Abstract
Background
Because of the deficiencies of traditional methods in multivalent rotavirus vaccine potency detection, a cell‐based quantitative RT‐qPCR assay (C‐QPA) was established and validated for specificity, precision, and accuracy.
Methods
In order to further validate the robustness of this method in actual titer detection, the linear range and the practical application under different conditions were tested using monovalent and trivalent rotavirus samples and standards.
Results
Results showed that the linear range was 2.0–6.5, 3.9–8.3, and 3.5–8.1 UI (unit of infectivity) for G2, G3, and G4, respectively. Besides, unknown sample with high titer exceeding the linear range can be calculated by dilution. The UIs of serotypes G2, G3, and G4 in monovalent and trivalent rotavirus samples showed a relative deviation ≤4.10%, and the monovalent samples of the same serotype with or without protective agents showed a relative deviation ≤4.28%; the coefficient of variation (CV) of at least 176 tests (548 individual runs) of 3 in vitro‐transcribed RNA standards with certain concentrations was not higher than 6.50%; the results of the trivalent samples tested by more than 149 times in 5 years (467 individual runs) showed the CVs lower than 12.66%; 15 samples detected by one laboratory showed a CV lower than 9.83%, while other three samples tested by two independent laboratories showed a CV lower than 6.90%.
Conclusion
In summary, the C‐QPA has good linearity, durability, repeatability, and reproducibility in practical application and has been proved by the authority to be widely used in the production, quality control and release of the recently licensed trivalent vaccine in China.
Keywords: C‐QPA, rotavirus, unit of infectivity, vaccine, validation
The linear range of C‐QPA. The correlation coefficients (R 2) of the three strains ranged from 0.97 to 0.98, indicating a good linearity between the UIs and the dilutions. According to the linear regression analysis, the linear range of the assay for G2 (LD9, red line), G3 (LS4/9, green line) and G4 (LH9, blue line) was 2.0–6.5, 3.9–8.3 and 3.5–8.1 UI, respectively.
1. INTRODUCTION
Rotavirus, a segmented double‐stranded RNA virus of the Reoviridae family, can cause severe gastroenteritis in young children. As the most common pathogen, group A rotaviruses caused 128,500 deaths and 258,000 episodes of diarrhea among children younger than 5 years in 2016. 1 The rotavirus genome is composed of 11 dsRNA segments and encodes 6 structural proteins and 6 non‐structural proteins. Rotavirus particles consist of three protein layers surrounding the core. The core consists of the viral RNA, VP1 and VP3, and the inner layer is formed by VP2. The intermediate layer consists of VP6, which is the most conserved protein among the structural proteins of rotavirus. The outer layer of the virus particle is formed by VP4 and VP7 proteins, which possess neutralization antigens and play an important role in virus entry and infection of the target cells. 2 , 3 , 4 Rotaviruses are classified into 8 groups or species (RVA–RVD and RVF–RVJ) based on the amino acid sequence of the structural protein, VP6. Although group A, B, C and H rotaviruses are associated with acute gastroenteritis in humans and animals, group A rotaviruses are the ubiquitous rotavirus group causing severe diarrhea in humans. A dual classification system based on the two outer capsid proteins, VP4 (P, protease‐sensitive) and VP7 (G, glycoprotein), has been used, 5 and at least 42 G serotypes and 58 P genotypes were classified. 6 Additionally, a whole‐genome classification based on nucleotide sequence is used and percent identity cut‐off values were established for each of the 11 genome segments assigned genotypes as follows: Gx‐P[x]‐Ix‐Rx‐Cx‐Mx‐Ax‐Nx‐Tx‐Ex‐Hx, denoting the VP7‐VP4‐VP6‐VP1‐VP2‐VP3‐NSP1‐NSP2‐NSP3‐NSP4‐NSP5/6 genes. Human rotavirus genomes are composed of two main genotype constellations: Wa‐like (genogroup 1: G1/3/4/9/12‐P[8]‐I1‐R1‐C1‐M1‐A1‐N1‐T1‐E1‐H1) and DS‐1‐like (genogroup 2: G2‐P[4]‐I2‐R2‐C2‐M2‐A2‐N2‐T2‐E2‐H2). A third less frequently genogroup, AU‐1‐like, is also detected in humans (genogroup 3: G3‐P[9]‐I3‐R3‐C3‐M3‐A3‐N3‐T3‐E3‐H3). 7 , 8
Because there is no specific treatment for RV infection, vaccine is considered to be the best way to prevent the infection, and thus, the WHO recommended rotavirus vaccination worldwide, especially in developing countries. 9 , 10 , 11 Two rotavirus vaccines, Rotarix (GlaxoSmithKline) and RotaTeq (Merck), have been successfully introduced in the National Immunisation Program (NIP) of more than 110 countries, drastically reducing the burden of rotavirus disease. Two other vaccines have more recently been developed in India: Rotavac (Bharat Biotech) and Rotasiil (Serum Institute of India). All of these four vaccines are prequalified by the WHO. 12 Besides, Rotavin‐M1 (POLYVAC, Vietnam), Lanzhou lamb rotavirus vaccine (LLR, Lanzhou Institute of Biological Products, China) and the trivalent live human–lamb reassortant rotavirus vaccine (LLR3, Lanzhou Institute of Biological Products, China) have been nationally licensed.
The Lanzhou lamb rotavirus vaccine (LLR) has been licensed in 2000, and it is an only domestic rotavirus vaccine at present. Over the past 23 years, more than 84 million doses of LLR have been lot released (data collected from Vaccine Lot Release System of National Institutes of Food and Drug Control, updated in March 2023). Despite lack of a well‐designed, randomized, placebo‐controlled clinical trial, the performance of LLR has not been evidenced, and the hospital‐based and population‐based case–control studies performed after commercialized showed that the vaccine efficacy of LLR against RVGE ranged from 35% to 73.3% around China. 13 , 14 Although the effectiveness of LLR was consistent with the four prequalified vaccines in low‐income countries, considering the significant differences in effectiveness, still existing burden and the serotype shifting of human rotavirus in China, a self‐developed alternative novel improved rotavirus vaccine with higher immunogenicity and independent intellectual property right should be the prospect.
Based on the safety and effectiveness of LLR and the segmented nature of the rotavirus genome which allows genetic reassortment occurring during mixed infection, 12 Lanzhou Institute of Biological Products Co., Ltd., China, has newly developed a reassortant rotavirus vaccine, live, oral trivalent (Vero cell). This domestic used trivalent rotavirus vaccine briefly named LLR3 was licensed in China recently in April 2023, becoming the second self‐developed rotavirus vaccine with independent intellectual property right, which was developed with lamb rotavirus strain LLR (G10P12) as backbone, and the VP7 encoding genome was replaced by the corresponding genome segment of human rotavirus G2 (LD9, G2P12), G3 (LS4/9, G3P2) and G4 (LH9, G4P12). A multi‐centered, double‐blind, placebo‐controlled phase III clinical trial was conducted in Henan province from July 2012 to June 2014; totally, 9998 infants from 6 to 13 weeks of age in 12 counties (field sites) were enrolled. After observation by two epidemic seasons, LLR3 provided 70.3% and 56.6% efficacies to severe rotavirus gastroenteritis and any‐severity rotavirus gastroenteritis caused by any serotype, 15 while 72% and 57% for Rotarix 16 and 78.9% and 69.3% for RotaTeq 17 in phase III clinical trial also performed in China, respectively. Also, the safety was evidenced similar to Rotarix and RotaTeq.
In terms of the quality control of the live multivalent rotavirus vaccine, especially the assay of the vaccine potency, it must be remembered that each candidate must be examined individually. The concentration of virus can be determined by infectivity titrations, for example, plaque‐forming unit (PFU), focus‐forming unit and cell culture infective dose 50% (CCID50) assays. However, as opposed to a single component, confirming the quantity of each rotavirus serotype in the final mixture of a multivalent vaccine is challenging, because conventional infectivity titrations are dependent on the observation of cytopathogenic effects (CPE) in mammalian cell culture and require an antibody specific to each of the vaccine components. However, not all the rotaviruses can produce clear CPE and it needs more than one week to produce obvious CPE. In addition, it is difficult to obtain reliable and reproducible results, because inoculated cell cultures often deteriorate before the presence of distinctive CPE. Moreover, these assays are typically time‐consuming, highly labor‐intensive and variable. Most importantly, owing to highly specific neutralizing antibodies for each of the components required in order to allow specific detection of the infectivity of the individual components, the application of these methods may pose a further level of complexity in the case of multivalent vaccine.
Quantitative real‐time polymerase chain reaction (qPCR), as a molecular method of measuring virus concentration that does not require the use of specific antibody, has been developed and widely used for the pathogen detection in clinical and environmental samples and recommended by the WHO for rapid viral pathogen detection. Although it is rapid, sensitive, time‐saving and can simultaneously detect different target sequences in one well, the results of qPCR indicate only the presence or absence of specific RNA/DNA sequences and do not provide any information about the virus infectivity.
To overcome the deficiencies of traditional potency tests and the limitations of inability to detect infectivity of qPCR in rotavirus detection, a one‐step TaqMan probe‐based quantitative RT‐qPCR assay combined with cell‐based method (C‐QPA) was established to determine the infectious rotavirus particles of the target virus in the novel trivalent reassortant rotavirus vaccine LLR3. 18 This rapid, specific and sensitive qualitative and quantitative analysis is very important in the diagnosis and monitoring of rotaviruses and especially in the quality control of each individual component in the production of the trivalent reassortant rotavirus vaccine LLR3. Briefly, this alternative method uses Vero cell monolayers, which are seeded in 96‐well plates and inoculated with dilutions of unknown samples and a trivalent reference standard with a known titer. Viral replication is allowed to proceed for 18 to 24 h, and the cells are then lysed by the addition of Triton X‐100 and one freeze–thaw cycle. Cell lysates are assayed by RT‐qPCR, and rotavirus viral RNA during replication in cells is quantified to calculate the relative potency result, the unit of infectivity (UI) by the parallel line analysis (Registration number: 2018SR211195). Three simplex RT‐qPCRs are performed employing primer/probe sets specific to each of the three reassortant.
In this study, a set of different monovalent and trivalent rotavirus samples and RNAs were used to further verify the linearity and practical application of the assay. Validation results showed the novel C‐QPA method has good linearity, durability, repeatability and reproducibility in practical application, especially in quantitating the infectious titers of the three different strains in the trivalent rotavirus vaccine simultaneously, and it has been listed in the regulations for the manufacture and verification of the recently licensed reassortant rotavirus vaccine, live, oral trivalent (Vero cell) (draft) and widely used in the production, quality control and release of the vaccine in China.
2. MATERIALS AND METHODS
2.1. Materials
2.1.1. Cells
Adult African green monkey kidney cells (Vero) were conserved by Lanzhou Institute of Biological Products Co., Ltd., and cultured in Eagle's Minimum Essential Medium (MEM) supplemented with 5% (v/v) heat‐inactivated fetal bovine serum and maintained in a 37°C incubator in 5% CO2. In addition, the working cell line ranges from 138 to 160 passages and no antibiotics are used during the cell culture procedure.
2.1.2. Viruses, monovalent and trivalent standards
The Genotype 2 (G2, LD9, G2P12), Genotype 3 (G3, LS4/9, G3P2), Genotype 4 (G4, LH9, G4P12) strains, monovalent and trivalent human–lamb reassortant rotavirus samples, and the trivalent standard were all prepared and provided by Lanzhou Institute of Biological Products Co., Ltd., China. The trivalent standard was prepared with the same three batches of virus stock solution (G2–G4) used in phase III clinical trials, and the titer ((lgCCID50/mL) of each component in trivalent rotavirus standard is 5.9 for LD9, 6.2 for LS4/9 and LH9, respectively.
2.1.3. In vitro‐transcribed RNAs
The type‐specific full‐length primers and positive recombinant plasmids pGEM‐T‐VP7 used to perform the in vitro transcription and the type‐specific in vitro‐transcribed RNAs responding to the vaccine genotype (G2–G4) were all designed and prepared by Lanzhou Institute of Biological Products Co., Ltd., China.
2.1.4. Reagents
The 7500 Real‐Time System and consumables, probes and primers were all from Applied Biosystems (American). MEM culture medium and trypsin were from Gibco (American). Heat‐inactivated fetal bovine serum was purchased from Royacel (China). T7 RNA Polymerase and One‐Step PrimeScript RT‐PCR Kit (Perfect Real Time) was from Takara (Japan). The DEPC‐treated water was purchased from Sangon Biotech (China).
2.2. Methods
2.2.1. A cell‐based RT‐qPCR assay (C‐QPA)
All of the monovalent and trivalent samples in the study were detected by the C‐QPA method described previously. 18 Samples were serially diluted by fivefold for 4 dilutions with serum‐free MEM (trypsin final concentration is 5 μg/mL) and allowed for trypsin digestion for 60–70 min in incubator with humidified 5% CO2 at 37°C. Subsequently, digested samples were added to Vero cells and cultured for 24 h at 37°C with 5% CO2. Day 1 post‐infection, 10 μL of 4.5% Triton X‐100 was added to each well for cell lysis, and then, the plate was freeze–thaw once. RT‐qPCR was followed with TaqMan primer/probe sets specific to each virus reassortant synthesized with different fluorescent molecules on the 5′ end and a quencher on the 3′ end (G2‐Probe: 5′VIC‐3′TAMRA; G3‐Probe: 5′FAM‐3′TAMRA; G4‐Probe: 5′NED‐3′MGB). The unit of infectivity (UI) of each sample was analyzed by parallel line analysis using Ct (cycle threshold) values generated by the software of the 7500 Real Time System.
2.2.2. The determination of linear range of C‐QPA
The three monovalent rotavirus samples (Monovalent 1:G2, Monovalent 1:G3 and Monovalent 1:G4) were serially diluted by 10‐fold for 6 dilutions, and each dilution was taken as one individual sample (D1–D6). The original concentration samples (D0) and the diluted samples (D1–D6) were tested by C‐QPA for 6 individual assays. The linear range of the method was determined by linear regression analysis.
2.2.3. The applicability of C‐QPA
Ten monovalent and trivalent rotavirus samples (Monovalent 4–12, Trivalent 1) were tested three times, respectively, to analyze the consistency of viral load of the same serotype in monovalent and multivalent samples and analyze the interference effect of protective agents. The viral loads of serotypes G2, G3 and G4 in Trivalent 1 were the same to that of Monovalent 4(G2), Monovalent 5(G3) and Monovalent 6(G4), respectively.
Three in vitro‐transcribed RNA standards specific for the VP7 gene of G2, G3 and G4 genotypes with a certain concentration were detected more than 176 times to test the variation of the RT‐qPCR procedure. In addition, one trivalent rotavirus standard (Trivalent 2) was detected for at least 149 individual assays during five years to determine the durability of C‐QPA.
Fifteen samples (Trivalent 3 to 17) were detected individually for three times by laboratory A, and the other three samples (Trivalent 18 to 20) were assayed 10 times by laboratory A and laboratory B (five times for each laboratory) to determine the application of the assay.
3. RESULTS
3.1. The determination of linear range of C‐QPA
As shown in Figure 1, the correlation coefficients (R 2) of the three strains ranged from 0.97 to 0.98, indicating a good linearity between the UIs and the dilutions. The UI of five dilutions of the three monovalent rotavirus samples is listed in Table 1. As shown in Table 1, all the CVs were lower than 5.97%, except for Monovalent 1‐D3 (17.46%) and Monovalent 1‐D4 (19.49%). In addition, the titer of the same sample calculated by D0, D1 and D2 showed a relative deviation ≤3.23% (Table 2).
FIGURE 1.
The linear range of C‐QPA.
TABLE 1.
UI of the three monovalent samples with different concentrations.
| Samples | Repeats | Mean ± SD | CV (%) | |||||
|---|---|---|---|---|---|---|---|---|
| 1 | 2 | 3 | 4 | 5 | 6 | |||
| Monovalent 1‐D0 | 6.5 | 6.6 | 6.4 | 6.6 | 6.6 | 6.4 | 6.5 ± 0.1 | 1.51 |
| Monovalent 1‐D1 | 5.5 | 5.5 | 5.4 | 5.6 | 5.6 | 5.6 | 5.5 ± 0.1 | 1.48 |
| Monovalent 1‐D2 | 4.4 | 4.4 | 4.2 | 4.5 | 4.4 | 4.5 | 4.4 ± 0.1 | 2.49 |
| Monovalent 1‐D3 | 3.8 | 2.9 | 2.6 | 3.5 | 3.2 | 2.4 | 3.1 ± 0.5 | 17.46 |
| Monovalent 1‐D4 | 2.1 | 1.5 | 1.7 | 2.2 | 2.6 | 1.9 | 2.0 ± 0.4 | 19.49 |
| Monovalent 2‐D0 | 8.3 | 8.3 | 8.3 | 8.2 | 8.3 | 8.1 | 8.3 ± 0.1 | 1.01 |
| Monovalent 2‐D1 | 7.3 | 7.0 | 7.3 | 6.9 | 7.2 | 6.2 | 7.0 ± 0.4 | 5.97 |
| Monovalent 2‐D2 | 6.0 | 6.4 | 6.1 | 6.1 | 6.0 | 6.3 | 6.2 ± 0.2 | 2.67 |
| Monovalent 2‐D3 | 5.0 | 5.1 | 5.0 | 4.9 | 5.0 | 5.0 | 5.0 ± 0.1 | 1.26 |
| Monovalent 2‐D4 | 3.7 | 3.8 | 3.8 | 4.0 | 3.9 | 4.1 | 3.9 ± 0.1 | 3.79 |
| Monovalent 3‐D0 | 8.4 | 8.1 | 8.3 | 7.8 | 7.9 | 7.8 | 8.1 ± 0.3 | 3.22 |
| Monovalent 3‐D1 | 7.4 | 7.1 | 7.0 | 6.9 | 6.7 | 6.8 | 7.0 ± 0.2 | 3.56 |
| Monovalent 3‐D2 | 6.2 | 6.0 | 5.9 | 5.7 | 6.0 | 5.6 | 5.9 ± 0.2 | 3.71 |
| Monovalent 3‐D3 | 4.9 | 4.8 | 4.8 | 4.4 | 5.0 | 4.8 | 4.8 ± 0.2 | 4.27 |
| Monovalent 3‐D4 | 3.6 | 3.5 | 3.5 | 3.1 | 3.6 | 3.5 | 3.5 ± 0.2 | 5.37 |
Abbreviation: CV, coefficient of variation.
TABLE 2.
Accuracy analysis of three monovalent samples detected by different dilutions.
| Parameters | Relative deviation (%) | ||
|---|---|---|---|
| G2 | G3 | G4 | |
| D0 & D1 | −0.26 | 3.23 | 0.83 |
| D0 & D2 | 1.79 | 1.21 | 1.86 |
3.2. The application of C‐QPA
The results of ten monovalent and trivalent rotavirus samples are shown in Table 3. The UIs of serotypes G2, G3 and G4 in monovalent and trivalent rotavirus samples showed a relative deviation ≤4.10%, and the monovalent samples of the same viral load with or without protective agents showed a relative deviation ≤4.28%.
TABLE 3.
Results of 9 monovalent samples and one trivalent sample tested by C‐QPA.
| Samples | Repeats | CV (%) | Relative deviation (%) | ||
|---|---|---|---|---|---|
| 1 | 2 | 3 | |||
| Monovalent 4 (G2) | 6.2 | 5.8 | 5.8 | 3.92 | 2.34 |
| Trivalent 1‐G2 | 6.2 | 6.4 | 5.7 | 6.05 | |
| Monovalent 5 (G3) | 6.1 | 6.0 | 6.4 | 3.02 | 4.10 |
| Trivalent 1‐G3 | 6.1 | 6.5 | 6.6 | 4.21 | |
| Monovalent 6 (G4) | 6.7 | 6.6 | 6.9 | 2.83 | 1.92 |
| Trivalent 1‐G4 | 6.7 | 7.0 | 6.9 | 2.26 | |
| Monovalent 7 (G2‐Yes) a | 5.9 | 6.3 | 6.7 | 6.73 | 0.21 |
| Monovalent 8 (G2‐No) a | 6.2 | 6.1 | 6.6 | 4.76 | |
| Monovalent 9 (G3‐Yes) a | 6.0 | 5.5 | 5.4 | 6.03 | 3.21 |
| Monovalent 10 (G3‐No) a | 5.4 | 6.0 | 5.9 | 5.44 | |
| Monovalent 11 (G4‐Yes) a | 6.7 | 6.5 | 6.8 | 2.28 | 4.28 |
| Monovalent 12 (G4‐No) a | 6.9 | 6.8 | 5.5 | 11.62 | |
Yes means the sample with protective agent. No means the sample without protective agent.
Table 4 shows the results of three RNA standards and the trivalent rotavirus sample (Trivalent 2) detected by RT‐qPCR and C‐QPA, respectively. The CVs of Ct values for each RNA standard were not higher than 6.50% among more than 176 assays (a subset of the 548 individual runs), while the CVs of UI (Trivalent 2) were lower than 12.66% from at least 149 individual tests (a subset of the 467 individual runs) during the five‐year period.
TABLE 4.
The durability of C‐QPA evaluated by in vitro‐transcribed RNA and trivalent standards.
| Parameters | RNA standards (Ct) | Trivalent 2 (UI) | ||||
|---|---|---|---|---|---|---|
| G2 (188) | G3 (176) | G4 (184) | G2 (149) | G3 (168) | G4 (150) | |
| Mean ± SD | 25.55 ± 1.57 | 21.59 ± 0.97 | 25.21 ± 1.64 | 5.7 ± 0.6 | 6.2 ± 0.7 | 6.0 ± 0.8 |
| CV (%) | 6.13% | 4.47% | 6.50% | 9.99% | 10.75% | 12.66% |
Abbreviations: Ct, threshold cycle; UI, unit of infectivity.
Eighteen trivalent samples (Trivalent 3–20) are shown in Table 5 and Table 6. Data analysis demonstrated that the CVs of the fifteen individual samples (Trivalent 3–17) tested by laboratory A were lower than 9.83%, while the CVs of the other three samples (Trivalent 18–20) tested by laboratory A and laboratory B were lower than 6.90%.
TABLE 5.
Results of fifteen individual samples tested by C‐QPA (laboratory A).
| Samples | G2 probe | G3 probe | G4 probe | |||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 1 | 2 | 3 | CV (%) | 1 | 2 | 3 | CV (%) | 1 | 2 | 3 | CV (%) | |
| Trivalent 3 | 6.1 | 5.8 | 5.3 | 6.67 | 6.1 | 6.7 | 6.1 | 5.49 | 6.2 | 6.7 | 6.0 | 6.24 |
| Trivalent 4 | 6.0 | 5.7 | 5.0 | 8.95 | 5.8 | 6.5 | 5.8 | 6.27 | 6.1 | 6.7 | 6.0 | 6.23 |
| Trivalent 5 | 4.6 | 5.4 | 4.7 | 8.45 | 6.4 | 6.6 | 7.0 | 4.60 | 5.6 | 5.9 | 5.6 | 3.14 |
| Trivalent 6 | 4.8 | 5.5 | 5.0 | 6.63 | 6.6 | 7.3 | 7.2 | 4.97 | 6.0 | 6.0 | 5.6 | 4.06 |
| Trivalent 7 | 4.2 | 5.2 | 4.8 | 9.83 | 6.4 | 6.9 | 6.8 | 4.01 | 5.7 | 6.0 | 5.9 | 2.44 |
| Trivalent 8 | 5.6 | 5.5 | 5.2 | 3.45 | 6.8 | 7.1 | 6.9 | 1.93 | 5.6 | 6.1 | 5.8 | 4.17 |
| Trivalent 9 | 4.6 | 5.0 | 4.8 | 3.65 | 6.7 | 6.7 | 6.8 | 1.04 | 5.2 | 5.7 | 6.0 | 7.20 |
| Trivalent 10 | 4.7 | 5.5 | 4.9 | 7.43 | 7.0 | 7.4 | 7.3 | 2.48 | 5.5 | 5.8 | 6.1 | 4.78 |
| Trivalent 11 | 4.9 | 5.6 | 5.3 | 6.63 | 6.7 | 7.3 | 6.9 | 4.30 | 5.6 | 6.3 | 5.9 | 6.00 |
| Trivalent 12 | 5.6 | 5.3 | 5.8 | 4.40 | 6.3 | 7.3 | 6.6 | 7.15 | 6.1 | 6.3 | 5.6 | 5.38 |
| Trivalent 13 | 5.4 | 5.2 | 5.8 | 5.64 | 6.0 | 7.0 | 6.5 | 7.75 | 5.8 | 5.8 | 5.7 | 0.86 |
| Trivalent 14 | 5.3 | 4.7 | 5.7 | 9.00 | 5.8 | 6.7 | 6.1 | 7.20 | 5.6 | 5.8 | 5.4 | 3.07 |
| Trivalent 15 | 4.6 | 5.2 | 4.8 | 6.86 | 6.2 | 6.9 | 7.0 | 6.60 | 5.6 | 5.8 | 5.5 | 3.14 |
| Trivalent 16 | 4.7 | 5.2 | 5.0 | 5.30 | 6.8 | 7.4 | 7.1 | 4.58 | 5.5 | 5.8 | 5.7 | 2.51 |
| Trivalent 17 | 5.4 | 6.1 | 5.9 | 6.56 | 6.2 | 6.6 | 6.1 | 3.95 | 6.4 | 6.4 | 6.7 | 2.92 |
Abbreviations: CV, coefficient of variation; UI, unit of infectivity.
TABLE 6.
Results of 3 trivalent samples tested in two laboratories by C‐QPA.
| Samples | Probes | Laboratory A (UI) | Laboratory B (UI) | CV(%) | ||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|
| 1 | 2 | 3 | 4 | 5 | 1 | 2 | 3 | 4 | 5 | |||
| Trivalent 18 | G2 | 5.9 | 5.4 | 6.1 | 5.9 | 5.4 | 5.8 | 6.1 | 5.6 | 6.3 | 6.6 | 6.56 |
| G3 | 6.3 | 6.2 | 6.6 | 6.1 | 6.1 | 6.5 | 6.7 | 6.1 | 6.7 | 6.7 | 4.11 | |
| G4 | 6.6 | 6.4 | 6.4 | 6.7 | 6.0 | 5.8 | 6.1 | 5.9 | 6.5 | 6.7 | 5.36 | |
| Trivalent 19 | G2 | 5.9 | 5.9 | 5.9 | 5.9 | 5.9 | 5.8 | 5.8 | 5.6 | 5.9 | 6.0 | 1.94 |
| G3 | 6.0 | 6.1 | 6.1 | 6.1 | 6.0 | 6.2 | 6.2 | 6.0 | 6.1 | 6.2 | 1.28 | |
| G4 | 6.0 | 6.0 | 6.2 | 6.2 | 6.1 | 5.7 | 5.4 | 6.1 | 5.7 | 5.7 | 4.54 | |
| Trivalent 20 | G2 | 5.4 | 5.4 | 5.6 | 5.5 | 5.5 | 5.5 | 5.4 | 5.6 | 5.5 | 5.4 | 1.44 |
| G3 | 6.6 | 6.6 | 6.6 | 6.7 | 6.6 | 5.8 | 5.6 | 6.1 | 5.9 | 5.8 | 6.90 | |
| G4 | 6.0 | 6.0 | 6.2 | 6.2 | 6.1 | 5.7 | 5.4 | 6.1 | 5.7 | 5.7 | 4.54 | |
Abbreviations: CV, coefficient of variation; UI, unit of infectivity.
4. DISCUSSION
The samples of Monovalent 1–3 with 7 concentrations (D0‐D6) were measured by C‐QPA method for 6 times. The results of the samples with 5 concentrations (D0–D4) from G2, G3 and G4 were retained for linear analysis, because the results of the samples with D5 and D6 concentrations could not guarantee that Ct values could meet the requirements of the method for the UI calculation each time (data were not shown). According to the linear regression analysis, the linear range of the assay for G2, G3 and G4 was 2.0–6.5, 3.9–8.3 and 3.5–8.1 UI, respectively. Although the CVs of the assay for Monovalent 1‐D3 (17.46) and Monovalent 1‐D4 (19.49) were much higher than other data, the linearity is satisfactory (R 2 ≥ 0.97). The reason for the bad coefficient of variation (CV) may be that the dilution is too high for Monovalent 1, and in that case, the Ct values cannot be detected or varied greatly and significantly influence the UI calculation.
The samples with the highest concentration in this linearity verification assay can be measured and are within the linear range. However, the upper limit of the linear range is limited to the high‐concentration samples. For example, the high‐concentration (D0) samples of G2 type are only 6.5. Therefore, in order to improve the upper limit of linear range of this method, further study should be conducted using higher concentration samples. In the comparison of the results of the same samples calculated by D0, D1 and D2, the relative deviation between the titers is less than 3.23%. It indicates that a sample with high titer exceeding the linear range can be calculated by dilution (10 or 100 times dilution).
The UIs of serotypes G2, G3 and G4 in monovalent and trivalent rotavirus samples showed a relative deviation ≤4.10%, and the monovalent samples of the same serotype with or without protective agents showed a relative deviation ≤4.28%. It indicates a good consistency between monovalent and trivalent rotaviruses of the same G serotype with same viral load and between the samples with or without protective agents. Therefore, there was no interference among the three mixed rotavirus strains, and the protective agents did not influence the titer detection.
Totally, 548 individual runs for three RNA standards (added only to the RT‐qPCR) showed CVs of Ct values not higher than 6.50%. The results of the trivalent rotavirus vaccine samples tested for more than 149 times during five years (a subset of the 467 individual runs) showed the CVs of UI were lower than 12.66%. These results indicate that there was a significant source of run‐to‐run variability in the raw output of the assay that was due to the RT‐qPCR portion of the assay, and the CVs can be easily controlled within 30% as reported by Todd Ranheim et al. 19 However, the CVs of UI were higher than that of the Ct, indicating the virus inoculation procedures also account for the variability of C‐QPA in certain extent.
Fifteen samples were detected individually for three times by laboratory A; the lower CVs (≤9.83%) indicate a good intermediate precision. Results of the other three samples detected five times per laboratory (laboratory A and laboratory B) were similar, with CVs lower than 6.90% (less than 25% reported by Todd Ranheim et al), indicating a good repeatability. In conclusion, all the data indicate that the assay has a good durability and performance.
In short, whether it is monovalent or trivalent sample, viral samples or RNAs, with or without protective agents, the CV of the assay was not larger than 12.66% (0.86% ~ 12.66%), indicating a good stability of the assay in the potency detection of the three vaccine strains.
Although each of the three vaccine strains can be detected by individual PCR at the presence of the other two strains, to be noted, the limitation of the assay is that it cannot detect all the three rotavirus strains in the same RT‐qPCR well simultaneously, in which case it is more cost‐effective for rotavirus potency detection if a triplex RT‐qPCR was established for the three different strains. Our results showed that when three pairs of primers and probes were added in the multiplex RT‐qPCR, some of the target genes lost their fluorescence (data were not shown). The multiplex RT‐qPCR needs to be further optimized to reduce the interference among the primers and probes for effective potency determination, identification and differentiation of these three strains.
5. CONCLUSIONS
In summary, the C‐QPA is being established for determination of the titers of bulk and final products during the vaccine production and has been joint validated and approved by the National Institutes for Food and Drug Control (NIFDC) of China for the release of the trivalent rotavirus vaccine LLR3. This method has good linearity, durability, repeatability and reproducibility in practical application for detecting the three target strains contained in the trivalent reassortant vaccine. The UIs of samples with high titer exceeding the linear range can be calculated by dilutions. Furthermore, there was no interference among the three mixed rotavirus strains, and the protective agents did not influence the titer detection. Finally, the CV of the assay was not larger than 12.66%. Thus, the infectious potency assay with good specificity and short detection cycle is valuable in evaluating the infectious potency of rotavirus during the production, quality control and release of the reassortant rotavirus vaccine, live, oral trivalent (Vero cell). In addition, the C‐QPA represents a major improvement over the conventional titer detection approaches for its reliability, repeatability, reproducibility and simple operation, especially in quantitating the infectious titers of different strains in multivalent rotavirus vaccine at the same time. The most important advantage of this method is that it uses the trivalent standards which were prepared with the same viral stock solution similar to the phase III clinical vaccine through the same preparation process, which can more directly reflect the titer relationship between the released vaccine and the clinical vaccine during vaccine release, ensuring the safety and effectiveness of the released vaccine. The trivalent rotavirus vaccine LLR3 has not been commercialized after approval so far and would be available in the fourth quarter. Moreover, although the C‐QPA was established by our department which is mainly focusing on the preclinical research, the results of the actual vaccine batches and the titration comparison between QPA and traditional method of the same batches will be detected and analyzed by the quality control department of our institute. What's more, this novel method can be easily applied to detect the potency of other live viruses in our own laboratory, such as respiratory syncytial virus and human parainfluenza virus type 3, and will be a more valuable tool for quality control and stability monitoring of the live virus vaccines in the future.
6. PATENTS
A parallel line analysis software copyright (Registration number: 2018SR211195) was obtained to calculate the unit of infectivity (UI) based on the work of this manuscript.
AUTHOR CONTRIBUTIONS
Yunjin Wang designed the study and participated in the experiments, data analysis and manuscript drafting. YueYue Liu, Hong Bao, Yueru Chen and Guiying Kou performed the experiments. Mingqiang Wang, Wen Huo and Wenzhu Guan carried out the data analysis. Xiongxiong Li, Xu Zhou and Shengfang Fu revised the final manuscript and helped in the submission of the manuscript. All authors read and approved the final manuscript.
FUNDING INFORMATION
Not applicable.
CONFLICT OF INTEREST STATEMENT
The authors declare that there is no conflict of interest.
ACKNOWLEDGEMENTS
We would like to express our utmost appreciation and gratitude to all the members of Division of Enteric Virus Vaccine, National Institutes for Food and Drug Control, for the provided support and assistance.
Wang Y, Liu Y, Bao H, et al. Application of the cell‐based RT‐qPCR assay (C‐QPA) for potency detection of the novel trivalent rotavirus vaccine in China. J Clin Lab Anal. 2023;37:e24989. doi: 10.1002/jcla.24989
Contributor Information
Xu Zhou, Email: wowodetougao123456@163.com.
Xiongxiong Li, Email: lixx985@163.com.
DATA AVAILABILITY STATEMENT
Data are included within the article.
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Associated Data
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Data Availability Statement
Data are included within the article.