Development and comparison of three cell-based potency assays for anti-respiratory syncytial virus monoclonal antibody.

There is an increasing demand for monoclonal antibody (mAb) therapies to confer passive immunity against viral diseases. Respiratory syncytial virus (RSV) is the most common cause of bronchiolitis, lower respiratory tract infections, and hospitalization in infants. Currently, there is no RSV vaccine but a humanized mAb available for high risk infants. MK-1654 is a fully human mAb with YTE mutation in the fragment crystallizable (Fc) region to extend the half-life in circulation. It binds to a highly conserved epitope of RSV Fusion protein with high affinity and neutralizes RSV infection. A functional cell-based assay is a regulatory requirement for clinical development, commercial release, and stability testing of MK-1654. In this study, we have evaluated three RSV neutralization assays to test the potency of MK-1654, including an imaging-based virus reduction neutralization test (VRNT) and two reporter virus-based assays (RSV-GFP and RSV-NLucP). All three methods showed good dose response curves of MK-1654 with similar EC50 values. RSV-NLucP method was chosen for further development because it is simple and can be easily adapted to quality control testing laboratories. After optimization, the RSV-NLucP assay was pre-qualified with good linearity, relative accuracy, intermediate precision, and specificity, therefore suitable for a cell-based potency assay.

Activation of IFN-β expression by a viral mRNA through RNase L and MDA5.

IFNs play a critical role in innate immunity against viral infections. Melanoma differentiation-associated protein 5 (MDA5), an RNA helicase, is a key component in activating the expression of type I IFNs in response to certain types of viral infection. MDA5 senses noncellular RNA and triggers the signaling cascade that leads to IFN production. Synthetic double-stranded RNAs are known activators of MDA5. Natural single-stranded RNAs have not been reported to activate MDA5, however. We have serendipitously identified a viral mRNA from parainfluenza virus 5 (PIV5) that activates IFN expression through MDA5. We provide evidence that the signaling pathway includes the antiviral enzyme RNase L. The L mRNA of PIV5 activated expression of IFN-ß. We have mapped the RNA to a region of 430 nucleotides within the L mRNA of PIV5. Our results indicate that a viral mRNA, with 5'-cap and 3'-poly (A), can activate IFN expression through an RNase L-MDA5 pathway.

Development of a Cell-Based Reporter Potency Assay for Live Virus Vaccines.

The rapid development of potency assays is critical in the development of life-saving vaccines. The traditional plaque assay or fifty percent tissue culture infectious dose (TCID50) assay used to measure the potency of live virus vaccines is time consuming, labor intensive, low throughput and with high variability. Described here is the development and qualification of a cell-based reporter potency assay for two vaccines for respiratory viral infection, one based on the recombinant vesicular stomatitis virus (rVSV) backbone, termed Vaccine 1 in this paper, and the other based on the measles virus vector, termed Vaccine 2. The reporter potency assay used a Vero E6 cell line engineered to constitutively express NanuLuc® luciferase, termed the VeroE6-NLuc or JM-1 cell line. Infection of JM-1 cells by a live virus, such as rVSV or measles virus, causes a cytopathic effect (CPE) and release of NanuLuc® from the cytoplasm into the supernatant, the amount of which reflects the intensity of the viral infection. The relative potency was calculated by comparison to a reference standard using parallel line analysis (PLA) in a log-log linear model. The reporter assay demonstrated good linearity, accuracy, and precision, and is therefore suitable for a vaccine potency assay. Further evaluation of the Vaccine 1 reporter assay demonstrated the robustness to a range of deliberate variation of the selected assay parameters and correlation with the plaque assay. In conclusion, we have demonstrated that the reporter assay using the JM-1 cell line could be used as a potency assay to support the manufacturing and release of multiple live virus vaccines.

Sumoylation of the P protein at K254 plays an important role in growth of parainfluenza virus 5.

The P protein of parainfluenza virus 5 (PIV5) is an essential cofactor of the viral RNA-dependent RNA polymerase. Phosphorylation of the P protein can positively or negatively regulate viral gene expression, depending on the precise phosphorylation sites. Sumoylation, a process of adding small ubiquitin-like modifier (SUMO) to proteins posttranslationally, plays an important role in regulating protein function. In this study, we have found that the P protein of PIV5 was sumoylated with SUMO1 in both transfected and infected cells. The K254 residue of the P protein is within a consensus sumoylation motif. Mutation of the P protein at K254 to arginine (P-K254R) reduced PIV5 minigenome activity, as well as the sumoylation level of the P protein. Incorporation of K254R into a recombinant PIV5 (rPIV5-P-K254R) resulted in a virus that grew to a lower titer and had lower levels of viral RNA synthesis and protein expression than wild-type PIV5, suggesting that sumoylation of the P protein at K254 is important for PIV5 growth. Biochemical studies did not reveal any defect of P-K254R in its interactions with viral proteins NP and L or formation of homotetramers. We propose that sumoylation of the P protein at K254 regulates PIV5 gene expression through a host protein.

Identification of a phosphorylation site within the P protein important for mRNA transcription and growth of parainfluenza virus 5.

The viral RNA-dependent RNA polymerase (vRdRp) of paramyxovirus consists of the large (L) protein and the phosphoprotein (P). P is heavily phosphorylated, and it is thought that the phosphorylation of P plays a role in regulating viral RNA synthesis. However, no phosphorylation site within the P protein in paramyxovirus has been identified as playing a positive role in viral RNA synthesis in virus infection. Using mass spectrometry analysis, the threonine residue at position 286 of P of parainfluenza virus 5 (PIV5) was found phosphorylated. Mutation of T286 to alanine (T286A), aspartic acid (T286D), or glutamic acid (T286E) reduced minigenome activity. Recombinant virus containing a mutation at the T286 position (rPIV5-P-T286A) grew slower than wild-type virus; viral mRNA synthesis and protein expression of rPIV5-P-T286A were delayed. Biochemical studies showed that the binding of NP or L protein with the P mutants or tetramer formation by the mutant P proteins was unaltered from that for wild-type P. While we failed to rescue rPIV5-P-T286E virus, several revertant viruses were obtained. All non-wild-type revertants had mutations at T286 and showed defects in both minigenome activity and viral growth. This is the first time that a phosphorylation site within the P protein in paramyxovirus has been found to play a positive role in viral mRNA synthesis and virus growth.

Virus reduction neutralization test and LI-COR microneutralization assay bridging and WHO international standard calibration studies for respiratory syncytial virus.

Background: Respiratory syncytial virus (RSV) vaccine is an unmet medical need. The virus reduction neutralization test (VRNT) was developed to replace the LI-COR microneutralization assay to measure RSV neutralization titers. Methods: A bridging study using selected V171 phase I samples and calibration studies using the WHO international standard antiserum to RSV were performed to compare VRNT and LI-COR. Results: From the bridging study, we showed good concordance between VRNT and LI-COR titers, and similar post-/prevaccination titer ratios. From the calibration studies, we can convert VRNT and LI-COR titers into similar IU/ml. Conclusion: The VRNT and LI-COR microneutralization assay correlate well and the titers can be standardized as similar IU/ml, enabling direct comparison of titers from different assays.

PLK1 down-regulates parainfluenza virus 5 gene expression.

The paramyxoviruses are a family of negative-sense RNA viruses that includes many important human and animal pathogens. Paramyxovirus RNA synthesis requires the viral phosphoprotein (P) and the large (L) protein. Phosphorylation of P is thought to regulate viral gene expression, though direct proof remains elusive. Recently, we reported that phosphorylation of a specific residue (Ser157) of the P protein of parainfluenza virus 5 (PIV5), a prototypical paramyxovirus, correlates with decreased viral gene expression and cytokine expression in infected cells. Here, we show that: Polo-like kinase 1 (PLK1), a serine/theronine kinase that plays a critical role in regulating the cell cycle, interacts with PIV5 P through the S157 residue; PLK1 inhibition increases viral gene expression; PLK1 over-expression inhibits viral gene expression; and PLK1 directly phosphorylates P in vitro, indicating that PLK1 down-regulates viral gene expression by phosphorylating P. Furthermore, we have determined the PLK1 phosphorylation site on P and found that mutant recombinant PIV5 whose P proteins cannot either bind to or be phosphorylated by PLK1 have similar phenotypes. Increased viral gene expression in PIV5 with mutations in the PLK1 binding/phosphorylation sites correlates with increased induction of cell death and cytokine expression, suggesting that PIV5 limits its viral gene expression to avoid these host effects. It is possible that targeting PLK1 will enhance host innate immune responses, leading to a novel strategy of clearing paramyxovirus infections quickly.

Development and qualification of a fast, high-throughput and robust imaging-based neutralization assay for respiratory syncytial virus.

Respiratory syncytial virus (RSV) is a common pathogen causing severe respiratory illness in infants and elder adults. The development of an effective RSV vaccine is an important unmet medical need and an area of active research. The traditional method for testing neutralizing antibodies against RSV in clinical trials is the plaque reduction neutralization test (PRNT), which uses 24-well plates and needs several days post infection to develop viral plaques. In this study, we have developed a virus reduction neutralization test (VRNT), which allows the number of RSV infected cells to be automatically counted by an imaging cytometer at one day post infection in 96-well plates. VRNT was found robust to cell seeding density, detection antibody concentration, virus input and infection time. By testing twenty human sera, we have shown good correlation between VRNT50 and PRNT50 titers for multiple RSV strains: A2, Long and 18537 (serotype B). To understand the VRNT performance, eight human serum samples with high, medium and low neutralization titers were selected for VRNT qualification. We have demonstrated that VRNT had good specificity, precision, linearity and relative accuracy. In conclusion, VRNT is a better alternative to PRNT in serum neutralization test for RSV vaccine candidates.

Rational design of multiple TB antigens TB10.4 and TB10.4-Ag85B as subunit vaccine candidates against Mycobacterium tuberculosis.

PURPOSE: Rational design of recombinant antigens TB10.4 and TB10.4-Ag85B as subunit vaccine candidates against Mycobacterium tuberculosis. The main purpose is to obtain a large quantity of soluble antigens. METHODS: Recombinant antigens were cloned in frame with the N-terminal thioredoxin and expressed in E. coli. The thioredoxin tag was removed by TEV protease. Nickel-affinity and size-exclusion chromatography were used to purify antigens to homogeneity. Antigen stability at different pH levels was studied by photon correlation spectrometry. Circular dichroism was used to probe antigen secondary structure and thermal stability. RESULTS: N-terminal thioredoxin fusion dramatically increased antigen solubility. Soluble TB10.4 and TB10.4-Ag85B were purified to homogeneity and obtained in milligram quantity. Co-expression of bacteria chaperons increased the yield of TB10.4-Ag85B. Soluble TB10.4 and TB10.4-Ag85B purified from the inclusion body showed a reversible structure change. However, Ag85B and soluble TB10.4-Ag85B showed a clear melting temperature, above which the secondary structure was lost dramatically. CONCLUSION: Soluble TB10.4 and TB10.4-Ag85B were purified from the E. coli in significant quantities. The methods to purify and characterize these recombinant antigens were established, which paved the way for further vaccine development based on these antigens.

AKT1-dependent activation of NF-kappaB by the L protein of parainfluenza virus 5.

Innate immunity plays a critical role in the control of viral infections. The induction of innate immune responses requires activation of transcription factors. In particular, NF-kappaB plays an essential role in activating the expression of cytokines involved in innate immunity such as beta interferon (IFN-beta) and interleukin-6 (IL-6). However, the mechanisms by which viruses activate NF-kappaB are poorly defined. Infection by parainfluenza virus 5 (PIV5), a prototypical member of the Paramyxoviridae family of Mononegavirales, has been shown to activate the expression of IFN-beta and IL-6. To examine how PIV5 induces this expression, we have examined the activation of NF-kappaB by PIV5 proteins. We have found that expression of PIV5 L protein alone is sufficient to activate NF-kappaB. The L protein of PIV5, the catalytic component of the viral RNA-dependent RNA polymerase, contains six domains that are conserved among all negative-stranded nonsegmented RNA viruses. We have mapped the region that activates NF-kappaB to the second domain, which is thought to be involved in RNA synthesis. The activation of NF-kappaB by L requires AKT1, a serine/threonine kinase, since AKT1 small interfering RNA, an AKT inhibitor as well as a dominant-negative mutant of AKT1, blocks this activation. Furthermore, we have found that L interacts with AKT1 and enhances its phosphorylation. We speculate that L may encode AKT1 kinase activity.

A single amino acid residue change in the P protein of parainfluenza virus 5 elevates viral gene expression.

Parainfluenza virus 5 (PIV5) is a prototypical paramyxovirus. The V/P gene of PIV5 encodes two mRNA species through a process of pseudotemplated insertion of two G residues at a specific site during transcription, resulting in two viral proteins, V and P, whose N termini of 164 amino acid residues are identical. Previously it was reported that mutating six amino acid residues within this identical region results in a recombinant PIV5 (rPIV5-CPI-) that exhibits elevated viral protein expression and induces production of cytokines, such as beta interferon and interleukin 6. Because the six mutations correspond to the shared region of the V protein and the P protein, it is not clear whether the phenotypes associated with rPIV5-CPI- are due to mutations in the P protein and/or mutations in the V protein. To address this question, we used a minigenome system and recombinant viruses to study the effects of mutations on the functions of the P and V proteins. We found that the P protein with six amino acid residue changes (Pcpi-) was more efficient than wild-type P in facilitating replication of viral RNA, while the V protein with six amino acid residue changes (Vcpi-) still inhibits minigenome replication as does the wild-type V protein. These results indicate that elevated viral gene expression in rPIV5-CPI- virus-infected cells can be attributed to a P protein with an increased ability to facilitate viral RNA synthesis. Furthermore, we found that a single amino acid residue change at position 157 of the P protein from Ser (the residue in the wild-type P protein) to Phe (the residue in Pcpi-) is sufficient for elevated viral gene expression. Using mass spectrometry and (33)P labeling, we found that residue S157 of the P protein is phosphorylated. Based on these results, we propose that phosphorylation of the P protein at residue 157 plays an important role in regulating viral RNA replication.

Akt plays a critical role in replication of nonsegmented negative-stranded RNA viruses.

The order Mononegavirales (comprised of nonsegmented negative-stranded RNA viruses or NNSVs) contains many important pathogens. Parainfluenza virus 5 (PIV5), formerly known as simian virus 5, is a prototypical paramyxovirus and encodes a V protein, which has a cysteine-rich C terminus that is conserved among all paramyxoviruses. The V protein of PIV5, like that of many other paramyxoviruses, plays an important role in regulating viral RNA synthesis. In this work, we show that V interacts with Akt, a serine/threonine kinase, also known as protein kinase B. Both pharmacological inhibitors and small interfering RNA against Akt1 reduced PIV5 replication, indicating that Akt plays a critical role in PIV5 replication. Furthermore, treatment with Akt inhibitors also reduced the replication of several other paramyxoviruses, as well as vesicular stomatitis virus, the prototypical rhabdovirus, indicating that Akt may play a more universal role in NNSV replication. The phosphoproteins (P proteins) of NNSVs are essential cofactors for the viral RNA polymerase complex and require heavy phosphorylation for their activity. Inhibition of Akt activity reduced the level of P phosphorylation, suggesting that Akt is involved in regulating viral RNA synthesis. In addition, Akt1 phosphorylated a recombinant P protein of PIV5 purified from bacteria. The finding that Akt plays a critical role in replication of NNSV will lead to a better understanding of how these viruses replicate, as well as novel strategies to treat infectious diseases caused by NNSVs.

Virus Reduction Neutralization Test: A Single-Cell Imaging High-Throughput Virus Neutralization Assay for Dengue.

Vaccine immunogenicity and clinical efficacy are often assessed by the measure of serum-neutralizing antibodies. The present gold standard for detecting neutralizing antibodies against many viruses, including dengue, is the plaque/focus reduction neutralization test (P/FRNT). The FRNT is a cell-based assay that inherits high variability, resulting in poor precision and has lengthy turnaround times. The virus reduction neutralization test (VRNT) is a high-throughput alternative to the standard low-throughput and laborious FRNT. The VRNT is similar to FRNT using unaltered wild-type virus and immunostaining, yet uses imaging cytometry to count virus-infected cells 1 day post-infection, reducing assay time and increasing overall throughput 15-fold. In addition, the VRNT has lowered variability relative to FRNT, which may be explained in part by the observation that foci overlap alters foci count and titer over time, in the FRNT. The ability to count one infected cell, rather than waiting for overlapping foci to form, ensures accuracy and contributes to the precision (7-25% coefficient of variation) and sensitivity of the VRNT. Results from 81 clinical samples tested in the VRNT and FRNT show a clear positive relationship. During sample testing, a 96-well plate edge effect was noted and the elimination of this edge effect was achieved by a simple plate seeding technique. The VRNT is an improvement to the current neutralization assays for its shortened assay time, increased precision and throughput, and an alternative to the P/FRNT.

Rescue of wild-type mumps virus from a strain associated with recent outbreaks helps to define the role of the SH ORF in the pathogenesis of mumps virus.

Mumps virus (MuV) causes acute infections in humans. In recent years, MuV has caused epidemics among highly vaccinated populations. The largest outbreak in the U.S. in the past 20 years occurred in 2005-2006 with over 5000 reported cases in which the majority of the cases was in vaccinated young adults. We sequenced the complete genome of a representative strain from the epidemic (MuV-IA). MuV-IA is a member of genotype G, the same genotype of MuV that was associated with the outbreak in the UK in 2004-2005. We constructed a reverse genetics system for MuV-IA (rMuV-IA), and rescued a virus lacking the open reading frame (ORF) of the SH gene (rMuV∆SH). rMuV∆SH infection in L929 cells induced increased NF-κB activation, TNF-α production and apoptosis compared to rMuV-IA. rMuV∆SH was attenuated in an animal model. These results indicated that the SH ORF of MuV plays a significant role in interfering with TNF-α signaling and viral pathogenesis during virus infection.

Phosphorylation of paramyxovirus phosphoprotein and its role in viral gene expression.

Paramyxoviruses include many important human and animal pathogens such as measles virus, mumps virus, human parainfluenza viruses, and respiratory syncytial virus, as well as emerging viruses such as Nipah virus and Hendra virus. The paramyxovirus RNA-dependent RNA polymerase consists of the phosphoprotein (P) and the large protein. Both of these proteins are essential for viral RNA synthesis. The P protein is phosphorylated at multiple sites, probably by more than one host kinase. While it is thought that the phosphorylation of P is important for its role in viral RNA synthesis, the precise role of P protein phosphorylation remains an enigma. For instance, it was demonstrated that the putative CKII phosphorylation sites of the P protein of respiratory syncytial virus play a role in viral RNA synthesis using a minigenome replicon system; however, mutating these putative CKII phosphorylation sites within a viral genome had no effect on viral RNA synthesis, leading to the hypothesis that P protein phosphorylation, at least by CKII, does not play a role in viral RNA synthesis. Recently, it has been reported that the phosphorylation state of the P protein of parainfluenza virus 5, a prototypical paramyxovirus, correlates with the ability of P protein to synthesize viral RNA, indicating that P protein phosphorylation does in fact play a role in viral RNA synthesis. Furthermore, host kinases PLK1, as well as AKT1 have been found to play critical roles in paramyxovirus RNA synthesis through regulation of P protein phosphorylation status. Beyond furthering our understanding of paramyxovirus RNA replication, these recent discoveries may also result in a new paradigm in treating infections caused by these viruses, as host kinases that regulate paramyxovirus replication are investigated as potential targets of therapeutic intervention.

Expression analysis of light-regulated genes isolated from a full-length-enriched cDNA library of Onosma paniculatum cell cultures.

Shikonin and its derivatives are formed in large amounts in dark-cultured Onosma paniculatum cells. In order to isolate and identify the genes regulating shikonin biosynthesis, we constructed and characterized a full-length-enriched cDNA library of dark-cultured cells by using the SMART (Switching Mechanism At 5'-end of RNA Transcript) cDNA synthesis and LD-PCR (long-distance PCR) strategies. The titer of the primary cDNA library was 1.04 x 10(6)pfu/mL with a recombination rate of 99.60%. Most of the cDNA inserts ranged from 1.0 to 2.5 kb, and 78.33% of the 76 randomly selected clones contained full-length coding regions. Expression analysis of randomly selected genes by small scale microarray revealed that 23 genes were down-regulated, including 17 genes with known functions, 2 genes with putative functions, and 4 novel genes, and that 3 genes were up-regulated (two-fold) in cells cultured under white light as compared with those cultured in the dark. Interestingly, two of the down-regulated genes, encoding aci-reductone dioxygenase (ARD)-like protein and ethylene responsive factor (ERF), are involved in ethylene biosynthesis and signal transduction, implying that ethylene might play an important role as a signal molecule in light-regulated shikonin formation. These data contribute to a better understanding of light-involvement in regulating the formation of plant secondary metabolites.