AACF
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NIAID's Antimicrobial Acquisition and
Coordinating Facility AACF |
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Submission Info Screening Assays |
Human Papilloma Virus Assay
A. Screening protocol for testing anti-viral activity of compounds against HPV-11 (selected other HPV types such HPV-40 are available) using QRT-PCR: Day 0 Plate 2 x 105 A431 cells/well in 6-well cluster dishes. Day 1 Add replicate aliquots of HPV-11 (HPV-40) to each well representing an MOI of 150 particles per cell. Control wells without virus will be included. Day 2-3 Dilutions of compound are added to triplicate cultures. Control wells receive media alone. Positive control compound will be HPMPC (cidofovir) at 300 μg/ml (activity defined in a previous study). Day 5 Cell cultures are harvested, lysed with Trizol reagent (GIBCO/BRL) and RNA prepared. Days 6-7 QRT-PCR is conducted to quantitate the proportion of viral E1^E4 transcripts and a cellular reference RNA for the TATA-binding protein (TBP). Anti-viral effects of compounds will be assessed as an EC50 value representing a 50% reduction in the amount of E1^E4 viral transcript when compared with cultures infected with HPV-11 alone. CC50 toxicity is calculated as the compound dose at which 50% of total cellular RNA is recovered. The assay procedure may be modified to test compounds with microbicidal activity if requested. This modification will be represented by drug addition to A431 cells at the same time as infectious virus. Figure 1. Theoretical plot of antiviral activity of compound ARB00-000 using the HPV-11 monolayer QRT-PCR assay.
Anti-viral activities are calculated from the plots using 50% maximum values. In the theoretical example above, anti-viral activity is determined from the E1^E4 plot (?), which represents an IC50 = 9ug/ml. The toxicity is determined from total RNA yield (), which represents an CC50 = 100ug/ml. From these two values, the Selectivity Index (SI) is determined from CC50/EC50 = 11. A third parameter can be determined from the cellular house-keeping gene TATA-box binding protein (TBP) (�), and in this example, would have a IC50 > 250ug/ml. This value can be somewhat misleading because this value can remain at non-drug levels even though there is some cellular cytotoxicity.
Since the CC50 value is a more stringent criteria to determine SI, we have initially decided that SI>5 would be significant for the detection of an anti-viral activity. This value is arbitrarily derived, but is predicative from the SI values that we have obtained from our positive control compound, cidofovir. B. Screening protocols for testing anti-viral activity of compounds against BPV-1 using an ELISA-based assay system. Compounds will be tested in triplicate cultures and follow the protocol outlined below: Day 0 Plate 3 x 103 C127 cells/well into wells of a 96-well flat-bottomed microculture plate. Day 1 Add replicate aliquots of BPV-1 to each well, representing approximately 100 focus-forming units. Control wells without virus will be included. Day 3 Dilutions of drug will be added to triplicate cultures of both BPV-1-infected and uninfected cultures. Control wells will receive media without compound. Positive control compound will be cidofovir at 5 μg/ml (activity defined in previous studies). Days 5-14 Cell cultures will be fed with medium and compound every 3-4 days. Day 14 Cell numbers and viability will be assessed using the MTS assay. Anti-viral effects of compounds will be calculated using the following formula to obtain % anti-viral activity: B&A/B&C × 100% = % Anti-viral activity A = O.D. of BPV-1-containing, compound-treated cultures B = O.D. of BPV-1 containing cell cultures C = O.D. of cultures of cells alone The EC50 value will represent a 50% reduction in the amount of O.D. values (MTS signal) of compound-treated virus-infected cultures when compared with cultures containing BPV-1 alone and cultures containing cells alone. As for Protocol A, the assay procedure may be modified to test compounds with microbicidal activity if requested. This modification will be represented by drug addition to C127 cells at the same time as infectious virus. Figure 2. Theoretical plot of anti-viral activity of compound ARB00-0000 using the BPV-1 monolayer ELISA assay. In this example, the anti-viral IC50 is determined as the 50% OD value that occurs between the values of virus-only (= 1.5) and cells-only (= 0.7) which is subsequently and O.D. of 1.1. Thus the anti-viral IC50 is 3ug/ml. The EC50 (anti-ptoliferative value as the culture period is 2 weeks, and cellular cytotoxicity can not be assessed in this culture period) is the 50% O.D. value that occurs between the cells-only (= 0.7) and no cells (= 0.0). Thus the EC50 value is determined for the O.D. value of 0.35, and in the above graph is 20ug/ml. From these values, an SI can be determined which in this example is 7.0.
As for the HPV-11 QRT-PCR assay, we would consider an SI>5.0 to be significant based upon our findings with the positive control agent, cidofovir. C. Screening protocols for testing anti-viral activity of compounds against HPV-31b using the organotypic (raft) culture system. 1. Cultures of CIN612, clone 9E cells are prepared from established protocols. Individual rafts are developed containing multi-layers of 9E cells growing on a collagen matrix impregnated with mitomycin C-treated fibroblast cells. 2. Rafts are treated with compound delivered into the cell culture media that can diffuse into the 9E multulayer. 3. Treatments will be continuous for the culture duration, which is a period of 10 days. 4. After 10 days culture, the 9E rafts will be harvested and assayed for HPV-31b DNA (measure of viral DNA replication) and E1^E4 viral transcription (viral function) using the QRT-PCR assay described for the HPV-11 monolayer assay system. 5. Primers will be prepared to quantitate HPV-31B DNA and RNA (E1^E4) and the quantitation compared to TBP. Anti-viral activity will be measured quantitatively as either or both a reduction in viral DNA and RNA when compared to placebo-treated 9E rafts. 6. A portion of each raft will be removed for histology (H&E, immunostaining for specific marker keratins [keratin 10, involucrin] ). 7. Viral DNA and RNA levels will be plotted against compound concentrations to determine EC50 (50% reduction in viral DNA and/or RNA), CC50 (50% reduction in yield of total RNA/DNA. We will consider an SI > 5 to be significant for anti-viral activity. D. In vivo assays systems for subsequent testing of promising compounds. 1. Two contracts (subcontracts) exist for the testing of anti-viral compounds in vivo. Dr William Bonnez (U of Rochester, NY) has established the immune compromised mouse xenograft system to test anti-viral activity of compounds to HPV-6 and HPV-11. Dr Neil Christensen (Penn State College of Medicine) has established the cottontail rabbit papillomavirus (CRPV) rabbit model for testing compounds topically, intralesionally and systemically in immune competent hosts. |
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