CpG 684: an effective adjuvant for the inactivated COVID-19 vaccine in mice

Aim: This study used CpG 684 as adjuvant of inactivated COVID-19 vaccine to detect a humoral and cellular immune response in mice. Materials & methods: We used 10 and 20 µg CpG 684 as adjuvants of an inactivated COVID-19 vaccine to immunize mice. IgG, IgG1, IgG2a, IgG2b and IgM binding antibodies were detected in serum by ELISA. The IFN-γ cytokine was detected by ELISPOT. Results: CpG 684 improved spike-specific IgG and IgM subtype binding antibodies and increased the neutralizing antibody titer against prototype, Delta and Beta strains. CpG 684 also improved cellular immune response. Conclusion: CpG 684 is an effective adjuvant for inactivated COVID-19 vaccine.

and CpG 10 μg; and the inactivated COVID-19 vaccine and CpG 20 μg.The immunizing dose of inactivated COVID-19 vaccine contained 1-μg vaccine antigen and 50-μg aluminum hydroxide.Mice were immunized twice with a 28-day interval.Serum and spleen cells were obtained two weeks after the last immunization.
All animal experiments were conducted in accordance with relevant guidelines and regulations.
ELISA assay for detecting SARS-COV-2 spike-specific binding antibody SARS-COV-2 wildtype strain spike protein (Sino Biological Inc., China) was coated in 0.1 M sodium bicarbonate buffer solution (pH 9.6).The final concentration of spike protein was 1 μg/ml.About 100 μl was seeded into each well of a 96-well high-binding ELISA plate (Costar; NY, USA).The plates were incubated overnight at 4 • C.After incubation, plates were washed three-times with PBST (phosphate-buffered saline [PBS] containing 0.05% Tween 20).The plates were locked with blocking solution (PBS containing 2% bovine serum albumin) and incubated for 1 h at 37 • C. Serum was diluted 1:1000 with blocking solution and 100 μl of diluted serum was added to each well, followed by incubation at 37 • C for 1 h.After incubation, the plates were washed five-times with PBST, and 100 μl of peroxidase-conjugated antimouse immunoglobulin G, G1, G2a, G2b, G3, A and M antibody were diluted 1:5,000 with blocking solution.respectively.About 100 μl antibody dilution solution was added to the plates and these were incubated at 37 • C for 1 h.The plates were then washed five-times with PBST solution and 100 μl of 3,3 ,5,5 -tetramethylbenzidine substrate was added to each well.The plates were incubated at room temperature for 5 min.The color reaction was terminated with 50 μl 2 mol/l sulfuric acid.The optical density at 450 nm was detected with an automated plate reader (Biotek Agilent, VT, USA).
Neutralizing antibody assay SARS-COV-2 prototype, Delta and Beta strain live viruses were used to detect neutralizing antibody titer from serum.Prior to the experiment, the serum was inactivated for 30 min at 56 • C. First, the serum samples of mice were diluted in the first two columns of a 96-well plate at 1:4 dilution with cell culture maintenance medium.
To each remaining well, 200 ul solution was added and the sample was diluted by twofold serial dilutions.Each well contained equal volumes of viral solution, for a final concentration of 100 TCID50/well.The plates were then incubated at 37 • C for 1 h.Vero cells were seeded in 96-well plates and reached about 80% confluence before infection.The incubated virus-serum mixtures were added to 96-well cell plates and incubated at 37 • C for 4 days.The cytopathic effect of each well was observed under a microscope and the neutralizing antibody titer against SARS-COV-2 was recorded as the highest dilution of serum that showed 50% inhibition activity against SARS-CoV-2.
ELISPOT assay for detecting IFN-γ A commercial kit (Mouse IFN-γ ELISPOT Pair, BD, USA) was used to detect IFN-γ secreted by splenic immune cells.The antibody to IFN-γ was then diluted at 1:200 dilution with PBS, 100 μl of diluted antibody solution was added to each well of an ELISPOT plate and the plate was incubated overnight at 4 • C.After incubation, the coating solution was discarded and the plate was washed once with 200 ul/well blocking solution (1640 solution containing 10% fetal bovine serum [FBS] and 1% L-glutamine).About 200 μl of blocking solution was then added to each well, and the plate was incubated for 2 h at room temperature.Splenic cells of vaccine-immunized mice were thawed with 1640 medium (1640 solution containing 10% FBS and 1% L-glutamine) and the cells were counted by a cell counter.Cell suspensions were prepared at 10 5 cells/ml density.About 100 ul of each cell suspension were then added to the wells of the ELISPOT plate.Each splenic cells suspension was added to three wells, one as a negative control and two as experiment samples.The experiment sample wells were stimulated by 20 μg SARS-COV-2 spike antigen peptide.The ELISPOT plate was then incubated at 37 • C and 5% CO 2 for 20 h.Cell suspension was the discarded and the wells were washed twice with 200 ul deionized water.The plate was then washed three-times with 200 ul PBST solution.The biotinylated antimouse IFN-γ antibody was diluted with dilution buffer (PBS containing 10% FBS) at a ratio of 1:250.About 100 ul diluted antibody solution was added to each well and the plate was incubated at room temperature for 2 h.The plate was washed three-times with 200 ul PBST solution.The streptavidin-HRP antibody was diluted with dilution buffer (PBS containing 10% FBS) at a ratio of 1:100.100 ul diluted antibody solution was added to each well and the plate was incubated at room temperature for 1 h.Streptavidin-HRP antibody solution was discarded and the plate was washed six-times with 200 ul PBST solution, 100 ul AEC substrate solution was added and spots were allowed to develop for 30 min in the dark.The substrate color reaction was stopped by washing the wells with deionized water.The plate was air-dried overnight at room temperature in the dark.The spots were counted by ELISPOT reader (AID, Germany).

Statistical analysis
Spike protein-specific binding antibody data were presented as mean ± standard deviation.The neutralizing antibody data were presented as geometric mean.The differences between the control group and the experimental groups were analyzed using an unpaired t-test.Data were considered to be statistically significant at p < 0.05.

Spike-specific IgG & IgG binding antibody response
After two doses of the vaccine, mice serum was tested for spike-specific IgG and IgG binding antibody response (Figure 1A).The results showed that CpG increased the IgG binding antibody response to the inactivated COVID-19 vaccine (Figure 1B).The IgG1 binding antibody response to inactivated COVID-19 vaccine and CpG 20 μg was significantly lower than to the inactivated COVID-19 vaccine and the inactivated COVID-19 vaccine and CpG 10 μg group (Figure 2A).There were no differences in the IgG2a binding antibody response among the three groups (Figure 2B).The IgG2a/IgG1 ratio in the inactivated COVID-19 vaccine and CpG 20 μg group was higher than in the inactivated COVID-19 vaccine and inactivated COVID-19 vaccine and CpG 10 μg group (Figure 2C).The data indicates that CpG changed the antibody subtype ratio in response to the inactivated COVID-19 vaccine and increased functional antibody ratio.The IgG2b binding antibody response to the inactivated COVID-19 vaccine was significantly lower than to the inactivated COVID-19 vaccine and CpG 20 μg and the inactivated COVID-19 vaccine and CpG 10 μg (Figure 2D).

Spike-specific IgM binding antibody response
There were significant differences in the IgM binding antibody response to inactivated COVID-19 vaccine and CpG 10 μg, the inactivated COVID-19 vaccine and CpG 20 μg and the inactivated COVID-19 vaccine (Figure 3).The IgM binding antibody response to the inactivated COVID-19 vaccine was significantly lower than the inactivated COVID-19 vaccine and CpG 10 μg and inactivated COVID-19 vaccine and CpG 20 μg.

Neutralizing antibody response
Neutralizing antibody titers against the prototype, Delta and Beta strains of SARS-COV-2 were detected by live virus cytopathic effect.The data showed that CpG increased the neutralizing antibody titer in all tested strains, with a significant difference in neutralizing antibodies against the prototype strain between the inactivated COVID-19 vaccine and the inactivated COVID-19 vaccine and CpG 20 μg (Figure 4A).The neutralizing antibody titer against the prototype strain in response to the inactivated COVID-19 vaccine and CpG 20 μg was 2.6-times higher than that to the inactivated COVID-19 vaccine (Table 1).The neutralizing antibody titer of CpG adjuvant-inactivated vaccines against Delta and Beta strains was higher than in the inactivated vaccine group.However, compared with the neutralizing antibody titer against the prototype strain, the decrease rate of neutralizing antibody titers against Delta and Beta strains in the CpG adjuvant-inactivated vaccine group was higher than that in the inactivated vaccine group (Table 1).

Cellular response
An ELISPOT assay was conducted to detect IFN-γ secreted by SARS-COV-2 vaccine immune splenic cells, and the data indicated that splenic cells of mice injected with the inactivated vaccine hardly secreted IFN-γ cytokines (Figure 5).CpG significantly increased IFN-γ spots, and the difference between the inactivated vaccine and both the inactivated vaccine and CpG 10 μg and the inactivated vaccine and CpG 20 μg was significant.

Discussion
An effective vaccine is the best way to control the transmission of SARS-COV-2, yet the protective effect of the inactivated vaccine could be improved.CpG has been proven to improve humoral and cellular immune response in vaccine research by inducing the innate immune response to improve antigen presentation [23,24].This study investigated the CpG adjuvant effect for the inactivated COVID-19 vaccine and found that CpG 684 can increase the neutralizing antibody and IgG2b and IgM binding antibody responses.The current opinion is that neutralizing antibodies correlate to protection rate of the SARS-COV-2 vaccine [5- 10].In this study, we found that CpG 684 increased the spike-specific IgG2b and IgM binding and neutralizing antibody response.A recent study of COVID-19 vaccines showed that IgM, IgA and IgG antibodies may play an important role in the protective antiviral response of the body [25].This change of the IgG2a/IgG1 ratio may contribute to an increase in neutralizing antibodies, as IgG2a/IgG1 ratio is an important indicator of T helper cell (Th) polarization: Th1 mediates the cellular immune response and mainly secretes IFN-γ, IL-2, TNF, α-IFN and stimulates B cells to produce IgG2a antibodies, whereas Th2 mediates the humoral immune response and mainly secrete IL-4, IL-5, IL-6, IL-10 and stimulate B cells to produce IgG1 antibodies [26][27][28][29].Many studies have shown that changes in the immune response from Th1 to Th2 improve vaccine protection [30][31][32].The shift in IgG2a/IgG1 ratio induced by the CpG adjuvant may result in the increase of neutralizing antibody titer observed in our study and may improve vaccine protection.It has been reported in other vaccine research that CpG can alter vaccine-induced IgG2a/IgG1 antibody ratio.
In terms of neutralizing antibodies, CpG adjuvants can enhance the neutralizing antibody response induced by the inactivated vaccine.The ratio between antigen and CpG has been correlated to neutralizing antibody titers [33], so optimizing the ratio of antigen and adjuvant may improve the neutralizing antibody titer of inactivated vaccines.The decrease rate of neutralizing antibodies against Delta and Beta variants in the CpG adjuvant-inactivated vaccine group was higher than that of the prototype strain, which may be because the antigen-presenting cells activated by CpG preferentially presented the dominant epitope of the prototypic strain.
The cellular immune response plays an important role in the immune protection of vaccines.An IFN-γ ELISPOT test was used to detect the cellular immune response to the CpG adjuvant and inactivated vaccine and indicated that the inactivated vaccine resulted in minimal secretion of IFN-γ cytokines.Although CpG 684 increased IFN-γ, the spot forming unit of IFN-γ induced by the CpG adjuvant-inactivated vaccine was still low.There may be two reasons for this.The first was that CpG 684 belongs to a B-type CpG, which trigger pDCs to differentiate and produce TNF-α and B cells to proliferate and secrete IgM.B-type CpGs are weak in inducing a cellular immune response.The second reason was that the number of splenic cells used in the ELISPOT test was 10 4 cells per well, and the number of splenic cells routinely used in the ELISPOT test is 10 5 -10 6 cells/ per well.Because the splenic cells underwent cryopreservation, the number of living cells decreased.

Figure 4 .
Figure 4.The neutralizing antibody titer of mice sera against prototype, Delta and Beta strains of SARS-COV-2.(A) The neutralizing antibody titer against prototype strain.(B) The neutralizing antibody titer against the Delta strain.(C) The neutralizing antibody titer against the Beta strain.**p < 0.01.