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Pathogen-free female BALB/c mice aged 6-8 weeks were obtained from Beijing Vital River Laboratory Animal Technology (Beijing, China) and maintained under pathogen-free conditions. All animal experiments in this study were approved by the Animal Experimental Ethical Committee of the Nation Institute for Viral Disease Control and Prevention (No. 20170606019-2). The mice were euthanized by CO2 inhalation.
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Influenza virus A/Hong Kong/1968 (H3N2 subtype) was used in the challenge infection. It was kindly supplied by Professor Earl Brown from the University of Ottawa. This virus was grown for 48 h in 9-11-day-old embryonated eggs at 37 ℃. Allantoic fluid was collected and stored at −80 ℃ until use.
H3N2 split vaccine (A/Hong Kong/4801/2014) (designated as sH3N2 vaccine) was donated by Yening Zou from Sinovac Biotech Ltd. Adjuvant of alum (aluminum hydroxide, 10 mg/mL) was purchased from Thermo ScientificTM (United States). Adjuvant of CTA1-DD was expressed in Escherichia coli (E. coil) BL21 (DE3) as described previously[22].
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The formulations of vaccine and adjuvant were as previous reported[21].
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Sixty mice were randomly assigned to five groups (Table 1). All animals were immunized twice, two weeks apart. Mice were anesthetized with CO2 before nasal immunization with 3 μg of H3N2 vaccine, either alone or with 5 μg of CTA1-DD in a final volume of 50 μL administered dropwise at 25 μL per nostril. Negative control mice were administered CTA1-DD alone or phosphate-buffered saline (PBS) via the i.n. route. Positive control mice were administered alum adjuvant plus H3N2 split vaccine via the intramuscular (i.m.) route. Two weeks later, mice received a second dose.
Table 1. Immunizations
Groups Adjuvant Antigen Route PBS - PBS i.n. vaccine alone - H3N2 split vaccine i.n. vaccine+alum Al(OH)3 H3N2 split vaccine i.m. vaccine+CTA1-DD CTA1-DD H3N2 split vaccine i.n. CTA1-DD alone CTA1-DD - i.n. On days 0, 14, 21, 28, and 35, blood samples were collected and serum were separated by centrifugation and stored at −20 ℃ prior to analysis. One week after boost, some mice were euthanized, and the spleens, bronchoalveolar lavages and vaginal lavages were collected.
For the challenge studies, at three weeks after boost, six mice from each group were anesthetized with CO2 and then infected with the minimal level of five times the 50% mouse lethal dose (5 × MLD50) of the mouse-adapted re-assortant influenza A H3N2 virus strain A/Hong Kong/1968 in PBS via the i.n. route. Body weight changes and mortality were monitored for 14 days after challenge.
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HI assays were performed as described previously[23]. In this study, 4 HA units of the H3N2 A/Hong Kong/4801/2014 virus and 1% guinea pig red blood cells were used.
Antibody titers of influenza virus-specific IgM, IgG, IgG1, IgG2a, and IgA were determined using enzyme-linked immunosorbent assays (ELISAs). In brief, the assays were performed in 96-well plates coated at 4 ℃ for 12 h with 200 ng/well antigen protein, and then blocked with 3% BSA. Serially diluted serum and mucosal samples were incubated at 37 ℃ for 2 h in the plates. Antibody titers were measured with HRP-conjugated goat anti-mouse IgM, IgG, IgG1, IgG2a, and IgA secondary antibodies (Sigma-Aldrich, St. Louis, MO, USA) and TMB substrate (Solarbio, Beijing, China). When the reaction was stopped with 2 mol/L H2SO4, the values at 450 nm were detected by an ELISA reader (Thermo Scientific, Vantaa, Finland) and endpoint titers were defined as the highest dilution of sample for which the optical density (OD) was at least twice that of the negative control samples.
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Two weeks after boost, the spleen samples were analyzed through interferon-γ (IFN-γ) and interleukin-4 (IL-4) ELISpot assays using an ELISpot kit (Dakewe, Beijing, China) according to the manufacturer's instructions.
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One-way ANOVA with Tukey's multiple comparison tests (GraphPad Prism v.5) were used for the analysis of significance in all experiments groups. In addition, the Kaplan-Meier test was used to compare percent survival among groups of mice. Statistical significance is presented as P-values: P < 0.05 (*), P < 0.01 (**) and P < 0.001 (***).
doi: 10.3967/bes2019.070
Intranasal Immunization Using CTA1-DD as a Mucosal Adjuvant for an Inactivated Influenza Vaccine
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Abstract:
Objective To evaluate the effect of intranasal immunization with CTA1-DD as mucosal adjuvant combined with H3N2 split vaccine. Methods Mice were immunized intranasally with PBS (negative control), or H3N2 split vaccine (3 μg/mouse) alone, or CTA1-DD (5 μg/mouse) alone, or H3N2 split vaccine (3 μg/mouse) plus CTA1-DD (5 μg/mouse). Positive control mice were immunized intramuscularly with H3N2 split vaccine (3 μg/mouse) and alum adjuvant. All the mice were immunized twice, two weeks apart. Then sera and mucosal lavages were collected. The specific HI titers, IgM, IgG, IgA, and IgG subtypes were examined by ELISA. IFN-γ and IL-4 were test by ELISpot. In addition, two weeks after the last immunization, surivival after H3N2 virus lethal challenge was measured. Results H3N2 split vaccine formulated with CTA1-DD could elicit higher IgM, IgG and hemagglutination inhibition titers in sera. Furthermore, using CTA1-DD as adjuvant significantly improved mucosal secretory IgA titers in bronchoalveolar lavages and vaginal lavages. Meanwhile this mucosal adjuvant could enhance Th-1-type responses and induce protective hemagglutination inhibition titers. Notably, the addition of CTA1-DD to split vaccine provided 100% protection against lethal infection by the H3N2 virus. Conclusion CTA1-DD could promote mucosal, humoral and cell-mediated immune responses, which supports the further development of CTA1-DD as a mucosal adjuvant for mucosal vaccines. -
Key words:
- Adjuvants /
- H3N2 /
- Influenza /
- Mucosal immunization /
- Split vaccine
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Figure 1. H3N2 HA-specific IgG and IgM are significantly enhanced by CTA1-DD adjuvant. Mice were immunized with PBS, CTA1-DD (5 μg/mouse), H3N2 split vaccine (3 μg/mouse) without or with CTA1-DD (5 μg/mouse)/alum adjuvant at days 0 and 14. Sera were collected on days 14, 21, 28, and 35. Then, H3N2 HA-specific IgG (A-D) and IgM (E-H) were determined by ELISA. Statistical analyses consisted of one-way ANOVA with Tukey's multiple comparison tests. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Figure 2. CTA1-DD increased the mucosal antibody responses and HI titers. Mice were immunized intranasally with PBS (negative control), or H3N2 split vaccine (3 μg/mouse) alone, or CTA1-DD (5 μg/mouse) alone, or H3N2 split vaccine (3 μg/mouse) plus CTA1-DD (5 μg/mouse). Positive control mice were immunized intramuscularly with H3N2 split vaccine (3 μg/mouse) and alum adjuvant. Three weeks after booster immunization, bronchoalveolar lavages (A) and vaginal lavages (B) were collected, and H3N2 HA-specific IgA was measured. Sera were also collected and HI titers (C) were assessed. The data are expressed as the mean ± SD (n = 6 mice per group). One-way ANOVA with Turkey's multiple test was used to analyze differences among groups. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Figure 3. CTA1-DD enhanced the Th-1-type response. Animals (n = 6) were immunized twice (day 0 and day 14) with PBS, CTA1-DD (5 μg/mouse), H3N2 split vaccine (3 μg/mouse) without or with CTA1-DD (5 μg/mouse)/alum adjuvant. Cytokine production from splenocytes derived from immunized mice was determined. Three weeks after the booster, spleens were collected and splenic lymphocytes were cultured. The production of IFN-γ (A) and IL-4 (B) were measured by ELISpot. Sera were also collected and IgG1 and IgG2a (C) titers were detected. The data are expressed as the mean ± SD (n = 6 mice per group). One-way ANOVA with Turkey's multiple was used to analyze differences among groups. *, P < 0.05; **, P < 0.01; ***, P < 0.001.
Figure 4. Protection of mice from lethal influenza virus. Mice were immunized with PBS, CTA1-DD (5 μg/mouse), H3N2 split vaccine (3 μg/mouse) without or with CTA1-DD (5 μg/mouse)/alum adjuvant at days 0 and 14. Three weeks after the last immunization, the immunized mice were challenged intranasally with 5 × MLD50 of the mouse-adapted re-assortant influenza A H3N2 virus strain A/Hong Kong/1968. Mice (n = 6) were monitored every two days for 14 days for body weight changes (A) and survival rates (B).
Table 1. Immunizations
Groups Adjuvant Antigen Route PBS - PBS i.n. vaccine alone - H3N2 split vaccine i.n. vaccine+alum Al(OH)3 H3N2 split vaccine i.m. vaccine+CTA1-DD CTA1-DD H3N2 split vaccine i.n. CTA1-DD alone CTA1-DD - i.n. -
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