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| **          | Mammalian and chicken cell substrates are used in      |
| Abstract:** | preparation of live, viral vaccines. In this case,     |
|             | since there are no viral inactivation or removal steps |
|             | during manufacture, extensive and rigorous cell        |
|             | substrate testing is done to ensure the absence of     |
|             | adventitious agents in the product. Since all cells    |
|             | contain endogenous retroviral sequences, some of which |
|             | may give rise to an infectious virus, the FDA has      |
|             | recommended use of highly sensitive and broadly        |
|             | reactive, PCR-based reverse transcriptase assays (such |
|             | as PERT) for cell substrate testing. In addition, PERT |
|             | testing is done on the harvest or final product to     |
|             | assure absence of retrovirus activation from the cell  |
|             | substrate due vaccine virus infection or production    |
|             | procedures. In general, PERT assays can provide        |
|             | confident data regarding the absence of extracellular  |
|             | reverse transcriptase (RT) activity in the case of     |
|             | mammalian cells. However, the interpretation of the    |
|             | PERT results have been difficult in cases where there  |
|             | is cross-reactivity in the assay due to release of     |
|             | cellular DNA polymerases during vaccine production or  |
|             | in the case of chicken cells where there is            |
|             | spontaneous release of defective retroviral particles  |
|             | in normal cells derived from any egg source, including |
|             | SPF. Additionally, the increased use of novel cell     |
|             | substrates, especially tumor-derived cells for new     |
|             | vaccines, has given impetus to further develop assays  |
|             | and strategies for enhanced and specific retrovirus    |
|             | detection. We have investigated retrovirus induction   |
|             | and use of a PERT assay for virus detection using a    |
|             | well-characterized mouse cell line. We now propose to  |
|             | employ these results to develop enhanced retrovirus    |
|             | detection strategies with assay standardization for    |
|             | detection of latent, endogenous retroviruses in        |
|             | vaccine cell substrates such as vero cells, primary    |
|             | chicken cells and a novel tumor cell substrate.        |
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| **Specific  | 1)  To develop experimental conditions and strategies  |
| aims:**     |     for activation of latent, endogenous retroviruses  |
|             |     in mammalian and chicken cell substrates. This     |
|             |     will aid in early selection of "clean" cell        |
|             |     substrates that are free of endogenous, infectious |
|             |     retroviruses and assure vaccine safety.            |
|             |                                                        |
|             | 2)  To evaluate retrovirus activation in mammalian and |
|             |     chicken cells using a PERT assay with defined,     |
|             |     reproducible enzyme standards, which will enable   |
|             |     use of the assay in different labs and provide     |
|             |     meaningful data.                                   |
|             |                                                        |
|             | 3)  To characterize the infectivity of induced         |
|             |     retroviruses from vaccine cell substrates using    |
|             |     various target cells including human.              |
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| **Ba        | All mammalian and avian species contain numerous       |
| ckground:** | copies of endogenous retroviral sequences as a part of |
|             | the normal cell genome. Although the majority of these |
|             | sequences are defective due to incomplete copies of    |
|             | the viral DNA, one or more complete copies of the      |
|             | retroviral genome may exist. Generally, endogenous     |
|             | retroviruses remain latent; however, they can be       |
|             | induced to produce infectious virus particles. The     |
|             | regulation of the expression of endogenous             |
|             | retroviruses is complex and multifactorial. In         |
|             | general, a variety of agents and conditions may induce |
|             | retroviruses including other viruses and various       |
|             | chemical, biological or immunological agents as well   |
|             | as stressful culture conditons. Therefore, the         |
|             | presence of latent, inducible, infectious retroviral   |
|             | DNAs in vaccine cell substrates is a safety concern,   |
|             | especially in case of live, viral vaccines where there |
|             | is little or no viral inactivation or removal during   |
|             | product manufacture. We have investigated and          |
|             | optimized retrovirus induction from a                  |
|             | well-characterized mouse cell line (Khan, A.S. et al., |
|             | 2001. Early detection of endogenous retroviruses in    |
|             | chemically-induced mouse cells. *Virus Res.*           |
|             | 79:39-45). We have further investigated the            |
|             | specificity of a PERT assay to distinguish retrovirus  |
|             | activation from increased cellular DNA polymerase      |
|             | production in chemically-treated cells. We plan to     |
|             | apply the information obtained from the mouse cells to |
|             | develop conditions and assays to evaluate mammalian    |
|             | and chicken vaccine cell substrates for endogenous     |
|             | retrovirus induction and detection. Evaluation of cell |
|             | substrates under optimized conditions of induction and |
|             | use of sensitive retrovirus detection assays will      |
|             | provide a sensitive detection strategy for early       |
|             | selection of cell substrates that are free of occult   |
|             | adventitious retrovirus agents, and thereby minimize   |
|             | product contamination due to the cell substrate during |
|             | the manufacturing process.                             |
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| **Study     | **Cells and Viruses:** K-BALB mouse cells will be the  |
| des         | positive control for induction. Vaccine cell           |
| ign/Plan:** | substrates for investigation include vero, primary     |
|             | chicken cells from eggs obtained from various sources  |
|             | and a human tumor cell line. For chicken cell studies, |
|             | endogenous and exogenous avian leukemia viruses (ALVs) |
|             | will be used as positive control. For mammalian cell   |
|             | studies, viruses of appropriate relevant species will  |
|             | be used. **Inducers:** Various chemicals including     |
|             | 5'-iodo-deoxyuridine (IdU) and azacytidine (AzaC);     |
|             | \[investigational\] viruses including Modified Virus   |
|             | Ankara (MVA) and influenza. **Assays:** STF-PERT assay |
|             | using murine leukemia virus (MLV) and avian leukemia   |
|             | virus (ALV) RT standards will be used detection of     |
|             | induced viruses *(Sears, J.F. and Khan, A.S. 2003.     |
|             | Single-tube fluorescent product-enhanced reverse       |
|             | transcriptase assay with Ampliwax (STF-PERT) for       |
|             | retrovirus quantitation. [J. Virol.                    |
|             | Methods]{.underline} 108:139-142);* virus induction    |
|             | will be confirmed by TEM (J. Muller); virus            |
|             | infectivity will be evaluated using various assays for |
|             | avian and mammalian retroviruses that are already      |
|             | established in the lab (Khan).                         |
|             |                                                        |
|             | **Study Design:** [General:]{.underline} 1)            |
|             | Determination of drug toxicity: cells will be exposed  |
|             | to a range of drug concentrations and evaluated for    |
|             | cytotoxic effect by microscopic examination and using  |
|             | the Roche Cytotoxicity Detection Kit (LDH); 2)         |
|             | Retrovirus induction in the supernatant will be        |
|             | evaluated by a modified PERT assay with enhanced       |
|             | retrovirus RT specificity. To assure assay             |
|             | reproducibility in different labs and generate         |
|             | confident and interpretable data, the PERT assay will  |
|             | be standardized using commercially available ALV and   |
|             | MLV RTs and SOPs will be developed for preparing and   |
|             | storage of enzyme standards with stability,            |
|             | sensitivity, and reproducibility data; 3) Retrovirus   |
|             | induction in the cells will be confirmed by TEM:       |
|             | particle type will be identified based upon physical   |
|             | characteristics; 4) Specific retrovirus activation     |
|             | will be evaluated in infectivity assays.               |
|             | [Specific:]{.underline} 1) In case of mammalian cells, |
|             | PERT assay will be modified to distinguish between     |
|             | cellular polymerases (which are released due to        |
|             | induction) and activated, endogenous, retrovirus RT.   |
|             | 2) In case of chicken cells: i) the baseline PERT      |
|             | activity produced from normal cells will be determined |
|             | by evaluating cells from various egg sources           |
|             | (including SPAFAS) and testing cell culture            |
|             | supernatant at various time points using a             |
|             | standardized PERT assay; ii) sensitivity of induced    |
|             | retrovirus detection from a chicken cell line known to |
|             | contain endogenous ALV will be evaluated using a PERT  |
|             | assay and infectivity assays. Spiking studies will be  |
|             | done to evaluate sensitivity of retrovirus detection   |
|             | in the background RT activity produced from normal     |
|             | chicken cells.                                         |
|             |                                                        |
|             | **Expected Outcome:** 1) Identification of conditions  |
|             | for endogenous retrovirus activation from vero cells   |
|             | and chicken cells, two cell substrates that are        |
|             | extensively used in live viral vaccine production. 2)  |
|             | Development of a standardized PERT assay to evaluate   |
|             | retroviruses in chicken cell substrates in different   |
|             | labs. 3) To provide guidance to vaccine sponsors for   |
|             | early, sensitive, and specific detection of            |
|             | retroviruses in cell substrates for vaccine safety.    |
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