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Optimized Interferon-gamma ELISpot Assay to Measure T Cell Responses in the Guinea Pig Model after Vaccination
Published on: January 20, 2019
Optimizing Microneutralization and IFN-γ ELISPOT Assays to Evaluate Mpox Immunity
Yinyi Yu1, Krystal Meza1, Chase Colbert1
1Division of Infectious Diseases, Allergy and Immunology, Saint Louis University, St. Louis, MO 63104, USA.
Background:
Available assays to measure pox virus neutralizing antibody titers are laborious and take up to 5 days. In addition, assays to measure T cell responses require the use of specific antigens, which may not be the same for all pox viruses. This study reports the development of robust assays for the measurement of mpox-specific neutralizing antibodies and IFN-γ-producing T-cell responses.
Methods:
Fourteen samples from 7 volunteers who received Modified Vaccinia Ankara-Bavarian Nordic (MVA-BN) were used. The focused reduction neutralization test (FRNT) was performed using the mpox-specific A29 monoclonal antibody. Optimization and further development of FRNT were conducted using the plaque reduction neutralization test (PRNT) as the gold standard. The mpox-specific IFN-γ ELISPOT assay was optimized using different mpox antigen preparations. Results with pre-vaccination samples were compared with post-vaccination samples using the Wilcoxon matched-pairs test.
Results:
Pre-vaccination and post-vaccination sera (n = 7) had FRNT50 (i.e., titers that inhibited at least 50% of the virus) of 109.1 ± 161.8 and 303.7 ± 402.8 (mean ± SD), respectively. Regression analysis of fold changes in FRNT50 and PRNT50 showed that the two assays closely agree (n = 25 tests on paired samples, R2 of 0.787). Using UV-inactivated mpox as an antigen, the number of IFN-γ spot-forming T cells (SFC) in pre-vaccination samples (16.13 ± 15.86, mean ± SD) was significantly lower than SFC in post-vaccination samples (172.9 ± 313.3, mean ± SD) with p = 0.0078.
Conclusions:
Our newly developed microneutralization test has a good correlation with PRNT. UV-inactivated mpox is an appropriate antigen for the ELISPOT assay that measures mpox cross-reactive T cells. These assays will be useful in future mpox vaccine studies.
Insights
New assays for mpox (monkeypox virus) neutralizing antibodies and T cell responses were developed. These robust assays show promise for future mpox vaccine efficacy studies.
Area of Science:
- Immunology
- Virology
- Vaccinology
Background:
- Current pox virus assays are time-consuming and may require specific antigens.
- There is a need for efficient methods to measure immune responses to mpox (monkeypox virus).
Purpose of the Study:
- To develop and validate robust assays for measuring mpox-specific neutralizing antibodies.
- To develop and validate robust assays for measuring mpox-specific T cell responses.
Main Methods:
- Focused Reduction Neutralization Test (FRNT) was optimized and compared to plaque reduction neutralization test (PRNT).
- Interferon-gamma (IFN-γ) ELISPOT assay was optimized using UV-inactivated mpox antigen.
- Assays were performed on samples from volunteers vaccinated with Modified Vaccinia Ankara-Bavarian Nordic (MVA-BN).
Main Results:
- The FRNT showed good agreement with PRNT (R² = 0.787).
- Post-vaccination sera showed significantly higher neutralizing antibody titers compared to pre-vaccination sera.
- The IFN-γ ELISPOT assay detected significantly higher T cell responses in post-vaccination samples.
Conclusions:
- A novel microneutralization test correlates well with PRNT for mpox antibodies.
- UV-inactivated mpox is suitable for ELISPOT assays to detect cross-reactive T cells.
- These developed assays are valuable tools for future mpox vaccine research.
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