Rapid detection of anti-Vaccinia virus neutralizing antibodies

Virology Journal Rapid detection of anti-Vaccinia virus neutralizing antibodies Marit Kramski 0 2 Anna Drozd 0 Gregor F Lichtfuss 0 1 Piotr W Dabrowski 0 Heinz Ellerbrok 0 0 Robert Koch-Institute, Centre for Biological Safety , Nordufer 20, 13353 Berlin , Germany 1 Burnet Institute , 85 Commercial Road, Melbourne , Australia 2 Department of Microbiology and Immunology, University of Melbourne , Royal Parade, Parkville, 3206 VIC , Australia 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. - Background 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 [1]. 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 [2]. 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) [3] or recombinant GFP expressing VACV strains [4,5]. Eyal et al. [6] 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 To quantify actively replicating virus, three OPV-specific reverse transcriptase real-time PCR assays were established. The OPV12/13 assay targets the gene for the VACV LE DNA-binding phosphoprotein involved in DNA replication and nucleotide metabolism. The other two OPV-specific real-time PCR assays, D8L and Rpo18 [7], are specific for the D8L membrane protein coding region of IMV particles and the 18-kDa RNA polymerase subunit gene, respectively. All three real-time PCR assays were OPV-specific, showed no cross-reactivity to cellular genes (data not shown) and therefore were used as a measure for replicating virus within cells. To standardize virus mRNA copies to an equal number of cells a cellular gene-specific c-myc real-time PCR assay was used. All assays have a linear detection range from 106 to 10 copies per reaction with an overall R2 of 0.98 and PCR efficiencies 95% (table 1), which are common features for many other real-time PCR assays used in virology and microbiology [8-10]. Results of intra- and inter-assay variability for plasmids standards were less than 1 CT (see details for OPV12/13 and c-myc assays in table 1) demonstrating a high degree of intraand inter-assay precision. 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 (...truncated)