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Development of a High-Content Orthopoxvirus Infectivity and Neutralization Assays.

Gates I, Olson V, Smith S, Patel N, Damon I, Karem K - PLoS ONE (2015)

Bottom Line: In addition to the traditional plaque reduction neutralization test (PRNT), newer higher throughput assays are based on neutralization of recombinant vaccinia virus, expressing reporter genes such as β-galactosidase or green fluorescent protein.However, PRNT assays are time consuming, labor intensive, and require considerable volume of serum sample for testing.Here, we describe the development of a high-throughput, cell-based imaging assay that can be used to measure neutralization, and characterize replication kinetics of various Orthopoxviruses, including variola, vaccinia, monkeypox, and cowpox.

View Article: PubMed Central - PubMed

Affiliation: Atlanta Research and Education Foundation, Decatur, Georgia, United States of America.

ABSTRACT
Currently, a number of assays measure Orthopoxvirus neutralization with serum from individuals, vaccinated against smallpox. In addition to the traditional plaque reduction neutralization test (PRNT), newer higher throughput assays are based on neutralization of recombinant vaccinia virus, expressing reporter genes such as β-galactosidase or green fluorescent protein. These methods could not be used to evaluate neutralization of variola virus, since genetic manipulations of this virus are prohibited by international agreements. Currently, PRNT is the assay of choice to measure neutralization of variola virus. However, PRNT assays are time consuming, labor intensive, and require considerable volume of serum sample for testing. Here, we describe the development of a high-throughput, cell-based imaging assay that can be used to measure neutralization, and characterize replication kinetics of various Orthopoxviruses, including variola, vaccinia, monkeypox, and cowpox.

No MeSH data available.


Related in: MedlinePlus

Average immune response for different vaccinee categories.Neutralization responses for each vaccinee category were grouped according to blood draw days. Calculated average and standard deviation values were based on the results from several vaccinee per each category.
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pone.0138836.g008: Average immune response for different vaccinee categories.Neutralization responses for each vaccinee category were grouped according to blood draw days. Calculated average and standard deviation values were based on the results from several vaccinee per each category.

Mentions: To further evaluate assay performance we used HC-OVNA derived EC50 neutralizing dilution values from correlation experiment and compared the changes in levels of serum neutralizing antibodies for different vaccinee categories. (Fig 8). The EC50 dilution results were grouped by vaccination status (categories A, B, C, D) and then by serum collection days. The EC50 results for each category were averaged and analyzed for days 0, 7, 21 and 6 months. For the first time vaccinee (Category A) we were expecting to see a significant increase in neutralizing antibodies by day 21 and decrease in antibody levels by 6 months [12, 13]. Consistent with our expectations the highest level of IgG response was observed on day 21, with EC50 at 1:960 serum dilutions (median 1241, range 348–1295). Neutralization responses from serum samples collected on days 0 and 7 were rated as non-neutralizing with EC50 values below a 1:40 dilution, since minimal to no neutralization activity was observed. By 6 months the amount of serum neutralizing antibodies was reduced, resulting in EC50 dilutions at 1:280 dilution (median 263, range 206–377).


Development of a High-Content Orthopoxvirus Infectivity and Neutralization Assays.

Gates I, Olson V, Smith S, Patel N, Damon I, Karem K - PLoS ONE (2015)

Average immune response for different vaccinee categories.Neutralization responses for each vaccinee category were grouped according to blood draw days. Calculated average and standard deviation values were based on the results from several vaccinee per each category.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4591290&req=5

pone.0138836.g008: Average immune response for different vaccinee categories.Neutralization responses for each vaccinee category were grouped according to blood draw days. Calculated average and standard deviation values were based on the results from several vaccinee per each category.
Mentions: To further evaluate assay performance we used HC-OVNA derived EC50 neutralizing dilution values from correlation experiment and compared the changes in levels of serum neutralizing antibodies for different vaccinee categories. (Fig 8). The EC50 dilution results were grouped by vaccination status (categories A, B, C, D) and then by serum collection days. The EC50 results for each category were averaged and analyzed for days 0, 7, 21 and 6 months. For the first time vaccinee (Category A) we were expecting to see a significant increase in neutralizing antibodies by day 21 and decrease in antibody levels by 6 months [12, 13]. Consistent with our expectations the highest level of IgG response was observed on day 21, with EC50 at 1:960 serum dilutions (median 1241, range 348–1295). Neutralization responses from serum samples collected on days 0 and 7 were rated as non-neutralizing with EC50 values below a 1:40 dilution, since minimal to no neutralization activity was observed. By 6 months the amount of serum neutralizing antibodies was reduced, resulting in EC50 dilutions at 1:280 dilution (median 263, range 206–377).

Bottom Line: In addition to the traditional plaque reduction neutralization test (PRNT), newer higher throughput assays are based on neutralization of recombinant vaccinia virus, expressing reporter genes such as β-galactosidase or green fluorescent protein.However, PRNT assays are time consuming, labor intensive, and require considerable volume of serum sample for testing.Here, we describe the development of a high-throughput, cell-based imaging assay that can be used to measure neutralization, and characterize replication kinetics of various Orthopoxviruses, including variola, vaccinia, monkeypox, and cowpox.

View Article: PubMed Central - PubMed

Affiliation: Atlanta Research and Education Foundation, Decatur, Georgia, United States of America.

ABSTRACT
Currently, a number of assays measure Orthopoxvirus neutralization with serum from individuals, vaccinated against smallpox. In addition to the traditional plaque reduction neutralization test (PRNT), newer higher throughput assays are based on neutralization of recombinant vaccinia virus, expressing reporter genes such as β-galactosidase or green fluorescent protein. These methods could not be used to evaluate neutralization of variola virus, since genetic manipulations of this virus are prohibited by international agreements. Currently, PRNT is the assay of choice to measure neutralization of variola virus. However, PRNT assays are time consuming, labor intensive, and require considerable volume of serum sample for testing. Here, we describe the development of a high-throughput, cell-based imaging assay that can be used to measure neutralization, and characterize replication kinetics of various Orthopoxviruses, including variola, vaccinia, monkeypox, and cowpox.

No MeSH data available.


Related in: MedlinePlus