A Reverse Transcription-Quantitative Polymerase Chain Reaction-based Microneutralization Assay for Assessing Human Cytomegalovirus-Neutralizing Antibody Activity

• Author / Creator

  Yu, Jiaao

• Human cytomegalovirus (HCMV) is a leading cause of sensorineural hearing loss and neurodevelopmental delays resulting from congenital infections and a major reason for morbidity and mortality in immunocompromised patients. To prevent these complications of HCMV infection, developing a prophylactic vaccine with high efficacy has been considered a top priority to public health. Neutralization assays are fundamental for evaluating neutralizing activities of antibodies in vaccine development, but traditional immunostaining-based neutralization assays are tedious and laborious, making it less suitable for screening large numbers of samples. A reverse transcription-quantitative polymerase chain reaction (RT-qPCR) -based microneutralization assay targeting the immediate-early gene transcript of HCMV was previously reported as an alternative solution for testing neutralizing activity in a timely manner. In a separate study, a cell-lysate-generation method was capable of simplifying RNA isolation steps for RT-qPCR analysis and reducing running costs, but both methods have not been well validated for clinical application.
  In this study, both methods were combined and evaluated with a laboratory-adapted HCMV clinical strain VR1814 with wide tropism in fibroblasts (MRC-5), epithelial cells (ARPE-19) and endothelial cells (HMEC-1). My goal was to assess neutralizing activities of human immunoglobulins (HIG) and monoclonal antibodies on clinical HCMV strains by comparing my combined RT-qPCR-based neutralization assay with immunostaining.
  My RT-qPCR assay had a sensitivity of 0.6 infectious units (IU)/reaction with a linear range from 104 to 1 IU/reaction. The assessment can be conducted as early as 20 h post-infection. High agreement was observed between the results of RT-qPCR and immunostaining assays in determination of antibody neutralization against VR1814 and clinical HCMV strains. In contrast to the laboratory-adapted strain VR1814, neutralization resistance to immunoglobulins and monoclonal antibodies was observed in HCMV in primary clinical samples of urine, milk and saliva.
  Thus, my RT-qPCR assay is a useful alternative method of assessing HCMV-neutralizing activity with higher accuracy and precision than the immunostaining assay. The neutralization resistance of HCMV from clinical specimens suggests that current monoclonal antibodies may be incapable of preventing CMV replication in vitro.

• Subjects / Keywords

  • Human Cytomegalovirus
  • RT-qPCR
  • Neutralization
  • Neutralizing antibodies

• Graduation date

  Spring 2020

• Type of Item

  Thesis

• Degree

  Master of Science

• DOI

  https://doi.org/10.7939/r3-02xm-c728

• License

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