Home Preclinical immunogenicity assessment of a cell-based inactivated whole-virion H5N1 influenza vaccine
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Preclinical immunogenicity assessment of a cell-based inactivated whole-virion H5N1 influenza vaccine

  • Zhegang Zhang , Zheng Jiang , Tao Deng , Jiayou Zhang , Bo Liu , Jing Liu , Ran Qiu , Qingmei Zhang , Xuedan Li , Xuanxuan Nian , Yue Hong , Fang Li , Feixia Peng , Wei Zhao , Zhiwu Xia , Shihe Huang , Shuyan Liang , Jinhua Chen , Changgui Li EMAIL logo and Xiaoming Yang EMAIL logo
Published/Copyright: September 26, 2022
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Open Life Sciences
From the journal Open Life Sciences Volume 17 Issue 1

Abstract

In influenza vaccine development, Madin–Darby canine kidney (MDCK) cells provide multiple advantages, including large-scale production and egg independence. Several cell-based influenza vaccines have been approved worldwide. We cultured H5N1 virus in a serum-free MDCK cell suspension. The harvested virus was manufactured into vaccines after inactivation and purification. The vaccine effectiveness was assessed in the Wuhan Institute of Biological Products BSL2 facility. The pre- and postvaccination mouse serum titers were determined using the microneutralization and hemagglutination inhibition tests. The immunological responses induced by vaccine were investigated using immunological cell classification, cytokine expression quantification, and immunoglobulin G (IgG) subtype classification. The protective effect of the vaccine in mice was evaluated using challenge test. Antibodies against H5N1 in rats lasted up to 8 months after the first dose. Compared with those of the placebo group, the serum titer of vaccinated mice increased significantly, Th1 and Th2 cells were activated, and CD8+ T cells were activated in two dose groups. Furthermore, the challenge test showed that vaccination reduced the clinical symptoms and virus titer in the lungs of mice after challenge, indicating a superior immunological response. Notably, early after vaccination, considerably increased interferon-inducible protein-10 (IP-10) levels were found, indicating improved vaccine-induced innate immunity. However, IP-10 is an adverse event marker, which is a cause for concern. Overall, in the case of an outbreak, the whole-virion H5N1 vaccine should provide protection.

Keywords: MDCK cells; humoral immune response; cellular immune response; H5N1; vaccine

1 Introduction

The H5N1 virus is a highly pathogenic avian influenza virus that causes infection and death in poultry and wild birds. It was initially found in Guangdong, China, in 1996 [1]. Humans can be infected by the H5N1 virus in the absence of an intermediate host. In Hong Kong, a total of 18 persons were infected with 6 deaths in 1997 [2]. The clinical signs of the patients were severe pneumonia and infectious multiorgan failure, with fever above 38°C. Upper respiratory symptoms, on the other hand, are not as severe as they are with seasonal influenza [3]. Vaccines are an efficient way to prevent H5N1 outbreaks. In 2020, a phase 2/3 clinical trial verified the safety and efficacy of an H5N1 whole-virion vaccine produced in egg embryos [4]. Although outbreaks of avian influenza might disrupt the availability of egg embryos, cell-based vaccine platforms are able to ensure vaccine production and supply. Easy scale-up, low batch variation, easy control of sterile conditions, and the absence of egg allergens are all advantages of the cell-based platform. Vero cells used to be a common vector for producing the H5N1 vaccine and have been approved by the EU [5,6,7,8]. In recent years, Madin–Darby canine kidney (MDCK) cells have been increasingly used in influenza vaccine manufacturing and are now available in United States, EU, and South Korea. According to reports, India is working on both seasonal influenza and avian influenza vaccines based on MDCK cells [9,10]. In terms of the influenza vaccine market in China, cell-based influenza vaccine has not been licensed in China (including Taiwan) and production capacity of egg-based vaccine cannot meet market demand, so cell-based influenza vaccine could fill a gap in the market. There are two main lines of MDCK cells, suspended and adherent MDCK cell lines (sMDCK and aMDCK). The sMDCK (fetal bovine serum [FBS] is not required) could provide greater yield and lower cost than aMDCK cells. Therefore, we acclimated a serum-free culture of sMDCK from above aMDCK for the H5N1 vaccine manufacturing. According to Kongsomros et al., the H5N1 virus spreads faster and has a greater titer between cells than the H1N1 virus, a feature that has also been found in human lung epithelial cells [11], indicating that MDCK cells might be better suited for H5N1 virus propagation than H1N1 virus propagation. More importantly, eggs might not be suitable for some specific viruses, such as some H3N2 strains, which cannot grow well in eggs, and wild type H5N1, which could kill egg embryos [12]. We cultured H5N1 virus on sMDCK for 72 h in a 40 L bioreactor. The harvested virus was inactivated and purified as a stock solution. Then, we evaluated the immunogenicity of the H5N1 vaccine. We used H5N1 viruses that had been propagated five times in the sMDCK cells for the challenge test because the limited conditions of the laboratory prevented us from using wild strains, and viruses that propagated in sMDCK were pathogenic in mammals. We discovered in earlier experiments that multi-propagation virus could cause significant reduction in body weight and clinical phenomena. Although the vaccine was not supplemented with adjuvants, the viral structural proteins and nucleic acids contained in the whole-virus particles in the vaccine may induce innate and cellular immunity, so the purpose of this study is to comprehensively assess the efficacy of the vaccine.

2 Materials and methods

2.1 Cells and viruses

sMDCK cells were cultured in VP and DMEM (1:2, with 1% glutamine, bovine serum free). The cells used in the TCID50 assay were aMDCK cells from the ATCC, USA. Influenza viruses (A/reassortant/NIBRG-14(Vietnam/1194/2004 × Puerto Rico/8/1934)) were purchased from NIBSC. Working virus seeds were propagated in 9 days specefic pathogen free (SPF) eggs, and the TCID50 of the virus was 105.67/0.1 mL. The virus strain used in the challenge test was propagated multiple times in sMDCK cells, and the TCID50 was 103.5/0.1 mL.

2.2 Vaccines

sMDCK cells were cultured in a 40 L bioreactor (Bio-510, NBS, USA) for 72 h and inoculated with virus prepared in SPF eggs at 33 ± 1°C. To remove host cell protein and DNA, the harvest was clarified by depth filtration (PALL, USA), treated with benzonase, and purified by CaptoTM Core 700 (GE, USA) and CaptoTM Q (GE, USA). β-Propiolactone (Serva, Germany) was added to inactivate the virus. The solution (containing 128 μg/mL hemagglutinin [HA]) was stored at 4°C in phosphate-buffered saline (PBS). The placebo in this study was the PBS that was used to dilute the solution.

2.3 Animals

Healthy female BALB/C mice and Wistar rats were supplied by the animal reproduction facilities of Wuhan Institute of Biological Products (WIBP) (experimental animal production license number: 110011216263344054) and kept in the BSL-2 laboratory at the animal facilities of WIBP. The animals were divided into different groups. The grouping details are shown in Tables 1 and 2.

Table 1

Animal groups for vaccine injection

Group Animal species Purpose Immunization Dose group (μg HA/500 µL) Sampling time (days post first vaccination) Sampling tissue Number of animals in each dose group Total
1 Wistar rat • Long-term antibody titer Two doses on days 0 and 28, each delivered IM 0a, 1, 2, 3.75, 7.5, 15, 30, 45 28, 42, 56, 84, 112, 140, 168, 196 Blood 5 40
2 BALB/c mice • Antibody titer 0, 0.5, 1, 2, 3.75, 7.5, 15 28, 42, 56 Blood 10 70
• IgG subclassb
3 BALB/c mice • Lymphocyte and cytokine detection 0, 0.5, 1,2, 3.75, 7.5, 15 28, 32, 56 Spleen 5 35
4 BALB/c mice • Early cytokine production induction Single dose on day 0, delivered IM 0, 3.75 0c, 3, 6, 12, 24, 48 h Blood / /d

a: Dose 0 indicates the placebo. Each dose group contained 5 rats; for example, the total number of rats in group no. 1 (rats) was 40.

b: Serum used for IgG detection was obtained from 3.75, 7.5, and 15 μg groups.

c: Samples were collected at hours post-vaccination, and “h” represents hours.

d: The number of animals in this group is detailed in section 2.4.

Table 2

Animal groups for the challenge test

Group Animal species Treatment before challenge Sampling time (days post-challenge) Sampling tissue
5 BALB/c mice Two doses on days 0 and 28, each delivered IM 6, 8, 10 Lungs

  1. Ethical approval: The research related to animal use has been complied with all the relevant national regulations and institutional policies for the care and use of animals, the guidelines of the Laboratory Animal Guidelines for Ethical Review of Animal Welfare (Standardization Administration of China, 2018). Animal studies were approved by the animal ethics committee of WIBP (Ethics No. WIBP-AⅡ332021001).

2.4 Challenge test

In the challenge test, the mice were divided into 3 subgroups: a placebo (n = 13, 10 for observation and 3 for dissection), a vaccine (same as above), and a blank group (n = 2, all for dissection). Mice were injected intramuscularly (IM) with two doses of vaccine (HA 3.75 μg/0.5 mL) or PBS (0.5 mL) on day 0 and day 28. On day 42 (14 days after boost), the mice were challenged with 100 μL of H5N1 virus via the respiratory tract. Body weight and temperature were monitored daily during the challenge phase, day 0–13 post challenge (PC). The mice were checked daily for signs of disease until the test endpoint (day 13 PC). For the dissection mice, the lungs were isolated on day 8 PC and day 11 PC, and the upper lobe of the right lung was sectioned for pathological examination.

2.5 Serological tests

The mice and rats were made to bleed via orbital veins. The blood was placed at 37°C for 2 h and centrifuged at 3,000 rpm for 10 min to obtain serum. The sera were stored at −80°C.

2.5.1 Hemagglutination inhibition (HI) assay

The HI assay was conducted based on the China National Influenza Center Standard Practice (Revised Version, 2007). Briefly, the sera were diluted with receptor-destroying enzyme (Sigma, USA), incubated at 37°C for 18 h, and inactivated at 56°C for 1 h. Sera were titrated 2-fold and incubated for 1 h with 4 hemagglutinating units (HAUs) of standard antigen. The initial dilution was 1:10. Then, 1% turkey erythrocytes were combined with the remaining virus. The titers of the HI assay are expressed as the reciprocal of the serum dilution that completely inhibited HA activity at 4 HAUs.

2.5.2 Microneutralization (MN) test

The MN assay was conducted based on the China National Influenza Center Standard Practice (Revised Version, 2007). Briefly, the serum samples were treated as described above. Then, 100 CCID50 H5N1 virus was incubated with diluted sera for 1 h at 37°C. Following incubation, the mixture was added to a confluent monolayer of aMDCK cells and incubated for 72 h at 33 ± 1°C. Plates were stained with 1% crystal violet and measured by a spectrophotometer. The titers of the MN assay were considered the reciprocal of the serum dilution that completely inhibited the virus activity.

2.5.3 Subtype classification of IgG

IgG1 and IgG2a subtypes in sera were detected by ELISA kits (Mouse IgG1 ELISA Kit and/Mouse IgG2a ELISA Kit, Bethyl). Dilution and detection procedures were conducted based on the manufacturer’s protocol. Briefly, the plates were precoated with an anti-mouse IgG1 or IgG2a antibody, and diluted samples and standards were added to the plate and incubated for 1 h. After incubation, horseradish peroxidase (HRP)-conjugated antibody, tetramethylbenzidine (TMB) substrate solution, and stop solution were added according to the protocol. The absorbance was read at 450 nm by a microplate reader (Thermo, USA). The concentration of IgG1 or IgG2a in the samples was calculated based on a standard curve.

2.6 Intracellular cytokine staining and multiplex cytokine detection assay

Mice were sacrificed and then bathed in 75% alcohol. The spleens were harvested and placed in prechilled medium (RPMI1640, 10% FBS). The spleens were milled individually on a 70 μm cell sieve. The cells were centrifuged (350×g, 5 min) and washed with PBS. Lymphocytes were isolated using lymphocyte isolation solution (Dakewe, China), and the lymphocytes were washed with ten volumes of PBS. The lymphocytes were resuspended to 107 cells/mL. H5N1 stock solution was added to 2 mL of lymphocytes. The 2 mL lymphocyte suspension with 5 μg/mL HA was divided equally into 2 parts for 2 tests. The supernatant of 1 mL of lymphocytes was collected 24 h after stimulation for the multiplex cytokine detection assay. The LEGENDplexTM Multi-Analyte Flow Assay Kit was used to detect the concentrations of interleukin-2 (IL)/4/5/6/9/10/13/17A/17F/22, interferon (IFN-γ), and Tumor necrosis factor-α (TNF), following the manufacturer’s instructions. Flow cytometry was performed with a Cytoflex S flow cytometer (Beckman, USA).

Two microliters of Brefeldin A were added to the other 1 mL of lymphocytes after 18 h of stimulation. After incubation for 20 h, the cells were collected for intracellular cytokine staining. In brief, the cells were washed twice with PBS. Then, the cells were stained with 1 μL/test Zombie (Aqua™ Fixable Viability Kit). The cells were sequentially stained with anti-CD3 (APC/Cyanine7 anti-mouse CD3, Biolegend), anti-CD4 (PercpCy5.5 anti-mouse CD4, Biolegend), and anti-CD8 (FITC anti-mouse CD8a, Biolegend) antibodies and incubated for 15 min. Then, 500 μL of fixation solution was added to each sample and thoroughly mixed to fix the cells for 20 min. Then, 1 mL of Perm (1×) was added to wash the cells twice, and then 100 μL of Perm was added to resuspend the cells and permeabilize the cell membrane. Premixed antibodies including anti-TNF-α (APC anti-mouse TNF-α, Biolegend) and anti-IFN-γ antibodies (Brilliant Violet 421™ anti-mouse IFN-γ, Biolegend) were added. The logic order of flow cytometry analysis was Zombie-CD3+ CD4+/CD4+ IFN-γ+ and Zombie-CD3+ CD8+/CD8+ TNF-α+ IFN-γ+. Then, the sample data were obtained from the flow cytometer.

Cytokine changes in the early stage were detected by a LEGENDplexTM Multi-Analyte Flow Assay Kit containing anti-IL-6, anti-IP-10, and anti-monocyte chemotactic protein-1 (MCP) antibodies on flow cytometer.

2.7 Clinical observation

Body weight, body temperature, and disease signs, such as back arched, decreased response to external stimuli, and hair erection, were recorded daily from 9:30 to 10:00 a.m. post challenge. All mice were anesthetized with tribromoethanol on day 13 PC.

2.8 Virus titer of lung tissue

The amount of lung tissue was weighed, and PBS (1 mg:1 mL) was added. TissueLyser II (Qiagen, Germany) was used to lyse the lung. After lysis, the cells were centrifuged at 800 rpm for 10 min, and the supernatant was collected for titer detection. The supernatant was titrated serially 10-folds until dilution 10−15 with VP medium (serum free, 2 μg/mL TPCK-trypsin). Gradient dilutions of homogenized tissue were added to monolayers of MDCK cells in a 96-well plate (initial dilution of 10−4) and incubated at 33 ± 1°C for 72 h. Plates were stained with 1% crystal violet and measured by a spectrophotometer. The viral titer in the lungs was estimated by the Reed & Muench Calculator.

2.9 Hematoxylin and eosin (H&E) staining and immunohistochemistry (IHC)

Pathology lung sections were prepared as described previously [13]. The steps of H&E staining were as follows. The sections were dipped into a Coplin jar containing hematoxylin and agitated for 30 s. The sections were rinsed in H2O for 1 min and then stained with 1% eosin Y solution for 10–30 s, with agitation. The sections were dehydrated with 95% alcohol twice and 100% alcohol twice for 30 s each. Alcohol was extracted with two changes in xylene. One drop of mounting medium was added, and the sections were covered with coverslips.

IHC steps were as follows. The sections were dewaxed, and antigen retrieval was performed using an electric pottery oven. We added 3% hydrogen peroxide to the sections and blocked the sections with mock rabbit serum for 30 min. The primary antibody (influenza anti A/Vietnam/1194/04 (H5N1) HA serum) 1:500, NIBSC, 07/148, UK) was added and incubated overnight at 4°C, and then the secondary antibody (1:500, HRP-rabbit anti-sheep IgG, Biodragon, BF03025, China) was added and incubated for 30 min at 37°C. The sections were rinsed with PBS, and TMB substrate was added. Then, the sections were restained, dehydrated, sealed, and dried. IHC images of sections were collected by a microscope.

2.10 Statistical analysis

Prism 9 (GraphPad Software, CA, USA) was used to perform the statistical analysis. P values were obtained using unpaired t test or two-way ANOVA nonparametric analysis. P values < 0.05 were considered statistically significant. P < 0.05 (*), P < 0.001 (**).

3 Results

3.1 The humoral immune response was significantly activated

The level of antibody secretion is the most direct embodiment of humoral immunity. Specific antibodies in rats can persist for at least 6 months after a boost. The antibody titer was obviously positively correlated with dose (Figure 1). Seroconversion was 100% in all vaccine groups, and antibody titers were far beyond 4-fold change than the placebo group, this scene was not observed for an inactivated quadrivalent split-virus seasonal influenza vaccine based on aMDCK cells (the data are not public). This result suggests that antibody induced by whole-virion particles can provide steady protection to rats for at least half a year.

Figure 1 Rats were boosted on day 28. The serum collected after vaccination was used in HI assay. Rats vaccinated with 2–15 μg HA induced strong neutralization antibody responses. Vaccine containing 2 μg HA induced high level antibody in rats for at least 6 months. Statistical analysis was performed using a two-way ANOVA.
Figure 1

Rats were boosted on day 28. The serum collected after vaccination was used in HI assay. Rats vaccinated with 2–15 μg HA induced strong neutralization antibody responses. Vaccine containing 2 μg HA induced high level antibody in rats for at least 6 months. Statistical analysis was performed using a two-way ANOVA.

The antibody titer peaked at day 42 and was at least 4-fold change than the placebo group at day 56 in mice (Figure 2). The MN results in all vaccine groups at 14 and 28 days after boost were also significantly higher than the placebo group (Figure 3). This finding shows that the specific antibodies in serum can effectively neutralize the virus after boost. The HI and MN results showed the same trend, and the geometric mean titer (GMT) increased with increasing vaccine dose. Since the amount of serum at day 28 was insufficient to complete two tests, the MN test for day 28 wase abandoned. We observed significant increases in antibody levels in both rats and mice, suggesting that the vaccine can highly efficiently induce persistent humoral immunity. According to the above results, the MN test showed a higher sensitivity, which may be a better gauge of vaccine effectiveness. Research by Ni et al. on influenza vaccines revealed the same results [14].

Figure 2 Mouse were boosted on day 28. The serum collected after vaccination was used in HI assay. There is no change in neutralization antibody after first dose. The antibody increased significantly 14 days after boost and decreased significantly 28 days after boost. Vaccine containing 15 μg HA induced highest GMT during the immunization.
Figure 2

Mouse were boosted on day 28. The serum collected after vaccination was used in HI assay. There is no change in neutralization antibody after first dose. The antibody increased significantly 14 days after boost and decreased significantly 28 days after boost. Vaccine containing 15 μg HA induced highest GMT during the immunization.

Figure 3 The serum used in microneutralization test were the same as in HI assay. The antibody increased significantly after immunization. There was a decrease in antibody levels, but not as significant as in HI assay.
Figure 3

The serum used in microneutralization test were the same as in HI assay. The antibody increased significantly after immunization. There was a decrease in antibody levels, but not as significant as in HI assay.

3.2 The cellular immune response was activated

IFN-γ and TNF-α can be secreted by activated CD8+ T cells and therefore used as indirect cytotoxicity markers for activated CD8+ T cells [15]. Both cytokines directly inhibit viral replication and activate immune cells [16]. On day 28, the proportion of CD8+ T cells secreting both TNF-α and IFN-γ in the spleen increased significantly (except for 7.5 μg group). On day 32, the proportion of CD8+ T cells secreting TNF-α and IFN-γ of vaccine groups was still higher than placebo group. On day 56, no difference was observed among the groups (Figure 4a). After the first dose, a large number of CD8+ T cells were activated and specific and nonspecific CD8+ T cells began to participate in the immune response. The changes after boost indicated that virus-specific CD8+ T cells were activated, and specific CD8+ T cells started to proliferate again, forming a population mainly composed of effector CD8+ T cells. After the antigen was removed, the CD8+ T cell population contracted due to apoptosis, leaving only a few memory T cells, and the number of CD8+ T cells returned to normal [17]. The total number of activated CD4+ T cells (IFN-γ+) was unchanged, and the activation was even stronger in the placebo group (Figure 4b).

Figure 4 (a) After the first dose, the activated CD8+ T cells were significantly enhanced in the spleen of the mice. The CD8+ T cells were also significantly activated (lower than the 1 ug and 15 ug groups) in the PBS group after boost. All CD8+ T cells decreased to no significant difference in day 56. (b) The differences in CD4+ T cells of splenocytes were not significant at day 28, 32, and 56.
Figure 4

(a) After the first dose, the activated CD8+ T cells were significantly enhanced in the spleen of the mice. The CD8+ T cells were also significantly activated (lower than the 1 ug and 15 ug groups) in the PBS group after boost. All CD8+ T cells decreased to no significant difference in day 56. (b) The differences in CD4+ T cells of splenocytes were not significant at day 28, 32, and 56.

The ratio of IgG2a to IgG1 can be used to describe the direction of the immune response. The subtype classification of IgG indicated the distribution of the Th cell response: IgG2a can represent the Th1 response, and IgG1 is commonly used to represent the humoral immune response. On day 28, there was no difference in the IgG2a/IgG1 ratio between the groups. The IgG2a/IgG1 ratio decreased more significantly in the placebo group than in the vaccine group at day 42, which suggested that boost inhibited part of the decline in IgG2a levels (Figure 5). In addition, there was a significant decrease in IgG2 levels and a significant increase in IgG1 levels in response to placebo stimulation, indicating that Th2 is the dominant immune response. However, the antigens in the vaccine changed this balance, which means that the role of the Th1 response was not diminished or its role was enhanced.

Figure 5 (a) The ratio of IgG2a/IgG1 decreased after boost. The immune response tended to the Th2 direction. This balance remained until day 56. (b and c) There is an antagonism between the Th1 and Th2, both of which showed a significant increase at day 42 after immunization. Both Th1 and Th2 began to exert immune function.
Figure 5

(a) The ratio of IgG2a/IgG1 decreased after boost. The immune response tended to the Th2 direction. This balance remained until day 56. (b and c) There is an antagonism between the Th1 and Th2, both of which showed a significant increase at day 42 after immunization. Both Th1 and Th2 began to exert immune function.

3.3 Changes in the levels of cytokines associated with the immune response

To further characterize the immune response, we detected the cytokine content in serum during immunization (Figure 6). These cytokines can be roughly divided into Th1-related cytokines, such as IFN-γ, TNF-α, and IL-2; Th2-related cytokines, such as IL-4/5/10/13; and Th17-related cytokines, such as IL-17A and IL-17F. The secretion level of these cytokines can indirectly explain the activation of relevant cells. IL-2 has an enhanced induction effect on virus-specific cellular immunity [18]. The expression of IL-2 was high before boost, indicating that Th1 cells were going to be activated, while IFN-γ and TNF-α levels did not change. However, after IL-2 levels returned to baseline, the increase in IFN-γ and TNF-α levels became significant, and Th1 cells were activated, which also explained why the decline in Th1 cells was inhibited after boost. The high level of Th2-related cytokines indicated that humoral immunity activation was significant after boost. IL-17A is a hallmark cytokine of Th17 cells [19]. IL-17F and IL-17A are located on the same chromosome, and IL-17F binds to IL-17A to form a dimer [20,21]. Since there were no significant changes in Th17 cell-related cytokine levels, we considered that Th17 cells do not play a critical role in this immune response.

Figure 6 On day 28, IL-2 levels in all vaccine groups (except for 7.5 μg) were significantly higher than those in the placebo group. After booster administration, IL-2 levels in all vaccine groups began to decline on day 32, and there was no difference between the vaccine groups and the placebo group on day 56 (a). IL-4 levels increased significantly on day 32 and declined to the level of the placebo group on day 56, except for those in the 7.5 and 15 μg groups (b). IL-5 levels in the vaccine groups were not different from those in the placebo group on day 28; those in the 7.5 and 15 μg groups were even lower. On day 32, IL-5 levels in the 0.5, 2, 3.75, and 7.5 μg groups were significantly increased compared with those in the placebo group, and on day 56, IL-5 levels in all vaccine groups declined to the level of the placebo group (c). There was no difference in IL-6 levels between the vaccine groups and the placebo group on day 28, but there was a significant increase in IL-6 levels in all vaccine groups on day 32, except for the placebo group. IL-6 levels decreased in all groups on day 56 and were still higher than those in the placebo group (d). IL-9 levels did not change significantly during the immunization period except at a dose of 3.75 μg (e). On day 32, IL-10 levels were significantly increased in all vaccine groups. IL-10 levels decreased in all vaccine groups on day 56 and were still higher than those in the placebo group (f). IL-13 levels increased significantly on day 32 and continuously increased until day 56, but there was no difference between the 15 μg and placebo groups (g). Significant changes in IL-22 were observed at day 28, but gradually decreased with time delay to no significant changes in the placebo group (h). At day 28, there was no significant difference in each group, but IL-17A levels increased after booster administration (j). IL-17F levels did not change significantly during the entire immunization process (i). There was no significant difference in IFN-γ levels between the vaccine groups and the placebo group on day 32 (except for the 0.5 dose group). IFN-γ levels in each dose group were significantly higher than those in the placebo group on days 28 and 56 (l). TNF-α levels in the placebo group were significantly decreased on day 28 and remained stable until day 56, while TNF-α levels in all dose groups were significantly increased at day 56 (k).
Figure 6

On day 28, IL-2 levels in all vaccine groups (except for 7.5 μg) were significantly higher than those in the placebo group. After booster administration, IL-2 levels in all vaccine groups began to decline on day 32, and there was no difference between the vaccine groups and the placebo group on day 56 (a). IL-4 levels increased significantly on day 32 and declined to the level of the placebo group on day 56, except for those in the 7.5 and 15 μg groups (b). IL-5 levels in the vaccine groups were not different from those in the placebo group on day 28; those in the 7.5 and 15 μg groups were even lower. On day 32, IL-5 levels in the 0.5, 2, 3.75, and 7.5 μg groups were significantly increased compared with those in the placebo group, and on day 56, IL-5 levels in all vaccine groups declined to the level of the placebo group (c). There was no difference in IL-6 levels between the vaccine groups and the placebo group on day 28, but there was a significant increase in IL-6 levels in all vaccine groups on day 32, except for the placebo group. IL-6 levels decreased in all groups on day 56 and were still higher than those in the placebo group (d). IL-9 levels did not change significantly during the immunization period except at a dose of 3.75 μg (e). On day 32, IL-10 levels were significantly increased in all vaccine groups. IL-10 levels decreased in all vaccine groups on day 56 and were still higher than those in the placebo group (f). IL-13 levels increased significantly on day 32 and continuously increased until day 56, but there was no difference between the 15 μg and placebo groups (g). Significant changes in IL-22 were observed at day 28, but gradually decreased with time delay to no significant changes in the placebo group (h). At day 28, there was no significant difference in each group, but IL-17A levels increased after booster administration (j). IL-17F levels did not change significantly during the entire immunization process (i). There was no significant difference in IFN-γ levels between the vaccine groups and the placebo group on day 32 (except for the 0.5 dose group). IFN-γ levels in each dose group were significantly higher than those in the placebo group on days 28 and 56 (l). TNF-α levels in the placebo group were significantly decreased on day 28 and remained stable until day 56, while TNF-α levels in all dose groups were significantly increased at day 56 (k).

3.4 The vaccine provided protection for mice against challenge

In the challenge test, significant weight loss was observed in the placebo group, while in contrast, the vaccine group maintained a stable weight (Figure 7). The change in body weight of the mice in the placebo group occurred on day 4 PC. The highest weight loss on day 7 PC was 30.15%. In the placebo group, eight mice were observed to have significant hair erection and arched back signs, in contrast with 0 mice in the vaccine groups. Significant increase in body temperature was observed in the vaccine group on day 4 PC.

Figure 7 The placebo group began to lose weight at day 4 PC, with the most significant weight loss at day 7 PC (a). Body temperature of the vaccinated group began to rise up to 38.6°C on day 4 PC (b).
Figure 7

The placebo group began to lose weight at day 4 PC, with the most significant weight loss at day 7 PC (a). Body temperature of the vaccinated group began to rise up to 38.6°C on day 4 PC (b).

Significant histopathology in pathology sections of the placebo group was observed (Figure 8). Relatively, histopathology was mild in the vaccine group at the beginning of infection. On day 11 PC, significant histopathology was observed in both the vaccine and placebo groups; however, the extent of lesions in the vaccine group was not as severe as the placebo group. Additionally, the positive cell, mean density, histochemistry score, and positive score results (Table 3) showed that fewer lung cells were infected in the vaccine group than in the placebo group and that smaller areas of lung tissue were infected (Figure 9). The viral titers in the lungs were consistent with these results (Figure 10). Viral titers in the vaccine group were all significantly lower than those in the placebo group during the infection period. Although the above results suggested that the vaccine cannot prevent virus infection in the lungs, it reduces the tissue damage caused by the virus by reducing the virus titer and number of infected areas and cells, thereby reducing clinical symptoms.

Figure 8 On day 8 PC, few lung lesions in the mice were discovered in the vaccine group, and there were no significant abnormalities in tissues (1A/1B/2A/2B). The alveolar wall was slightly thickened, along with a small amount of granulocyte infiltration (blue arrow) (3A/3B). Significant lesions appeared in the lungs of mice in the placebo group, and it was more common for lymphocytes to show focal infiltration around the blood vessels (blue arrow). Inflammatory cell infiltration was noted in a small number of alveolar cavities (green arrow). Due to local bleeding, red blood cells are visible in the alveolar cavity (yellow arrow) (1C/1D). The alveolar wall was slightly thickened, along with a small amount of granulocyte infiltration (blue arrow) (2C/2D). It was more common that congestion was seen in the blood vessels (yellow arrow). The alveolar wall was slightly thickened, along with a small amount of granulocyte infiltration (blue arrow) (3C/3D). On day 11 PC, lesions also developed in the lungs of mice in the vaccine group. There were multiple congestion sites in blood vessels and alveolar wall capillaries (yellow arrow). Few perivascular lymphocytes showed focal infiltration (blue arrow) (1E/1F). A large area of the alveolar wall was moderately thickened with a small amount of granulocyte infiltration (blue arrow) (2E/2F). Few perivascular lymphocytes showed small focal infiltrates (blue arrow) (3E/3F). However, more severe lesions were observed on day 11 PC in the placebo group, with multiple congested sites in the blood vessels and alveolar wall capillaries (yellow arrow). There were perivascular edema and loose connective tissue, accompanied by a small amount of lymphocyte infiltration (purple arrow) (1G/1H). A large area of alveolar wall was severely thickened, with a small amount of granulocyte infiltration (blue arrow). Inflammatory cells showed diffuse infiltration in multiple alveolar cavities (green arrow). It was more common that perivascular lymphocytes showed focal infiltration (purple arrow) (2G/2H). There was a large area of tissue necrosis (red arrow), with structural disorder. It was more common that eosinophilic homogenates appeared in the alveolar cavities (green arrow), surrounded and accompanied by a small amount of inflammatory cell infiltration. Inflammatory cell infiltration was seen in multiple alveolar cavities (blue arrow). There were perivascular edema and loose connective tissue, with a small amount of lymphocyte infiltration (purple arrow) (3G/3H). No significant abnormalities were found in the lung tissue of mice in the blank group (1I/1J/2I/2J).
Figure 8

On day 8 PC, few lung lesions in the mice were discovered in the vaccine group, and there were no significant abnormalities in tissues (1A/1B/2A/2B). The alveolar wall was slightly thickened, along with a small amount of granulocyte infiltration (blue arrow) (3A/3B). Significant lesions appeared in the lungs of mice in the placebo group, and it was more common for lymphocytes to show focal infiltration around the blood vessels (blue arrow). Inflammatory cell infiltration was noted in a small number of alveolar cavities (green arrow). Due to local bleeding, red blood cells are visible in the alveolar cavity (yellow arrow) (1C/1D). The alveolar wall was slightly thickened, along with a small amount of granulocyte infiltration (blue arrow) (2C/2D). It was more common that congestion was seen in the blood vessels (yellow arrow). The alveolar wall was slightly thickened, along with a small amount of granulocyte infiltration (blue arrow) (3C/3D). On day 11 PC, lesions also developed in the lungs of mice in the vaccine group. There were multiple congestion sites in blood vessels and alveolar wall capillaries (yellow arrow). Few perivascular lymphocytes showed focal infiltration (blue arrow) (1E/1F). A large area of the alveolar wall was moderately thickened with a small amount of granulocyte infiltration (blue arrow) (2E/2F). Few perivascular lymphocytes showed small focal infiltrates (blue arrow) (3E/3F). However, more severe lesions were observed on day 11 PC in the placebo group, with multiple congested sites in the blood vessels and alveolar wall capillaries (yellow arrow). There were perivascular edema and loose connective tissue, accompanied by a small amount of lymphocyte infiltration (purple arrow) (1G/1H). A large area of alveolar wall was severely thickened, with a small amount of granulocyte infiltration (blue arrow). Inflammatory cells showed diffuse infiltration in multiple alveolar cavities (green arrow). It was more common that perivascular lymphocytes showed focal infiltration (purple arrow) (2G/2H). There was a large area of tissue necrosis (red arrow), with structural disorder. It was more common that eosinophilic homogenates appeared in the alveolar cavities (green arrow), surrounded and accompanied by a small amount of inflammatory cell infiltration. Inflammatory cell infiltration was seen in multiple alveolar cavities (blue arrow). There were perivascular edema and loose connective tissue, with a small amount of lymphocyte infiltration (purple arrow) (3G/3H). No significant abnormalities were found in the lung tissue of mice in the blank group (1I/1J/2I/2J).

Table 3

IHC assay

Sample Positive cell ratioa (%) Mean densityb H scorec Positive scored Mean positive score
Day 8 Vaccinated 13.74 0.68 19.40 1 2.3
33.84 0.65 46.17 2
30.29 0.68 49.56 4
Unvaccinated 39.96 0.67 62.85 4 3.3
45.21 0.63 74.87 4
35.31 0.70 51.24 2
Day 11 Vaccinated 29.60 0.59 43.25 2 1.7
33.29 0.63 45.64 2
10.16 0.66 12.79 1
Unvaccinated 76.82 0.72 129.81 8 6.7
80.76 0.69 144.06 8
42.68 0.59 71.61 4
Blank 24.68 0.72 36.77 1 1
24.06 0.71 35.85 1

a: Positive cell ratio: (number of positive cells/number of total cells) × 100%.

b: Mean density: number of positive cells/area of tissue.

c: H score: histochemistry score, semiquantitative value of the number of positive cells and staining intensity.

d: Positive score: staining intensity score multiplied by the positive cell ratio score. Score of staining intensity: unstaining presented negative (0 points), pale yellow presented weakly positive (1 point), yellow presented moderately positive (2 points), and brown-yellow presented strongly positive (3 points). Score of the positive cell ratio: 0–5% (0 points), 6–25% (1 point), 26–50% (2 points), 51–75% (3 points), and > 75% (4 points). The larger the positive score is, the stronger the comprehensive positive intensity.

Figure 9 Each image is divided into two parts (left and right). The left part is an image selected from the immunohistochemical slices using a histologic section digital scanner, while the right part is an automatically analyzed left image by Image Analysis System (Servicebio, China). The nuclei are blue and the proteins of H5N1 virus are brownish yellow or dark brown. The positive cell ratio, mean density, histochemistry score, and positive score were calculated.
Figure 9

Each image is divided into two parts (left and right). The left part is an image selected from the immunohistochemical slices using a histologic section digital scanner, while the right part is an automatically analyzed left image by Image Analysis System (Servicebio, China). The nuclei are blue and the proteins of H5N1 virus are brownish yellow or dark brown. The positive cell ratio, mean density, histochemistry score, and positive score were calculated.

Figure 10 PBS (1 mg:1 mL) was added according to the mass of the lungs. The milled tissue fluid was stored at −80°C after centrifugation. Viral titer testing was performed concurrently after the last sampling. The CCID50 results showed that the viral load in the lung was significantly higher in the placebo group than vaccine group.
Figure 10

PBS (1 mg:1 mL) was added according to the mass of the lungs. The milled tissue fluid was stored at −80°C after centrifugation. Viral titer testing was performed concurrently after the last sampling. The CCID50 results showed that the viral load in the lung was significantly higher in the placebo group than vaccine group.

3.5 IP-10 levels increased in the early stage

More than half of the IL-6 and MCP-1 data were below the limit of detection; therefore, the data were not further analyzed. No significant changes in IP-10 levels were observed between 0 and 3 h after vaccination. The vaccinated group showed a significant change in IP-10 level compared to the placebo group at 6 and 12 h. This difference disappeared in the following 24 and 48 h. IP-10 secretion in the placebo group was consistently stable (approximately 200 pg/mL); on the other hand, the concentration in the vaccine group reached 610.3 pg/mL at 12 h (Figure 11).

Figure 11 The time for blood sampling was at 0, 3, 6, 12, 24, and 48 h after injection (n ≥ 5 per time point). IP-10 appeared to have significant changes at 6 h after injection and reached the peak at 12 h. Then, it dropped to a normal level in 12 h.
Figure 11

The time for blood sampling was at 0, 3, 6, 12, 24, and 48 h after injection (n ≥ 5 per time point). IP-10 appeared to have significant changes at 6 h after injection and reached the peak at 12 h. Then, it dropped to a normal level in 12 h.

4 Discussion

Approximately 50% of patients with symptoms after H5N1 infection have died [22]. Although there have not been large outbreaks of H5N1 in the population in recent years, a stockpile of the H5N1 vaccine is essential. Currently, the effectiveness of influenza vaccines of clinical trials are mainly assessed by serological tests, which represent the level of vaccine-induced neutralizing antibodies [23,24]. However, HI assay results mainly showed the binding of specific antibody to antigen. The results of MN experiment revealed the neutralization ability of antibody. Despite the difference in GMT, the results of both tests showed that the vaccine was effective in mice. The two tests conducted at the same time helped us to comprehensively assess the level of humoral immunity in animals. On the other hand, the significant increase in IgG1 content and proportion indicated that Th2 cells were significantly activated after antigen stimulation. Not surprisingly, the levels of Th2-related cytokines such as IL-4, IL-5, IL-10, and IL-13 increased. IL-4, IL-5, and IL-13 are the hallmark cytokines produced in the Th2 immune response and play a key role in immunity against pathogenic microorganisms [25]. Th2 cells also activate macrophages alternatively by expressing IL-4 and IL-13. IL-10 is secreted by Th2 cells to inhibit Th1 cell function [26]. In addition, IL-10 also inhibits chemokines and costimulatory molecules, regulates immunoreaction, and prevents immunopathology [27,28,29]. Th2 cells are activated to help B cells perform their functions. These results indicated that the vaccine induced a high level of specific antibody expression, and humoral immunity began to provide protection. Although the threshold of protection provided by the vaccine is currently expressed as GMT ≥40, the high HI titer cannot guarantee the protection against infection. Indeed, according to the test results, the H5N1 virus can still infect mice with antibody titers higher than 40 and cause lesions in the lungs. There is also a controversy about whether a GMT ≥40 is suitable for the elderly population [30,31,32]. In addition, antigen drift and shift of influenza viruses often cause vaccines to be ineffective against epidemic strains. We therefore focused on the effect of this vaccine on cellular immunity. Some structural proteins and genetic material of live viruses can induce cellular immunity, which can effectively prevent attack by heterologous viruses [33]. IgG2a reflected the activated degree of Th1 cells activation, and IgG1 reflected Th2 cells. The activation of Th1 and Th2 cells reflected the response intensity of cellular immunity and humoral immunity, respectively. So the content of IgG2a and IgG1 reflected the strength of cellular immunity and humoral immunity, while the ratio reflected how the balance of humoral immunity and cellular immunity changes in mice after vaccination [34,35,36]. We discovered that the decline in IgG2a levels was inhibited after boost, indicating that Th1 cells were activated. This result was also supported by a large increase in the levels of TNF-α, which is related to Th1 cells. A mouse experiment showed that CD4+ T cells can provide protection against viruses independent of B cells and CD8+ T cells [37]. Th1 cells can also activate CD8+ T cells and natural killer (NK) cells to help clear intracellular infections [38]. Th1 cells modulate lymph node recruitment of CD8+ T cells, bringing the CD8+ T cells into contact with antigens provided by antigen-presenting cells and thereby activating virus-specific CD8+ T cells [39,40]. In addition to the role of CD4+ T cells, specific CD8+ T cells require antigen activation. Whole-virion particles contain whole viral proteins, which include Matrix Protein, NP, PB1, PB2, PA, etc., all of which are important targets for virus-specific CD4+ and CD8+ T cell recognition. For example, CD8+ cytotoxic T lymphocytes (CTLs) among CD8+ T cells are an important component of cellular immunity in the fight against viral infection [41], and the main function of CTLs is to recognize and kill virus-infected cells by releasing perforin and granzyme B to induce apoptosis [42]. We observed activated CD8+ T cells in the 1 and 15 μg groups. The other dose groups did not show significant differences, but their mean values were higher than those of the placebo group, which might be related to specific proteins of the H5N1 virus. A study on avian influenza showed that highly conserved amino acid residues at residues 58–66 on M1 and 383–391 on NP of the 2009 human H1N1 pandemic virus enhanced the specific recognition by CTLs [43]. M and NP in the H5N1 virus also contain this sequence, so the activation of CD8+ T cells in this study might be caused by those residues. On the other hand, unlike humoral immunity, cellular immunity can provide prolonged protection after vaccination [44,45,46,47]. T cell-mediated immune responses provide a primary/extensive level of protection against influenza infection, and the precise molecular mechanisms behind their protective effects are not known [48]. In summary, after vaccination, both Th1 and Th2 cells are activated and participate in the immune response, and some CD8+ T cells are activated, while humoral immunity is activated and begins to induce the secretion of high titers of neutralizing and long-lived antibodies. The challenge test was a comprehensive assessment of the vaccine’s effectiveness, and the results indicate that the vaccine reduces clinical symptoms in mice. The lungs of the mice were infected in both the vaccine and placebo groups, but the degree of infection was different. The vaccine groups showed no obvious clinical symptoms, i.e., precipitous weight loss, vertical hair, or bowed back. Interestingly, the mice in the vaccine groups developed fever, which may be due to the increased inflammatory response caused by the re-exposure to the antigen in the immunized mice. In conclusion, the vaccine provides protection by reducing symptoms after infection.

In addition, some indicators in the early stage of vaccination are also worthy of attention. Respiratory viruses cause significant changes in the levels of cytokines or chemokines such as IL-6, MCP-1, and IP-10 during infection [49]. IP-10 is widely produced by various cell types on stimulation, including monocytes, T lymphocytes, NK cells, endothelial cells, and stromal cells, among others, where monocytes are responsible for the greatest proportion of IP-10 expression. During the viral infection, the activation of NK cells and monocytes, which function as virus-clearing cells in innate immunity, can be represented by IP-10 expression [50,51]. However, IP-10 is also a marker of adverse event, and could compromise the vaccine’s safety, overexpression of IP-10 can cause acute asthma or other adverse reactions [52]. IP-10 induced by IFN-γ in serum is a biomarker of the severity of acute respiratory infections [53]. It has been shown that the cytokine storm caused by H5N1 may be due to mutations in HA and neuraminidase (NA) glycosylation and sialylation that enhance immune-mediated pathology [49]. Of cause, IP-10 decreased to normal levels after 12 h, suggesting that the acute response may be short-lived. Unfortunately, the changes in IL-6 and MCP-1 levels were not captured in this experiment because the levels were too low. Significant alterations in the levels of all three cytokines generated by an inactivated whole-virion vaccine were detected in C57BL/6 mice in a study by Sekiya et al., which was not observed with a split virus vaccine formulation [54]. Further clinical trials should focus on the dual characteristics of IP-10. The efficacy of the H5N1 vaccine can be further evaluated by a NA inhibition assay. In addition to immunogenicity, the stability and safety of cell-based influenza vaccines also need further study. With the receding of the SARS-COV-2 epidemic, social distance, wearing masks, washing hands, and other measures have also been gradually removed, and a new round of influenza virus outbreaks has also emerged in China [55]. Outbreaks of seasonal influenza viruses such as H3N2 have occurred in several parts of China (https://www.chinacdc.cn/). Although avian influenza viruses such as H5N1 and H7N9 have not been widespread for a decade, they are still evolving in wild birds and could cause a huge social and economic burden once spread among population. Currently, H5N1 vaccines under development are mainly formulated with adjuvants such as MF59 and AS03, which are not yet licensed in China [56,57]. H5N1 vaccines that retain intact virus particles provide long-lasting and cellular immune-inducing protection without adjuvants, and might prevent death from the virus. The results of this study will be submitted to the China Food and Drug Administration for clinical application of H5N1 vaccine.

# Co-first author.

  1. Funding information: This research was supported by Ministry of Science and Technology of the People’s Republic of China (No. 2010AA022905).

  2. Conflict of interest: Authors state no conflict of interest.

  3. Data availability statement: The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.

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Received: 2022-04-26
Revised: 2022-07-06
Accepted: 2022-07-17
Published Online: 2022-09-26

© 2022 Zhegang Zhang et al., published by De Gruyter

This work is licensed under the Creative Commons Attribution 4.0 International License.

Articles in the same Issue

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  3. The mother of all battles: Viruses vs humans. Can humans avoid extinction in 50–100 years?
  4. Knockdown of G1P3 inhibits cell proliferation and enhances the cytotoxicity of dexamethasone in acute lymphoblastic leukemia
  5. LINC00665 regulates hepatocellular carcinoma by modulating mRNA via the m6A enzyme
  6. Association study of CLDN14 variations in patients with kidney stones
  7. Concanavalin A-induced autoimmune hepatitis model in mice: Mechanisms and future outlook
  8. Regulation of miR-30b in cancer development, apoptosis, and drug resistance
  9. Informatic analysis of the pulmonary microecology in non-cystic fibrosis bronchiectasis at three different stages
  10. Swimming attenuates tumor growth in CT-26 tumor-bearing mice and suppresses angiogenesis by mediating the HIF-1α/VEGFA pathway
  11. Characterization of intestinal microbiota and serum metabolites in patients with mild hepatic encephalopathy
  12. Functional conservation and divergence in plant-specific GRF gene family revealed by sequences and expression analysis
  13. Application of the FLP/LoxP-FRT recombination system to switch the eGFP expression in a model prokaryote
  14. Biomedical evaluation of antioxidant properties of lamb meat enriched with iodine and selenium
  15. Intravenous infusion of the exosomes derived from human umbilical cord mesenchymal stem cells enhance neurological recovery after traumatic brain injury via suppressing the NF-κB pathway
  16. Effect of dietary pattern on pregnant women with gestational diabetes mellitus and its clinical significance
  17. Potential regulatory mechanism of TNF-α/TNFR1/ANXA1 in glioma cells and its role in glioma cell proliferation
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  19. Quercetin inhibits cytotoxicity of PC12 cells induced by amyloid-beta 25–35 via stimulating estrogen receptor α, activating ERK1/2, and inhibiting apoptosis
  20. Nutrition intervention in the management of novel coronavirus pneumonia patients
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  22. Bmi-1 directly upregulates glucose transporter 1 in human gastric adenocarcinoma
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  171. Erratum
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  173. Erratum to “A two-microRNA signature predicts the progression of male thyroid cancer”
  174. Retraction
  175. Retraction of “Lidocaine has antitumor effect on hepatocellular carcinoma via the circ_DYNC1H1/miR-520a-3p/USP14 axis”
https://doi.org/10.1515/biol-2022-0478
Keywords for this article
MDCK cells; humoral immune response; cellular immune response; H5N1; vaccine
Creative Commons
BY 4.0

Articles in the same Issue

  1. Biomedical Sciences
  2. Effects of direct oral anticoagulants dabigatran and rivaroxaban on the blood coagulation function in rabbits
  3. The mother of all battles: Viruses vs humans. Can humans avoid extinction in 50–100 years?
  4. Knockdown of G1P3 inhibits cell proliferation and enhances the cytotoxicity of dexamethasone in acute lymphoblastic leukemia
  5. LINC00665 regulates hepatocellular carcinoma by modulating mRNA via the m6A enzyme
  6. Association study of CLDN14 variations in patients with kidney stones
  7. Concanavalin A-induced autoimmune hepatitis model in mice: Mechanisms and future outlook
  8. Regulation of miR-30b in cancer development, apoptosis, and drug resistance
  9. Informatic analysis of the pulmonary microecology in non-cystic fibrosis bronchiectasis at three different stages
  10. Swimming attenuates tumor growth in CT-26 tumor-bearing mice and suppresses angiogenesis by mediating the HIF-1α/VEGFA pathway
  11. Characterization of intestinal microbiota and serum metabolites in patients with mild hepatic encephalopathy
  12. Functional conservation and divergence in plant-specific GRF gene family revealed by sequences and expression analysis
  13. Application of the FLP/LoxP-FRT recombination system to switch the eGFP expression in a model prokaryote
  14. Biomedical evaluation of antioxidant properties of lamb meat enriched with iodine and selenium
  15. Intravenous infusion of the exosomes derived from human umbilical cord mesenchymal stem cells enhance neurological recovery after traumatic brain injury via suppressing the NF-κB pathway
  16. Effect of dietary pattern on pregnant women with gestational diabetes mellitus and its clinical significance
  17. Potential regulatory mechanism of TNF-α/TNFR1/ANXA1 in glioma cells and its role in glioma cell proliferation
  18. Effect of the genetic mutant G71R in uridine diphosphate-glucuronosyltransferase 1A1 on the conjugation of bilirubin
  19. Quercetin inhibits cytotoxicity of PC12 cells induced by amyloid-beta 25–35 via stimulating estrogen receptor α, activating ERK1/2, and inhibiting apoptosis
  20. Nutrition intervention in the management of novel coronavirus pneumonia patients
  21. circ-CFH promotes the development of HCC by regulating cell proliferation, apoptosis, migration, invasion, and glycolysis through the miR-377-3p/RNF38 axis
  22. Bmi-1 directly upregulates glucose transporter 1 in human gastric adenocarcinoma
  23. Lacunar infarction aggravates the cognitive deficit in the elderly with white matter lesion
  24. Hydroxysafflor yellow A improved retinopathy via Nrf2/HO-1 pathway in rats
  25. Comparison of axon extension: PTFE versus PLA formed by a 3D printer
  26. Elevated IL-35 level and iTr35 subset increase the bacterial burden and lung lesions in Mycobacterium tuberculosis-infected mice
  27. A case report of CAT gene and HNF1β gene variations in a patient with early-onset diabetes
  28. Study on the mechanism of inhibiting patulin production by fengycin
  29. SOX4 promotes high-glucose-induced inflammation and angiogenesis of retinal endothelial cells by activating NF-κB signaling pathway
  30. Relationship between blood clots and COVID-19 vaccines: A literature review
  31. Analysis of genetic characteristics of 436 children with dysplasia and detailed analysis of rare karyotype
  32. Bioinformatics network analyses of growth differentiation factor 11
  33. NR4A1 inhibits the epithelial–mesenchymal transition of hepatic stellate cells: Involvement of TGF-β–Smad2/3/4–ZEB signaling
  34. Expression of Zeb1 in the differentiation of mouse embryonic stem cell
  35. Study on the genetic damage caused by cadmium sulfide quantum dots in human lymphocytes
  36. Association between single-nucleotide polymorphisms of NKX2.5 and congenital heart disease in Chinese population: A meta-analysis
  37. Assessment of the anesthetic effect of modified pentothal sodium solution on Sprague-Dawley rats
  38. Genetic susceptibility to high myopia in Han Chinese population
  39. Potential biomarkers and molecular mechanisms in preeclampsia progression
  40. Silencing circular RNA-friend leukemia virus integration 1 restrained malignancy of CC cells and oxaliplatin resistance by disturbing dyskeratosis congenita 1
  41. Endostar plus pembrolizumab combined with a platinum-based dual chemotherapy regime for advanced pulmonary large-cell neuroendocrine carcinoma as a first-line treatment: A case report
  42. The significance of PAK4 in signaling and clinicopathology: A review
  43. Sorafenib inhibits ovarian cancer cell proliferation and mobility and induces radiosensitivity by targeting the tumor cell epithelial–mesenchymal transition
  44. Characterization of rabbit polyclonal antibody against camel recombinant nanobodies
  45. Active legumain promotes invasion and migration of neuroblastoma by regulating epithelial-mesenchymal transition
  46. Effect of cell receptors in the pathogenesis of osteoarthritis: Current insights
  47. MT-12 inhibits the proliferation of bladder cells in vitro and in vivo by enhancing autophagy through mitochondrial dysfunction
  48. Study of hsa_circRNA_000121 and hsa_circRNA_004183 in papillary thyroid microcarcinoma
  49. BuyangHuanwu Decoction attenuates cerebral vasospasm caused by subarachnoid hemorrhage in rats via PI3K/AKT/eNOS axis
  50. Effects of the interaction of Notch and TLR4 pathways on inflammation and heart function in septic heart
  51. Monosodium iodoacetate-induced subchondral bone microstructure and inflammatory changes in an animal model of osteoarthritis
  52. A rare presentation of type II Abernethy malformation and nephrotic syndrome: Case report and review
  53. Rapid death due to pulmonary epithelioid haemangioendothelioma in several weeks: A case report
  54. Hepatoprotective role of peroxisome proliferator-activated receptor-α in non-cancerous hepatic tissues following transcatheter arterial embolization
  55. Correlation between peripheral blood lymphocyte subpopulations and primary systemic lupus erythematosus
  56. A novel SLC8A1-ALK fusion in lung adenocarcinoma confers sensitivity to alectinib: A case report
  57. β-Hydroxybutyrate upregulates FGF21 expression through inhibition of histone deacetylases in hepatocytes
  58. Identification of metabolic genes for the prediction of prognosis and tumor microenvironment infiltration in early-stage non-small cell lung cancer
  59. BTBD10 inhibits glioma tumorigenesis by downregulating cyclin D1 and p-Akt
  60. Mucormycosis co-infection in COVID-19 patients: An update
  61. Metagenomic next-generation sequencing in diagnosing Pneumocystis jirovecii pneumonia: A case report
  62. Long non-coding RNA HOXB-AS1 is a prognostic marker and promotes hepatocellular carcinoma cells’ proliferation and invasion
  63. Preparation and evaluation of LA-PEG-SPION, a targeted MRI contrast agent for liver cancer
  64. Proteomic analysis of the liver regulating lipid metabolism in Chaohu ducks using two-dimensional electrophoresis
  65. Nasopharyngeal tuberculosis: A case report
  66. Characterization and evaluation of anti-Salmonella enteritidis activity of indigenous probiotic lactobacilli in mice
  67. Aberrant pulmonary immune response of obese mice to periodontal infection
  68. Bacteriospermia – A formidable player in male subfertility
  69. In silico and in vivo analysis of TIPE1 expression in diffuse large B cell lymphoma
  70. Effects of KCa channels on biological behavior of trophoblasts
  71. Interleukin-17A influences the vulnerability rather than the size of established atherosclerotic plaques in apolipoprotein E-deficient mice
  72. Multiple organ failure and death caused by Staphylococcus aureus hip infection: A case report
  73. Prognostic signature related to the immune environment of oral squamous cell carcinoma
  74. Primary and metastatic squamous cell carcinoma of the thyroid gland: Two case reports
  75. Neuroprotective effects of crocin and crocin-loaded niosomes against the paraquat-induced oxidative brain damage in rats
  76. Role of MMP-2 and CD147 in kidney fibrosis
  77. Geometric basis of action potential of skeletal muscle cells and neurons
  78. Babesia microti-induced fulminant sepsis in an immunocompromised host: A case report and the case-specific literature review
  79. Role of cerebellar cortex in associative learning and memory in guinea pigs
  80. Application of metagenomic next-generation sequencing technique for diagnosing a specific case of necrotizing meningoencephalitis caused by human herpesvirus 2
  81. Case report: Quadruple primary malignant neoplasms including esophageal, ureteral, and lung in an elderly male
  82. Long non-coding RNA NEAT1 promotes angiogenesis in hepatoma carcinoma via the miR-125a-5p/VEGF pathway
  83. Osteogenic differentiation of periodontal membrane stem cells in inflammatory environments
  84. Knockdown of SHMT2 enhances the sensitivity of gastric cancer cells to radiotherapy through the Wnt/β-catenin pathway
  85. Continuous renal replacement therapy combined with double filtration plasmapheresis in the treatment of severe lupus complicated by serious bacterial infections in children: A case report
  86. Simultaneous triple primary malignancies, including bladder cancer, lymphoma, and lung cancer, in an elderly male: A case report
  87. Preclinical immunogenicity assessment of a cell-based inactivated whole-virion H5N1 influenza vaccine
  88. One case of iodine-125 therapy – A new minimally invasive treatment of intrahepatic cholangiocarcinoma
  89. S1P promotes corneal trigeminal neuron differentiation and corneal nerve repair via upregulating nerve growth factor expression in a mouse model
  90. Early cancer detection by a targeted methylation assay of circulating tumor DNA in plasma
  91. Calcifying nanoparticles initiate the calcification process of mesenchymal stem cells in vitro through the activation of the TGF-β1/Smad signaling pathway and promote the decay of echinococcosis
  92. Evaluation of prognostic markers in patients infected with SARS-CoV-2
  93. N6-Methyladenosine-related alternative splicing events play a role in bladder cancer
  94. Characterization of the structural, oxidative, and immunological features of testis tissue from Zucker diabetic fatty rats
  95. Effects of glucose and osmotic pressure on the proliferation and cell cycle of human chorionic trophoblast cells
  96. Investigation of genotype diversity of 7,804 norovirus sequences in humans and animals of China
  97. Characteristics and karyotype analysis of a patient with turner syndrome complicated with multiple-site tumors: A case report
  98. Aggravated renal fibrosis is positively associated with the activation of HMGB1-TLR2/4 signaling in STZ-induced diabetic mice
  99. Distribution characteristics of SARS-CoV-2 IgM/IgG in false-positive results detected by chemiluminescent immunoassay
  100. SRPX2 attenuated oxygen–glucose deprivation and reperfusion-induced injury in cardiomyocytes via alleviating endoplasmic reticulum stress-induced apoptosis through targeting PI3K/Akt/mTOR axis
  101. Aquaporin-8 overexpression is involved in vascular structure and function changes in placentas of gestational diabetes mellitus patients
  102. Relationship between CRP gene polymorphisms and ischemic stroke risk: A systematic review and meta-analysis
  103. Effects of growth hormone on lipid metabolism and sexual development in pubertal obese male rats
  104. Cloning and identification of the CTLA-4IgV gene and functional application of vaccine in Xinjiang sheep
  105. Antitumor activity of RUNX3: Upregulation of E-cadherin and downregulation of the epithelial–mesenchymal transition in clear-cell renal cell carcinoma
  106. PHF8 promotes osteogenic differentiation of BMSCs in old rat with osteoporosis by regulating Wnt/β-catenin pathway
  107. A review of the current state of the computer-aided diagnosis (CAD) systems for breast cancer diagnosis
  108. Bilateral dacryoadenitis in adult-onset Still’s disease: A case report
  109. A novel association between Bmi-1 protein expression and the SUVmax obtained by 18F-FDG PET/CT in patients with gastric adenocarcinoma
  110. The role of erythrocytes and erythroid progenitor cells in tumors
  111. Relationship between platelet activation markers and spontaneous abortion: A meta-analysis
  112. Abnormal methylation caused by folic acid deficiency in neural tube defects
  113. Silencing TLR4 using an ultrasound-targeted microbubble destruction-based shRNA system reduces ischemia-induced seizures in hyperglycemic rats
  114. Plant Sciences
  115. Seasonal succession of bacterial communities in cultured Caulerpa lentillifera detected by high-throughput sequencing
  116. Cloning and prokaryotic expression of WRKY48 from Caragana intermedia
  117. Novel Brassica hybrids with different resistance to Leptosphaeria maculans reveal unbalanced rDNA signal patterns
  118. Application of exogenous auxin and gibberellin regulates the bolting of lettuce (Lactuca sativa L.)
  119. Phytoremediation of pollutants from wastewater: A concise review
  120. Genome-wide identification and characterization of NBS-encoding genes in the sweet potato wild ancestor Ipomoea trifida (H.B.K.)
  121. Alleviative effects of magnetic Fe3O4 nanoparticles on the physiological toxicity of 3-nitrophenol to rice (Oryza sativa L.) seedlings
  122. Selection and functional identification of Dof genes expressed in response to nitrogen in Populus simonii × Populus nigra
  123. Study on pecan seed germination influenced by seed endocarp
  124. Identification of active compounds in Ophiopogonis Radix from different geographical origins by UPLC-Q/TOF-MS combined with GC-MS approaches
  125. The entire chloroplast genome sequence of Asparagus cochinchinensis and genetic comparison to Asparagus species
  126. Genome-wide identification of MAPK family genes and their response to abiotic stresses in tea plant (Camellia sinensis)
  127. Selection and validation of reference genes for RT-qPCR analysis of different organs at various development stages in Caragana intermedia
  128. Cloning and expression analysis of SERK1 gene in Diospyros lotus
  129. Integrated metabolomic and transcriptomic profiling revealed coping mechanisms of the edible and medicinal homologous plant Plantago asiatica L. cadmium resistance
  130. A missense variant in NCF1 is associated with susceptibility to unexplained recurrent spontaneous abortion
  131. Assessment of drought tolerance indices in faba bean genotypes under different irrigation regimes
  132. The entire chloroplast genome sequence of Asparagus setaceus (Kunth) Jessop: Genome structure, gene composition, and phylogenetic analysis in Asparagaceae
  133. Food Science
  134. Dietary food additive monosodium glutamate with or without high-lipid diet induces spleen anomaly: A mechanistic approach on rat model
  135. Binge eating disorder during COVID-19
  136. Potential of honey against the onset of autoimmune diabetes and its associated nephropathy, pancreatitis, and retinopathy in type 1 diabetic animal model
  137. FTO gene expression in diet-induced obesity is downregulated by Solanum fruit supplementation
  138. Physical activity enhances fecal lactobacilli in rats chronically drinking sweetened cola beverage
  139. Supercritical CO2 extraction, chemical composition, and antioxidant effects of Coreopsis tinctoria Nutt. oleoresin
  140. Functional constituents of plant-based foods boost immunity against acute and chronic disorders
  141. Effect of selenium and methods of protein extraction on the proteomic profile of Saccharomyces yeast
  142. Microbial diversity of milk ghee in southern Gansu and its effect on the formation of ghee flavor compounds
  143. Ecology and Environmental Sciences
  144. Effects of heavy metals on bacterial community surrounding Bijiashan mining area located in northwest China
  145. Microorganism community composition analysis coupling with 15N tracer experiments reveals the nitrification rate and N2O emissions in low pH soils in Southern China
  146. Genetic diversity and population structure of Cinnamomum balansae Lecomte inferred by microsatellites
  147. Preliminary screening of microplastic contamination in different marine fish species of Taif market, Saudi Arabia
  148. Plant volatile organic compounds attractive to Lygus pratensis
  149. Effects of organic materials on soil bacterial community structure in long-term continuous cropping of tomato in greenhouse
  150. Effects of soil treated fungicide fluopimomide on tomato (Solanum lycopersicum L.) disease control and plant growth
  151. Prevalence of Yersinia pestis among rodents captured in a semi-arid tropical ecosystem of south-western Zimbabwe
  152. Effects of irrigation and nitrogen fertilization on mitigating salt-induced Na+ toxicity and sustaining sea rice growth
  153. Bioengineering and Biotechnology
  154. Poly-l-lysine-caused cell adhesion induces pyroptosis in THP-1 monocytes
  155. Development of alkaline phosphatase-scFv and its use for one-step enzyme-linked immunosorbent assay for His-tagged protein detection
  156. Development and validation of a predictive model for immune-related genes in patients with tongue squamous cell carcinoma
  157. Agriculture
  158. Effects of chemical-based fertilizer replacement with biochar-based fertilizer on albic soil nutrient content and maize yield
  159. Genome-wide identification and expression analysis of CPP-like gene family in Triticum aestivum L. under different hormone and stress conditions
  160. Agronomic and economic performance of mung bean (Vigna radiata L.) varieties in response to rates of blended NPS fertilizer in Kindo Koysha district, Southern Ethiopia
  161. Influence of furrow irrigation regime on the yield and water consumption indicators of winter wheat based on a multi-level fuzzy comprehensive evaluation
  162. Discovery of exercise-related genes and pathway analysis based on comparative genomes of Mongolian originated Abaga and Wushen horse
  163. Lessons from integrated seasonal forecast-crop modelling in Africa: A systematic review
  164. Evolution trend of soil fertility in tobacco-planting area of Chenzhou, Hunan Province, China
  165. Animal Sciences
  166. Morphological and molecular characterization of Tatera indica Hardwicke 1807 (Rodentia: Muridae) from Pothwar, Pakistan
  167. Research on meat quality of Qianhua Mutton Merino sheep and Small-tail Han sheep
  168. SI: A Scientific Memoir
  169. Suggestions on leading an academic research laboratory group
  170. My scientific genealogy and the Toronto ACDC Laboratory, 1988–2022
  171. Erratum
  172. Erratum to “Changes of immune cells in patients with hepatocellular carcinoma treated by radiofrequency ablation and hepatectomy, a pilot study”
  173. Erratum to “A two-microRNA signature predicts the progression of male thyroid cancer”
  174. Retraction
  175. Retraction of “Lidocaine has antitumor effect on hepatocellular carcinoma via the circ_DYNC1H1/miR-520a-3p/USP14 axis”
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