Enhances antibody titer and T cell activity of quadrivalent influenza vaccine by combination with CpG HP021

animals, vaccines, viruses

We purchased 6- to 8-week-old BALB/c mice and 10-week-old rats from Shanghai Slac Laboratory Animal Co., Ltd (Shanghai, China). CpG HP021 adjuvant was prepared by Jiangsu Taipurui Biotechnology Co., Ltd (Jiangsu Province, China). TCGCAACGTTGCCTTCGAAGG-3' is the sequence of CpG HP021, a newly synthesized adjuvant, patent application number CN 117,568,339. Quadrivalent influenza vaccines for the 2022-2023 season (A/Victoria/2570/2019, A/Darwin/September 2021, B/Austria/1,359,417/2021, B/Phuket/3073/2013) and influenza viruses for vaccine evaluation Cross-reactivity (A/California/07/2009, A/Michigan/45/2015, A/GM/SWL1536/2019, A/Wisconsin/15/09, A/Texas/50/2012, B/Florida/4/2006, B/Massachusetts/02/2012, B/ (Brisbane/60/2008, B/Washington/02/2019) These are Zhejiang Tianyuan Bio-Pharmaceutical Co., Ltd (Zhejiang, China).

immunity

BALB/c mice (n = 12 in each group) were administered two doses (days 0 and 14) of 50 μL (total 100 μL) containing IIV4 (3 μg each) in each paw. HA) in the range of CpG HP021 (0, 10, 20, 40, 80, or 160) μg) (Table 1). The control group received phosphate buffered saline (PBS). SD rats (n = 6 in each group) were also immunized by IM injection into the hindlimbs, receiving two doses (days 0 and 14) of 100 µL (total 200 µL) containing IIV4 (6 µg each HA) in each leg. ) was administered. CpG HP021 dose range (0, 50, 100, 200, or 400) µg), the control group was immunized with PBS (Table 2). The vaccinated mice and SD rats were euthanized using carbon dioxide asphyxiation, followed by cervical dislocation. Blood samples were collected before and on days 14, 21, 28, and 56 after the first immunization. Animals were sacrificed on days 28 and 56 after the first immunization, and spleens were collected for cell reaction testing.

ethics statement

All animal experiments were conducted in accordance with the Zhejiang Provincial Guide for the Care and Use of Laboratory Animals, and this study was approved by the Ethics Committee of Zhejiang Chinese Medical University (ethics approval number IACUC-202310–29). All steps of the study were performed in accordance with ARRIVE guidelines.

HI assay

HI tests were performed as previously described.⁵⁰. Briefly, 200 μL of receptor-disrupting enzyme was mixed with 50 μL of mouse serum and incubated overnight at 37 °C. After inactivation at 56 °C for 1 h, the mixture was diluted using PBS to an initial concentration of 1:20, followed by 2-fold serial dilutions. Viruses were titrated using 1% fresh chicken red blood cells (RBCs). Then, 4 HA units of virus were added to a 96-well U-bottom plate containing diluted serum. After incubation for 30 min at room temperature (RT), 1% chicken RBCs were added and incubated for 30 min at RT. HI titer was defined as the reciprocal of the highest serum dilution that could prevent RBC hemagglutination. SCR is defined as a prevaccination HI titer of less than 1:20 and a HI titer of 1:40 or more on day 28 after the first immunization, or a prevaccination HI titer of 1:20 or more and a HI titer of 28 days after the first immunization. defined as the proportion of animals with a 4-fold or greater increase in titer. . GMI is calculated by dividing the post-vaccination GMT by the pre-vaccination GMT.

Elisa

ELISA was performed as previously described ²⁶. Briefly, influenza antigens (0.5 μg/mL) were added to 96-well plates and stored overnight at 4 °C. Plates were blocked using confinement solution for 2 h. Serum samples were serially diluted 2-fold from a starting dilution of 1:1000 and added to the plate. Plates were incubated for 2 hours. Plates were then incubated with biotin anti-mouse IgG (catalog no. 1036-08), biotin anti-mouse IgG1 (catalog no. 1071-08), or biotin anti-mouse IgG2a (catalog no. 1081-08). 08) (all from Southern Biotech) 1 hour at room temperature. The plates were then incubated with streptavidin (HRP) for 40 min. After adding 100 μL of 3,30,5,50-tetramethylbiphenyl anhydride for 5 minutes, the reaction was stopped with 2M sulfuric acid. An enzyme labeling device was utilized to measure the absorbance at 450 nm. Antibody titer is the reciprocal of the highest dilution of antibody measured when the ratio of the optical density (OD) value of the test antibody to the OD value of the negative control is 2.1 or greater.

Erispot

Mouse IFN-γ ELISpotPLUS kit (Mabtech, catalog number 3321-4HPW-10) and mouse TNF-α ELISpotPLUS kit (Mabtech, catalog number 3511-4HPW-10) were used to assess T cell responses in mice. Rat. The Rat IFN-γ ELISPOT Kit (U-CyTech, Cat. No. CT079-PR5) and the Rat IL-4 ELISPOT Kit (U-CyTech, Cat. No. CT081-PR2) were used to assess T cell responses in rats. Ta. SD rat. 4 x 10 in 96-well plate⁵ Freshly obtained splenocytes were added to each well. Plates were incubated in 1640 medium with or without 5 μg/mL influenza antigen for 20 h at 37 °C and 5% CO .₂. The ELISpot plates were then stained according to the manufacturer's instructions. Plates were then read using an ELISPOT® Spot Imaging Analyzer.

MSD cytokine analysis

Serum samples from BALB/c mice were collected 28 days after the first vaccination. After fresh separation, 8 x 10⁵ Splenocytes per well of mice and SD rats were stimulated with 5 μg/mL influenza antigen for 28 hours. Subsequently, the supernatant was collected. V-PLEX Pro-Inflammatory Panel 1 (Mouse) Kit (Meso Scale Diagnostics, Cat. No. K15048D) and V-PLEX Pro-Inflammatory Panel 2 (Rat) Kit (Cat. No. K15059D-1).

flow cytometry

Mouse splenocytes were stained with Fixable Viability Stain 700. Cells were then stained with FITC anti-mouse CD3, BV605 anti-mouse CD8, phycoerythrin (PE)-cy7 anti-mouse CD4, and BV510 anti-mouse for 30 min at 4 °C. -Mouse CD62L, and BUV395 anti-mouse CD44 antibodies from BD Pharmingen. The stained cells were then examined on a CytoFLEX flow cytometer. Some mouse spleen cells were collected and cultured in 1640 medium with or without 5 μg/mL influenza antigen for 10 hours. Cells were then incubated with Fixable Viability Stain 700 and FITC anti-mouse CD3, BV510 anti-mouse CD4 and BV605 anti-mouse CD8 for 30 min at 4 °C. Fixation/permeabilization solution was added and incubated for 30 min at 4 °C, followed by the addition of Percp-cy5.5 IFN-γ. The samples were then incubated at 4 °C for 40 min. The stained cells were then examined on a CytoFLEX flow cytometer.

Statistical considerations

Data were analyzed using GraphPad Prism 10.2 software. Data analysis was performed using T-test, one-way analysis of variance (ANOVA) with Tukey's test, or Kruskal-Wallis test with post-hoc Dunn's multiple. A P value ≤ 0.05 is considered significant. In the figure, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.