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Whole-animal Imaging and Flow Cytometric Techniques for Analysis of Antigen-specific CD8+ T Cell Responses after Nanoparticle Vaccination
Published on: April 29, 2015
Flow cytometric analysis of cellular changes in mice after intradermal inoculation with a liposome-iscom adjuvanted
F San Gil1, B Turner, A Mullbacher
1NSW Agriculture, Elizabeth Macarthur Agricultural Institute, Camden, Australia.
Insights
Vaccine formulation impacts leucocyte homeostasis, with adjuvant SAMA4 complexed with antigens inducing significant immune responses and cell mobilization. These changes are crucial for effective vaccination, not merely coincidental.
Area of Science:
- Immunology
- Vaccinology
- Cell Biology
Background:
- Understanding leucocyte homeostasis changes is vital for interpreting vaccine efficacy.
- The biological relevance of systemic changes from vaccine formulations requires context of immunological outcomes.
Purpose of the Study:
- To quantify leucocyte subset changes induced by different vaccine formulations in mice.
- To compare the effects of antigen, adjuvant, and antigen-adjuvant complexes on immune cells.
Main Methods:
- Flow cytometry was used to analyze leucocyte subsets in mice.
- Mice were intradermally immunized with SAMA4 (adjuvant), outer membrane proteins (OMP) from Actinobacillus pleuropneumoniae, SAMA4-adjuvanted OMP, or PBS (control).
- Histological examination of spleen sections and analysis of circulating cells in blood and lungs were performed.
Main Results:
- SAMA4-adjuvanted OMP generated strong antibody and cytotoxic T-cell responses.
- Significant splenomegaly, granulocytosis, monocytosis, and megakaryocytosis were observed in the OMP vaccine group, linked to splenic hematopoiesis.
- Increased circulating granulocytes and monocytes, and lung granulocytes were noted in the OMP vaccine group; no changes in T- and B-cell numbers were detected.
- Thymocyte numbers drastically decreased in the OMP vaccine group within 3 days.
- Similar cellular responses occurred with ovalbumin complexed with SAMA4, indicating adjuvant-antigen synergy, not toxicity.
Conclusions:
- Complexing antigens with SAMA4 adjuvant induces significant leucocyte homeostasis changes.
- These observed changes are associated with strong adaptive immune responses, suggesting their importance for effective vaccination.
- The adjuvant effects of SAMA4 stem from synergistic antigen-adjuvant interaction.
Abstract:
As it is not known what changes to leucocyte homeostasis are mandatory for effective adjuvant action, the biological relevance of systemic changes elicited by different vaccine formulations can only be interpreted in the context of the immunological outcomes. We used flow cytometry to quantify the changes in leucocyte subsets induced in mice intradermally immunized with SAMA4 (adjuvant group), outer membrane proteins (OMP) purified from Actinobacillus pleuropneumoniae (OMP antigen group), SAMA4 adjuvanted OMP (OMP vaccine group), or phosphate-buffered saline (PBS: control group). This approach allowed direct comparisons to be made between the effects of antigen, adjuvant or antigen-adjuvant complexes on immune effector cell populations. Antigens complexed with the liposome-iscom hybrid adjuvant, SAMA4, generated strong antibody responses and cytotoxic T-cell activity in animals immunized intradermally, reflecting remobilization and recruitment of specific cell populations. Splenomegaly, due to granulocytosis, monocytosis and megakaryocytosis, was most prominent in the OMP vaccine group. Histological examination of spleen sections confirmed that these changes were due primarily to splenic haematopoiesis. Circulating numbers of granulocytes and monocytes increased significantly (P < 0.05) in the blood of the OMP vaccine group, as did granulocyte numbers in the lungs (P < 0.05). No changes in T- and B-cell numbers were detected by flow cytometry in the spleens, lungs or blood over the 28-day period in any treatment group. Thymocyte numbers (predominantly CD4+CD8+ cells) in the OMP vaccine group fell by 95% within 3 days of immunization. Identical cellular responses were obtained when an innocuous antigen, ovalbumin, was complexed with SAMA4 instead of OMP, thus demonstrating that the adjuvant effects of SAMA4 were due to synergistic interaction between antigen and adjuvant and not due to the presence of toxic components. The association of strong adaptive immune responses with such complex changes in leucocyte homeostasis induced by complexing adjuvant and antigen suggested that the changes were important for effective vaccination and were not purely circumstantial.
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