Optimizing Microneutralization and IFN-γ ELISPOT Assays to Evaluate Mpox Immunity
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...
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| Format: | Article |
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MDPI AG
2024-12-01
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| Series: | Vaccines |
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| Online Access: | https://www.mdpi.com/2076-393X/13/1/27 |
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| author | Yinyi Yu Krystal Meza Chase Colbert Daniel F. Hoft Anna Jaunarajs Azra Blazevic Sharon E. Frey Getahun Abate |
| author_facet | Yinyi Yu Krystal Meza Chase Colbert Daniel F. Hoft Anna Jaunarajs Azra Blazevic Sharon E. Frey Getahun Abate |
| author_sort | Yinyi Yu |
| collection | DOAJ |
| description | 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 (<i>n</i> = 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 (<i>n</i> = 25 tests on paired samples, R<sup>2</sup> 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 <i>p</i> = 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. |
| format | Article |
| id | doaj-art-9527883aebd04b53ae10a87971bc4a0f |
| institution | Kabale University |
| issn | 2076-393X |
| language | English |
| publishDate | 2024-12-01 |
| publisher | MDPI AG |
| record_format | Article |
| series | Vaccines |
| spelling | doaj-art-9527883aebd04b53ae10a87971bc4a0f2025-01-24T13:51:42ZengMDPI AGVaccines2076-393X2024-12-011312710.3390/vaccines13010027Optimizing Microneutralization and IFN-γ ELISPOT Assays to Evaluate Mpox ImmunityYinyi Yu0Krystal Meza1Chase Colbert2Daniel F. Hoft3Anna Jaunarajs4Azra Blazevic5Sharon E. Frey6Getahun Abate7Division of Infectious Diseases, Allergy and Immunology, Saint Louis University, St. Louis, MO 63104, USADivision of Infectious Diseases, Allergy and Immunology, Saint Louis University, St. Louis, MO 63104, USADivision of Infectious Diseases, Allergy and Immunology, Saint Louis University, St. Louis, MO 63104, USADivision of Infectious Diseases, Allergy and Immunology, Saint Louis University, St. Louis, MO 63104, USAEMMES, Rockville, MD 20850, USADivision of Infectious Diseases, Allergy and Immunology, Saint Louis University, St. Louis, MO 63104, USADivision of Infectious Diseases, Allergy and Immunology, Saint Louis University, St. Louis, MO 63104, USADivision of Infectious Diseases, Allergy and Immunology, Saint Louis University, St. Louis, MO 63104, USABackground: 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 (<i>n</i> = 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 (<i>n</i> = 25 tests on paired samples, R<sup>2</sup> 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 <i>p</i> = 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.https://www.mdpi.com/2076-393X/13/1/27mpoxvacciniaFRNTmicroneutralizationPRNTIFN-γ ELISPOT |
| spellingShingle | Yinyi Yu Krystal Meza Chase Colbert Daniel F. Hoft Anna Jaunarajs Azra Blazevic Sharon E. Frey Getahun Abate Optimizing Microneutralization and IFN-γ ELISPOT Assays to Evaluate Mpox Immunity Vaccines mpox vaccinia FRNT microneutralization PRNT IFN-γ ELISPOT |
| title | Optimizing Microneutralization and IFN-γ ELISPOT Assays to Evaluate Mpox Immunity |
| title_full | Optimizing Microneutralization and IFN-γ ELISPOT Assays to Evaluate Mpox Immunity |
| title_fullStr | Optimizing Microneutralization and IFN-γ ELISPOT Assays to Evaluate Mpox Immunity |
| title_full_unstemmed | Optimizing Microneutralization and IFN-γ ELISPOT Assays to Evaluate Mpox Immunity |
| title_short | Optimizing Microneutralization and IFN-γ ELISPOT Assays to Evaluate Mpox Immunity |
| title_sort | optimizing microneutralization and ifn γ elispot assays to evaluate mpox immunity |
| topic | mpox vaccinia FRNT microneutralization PRNT IFN-γ ELISPOT |
| url | https://www.mdpi.com/2076-393X/13/1/27 |
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