Enhancing antibody levels and T cell activity of quadrivalent influenza vaccine by combining it with CpG HP021

Abstract

Influenza virus infections are a serious danger to people's health worldwide as they are responsible for seasonal flu outbreaks. There is an urgent need to improve the effectiveness and durability longevity of the immune response to influenza vaccines. We synthesized the CpG HP021 and examined the impact of it on the immune response to an influenza vaccine. In BALB/c mice, hemagglutination inhibition (HI) titers to the vaccine were increased four- to eightfold against H1N1, H3N2, BV, and BY viruses by 3 μg IIV4 + 40 μg CpG HP021 compared with those of the non-adjuvanted IIV4 group, and the CpG HP021 group had a broader HI activity. Additionally, the immune response was directed towards Type 1 T helper (Th1) cells due to the CpG HP021 adjuvant. The CpG HP021-adjuvanted IIV4 induced a higher number of T cells secreting interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α), and increased the percentage of effector memory T cells in mice. In SD rats, the immune responses induced by IIV4 with CpG HP021 were similar to those in BALB/c mice. The development of CpG HP021 may expand the options for adjuvants in vaccines against infectious diseases.

Keywords: Antibody; CpG adjuvant; Hemagglutination inhibition; Quadrivalent influenza vaccine; T cell immunity.

Fig. 1

HI titers and GMI at day 28 after first immunization in BALB/c mice. (a-d) The serum HI titers were detected by HI against H1N1, H3N2, BV, and BY at day 28. The data represent geometric mean with 95% CI. (e) Dynamic changes in GMI at various time points. (f) The SCRs for H1N1, H3N2, BY, and BV types. The dotted line indicates HI titers of 1:40 which are suggested to indicate a probability of clinical protection of at least 50%. This titer serves as a threshold for predicting protection. N = 9 to 12. *p < 0.05, **p < 0.01, ***p < 0.001.

Fig. 2

The levels of antibody subtypes after IIV4 immunization in mice measured at day 28. (a-d) Total IgG levels were assessed using ELISA with split H1N1, H3N2, B/Victoria, and B/Yamagata viruses. IgG1 titers (e), IgG2a titers (f), and the ratio of IgG1 to IgG2a titers (g) were assessed using ELISA with split H1N1 viruses. *P < 0.05, **P < 0.01, ***P < 0.001. N = 6.

Fig. 3

Cross-reactive antibody responses at day 28 after first immunization in BALB/c mice.(a-d) HI antibody responses against the representative heterologous strains. The data represent geometric mean with 95% CI. The dotted line indicates HI titers of 1:40 which are suggested to indicate a probability of clinical protection of at least 50%. This titer serves as a threshold for predicting protection. N = 6. *P < 0.05, **P < 0.01, and ***P < 0.001.

Fig. 4

T cell responses and cytokine levels in the supernatant of splenic lymphocytes at day 28 in BALB/c mice. (a-b) IFN-γ ELISpot test following antigen-specific splenic stimulation in mice. (c) TNF-α ELISpot test following antigen-specific splenic stimulation in mice. (d) Multiplex cytokine analysis of splenic lymphocyte supernatant stimulated by peptides. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. N = 6.

Fig. 5

Flow cytometry assay identifying TEM cells and the production of IFN-γ by T cells at day 28 after first immunization in BALB/c mice. (a-b) The proportion of memory cells among lymphocytes was analyzed across the different groups at day 28. (c) Assessments of the IFN-γ producing CD8⁺ T cells from immunized mice. *P < 0.05, **P < 0.01, ***P < 0.001. N = 5 to 6.

Fig. 6

HI titers and T cell responses at day 28 after the first immunization in SD rats. (a) The serum HI titers were detected by HI against H1N1, H3N2, BV, and BY at day 28. The data represent geometric mean with 95% CI. (b-d) T cell responses were measured by IFN-γ ELISpot assays for various groups. (e–g) T cell responses were assessed by IL-4 ELISpot. Compared with 6 μg of IIV4, *P < 0.05, ***P < 0.001, ****P < 0.0001. N = 3 to 6.