 (A), L1P5 (B), XY165 (C), and LY1 (D). IgG titers in the ferrets detected by ELISA against SC14 (E), FS17 (F), HB16(G), and GD18 (H). HAI antibody titers in the ferrets against SC18 (I), L1P5 (J), XY165 (K), LY1 (L), and RE-11 (M). The data were analyzed by two-way ANOVA methods. * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and **** indicates P < 0.0001.

The IgG titers in the serum of ferrets from all groups were measured against Clade 2.3.4.4 H5 HA proteins, which were used as coating antigens. The results showed that H5 CAIVs induced the IgG ELISA titers on days 7, 14, 21, and 42 (Fig. 8E through H). Notably, the mock ferrets had a lower detection limit than all the immunized groups.

When detecting the HAI antibody titers, we utilized SC18, L1P5, XY165, LY1, and RE-11 as test antigens. The HAI assay results showed moderate antibody titers (Fig. 8I through M).

The ELISpot results showed an increase in the levels of IFN-γ-secreting cells when using different Clade 2.3.4.4 antigens (SC14, FS17, HB16, and GD18). In the challenge study with wild-type L1P5, the IFN-γ levels in the immunized group showed a significantly higher increase than those in the mock group (Fig. 9A). Moreover, in the case of the LY1 challenge study, the IFN-γ levels were significantly increased, particularly in the rA/B-GD18 ca group (Fig. 9B). In addition, we measured the IFN-γ cytokine levels in the supernatants of the splenocytes. The IFN-γ cytokine levels against L1P5 or LY1 were increased compared to the mock group (Fig. 9C and D).

Fig 9 — Cellular immune response in ferrets. The splenocytes were isolated from the group of ferrets to measure the secreted IFN-γ cytokine levels. The purified HA proteins belonging to H5 CAIVs were used as test antigens. IFN-γ-secreting cells of the splenocytes challenged with L1P5 (A) or LY1 (B). The IFN-γ cytokines in the supernatants challenged with L1P5 (C) or LY1 (D). The data are shown as mean ± SD. The data were analyzed by one-way ANOVA methods. * indicates P < 0.05, ** indicates P < 0.01, *** indicates P < 0.001, and **** indicates P < 0.0001.

In summary, these results indicated that H5 CAIVs induced a humoral immune response in ferret models.

Vaccination with H5 CAIVs reduces virus titers in ferrets

Two representative Clade 2.3.4.4b and 2.3.4.4h H5 candidate vaccines that circulated in China were chosen to study in ferrets. To evaluate the immunogenicity and cross-protection of two H5 CAIVs in ferrets, we challenged the ferrets with L1P5 and LY1 wild-type H5 AIVs. The design of the ferret study is shown in Fig. 10A.

Fig 10 — Cross-protection in the ferrets against wild-type Clade 2.3.4.4b and 2.3.4.4h H5 AIVs. The ferrets were immunized and inoculated with rA/B-FS17 ca, rA/B-GD18 ca, or PBS. On day 42, all the ferrets were challenged with L1P5 and LY1. The nasal fluid washes and throat swabs were titered against L1P5 and LY1 following by EID₅₀ protocol. The ferrets were euthanized, and tissues were collected on day 47. Immunogenicity and protective efficacy study design (A). Body weight loss (B) or body temperatures (C) of the ferrets challenged with L1P5. Virus titers in the ferrets against L1P5 from the nasal fluid swabs (D) and throat swabs (E). The ferrets were challenged with LY1 and observed for body weight loss (F) or body temperature changes (G). Virus titers in the ferrets against LY1 from the nasal fluid swabs (H) and throat swabs (I). The tissues were collected and titered when challenged with L1P5 (J) or LY1 (K). The data were shown as the mean ± SD. The data were analyzed by one-way ANOVA with Dunnett’s multiple comparisons test or Kruskal-Wallis with Dunn’s multiple comparisons test. * indicates P < 0.05. ** indicates P < 0.01, *** indicates P < 0.001, and **** indicates P < 0.0001.

We evaluated the safety of the H5 CAIVs in ferrets. The results are shown in Fig. S6. The ferrets inoculated with rA/B-FS17 ca or rA/B-GD18 ca vaccines showed no body weight loss and a maximum body temperature of 39.5°C (Fig. S6A and B). Fewer virus titers were observed in the nasal fluid washes and throat swabs (Fig. S6C and D). No virus was detected in the tissues of ferrets, including the lung, nasal turbinate, trachea, heart, brain, liver, spleen, kidney, and intestine (Fig. S6E).

When challenged with L1P5, slight body weight loss was observed in the immunized ferrets, while the mock ferrets showed a severe body weight loss of up to 7%–8% (Fig. 10B). The body temperatures in the immunized ferrets were no more than 39.5°C. However, in the mock group of ferrets, a significant increase in body temperature up to 40°C was observed (Fig. 10C). Furthermore, the nasal fluid washes and throat swabs were collected from the mice in all groups and titered. No virus titers were obtained from the nasal fluid washes and throat swabs from the immunized ferrets (Fig. 10D and E). However, virus titers were detected in the mock ferrets from days 43 to 47 (days 1 to 5 post challenge infection).

When challenged with LY1, slight body weight loss was observed in the immunized ferrets, while a severe body weight loss was observed in the mock ferrets (Fig. 10F). The body weight loss of the ferrets was not as severe as observed in the mice model. This may be because the ferrets were challenged with HPAI that lacked the known signatures of mammalian adaptation. The body temperatures in the immunized mice were below 39°C, and an increase in body temperatures up to 40°C was observed in the mock group (Fig. 10G). No virus titers were observed in the nasal fluid washes and throat swabs in the immunized ferrets. However, the virus titers were obtained from the nasal fluid washes (Fig. 10H) and throat swabs of the mock ferrets (Fig. 10I).

After the ferrets were challenged, the virus titers of the tissues were evaluated on day 5. High virus titers were obtained in the lungs, nasal turbinates, and tracheas in the mock ferrets, while no virus titers were detected in the immunized ferrets against L1P5 (Fig. 10J) or LY1 (Fig. 10K). Moreover, lower virus titers were detected in the liver, spleen, and intestine from the mock group.

In summary, our results showed that the H5 vaccines reduced the virus titers of the tissues in the ferrets and provided a cross-protection against different H5 wild-type AIVs.

DISCUSSION

Over the past decade, Clade 2.3.4.4 H5Nx AIVs have become the primary circulating subtype in poultry and wild birds worldwide. Human-infected H5N6 cases started occurring in 2014, and most identified cases have a contact history in living poultry markets (8, 35). Most prepandemic Clade 2.3.4.4 H5Nx influenza vaccines in humans were studied in a preclinical setting (36, 37) using inactivated vaccines, live influenza vaccines, virus-like particle vaccines, and virus-vector vaccines. However, high possibilities exist that they do not match the new circulating clades of H5 subtype influenza viruses.

The live attenuated influenza vaccines have the advantage of providing a pan-protection immune response. In recent H5Nx live influenza candidate vaccines, nearly all the master donors were from IAV. Therefore, these candidate vaccines may bear the concern regarding reassigning H5N1, H5N6, H1N1, and other circulating IAVs. Herein, we designed a novel chimeric Clade 2.3.4.4 H5 CAIVs based on cold-adapted IBV donors. Four representative Clade 2.3.4.4 H5 influenza viruses were selected in China, and their antigenic relatedness was found to be different. No natural reassortment between IAV and IBV has been reported to date. Hence, our vaccine cannot reassort with circulating H5Nx influenza viruses in humans and poultry, delivering greater safety than the IAV master donors. In addition, some reports indicate that the chimeric viruses exhibit a more attenuated phenotype than the wild-type influenza viruses (over 10-fold). Therefore, our chimeric vaccine afforded a lower risk of reassortment when coinfected with wild-type IBV.

Four Clade 2.3.4.4 H5 CAIVs developed herein can provide cross-protection after two doses immunization in mice. However, when immunized with one dose of H5 CAIVs, the mice only had partial protection. Several factors may be responsible for this phenomenon. First, this may result from the lack of parent wild-type H5Nx AIVs. The amino acids in the HA of the H5 influenza virus influence antigenicity and immunogenicity (38). Some amino acids in the HA of the four Clade 2.3.4.4 challenge influenza viruses were different from those of the four Clade 2.3.4.4 H5 candidate vaccines. Therefore, these differences may influence the antigenicity of the H5 CAIVs. Second, our H5 candidate vaccines produce stronger antibody responses on two-dose inoculation than those obtained after one-dose inoculation. Hence, the two-dose group mice showed broader protective efficacy.

Mucosal vaccines have been widely used and involve an interesting strategy in vaccine development. IgA antibodies at the mucosal surfaces provide broad protection. Live influenza vaccines induce IgA antibody responses following immunization (39, 40). Notably, nasal IgA antibodies have been detected in children or adults inoculated with live cold-adapted attenuated influenza vaccines. Moreover, influenza virus vector-based intranasal SARS-CoV-2 vaccine induces IgA antibodies in animal models (41). Our Clade 2.3.4.4 H5 CAIVs induced moderate IgA antibodies in the lung fluids of mice in this study (Fig. 4G and H). This can properly offer an advantage when these H5 influenza vaccines are used in humans.

The live influenza vaccines also incite cellular immune responses. Cellular immunity provides broader protection against pathogens. Some reports revealed that the live influenza vaccines induce CD4⁺ and/or CD8⁺ T-cell immunity after vaccination (11, 42). Herein, we detected IFN-γ and other cytokines by the intracellular cytokine staining (ICS) assay. We tested the cellular immune responses 10-day-post-immunization when the T-cell immunity was expected to attain higher levels. In mice models, H5 CAIVs produce CD4⁺ and CD8⁺ T-cell cellular immunity against special H5 antigens. However, the ICS assays were not conducted in the ferret model because of the lack of adequate standard immunologic reagents for ferrets and the complexity of operations. Instead, we used ELISpot methods to measure the IFN-γ cytokines in the splenocytes of ferrets. Recently, the tissue-resident memory T cells have been studied in influenza vaccines (43 – 45). The live influenza vaccines produce lung-resident memory T cells for heterologous protection. Therefore, in-depth research on lung-resident memory T cells needs to be conducted in the future.

Different types of vaccines demonstrate their unique advantages. Inactivated influenza vaccines offer protection primarily through neutralization antibodies. The live influenza vaccines produce moderate humoral antibodies, but the antibody titers may be lower than that of the inactivated influenza vaccines. To detect the humoral antibody responses induced by H5 CAIVs, the mice sera were collected and tested for HAI, MN, and IgG antibody titers. Different subclades or commercial antigens were chosen as test antigens in these assays to test the cross-protection in the mice model. The obtained results suggested that H5 CAIVs produce cross-reactive antibodies. Furthermore, they indicate that using the same subclade H5 antigens may produce cross-humoral antibody titers. Therefore, we used a protein microarray assay to measure cross-reactive antibodies against different subclades of H5 HA antigens. The protein microarray methods have been used in the influenza vaccination of humans and SARS-CoV-2 serum studies (46, 47). Unlike ELISA, these are high-throughput detecting methods and facilitate a better understanding of the specificity of the cross-reactive responses. Higher cross-reactive HA IgG antibody levels were produced by four H5 CAIVs, which indicate that we may modify the HA structure to design a novel universal H5 candidate vaccine against different clades of H5Nx AIVs.

The protective efficacy results from the mice and ferret studies showed cross-protection when challenged with the same subclade of H5Nx wild-type AIVs. These conclusions were confirmed by the virus titers from the lungs and results obtained from studying the pathology of the lungs. The ferrets are the standard testing animal models in evaluating the influenza vaccine recommended by WHO. However, in the preclinical study of some influenza vaccines, nonhuman primate animal models presenting human-like clinical symptoms were used (48, 49). We were not able to use such animal models because of some limitations such as the high prices and requirement of special facilities and cautionary measures, particularly after the SARS-CoV-2 pandemic. However, in future studies, we may test the protective efficacy of these H5 CAIVs in nonhuman primates. Currently, the development of universal influenza vaccines is one of the primary strategies in influenza vaccine research. The live influenza vaccine may also be used as a universal influenza vaccine (13, 50, 51). Our H5 CAIVs could induce systemic immune responses; therefore, we plan to study these H5 CAIVs against other Clade H5 wild-type AIVs in future studies. This will provide valuable insights toward studying the broad-spectrum H5 influenza virus vaccines.

In summary, we designed novel chimeric H5 CAIVs and studied their safety, immunogenicity, and protective efficacy in mice and ferret models. Our findings reveal that H5 CAIVs provide cross-protection in animal models. Therefore, our vaccine could be a novel H5 candidate vaccine that can be used to combat the Clade 2.3.4.4 H5Nx pandemic.

MATERIALS AND METHODS

Cells, proteins, and viruses

MDCK and human embryonic kidney cells (293T) were obtained from American Type Culture Collection (ATCC, USA) and cultured as described in a previous report (33). MDCK cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, USA) supplemented with 5% fetal calf serum (FCS). 293T cells were cultured in DMEM supplemented with 10% FCS, with 2 mM glutamine, 10 mM HEPES, and 100 mg/mL streptomycin or 100 IU/mL penicillin. The B/Vienna/1/99 (BV99) virus is a public cold-adapted master donor virus for IBVs that was made public in a patent (GenBank number: AX399742–AX399749) (52). Four Clade 2.3.4.4 H5N6 influenza candidate vaccine viruses circulating in China (Clade 2.3.4.4a, Clade 2.3.4.4b, Clade 2.3.4.4d, and Clade 2.3.4.4h) were selected, which were recommended by WHO global strategy for pandemic preparedness. The purified HA proteins expressed in the insect expression systems of the four Clade 2.3.4.4 H5 candidates were purchased from Sino Biological. The RE-11 antigen for hemagglutinin inhibition (HAI) assays was purchased from the Harbin National Engineering Research Centre of Veterinary Biologics Corporation in China. We propagated influenza viruses in 8- to 10-day-old embryonated chicken eggs for the IBV and in 9- to 11-day-old embryonated chicken eggs for the IAV. In addition, four wild-type Clade 2.3.4.4 H5Nx AIVs were used in this study. The Clade 2.3.4.4a H5N6 influenza virus A/cat/Sichuan/SC18/2014 (abbreviated as SC18) was isolated in 2014 from a cat in Sichuan Province, China (53). The mouse-adapted Clade 2.3.4.4b H5N8 A/mallard/Shanghai/SH-9/2013 (abbreviated as L1P5) was obtained after five passages in BALB/c mice (54). The Clade 2.3.4.4d H5N6 influenza virus A/poultry/China/xy165.4/2016 (abbreviated as XY165) was isolated in 2016 in Hubei province, China (55). Furthermore, the Clade 2.3.4.4h H5N6 influenza virus A/Whooper Swan/Xinjiang/ly1/2020 (abbreviated as LY1) was isolated in 2020 in Xinjiang province, China.

Generation of chimeric cold-adapted vaccine by reverse genetics

The eight-plasmid reverse genetics system of BV99 was constructed by a pBD vector (a bidirectional transcription vector) as previously described (33). The HA of the H5N6 candidate vaccine viruses was modified by deleting the multipolybasic cleavage amino acids and obtaining the ectodomain of H5 HA with the signal peptide, the transmembrane domain, and cytoplasmic domain of BV99. To improve the efficacy of the rescued virus, we retained the last five amino acids of IBV. Infusion cloning methods were used to construct the chimeric A/B HA plasmids and the primers are shown in Table S1. In brief, the chimeric HA plasmid and the other genes of BV99 were cotransfected with 293T cells and MDCK cells at 33°C with 5% CO₂. Each plasmid of 600 ng was cotransfected with lipofectamine 3000 in a volume of 10 µL in six-well plates. After 12 h incubation, the liquids in the plates were replaced with Opti-MEM with 0.2 µg/mL TPCK (L-1-tosylamide-2-phenylethyl chloromethyl ketone)-treated trypsin. The six-well plates were incubated 3 days at 33°C with 5% CO₂. Then, the supernatants were collected and inoculated in MDCK monolayer six-well plates or 8- to 10-day-old embryonated chicken eggs. To confirm the evaluation of the rescued viruses, 1% chicken red blood cells were used. The four H5 influenza chimeric vaccines are abbreviated as rA/B-SC14 ca (Clade 2.3.4.4a), rA/B-FS17 ca (Clade 2.3.4.4b), rA/B-HB16 ca (Clade 2.3.4.4d), and rA/B-GD18 ca (Clade 2.3.4.4h). Furthermore, the whole genomes of the rescued vaccines were confirmed using PCR amplification and Sanger sequencing. The PCR amplification primers are listed in Table S2.

Biological characteristics and growth kinetics of H5 CAIVs

We evaluated the cold-adapted phenotype, temperature sensitivity phenotype, and growth curves of the rescued H5 candidate vaccines. The temperature sensitivity and cold-adapted phenotype were determined by 50% egg infective dose (EID₅₀) titers at different temperatures (27, 33, and 39℃). The virus was considered to be temperature-sensitive when the virus titer at 39°C was lower by three log₁₀ EID₅₀ than that at 33°C. The viruses were considered cold-adapted if the titer was no more than three log₁₀ EID₅₀ lower at 27°C that at 33°C.

The growth curves were evaluated by 50% tissue culture infectious doses (TCID₅₀) in 12-well MDCK monolayer plates. The plates were cultured with the rescued vaccines at a multiplicity of infection of 0.01. The supernatants were collected at different intervals (12, 24, 36, 48, 60, and 72 h). Four H5 candidate vaccines were incubated in 96-well MDCK monolayer plates for 3 days at 33°C with 5% CO₂ . After incubation, the supernatants of each well were collected, and the final results were determined using 0.5% chicken red blood cells.

Mouse study design

The specific-pathogen-free, 4–6 weeks old, female BALB/c mice were used for the evaluation of safety, immunogenicity, and protective efficacy of four Clade 2.3.4.4 H5 CAIVs. The mice were purchased from the Experimental Animal Center of Charles River. They were anesthetized using isoflurane. When evaluating the safety of the four H5 CAIVs, the immunized group mice (n = 11) were intranasally inoculated at 10⁵ EID₅₀ in a volume of 50 µL. The mice in the mock group (n = 5) were intranasally inoculated with an equal volume of PBS. Body weight changes and survival in 14 days following inoculation were recorded for all the mice groups. The mice were euthanized on days 3 and 6, and the lung tissues were collected. On day 3, the nasal turbinate, brain, heart, liver, spleen, and intestine of the euthanized mice were also collected . The virus titers of the tissues were determined using EID₅₀ in 8- to 10-day-old embryonated chicken eggs.

The mice were divided into one- and two-dose groups to study the immunogenicity. In the one-dose group, the immunized group of mice was inoculated intranasally at 10⁵ EID₅₀ in a volume of 50 µL, 10 mice per group. The mock group mice (n = 10) were intranasally inoculated with the same volume of PBS. In the two-dose group, the immunized group mice (n = 10) were intranasally inoculated at 10⁵ EID₅₀ in 50 µL at day 0 and day 21. The mice in the mock group (n = 10) were inoculated with PBS. The samples were collected on day 21 from the one-dose group or day 42 in the two-dose group. The blood was collected from the retro-orbital plexus after anesthesia and used to detect HAI, microneutralization (MN), and IgG antibodies. In addition, the lung lavage fluids were collected to detect mucosal IgA antibodies. After anesthesia, an 18G intravenous indwelling cannula was introduced into the trachea, and 500 µL PBS was injected into the lungs. Then, the syringe was pulled out, and the lungs were massaged carefully. The splenocytes were collected using ELISpot and Luminex assays to detect cellular immunity responses. Furthermore, the intracellular cytokine staining assay was conducted in the splenocytes after 10 days of booster immunization.

The mice in the one- and two-dose groups (n = 11) were challenged with H5Nx wild-type influenza viruses (10^3.63 EID₅₀ for SC18, 10^2.25 EID₅₀ for L1P5, 10^7.5 EID₅₀ for XY165, and 10^7.5 EID₅₀ for LY1) in a 50 µL volume. After the challenge, the mice in all groups were observed for 14 days, and their body weight and survival were recorded. The mice were euthanized on day 3 and day 6, and their lungs and nasal turbinates were collected to detect the virus titers. On day 3, the lungs from the two-dose group mice were collected in 10% formalin/PBS to perform histological lesion assays. The lungs were stained with HE, and IHC staining was also performed. The tissue sections were incubated with the primary antibody of anti-Influenza A Virus nucleoprotein antibody (AA5H) (1:1,000, ab20343; Abcam). When the body weights of the mice decreased by 25%, the mice were humanely euthanized.

Ferret study design

Female ferrets (4- to 6-month-old) were purchased from Wuxi Cay Ferret Farm, Jiangsu, China. In this study, the ferret serum was measured for antibodies against the IAV using the HAI assay. All ferrets had free access to standard ferret food and water. The ferrets were anesthetized with 1 mg/kg Zoletil 50 (Virbac, Carros, France) and 1 mg/kg xylazine when inoculating with H5 candidate vaccines and wild-type H5Nx influenza viruses. Temperature chips (UID, USA) were transplanted in the ferrets to record the body temperature. Then, we evaluated Clade 2.3.4.4b and 2.3.4.4h H5 CAIVs in the ferret model. In the safety evaluation experiment, the immunized ferrets (n = 6) were intranasally inoculated with 10⁵ EID₅₀ in 500 µL (250 µL in each nostril). The mock group ferrets (n = 6) were intranasally inoculated with 500 µL PBS (250 µL in each nostril). On day 3, the ferrets (n = 3) were euthanized, and tissues were collected from the lungs, nasal turbinates, trachea, brain, heart, liver, spleen, kidney, and intestine. The virus titers of the tissues were determined using EID₅₀. We observed the body weights and body temperatures of the ferrets for 14 days. Furthermore, we collected nasal fluid washes and throat swabs on days 1, 3, 5, 7, and 9.

For the immunogenicity and protective efficacy experiments, the ferrets were divided into three groups (n = 4): rA/B-FS17 ca group, rA/B-GD18 ca group, and mock groups. The ferrets were intranasally inoculated with 10⁵ EID₅₀ of H5 CAIVs in 500 µL (250 µL in each nostril) on days 0 and 21. The mock group was inoculated with an equal volume of PBS. The serum was collected on days 7,14, 21, and 42. We used a needle and syringe in blood collection tubes and drew the blood from the anterior vena cava of ferrets. After the second immunization on day 21, all the groups were challenged with H5Nx wild-type influenza viruses (10^6.0 EID₅₀ of A/mallard/Shanghai/SH-9/2013 H5N8 AIV and 10^7.5 EID₅₀ of A/Whooper Swan/Xinjiang/ly1/2020 H5N6 AIV) in a volume of 500 µL (250 µL in each nostril). L1P5 was a mouse-adapted AIV that was lethal to mouse. However, because L1P5 was not lethal to ferrets, the ferrets were infected with a higher inoculation amount in the challenge study. After the challenge, the body weights and temperatures were recorded, and nasal fluid washes, and throat swabs were collected from all groups. On day 5, all the ferrets were euthanized, and the lungs, nasal turbinates, and other areas (the trachea, brain, heart, liver, spleen, kidney, and intestine) were collected for virus load detection. The splenocytes of the ferrets were obtained and used to detect the IFN-γ-secreting cells using the ELISpot assay and IFN-γ cytokine levels in the supernatants using ELISA.

Virus titrations in MDCK cells, embryonated chicken eggs and tissues

The H5 influenza candidate vaccines or wild-type H5Nx AIVs used in this study were titrated in MDCK cells or 8- to 10-day-old embryonated chicken eggs. Virus titers of the tissues were measured. The tissues in mice or ferrets were collected at different time points and homogenized with the TissueLyser (QIAGEN, Germany) in 1 mL of DMEM, followed by centrifugation for 10 min at 5,000 rpm. We also used a 10-fold dilution series of homogenization with DMEM (containing 1% antibiotic-antimycotic mixture) in a volume of 100 µL and inoculated 8- to 10-day-old embryonated chicken eggs. The IAVs were titered and incubated for 48 h at 37°C, but IBV needed to be incubated for 48–72 h at 33°C. Then, the embryonated chicken eggs were stored for 4–6 h at 4°C. The allantoic fluids were harvested after 4–6 h and used to detect the final results of HA titers. The HA assays were performed with 0.5% chicken erythrocytes. The virus titer was calculated using the Reed and Muench method (33).

To evaluate the end-point viral titration of the H5 candidate vaccines in MDCK cells, the monolayers of cells were prepared in 96-well culture plates. The vaccines were diluted by 10 folds and inoculated into the plates with 100 µL/well. Then, the plates were incubated at 37°C for IAV or 33°C for IBV for 1 h under 5% CO₂. After incubation, the plates were supplied with 200 µL/well Opti-MEM with 2 µg/mL TPCK-treated trypsin and incubated with 5% CO₂ for 2–3 days. The final results were confirmed positively by the HA assays with 0.5% chicken erythrocytes. The virus titers of the H5 candidate vaccines were calculated according to the Reed and Muench method.

Hemagglutinin inhibition assay and microneutralization assay

The HAI assay was conducted as previously described (33). The serum of mice or ferrets was collected and pretreated with receptor-destroying enzymes (RDE, Denka Seiken, Tokyo, Japan) for 16–18 h at 37°C and then heat-inactivated for 30 min at 56°C. The commercial influenza antigen RE-11 belongs to Clade 2.3.4.4d. The inactivated H5 wild-type viruses were used as test antigens in the HAI assay. The serum was twofold serially diluted in PBS in a V-bottom, 96-well microplate. The diluted serum and four HA units were incubated at 22–25°C for 30 min. Furthermore, 0.5% chicken red blood cells were used to determine the HAI assay titers.

The MN antibody titers were determined as described in a previous report (33). The serum was handled with RDE (1:4) and mixed with H5Nx wild-type influenza viruses (SC18, L1P5, XY165, and LY1) at 100 TCID₅₀ for 1 h at 37°C. Then, MDCK monolayer cells in 96-well plates (5 × 10⁵ cells/well) were washed with a reduced serum medium (Opti-MEM), and 100 µL mixed samples were added per well at 37°C and incubated for 1 h. After incubation, the liquids in the plates were discarded and replaced with Opti-MEM, containing streptomycin, penicillin, and 2 µg/mL TPCK-treated trypsin under a 5% CO₂ atmosphere for approximately 2–3 days. The titers of neutralizing antibodies were defined as the highest dilution of serum that led to complete neutralization, as determined by the HA test. The HA assays were performed using 0.5% chicken red blood cells.

Enzyme-linked immunosorbent assay

ELISA was used to measure the IgG or IgA antibodies in serum or lung lavage fluids and evaluate the cytokine levels in the supernatants of splenocytes in mice or ferrets. Purified HA proteins were used as capture antigens in microplates. The standard operating procedures in the ELISA were performed as previously described (33). In brief, microplates (high-sorbent, 96-well plate) were coated with 100 µL/well of 5 µg/mL H5 vaccine HA protein at 4°C overnight. Thereafter, the coated plates were washed with PBS-T and incubated with 1% bovine serum albumin in PBS for 1 h at 37°C. The serum was diluted 1:20 for IgG antibody detection, and the lung lavage fluids were diluted 1:10 for IgA antibody detection. The plates were washed three times with PBS-T (150 µL/well). Then, the plates were incubated with goat anti-mouse IgG H&L (1:100,000, ab6789; Abcam), goat anti-mouse IgA H&L (1:100,000, ab97235; Abcam) , or goat Anti-Ferret IgG H&L (1:100,000, ab112770; Abcam) for 1 h at 37°C. After incubation, the plates were washed three times with PBS-T. Thereafter, the tetramethylbenzidine substrate solution was added, and after 30 min, a stopping solution was added. The optical density was measured at 450 nm with a plate reader (Bio-Rad, Hercules, CA, USA). Notably, wells were considered positive when the OD₄₅₀ value was at least twofold greater than that of the control wells. The IgA and IgG antibody titers were determined by measuring the OD₄₅₀ of the highest dilution of the serum with OD₄₅₀.

Protein microarray assay

Serum was collected from the immunized mice on day 21 in the one-dose group or on day 42 in two-dose groups. The protein microarray assay was used to detect the IgG antibodies in the serum. The different clades of H5 influenza HA proteins were purchased from Sino Biological Inc. (Beijing, China) and Cambridge Biologics (Suzhou, China). The experimental procedures were conducted as previously described (33). In brief, the HA antigens were reconstituted at a 10 µg/mL concentration with PBS and then printed on the microarray slides. Thereafter, the arrays were incubated with a blocking buffer (PBS with 0.001% Tween-20 and 1% BSA) for 30 min, followed by washing the arrays three times with TBST buffer (20 mM Tris-HCl, pH 7.6, 150 mM NaCl, 0.05% Tween-20). The diluted serum (1:300) was added to the arrays (100 µL/slide), and the arrays were sealed and incubated for 1 h at 60 rpm in the dark. The goat-anti-mouse IgG H&L (Cy5 ) preadsorbed (ab6563; Abcam) was added and incubated for 30 min. After incubation, the arrays were washed three times with 100 µL TBST per washing, air-dried, and centrifuged at 60 rpm for 10 min. Afterward, the slides were imaged using an ArrayCam imager to measure the fluorescence signals. Mean fluorescence intensity was used to analyze the final results. Data analysis was performed using the GraphPad Prism software.

ELISpot assay and Luminex assay

The ELISpot assay in the splenocytes of mice or ferrets was performed to assess the IFN-γ or IL-4 cytokine-secreting cell levels. The splenocytes were isolated from mice or ferrets at different time points and resuspended in 10% FCS RPMI 1640 medium. Precoated IFN-γ or IL-4 ELISpot plates (Mabtech AB, Stockholm, Sweden) were incubated with splenocytes at 1 × 10⁶ cells/mL. The purified H5 HA proteins were used to stimulate at a final concentration of 10 µg/mL in 96-well plates. The plates were washed four times with sterile PBS and incubated with 10% FCS RPMI 1640 medium for at least 30 min at 22–25°C. Then, the plates were incubated for 36 h at 37°C. Thereafter, the plates were washed five times with PBS and developed by adding the detection antibody BVD6-24G2-biotin (dilution, 1:1,000), R4-6A2-biotin (dilution, 1:1,000), or MTF19-biotin (dilution, 1:1,000) for 2 h, followed by the streptavidin-ALP (dilution, 1:1,000) and BCIP/NBT-plus. Next, the plates were air-dried and stored in the dark at 22–25°C, and spots were read after 1 week by an ELISpot reader (Multispotreader Spectrum, AID, Strasberg, Germany).

Luminex assay was performed to measure the secreted cytokine levels in the supernatants of splenocyte cultures in all groups. The splenocytes of mice were isolated as described in the case of ELISpot assay. The splenocytes in six-well plates were stimulated by purified H5 HA proteins at a final concentration of 10 µg/mL. After incubation under a 5% CO₂ atmosphere at 37°C, the culture supernatants were collected, centrifuged at 2,000 rpm for 10 min, and stored at −80°C. The supernatants of splenocytes were measured by a Th1/Th2 Cytokine 11-Plex Mouse Panel kit (Cat. No. EPX110-20820-901, Invitrogen) using a Luminex 200 system (Luminex, USA).

Intracellular cytokine staining assay

The intracellular cytokine staining (ICS) assay was performed using flow cytometry. The spleens from the mice from all the groups were sampled and isolated on day 10 after the second immunization. The splenocytes were cultured at 5 × 10⁵ cells/well for 16–18 h in the 96-well U plates. The purified HA proteins from H5N6 at a final concentration of 10 µg/mL were used as stimulating antigens. The plates were treated with 1 µg/mL of the protein transport inhibitor BD GolgiPlug (555029, BD Biosciences). Thereafter, the plates were washed and added the solution in the Zombie NIR Fixable Viability Kit (423106, Biolegend), and the plates were kept at 4° for 30 min in the dark. Then, the plates were washed the staining buffer three times (200 µL/well). The splenocytes were then stained with antibodies containing APC anti-Mouse CD45 (147708, Biolegend), mCD4-R711-FITC (50134-R711-F, Sino Biological), and Brilliant Violet 785 anti-Mouse CD8a (100750, Biolegend). After the staining, the plates were incubated at 4°C for 30 min in the dark. Next, the plates were washed with the staining buffer, and the fixation/permeabilization solution (554714, BD Biosciences) was added at 4°C for 20 min in the dark. After washing, the BV421 anti-Mouse IFN-γ (505830, Biolegend), BV605 rat anti-mouse IL2 (563911, BD Biosciences), and PE-Cy7 rat anti-mouse TNF (557644, BD Biosciences) were added to the plates. Thereafter, the stained cells were resuspended and measured using the Nonocyte flow cytometry system (ACEA Biosciences, USA). The obtained data were analyzed by NovoExpress software and plotted using the GraphPad Prism 8.0 software. A representative flow cytometry gating strategy for the mock mice is shown in Fig. S3. The flow cytometry panel is shown in Table S4.

Statistical analysis

The data obtained in this study were analyzed using GraphPad Prism 8.0 software (GraphPad Software, San Diego, CA, USA). When comparing more than two groups, the data were analyzed by one-way ANOVA with Dunnett’s multiple comparisons test, Brown-Forsythe ANOVA test, or Welch’s ANOVA test. When the data were not normally distributed, they were analyzed using the Kruskal-Wallis test. When comparing more than two groups at different time points, the data were analyzed by two-way ANOVA with Tukey’s multiple comparisons test. The data were expressed as the mean ± SD.

ACKNOWLEDGMENTS

We thank Dr. Hualan Chen (Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences) for providing bidirectional transcription vector pBD. We thank Creative Biochip company for providing the protein microarray technical support.

This work was supported by the National Science and Technology Major Project of China (2020ZX10001-016-003).

Contributor Information

Xianzhu Xia, Email: xiaxzh@cae.cn.

Yuwei Gao, Email: yuwei0901@outlook.com.

Kanta Subbarao, The Peter Doherty Institute for Infection and Immunity, Melbourne, Victoria, Australia .

ETHICS APPROVAL

All live animal work was performed following guidelines from the Animal Welfare and Ethics Committee of the Changchun Veterinary Research Institute of the Chinese Academy of Agricultural Sciences (IACUC of AMMS-11-2019-002). The environment and housing facilities satisfied China's National Standards of Laboratory Animal Requirements (GB 14925-2010). If the body weight of a mouse decreased by over 25% when challenged, then that mouse was euthanized. In the ferret challenge study, if any ferret had severe clinical diseases, displayed more than 20% weight loss, showed neurological symptoms, or was hardly breathing, the animal was euthanized by cardiac bleeding with ketamine (5 mg/kg) and medetomidine (0.1 mg/kg).

All the recombinant influenza B viruses and wild-type H5Nx HPAI experiment protocols were approved by the institutional Biosecurity Committee. All live H5Nx influenza virus experiments were approved and performed in an animal biosecurity level 3 laboratory. The members of the biosecurity office regularly provided oversight on the research, experiments, facilities, and security to ensure biosecurity. All the experiments were well conducted and supervised by experienced researchers.

SUPPLEMENTAL MATERIAL

The following material is available online at https://doi.org/10.1128/jvi.01101-23.

Figures S1-S6, Tables S1-S4. jvi.01101-23-s0001.pdf.

Supplemental materials.

Click here for additional data file.^{(2.4MB, pdf)}

DOI: 10.1128/jvi.01101-23.SuF1

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REFERENCES

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DOI: 10.1128/jvi.01101-23.SuF1

[B1] 1. Webster RG, Hulse DJ. 2004. Microbial adaptation and change: avian influenza. Rev Sci Tech 23:453–465. doi: 10.20506/rst.23.2.1493 [DOI] [PubMed] [Google Scholar]

[B2] 2. World Health Organization/World Organisation for Animal Health/Food and Agriculture Organization (WHO/OIE/FAO) H5N1 Evolution Working Group . 2014. Revised and updated nomenclature for highly pathogenic avian influenza A (H5N1) viruses. Influenza Resp Viruses 8:384–388. doi: 10.1111/irv.12230 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B3] 3. Yamaji R, Saad MD, Davis CT, Swayne DE, Wang D, Wong FYK, McCauley JW, Peiris JSM, Webby RJ, Fouchier RAM, Kawaoka Y, Zhang W. 2020. Pandemic potential of highly pathogenic avian influenza clade 2.3.4.4 A(H5) viruses. Rev Med Virol 30:e2099. doi: 10.1002/rmv.2099 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B4] 4. Zhang R, Chen T, Ou X, Liu R, Yang Y, Ye W, Chen J, Yao D, Sun B, Zhang X, Zhou J, Sun Y, Chen F, Wang SP. 2016. Clinical, epidemiological and virological characteristics of the first detected human case of avian influenza A(H5N6) virus. Infect Genet Evol 40:236–242. doi: 10.1016/j.meegid.2016.03.010 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B5] 5. Pyankova OG, Susloparov IM, Moiseeva AA, Kolosova NP, Onkhonova GS, Danilenko AV, Vakalova EV, Shendo GL, Nekeshina NN, Noskova LN, Demina JV, Frolova NV, Gavrilova EV, Maksyutov RA, Ryzhikov AB. 2021. Isolation of clade 2.3.4.4B A(H5N8), a highly pathogenic avian influenza virus, from a worker during an outbreak on a poultry farm. Euro Surveill 26:2100439. doi: 10.2807/1560-7917.ES.2021.26.24.2100439 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B6] 6. Shi W, Gao GF. 2021. Emerging H5N8 avian influenza viruses. Science 372:784–786. doi: 10.1126/science.abg6302 [DOI] [PubMed] [Google Scholar]

[B7] 7. Jiang W, Dong C, Liu S, Peng C, Yin X, Liang S, Zhang L, Li J, Yu X, Li Y, Wang J, Hou G, Zeng Z, Liu H. 2022. Emerging novel reassortant influenza A(H5N6) viruses in poultry and humans China, 2021. Emerg Infect Dis 28:1064–1066. doi: 10.3201/eid2805.212163 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B8] 8. Li J, Fang Y, Qiu X, Yu X, Cheng S, Li N, Sun Z, Ni Z, Wang H. 2022. Human infection with avian-origin H5N6 influenza a virus after exposure to slaughtered poultry. Emerg Microbes Infect 11:807–810. doi: 10.1080/22221751.2022.2048971 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B9] 9. Zhu W, Li X, Dong J, Bo H, Liu J, Yang J, Zhang Y, Wei H, Huang W, Zhao X, Chen T, Yang J, Li Z, Zeng X, Li C, Tang J, Xin L, Gao R, Liu L, Tan M, Shu Y, Yang L, Wang D. 2022. Epidemiologic, clinical, and genetic characteristics of human infections with influenza A(H5N6) viruses, China. Emerg Infect Dis 28:1332–1344. doi: 10.3201/eid2807.212482 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B10] 10. World Health Organization . 2022. Regional office for the Western P. 2022. avian influenza weekly update. , WHO Regional Office for the Western Pacific, Manila. [Google Scholar]

[B11] 11. Mohn KG-I, Smith I, Sjursen H, Cox RJ. 2018. Immune responses after live attenuated influenza vaccination. Hum Vaccin Immunother 14:571–578. doi: 10.1080/21645515.2017.1377376 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B12] 12. Subbarao K. 2021. Live attenuated cold-adapted influenza vaccines. Cold Spring Harb Perspect Med 11:a038653. doi: 10.1101/cshperspect.a038653 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B13] 13. Ullah S, Ross TM. 2022. Next generation live-attenuated influenza vaccine platforms. Expert Rev Vaccines 21:1097–1110. doi: 10.1080/14760584.2022.2072301 [DOI] [PubMed] [Google Scholar]

[B14] 14. Belshe RB, Nichol KL, Black SB, Shinefield H, Cordova J, Walker R, Hessel C, Cho I, Mendelman PM. 2004. Safety, efficacy, and effectiveness of live, attenuated, cold-adapted influenza vaccine in an indicated population aged 5-49 years. Clin Infect Dis 39:920–927. doi: 10.1086/423001 [DOI] [PubMed] [Google Scholar]

[B15] 15. Brooks WA, Zaman K, Lewis KDC, Ortiz JR, Goswami D, Feser J, Sharmeen AT, Nahar K, Rahman M, Rahman MZ, Barin B, Yunus M, Fry AM, Bresee J, Azim T, Neuzil KM. 2016. Efficacy of a Russian-backbone live attenuated influenza vaccine among young children in Bangladesh: a randomised. The Lancet Global Health 4:e946–e954. doi: 10.1016/S2214-109X(16)30200-5 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B16] 16. Mendelman PM, Rappaport R, Cho I, Block S, Gruber W, August M, Dawson D, Cordova J, Kemble G, Mahmood K, Palladino G, Lee MS, Razmpour A, Stoddard J, Forrest BD. 2004. Live attenuated influenza vaccine induces cross-reactive antibody responses in children against an a/Fujian/411/2002-like H3N2 Antigenic variant strain. Pediatr Infect Dis J 23:1053–1055. doi: 10.1097/01.inf.0000143643.44463.b1 [DOI] [PubMed] [Google Scholar]

[B17] 17. Baxter R, Eaton A, Hansen J, Aukes L, Caspard H, Ambrose CS. 2017. Safety of quadrivalent live attenuated influenza vaccine in subjects aged 2-49years. Vaccine 35:1254–1258. doi: 10.1016/j.vaccine.2017.01.062 [DOI] [PubMed] [Google Scholar]

[B18] 18. Zhou B, Meliopoulos VA, Wang W, Lin X, Stucker KM, Halpin RA, Stockwell TB, Schultz-Cherry S, Wentworth DE. 2016. Reversion of cold-adapted live attenuated influenza vaccine into a pathogenic virus. J Virol 90:8454–8463. doi: 10.1128/JVI.00163-16 [DOI] [PMC free article] [PubMed] [Google Scholar]

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PERMALINK

Clade 2.3.4.4 H5 chimeric cold-adapted attenuated influenza vaccines induced cross-reactive protection in mice and ferrets

Weiyang Sun

Jiaqi Xu

Zhenfei Wang

Dongxu Li

Yue Sun

Menghan Zhu

Xiawei Liu

Yuanguo Li

Fangxu Li

Tiecheng Wang

Na Feng

Zhendong Guo

Xianzhu Xia

Yuwei Gao

Roles

ABSTRACT

IMPORTANCE

INTRODUCTION

RESULTS

Construction and biological characteristics of Clade 2.3.4.4 H5 CAIVs

Fig 1.

TABLE 1.

Fig 2.

Vaccination with H5 CAIVs induces a cross-reactive antibody response in mice

Fig 3.

Fig 4.

Vaccination with H5 CAIVs induces cellular immune responses in mice

Fig 5.

Vaccination with H5 CAIVs reduces body weight and virus titers of the tissues after challenge in mice

Fig 6.

Fig 7.

Vaccination with H5 CAIVs induces humoral and cellular immune responses in ferrets

Fig 8.

Fig 9.

Vaccination with H5 CAIVs reduces virus titers in ferrets

Fig 10.

DISCUSSION

MATERIALS AND METHODS

Cells, proteins, and viruses

Generation of chimeric cold-adapted vaccine by reverse genetics

Biological characteristics and growth kinetics of H5 CAIVs

Mouse study design

Ferret study design

Virus titrations in MDCK cells, embryonated chicken eggs and tissues

Hemagglutinin inhibition assay and microneutralization assay

Enzyme-linked immunosorbent assay

Protein microarray assay

ELISpot assay and Luminex assay

Intracellular cytokine staining assay

Statistical analysis

ACKNOWLEDGMENTS

Contributor Information

ETHICS APPROVAL

SUPPLEMENTAL MATERIAL

REFERENCES

Associated Data

Supplementary Materials

ACTIONS

PERMALINK

RESOURCES

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