Chronic hypoxia modulates vaccine adjuvant-induced specific immune responses: QML maintains stability while CpG significantly enhances

Abstract
Objective
High-altitude chronic hypoxia can alter the immune status, yet the immune regulatory characteristics of various vaccine adjuvants in this environment remain to be fully elucidated. Accordingly, emphasis is placed on evaluating the regulatory effects of chronic hypoxia on specific humoral, cellular, and memory T cell responses elicited by different adjuvants.

Methods
Using ovalbumin (OVA) as the antigen, Th2-biased adjuvant aluminum hydroxide (Alum), Th1-biased adjuvant CpG, and Th1/Th2-balanced adjuvant QML were combined for intramuscular immunization in mice on days 0 and 14. Th1-biased adjuvant CpG, or Th1/Th2-balanced adjuvant QML for intramuscular immunization on day 0 and 14 (n=16). A total of 160 SPF female C57BL/6 mice (6 to 8 weeks old, weighing 16 to 18 g) were randomly divided into 5 groups (n=32): PBS group (sterile PBS), OVA group (5 μg OVA), OVA+Alum group (5 μg OVA and 100 μg Alum), OVA+CpG group (5 μg OVA and 20 μg CpG), and OVA+QML group (5 μg OVA and 50 μL QML). Each group was further divided into normoxia control (n=16, housed in 21% O₂ environment) and chronic hypoxia (n=16, simulated altitude 5 800 m) subgroups. On days 14 and 60 after the last immunization, the serum levels of specific IgG, IgG1, and IgG2c antibodies were measured by ELISA; enzyme-linked immunospot assay (ELISpot) was used to detect the numbers of cells secreting IFN-γ, IL-4, and IL-17A in the splenocytes. On day 14 after the last immunization, flow cytometry was performed to determine the proportions of CD4⁺IFN-γ⁺, CD4⁺IL-4⁺, CD4⁺IL-17A⁺, CD8⁺IFN-γ⁺, and CD8⁺IL-2⁺ cell subsets in the spleen.

Results
Chronic hypoxia significantly inhibited the ability of OVA alone to induce IgG antibodies (P<0.01), and all 3 adjuvants reversed this effect. QML exerted a stable and superior enhancing effect on serum-specific IgG, IgG1, and IgG2c levels compared to CpG (P<0.05). Notably, CpG induced significantly higher levels of total IgG (P<0.01) and IgG1 (P<0.05) in the chronic hypoxia group than in the corresponding normoxia group at days 60 after the final immunization. For cellular immunity, QML stably increased the proportions of CD4⁺IFN-γ⁺ and CD4⁺IL-17A⁺ cells, as well as the secretion levels of IFN-γ and IL-4, under both normoxic and hypoxic conditions. In contrast, the T cell regulatory effects of CpG were hypoxia-dependent, with significant enhancement of IFN-γ-secreting cell numbers in the hypoxia group at both short-term (14 d, P<0.01) and long-term (60 d, P<0.05) time points. Analysis of memory T cells showed that all 3 adjuvants maintained CD4⁺Tcm levels under hypoxia. Additionally, CpG increased the proportions of CD8⁺Tcm (P<0.05) and CD8⁺Tem (P<0.01) in the hypoxic group, while Alum only specifically elevated CD4⁺Tem levels under hypoxic conditions (P<0.05).

Conclusion
QML stably induces comprehensive immune responses under both normoxic and hypoxic conditions, while CpG exhibits advantages in Th1-type response enhancement and CD8⁺ memory T cell expansion under chronic hypoxia, providing experimental evidence for adjuvant selection in chronic hypoxic environments such as high-altitude regions.