Rapid detection of anti-Vaccinia virus neutralizing antibodies
© Kramski et al; licensee BioMed Central Ltd. 2011
Received: 22 November 2010
Accepted: 25 March 2011
Published: 25 March 2011
Increasing infections with Monkeypox and Cowpox viruses pose a continuous and growing threat to human health. The standard method for detecting poxvirus neutralizing antibodies is the plaque-reduction neutralization test that is specific but also time-consuming and laborious. Therefore, a rapid and reliable method was developed to determine neutralizing antibody titers within twelve hours. The new assay measures viral mRNA transcription as a marker for actively replicating virus after incomplete neutralization using real-time PCR.
The increasing number of humans infected with zoonotic orthopox viruses (OPV) such as Cowpox and Monkeypox virus poses a continuous and increasing threat to human health . Thus, efforts to develop new vaccines against OPV infections remain important. Natural immune response and efficacy of vaccines are characterized through their capacity to induce neutralizing antibodies. The standard diagnostic method to determine OPV neutralizing antibodies in plasma or serum samples is the plaque-reduction neutralization test (PRNT). It quantifies neutralizing antibodies by measuring the reduction of virus-induced plaques where one infectious virus particle is directly related to one virus-formed plaque. PRNT is the gold standard because it is specific, direct and reproducible . However the PRNT suffers from long turn-around times (several days), is laborious and uses an operator-error prone manual readout based on calculating the neutralization titer from the number of plaques counted. Due to long incubation times of the infected cell cultures necessary to allow plaque formation, anti-co-agglutinants like EDTA and plasma components can interfere with the cell monolayer and affect plaque formation, especially in low plasma dilutions. While pre-dilution of plasma might reduce these effects, it also reduces sensitivity of the PRNT and low titers of neutralizing antibodies might be missed.
Recently, four alternative methods were described to determine neutralizing anti-Vacinia virus (VACV) antibodies using either a beta-galactosidase expressing VACV Western Reserve strain (WR)  or recombinant GFP expressing VACV strains [4, 5]. Eyal et al.  measured remaining infectivity by enzyme immunoassay using VACV strains WR and Lister Elstree (LE). These assays are designed for large-scale screening but still are time consuming. Additionally, three of them require the use of specific recombinant VACV strains.
The assay presented here uses VACV LE and human VACV immunoglobulin (HIVIG) as a model system and quantifies neutralizing anti-VACV antibodies by combining the classic PRNT with a OPV-specific real-time PCR (designated NT-PCR) allowing quantification of replicating virus within a few hours after infection of the host cell.
Results and discussion
Validation of real-time PCR assays
Variability and efficiency of OPV12/13 and c-myc real-time PCR assays.
PCR Efficiency (%) **
17.58 ± 0.23
17.42 ± 0.20
20.33 ± 0.16
20.55 ± 0.20
24.13 ± 0.15
24.34 ± 0.21
27.01 ± 0.08
27.42 ± 0.41
31.22 ± 0.08
31.24 ± 0.38
34.42 ± 0.05
34.96 ± 0.47
19.63 ± 0.04
19.74 ± 0.46
23.23 ± 0.28
23.09 ± 0.31
26.40 ± 0.05
26.61 ± 0.29
29.53 ± 0.02
29.65 ± 0.55
34.73 ± 0.49
33.76 ± 0.97
36.74 ± 0.36
36.39 ± 0.58
Evaluation of HVIG neutralizing antibody titers with standard PRNT
Neutralizing antibody titers of HVIG (VIG and Omrigam) were first determined with the standard PRNT. The PRNT titer is defined as the antibody dilution resulting in 50% plaque reduction. HVIG preparations were tested using 4.4 × 101 pfu/well VACV LE and 1 h, 2 h or 3 h of incubation for virus neutralization. For both, VIG and Omrigam, the mean neutralizing PRNT titer from three independent measurements was 1:320± one dilution step. VIG neutralizing titers varied depending on neutralization time: 1:160 (n = 1/3 for 1 h neutralization, n = 1/3 for 2 h neutralization), 1:320 (n = 2/3 for 1 h, 2 h and 3 h neutralization) and 1:640 (n = 1/3 for 3 h neutralization). The PRNT titer for Omrigam was always 1:320 (n = 3 for 1 h, 2 h and 3 h of neutralization). As both HVIG showed the same neutralizing activity in the PRNT, VIG was used for establishment of the NT-PCR assay for reason of availability.
The NT-PCR assay
The NT-PCR assay principle is based on measuring actively replicating VACV LE by quantifying virus mRNA levels in infected Vero E6 cells. Since cell numbers per well, RNA integrity and quantity can vary all virus copy numbers were normalized against 106 copies of the cellular reference gene transcript of c-myc. The c-myc gene is expressed constitutively and independently from experimental conditions and sample treatment, different cell types and developmental stages. It is not affected by the infection with different OPVs (A. Nitsche, personal communication). After pre-incubation of VACV LE with VIG fewer cells became infected compared to VACV LE alone resulting in a decreased virus mRNA copy number due to neutralization of infectious virus. For VACV LE incubated with negative human serum virus mRNA copy numbers were set to 100%. Based on the mRNA copy numbers determined for each antibody dilution the percentage of replicating virus was calculated in comparison to the 100% expression in virus control sample.
For the NT-PCR assay virus concentrations of 4.4 × 101, 2.2 × 102 and 4.4 × 102 pfu/well were tested using experimental conditions similar to the standard PRNT with 1 h neutralization time and 24 h for infection and replication in order to compare both assays. Using 4.4 × 101 and 2.2 × 102 pfu/well of VACV LE, levels of viral mRNA were below detection threshold of the OPV12/13 real-time PCR assay (< 101 copies) for antibody dilutions 1:40-1:80. For antibody dilutions ≥ 1:160 copy numbers varied between 101 and 103 copies. No correlation between virus replication and antibody dilution was observed. In contrast, when using a virus dose of 4.4 × 102 pfu/well, ≥ 101 OPV12/13 copies were detected for all antibody dilutions tested. A significant increase of virus replication could be detected for an antibody dilution of ≥ 1:640. Therefore, an infectious dose of 4.4 × 102 pfu was selected for further experiments.
To reduce handling time, neutralization efficacy was evaluated after 1 h, 2 h and 3 h of co-incubation of VACV LE with the VIG antibodies. For 1 h and 3 h of co-incubation no significant correlation was detected due to high variability of virus replication for all antibody dilutions. After 2 h a significant increase of virus replication was observed correlating with increasing antibody dilution. Virus mRNA copy numbers revealed ≤ 3x102 OPV12/13 copies for antibody dilutions < 1:640 whereas copy numbers for dilutions ≥ 1:640 increased at least twofold (data not shown). A visual cut-off was set for a dilution of 1:640 and neutralization time of 2 h was chosen for further validation.
While a complete replication cycle of OPV takes between 9 and 24 h [11, 12], early gene expression is detectable by real-time PCR less than 30 min after infection . In initial experiments virus was allowed to replicate for 24 h to ensure one complete replication cycle. However, to reduce assay time, expression of viral genes was evaluated after 5 h and 7 h of virus infection. Additionally to the OPV12/13 assay, two other real-time PCR assays (Rpo18, D8L) were tested simultaneously. While all three assays target genes highly conserved between OPV species only OPV12/13 and Rpo18 target genes directly involved in virus replication. Although all tested OPV-specific real-time PCR assays have similar characteristics in terms of sensitivity, amplification efficiency and specificity no significant correlation between antibody dilution and virus neutralization could be seen with the D8L real-time PCR assay (data not shown). Thus, the D8L assay was not used for further experiments. In contrast, after 7 h but not after 5 h of virus replication, the Rpo18 and OPV12/13 real-time PCR assays were successfully and consistently detecting increased viral mRNA levels for antibody dilutions ≥ 1:1280 and 1:640, respectively, indicating reduced virus neutralization. Using the OPV12/13 and Rpo18 assay a significant correlation between antibody dilution and virus replication was detected. The results for OPV12/13 and Rpo 18 suggest that it is important to use a real-time PCR that targets a gene directly involved in virus replication. As the OPV12/13 assay was originally designed to detect the DNA binding phosphoprotein of VACV the OPV12/13 and an infection time of 7 h was selected for further validation of the NT-PCR.
Calculation of the neutralizing antibody titer determined with NT-PCR assay (1.) and its intra- and inter-assay variability (2.)
1. Calculation of the NT-PCR assay
Σ R2single well*
2. Inter-and intra-assay precision
respective NT-PCR titer for each assay
1:640± one dilution
1:640± one dilution
Serum samples from VACV LE vaccinated individuals analyzed by PRNT and NT-PCR.
Although further evaluation with clinical samples is needed this preliminary evaluation indicates that it will be suitable for routine diagnostic use especially if results are needed rapidly, if plasma components interfere with the cell culture or if neutralizing antibodies to viruses with no or poor cytopathic effect in cell culture (no need for the detection of plaques) need to be analyzed. It should be easily adaptable to other DNA viruses as soon as a suitable real-time PCR assay is available to detect viral mRNA. For DNA viruses the detection of viral mRNA indicates the presence of actively replicating virus in the cell limiting the potential effect of contaminating DNA from the genome of the inoculated virus just sticking to the cell surface. Also, the assay should be adaptable to RNA viruses if active virus replication could be determined and distinguished from mere contamination from viral genomic RNA. Assay conditions need to be adapted for each new virus, however, this is also true for standard PRNT assays.
Taken together, the new and rapid NT-PCR assay can be completed within 12 h while the turn-around time of the standard PRNT is at least 4 days. Final NT-PCR conditions are: 4.4 × 102 pfu/well VACV LE, 2 h of neutralization time, 7 h of infection and replication time and final quantitative examination of virus mRNA copies in cells by quantitative OPV12/13 real-time PCR.
The NT-PCR assay is a fast, reliable and sensitive tool for the detection of VACV neutralizing antibodies. It will be suitable for routine diagnostic and could pose a valuable alternative to the standard PRNT assay. The low detection limit and the high degree of accuracy for the detection of replicating virus by real-time PCR resulted in high reproducibility and short assay turn-around time. This assay can be performed with all wild-type OPV strains without the need of particular VACV reporter strains. It may also be beneficial for viruses with no or poor cytopathic effect in cell culture.
Cells, viruses and antibodies
VACV LE was propagated in Hep2 cells (ECACC: 86030501). Vero E6 cells (ECACC: 85020205) were used for virus stock titrations, for standard PRNT and NT-PCR. Overlay medium was 1.6% carboxymethylcellulose (CMC). All cells were maintained according to ECACC recommendations.
VACV LE (Bavarian Nordic, Martinsried, Germany) (4.4 × 108 pfu/mL), human Vaccinia virus immunoglobulin (HVIG) (VIG antibodies, BER/FDA Bethesda, MD, USA and Omrigam 5%, G2H50CN HO4021, Israel, 2003) and sera from six individuals vaccinated with VACV LE were used. All sera were tested for absence of cytotoxicity.
For standard PRNT 8.5 × 104 cells/well of Vero E6 cells were seeded into 48-well plates and incubated over night. HVIG and sera were twofold serially diluted in cell culture medium from 1:40 to 1:2560 and mixed with an equal volume of VACV LE to 4.4 × 101 pfu/100 μL. Medium served as negative control. After 1 h 100 μL of serum-virus mixture was added to cells in 100 μL cell culture medium. Plates were centrifuged (15 min, 200 × g) and virus was allowed to adsorb for 1 h at 37°C. Next, cells were overlaid with 400 μL overlay medium and incubated for 4 days. Medium was aspirated and cells were fixed with 4% formaldehyde for 30 min, stained with naphthalen blue black and washed once with tap water. Plaques were counted and the titer for 50% plaque reduction was calculated referred to VACV-negative serum.
To optimize the NT-PCR assay, three VACV LE concentrations (4.4 × 102, 2.2 × 102 and 4.4 × 101 pfu/well), a neutralization time between 1 h, 2 h and 3 h, total incubation time for virus entry and initial replication of 24 h, 7 h or 5 h and three different viral target sequences for OPV-specific real-time PCR were investigated. All other preceding steps were performed according to the standard PRNT. After incubation, medium was aspirated and cells were washed with PBS, trypsinized, washed twice with PBS and centrifuged (5 min, 250 × g). Cell pellet was immediately resuspended in 700 μL RLT buffer and RNA was extracted using the RNeasy Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's protocol. Residual DNA was eliminated by DNase treatment (Turbo DNase Kit; Ambion, Hamburg, Germany). cDNA was synthesized in a total volume of 20 μL containing 500 ng Oligo dT(18) primer, 500 ng calf thymus DNA and 10 μL RNA. No RNase inhibitors were added as all RNA samples were processed immediately after extraction. After 5 min at 65°C a mix consisting of 4 μL 5 × buffer, 2 μL 0.1 M DTT, 0.4 μL 25 mM dNTP and 200 U Superscript II reverse transcriptase (Invitrogen, Karlsruhe, Germany) was added.
Details of oligo nucleotides used in PCR reactions
Oligonucleotide sequence 5'→3'
OPV 12/13 /MPXV
F-CCTTCTATAGATCTGAGAAT NQF MGB
rpo 18 R
D8L/IMV membrane protein
Statistical analyses were done using GraphPad's Prism software (Version 5.0a) and a repeated measures ANOVA test and "Tukey's Multiple Comparison Test" was used for statistical analysis and p-value ≤ 0.05 was regarded as significant.
List of Abbreviations
human Vaccina virus immunoglobulin
plaque-reduction neutralization test
- VACV LE:
Vaccinia virus Lister Elstree
The study was funded by the German Federal Ministry of Health (Foko 1-121-42261). The authors are grateful to Andreas Nitsche for help in designing real-time PCR assays, to Janine Reiche and Jung-Won Sim-Brandenburg for excellent technical assistance, and to Ursula Erikli for copy editing.
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