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. 2024 Apr 6;12(4):742.
doi: 10.3390/microorganisms12040742.

Evaluation of a Reverse Transcription-Quantitative Polymerase Chain Reaction (RT-qPCR)-Based Microneutralization Assay for Assessing Clinical Human Cytomegalovirus-Neutralizing Antibody Activity

Jiaao Yu  1 , Maria E Hasing  1 , Jutta K Preiksaitis  2 , Xiaoli Pang  1   3
Affiliations

Evaluation of a Reverse Transcription-Quantitative Polymerase Chain Reaction (RT-qPCR)-Based Microneutralization Assay for Assessing Clinical Human Cytomegalovirus-Neutralizing Antibody Activity

Jiaao Yu et al. Microorganisms. .

Abstract

Development of a vaccine for human cytomegalovirus (hCMV) is critical because of the severe consequences of infection in congenitally infected newborns and immunocompromised patients. The assessment of hCMV-neutralizing antibody activity is crucial for vaccine development. This study evaluated a RT-qPCR assay targeting the immediate-early gene transcript of hCMV for determining microneutralizing antibody activity. The assay was evaluated for sensitivity, specificity, and precision using endotheliotropic clinical isolate VR1814 that infects fibroblasts, epithelial, and endothelial cells. The RT-qPCR-based neutralization assay was compared with an immunostaining-based neutralization assay using virions present in hCMV-positive urine, saliva, and breast-milk samples. Our results showed that hCMV replication was detectable at 20 h post-infection with a limit of detection of 1 infectious units (IU)/reaction. The RT-qPCR assay had a dynamic range of 1 to 1.0 × 104 IU/reaction, with coefficients of variation ranging from 0.94% to 15.08%. The RT-qPCR results were in high agreement with the immunostaining assay for hCMV-antibody neutralization assessment. Overall, the RT-qPCR neutralization assay is a reliable, rapid, efficient, and sensitive alternative method for evaluating hCMV-neutralizing activity in vitro.

Keywords: RT-qPCR; human cytomegalovirus; immunostaining assay; neutralization; viral mRNA.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
A standard curve of the one-step RT-qPCR assay. The standard RNA from the hCMV immediate-early gene UL123 was extracted from 10-fold serial dilutions of hCMV ranging from 1 to 104 IU/reaction and amplified in the one-step RT-qPCR assay. The quantity of infectious particles was plotted against average cycle numbers.
Figure 2
Figure 2
VR1814 proliferation in MRC-5, ARPE-19 and HMEC-1. 1000 IU/well of VR1814 were seeded in MRC-5, ARPE-19, and HMEC-1 in 96-well plates. Culture medium and monolayers were collected at 0, 1, 3, 5, 8, 10, 13, 16, 18, and 22 h.p.i. and analyzed by RT-qPCR with primers targeting the hCMV IE1 gene transcript. Each point represents the mean with the standard deviation of the data set (n = 4).
Figure 3
Figure 3
RNA yields of hCMV from clinical samples at 20 vs. 48 h.p.i. by RT-qPCR. 200 μL of hCMV positive samples (3 breast milk, 1 saliva and 2 urine) were used to inoculate MRC-5, ARPE-19, and HMEC-1 in 96-well plates. Culture medium and monolayers were harvested at 20 and 48 h.p.i. and analyzed by RT-qPCR.
Figure 4
Figure 4
Correlation between the RT-qPCR and immunostaining assays in determining infectious hCMV virions. A series of diluted VR1814 was used to inoculate MRC-5 cells in two 96-well plates. The cells in each plate were analyzed using either RT-qPCR or immunostaining. The results were plotted on a scatter plot, with data from the immunostaining assay on the y-axis and data from the RT-qPCR assay on the x-axis. A regression line was drawn based on the data (n = 6).
Figure 5
Figure 5
Neutralization efficacy curves of VR1814 generated by (A) RT-qPCR-based and (B) immunostaining-based neutralization assays. A 2-fold serial dilution of IgG Hizentra from 50 mg/mL to 0.38 μg/mL was incubated with an equal volume of 1000 IU of VR1814 for 1 h. The mixture was used to inoculate MRC-5, ARPE-19 and HMEC-1 in 96-well plates and analyzed by (A) RT-qPCR and (B) immunostaining respectively (n = 3).
Figure 6
Figure 6
Percentages of antibody neutralization efficacy of hCMV from a representative urine sample and VR1814 detected by (A) immunostaining-based and (B) RT-qPCR-based neutralization assays. IgG Cytogam at 1280 (high) and 640 μg/mL (low), anti-gB #1 at 50 and 10 μg/mL, anti-gB #2 at 50 and 7.5 μg/mL, anti-gH at 50 and 35 μg/mL, and anti-pentamer at 50 and 25 μg/mL were incubated with equal volume of urine for 1 h and then used to inoculate MRC-5 in 96-well plates. The 96-well plates were assessed by (A) RT-qPCR and (B) immunostaining respectively after incubation (n = 3).
Figure 6
Figure 6
Percentages of antibody neutralization efficacy of hCMV from a representative urine sample and VR1814 detected by (A) immunostaining-based and (B) RT-qPCR-based neutralization assays. IgG Cytogam at 1280 (high) and 640 μg/mL (low), anti-gB #1 at 50 and 10 μg/mL, anti-gB #2 at 50 and 7.5 μg/mL, anti-gH at 50 and 35 μg/mL, and anti-pentamer at 50 and 25 μg/mL were incubated with equal volume of urine for 1 h and then used to inoculate MRC-5 in 96-well plates. The 96-well plates were assessed by (A) RT-qPCR and (B) immunostaining respectively after incubation (n = 3).
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