Antiviral Discovery and Development

We screen compounds prophylactically for antiviral activity against SARS CoV-2 using a cell-based assay based on the publication from the Jonsson lab: "Development and Validation of a High-Throughput Screen for Inhibitors of SARS CoV and Its Application in Screening of a 100,000-Compound Library." J Biomol Screen 2007 12: 33. DOI: 10.1177/1087057106296688. We invite you to learn more about Dr. Jonsson’s HTS program. 

Is a positive control run for HTS assay? Yes. HTS plates are examined for acceptability in each run using Z score and CV statistics. HTS assay validation is managed according to the NIH Assay Guidance Manual. From this table, we calculate the Z score, CV, S/B and S/N for each plate. The positive control is the cells only and the negative control is virus. This are set to 100 and 0 respectively to normalize the data. In line with the HTS approach, an inhibitor is added to each plate at a single dose. This compound's identity will not be provided as it is not required for review of the data. Any plate that doesn’t meet the criteria below will be rerun for no cost.

Parameter	Definition	Range
Z value	1-(3* s.d. of cell control (σc) + 3* s.d. of the virus control (σv)/ [mean cell control signal (µc) - mean virus control signal (µv)].	> 0.5 A value of 1.0 is the maximum
signal/background (S/B)	mean cell control signal (µc)/mean virus control signal (µv)	>10
signal/noise (S/N)	mean cell control signal (µc) - mean virus control signal (µv) / (s.d. of the cell control signal (σc)2 - s.d. of the virus control signal (σv)	>3
Coefficient of Variance (CV)	Percentage of s.d. per mean of cell or virus control	<10%
Correlation coefficient (r2)	Does a straight line fit the data? ∑[(x0 - xi)2 + (y0 - yi)2]1/2 x0 and y0 = line of best fit xi and yi = coordinates for each data point	>0.7

SCREENING FREQUENCY:

• Screening is conducted on a first come, first served basis. 
• High Throughput Screening (HTS): Monday, Tuesday, and/or Wednesday, depending on volume and other studies. Compounds need to arrive at least 2 days/weeks prior to the screening date. Assays will be scheduled according to holidays and volume of incoming work. 

SHIPPING INFORMATION:

• Send by FEDEX or UPS to:

To run this assay, it is essential to know the cytotoxicity of the compound in the cell line chosen. Without the information the assay’s data is not qualified.

For titer reduction assays, Vero E6 TMPRESS or A549 ACE2 or any other cells (primary, etc.) are grown in 12 to 24-well plates overnight and then infected with virus at a MOI of 0.1 diluted in the appropriate media (depends on the cell line). The cells are infected for one hour of adsorption at 37C, cells are washed with PBS twice and media containing 5 μM of compound (or as directed) is added to each well. After two days, the supernatant is harvested and the amount of virus in each well is measured using a TCID50 or plaque assay (as directed).

Drug can also be added 2 hours prior for prophylactic assays which we recommend people to test first. 

Drug concentration can be modified as needed and multiple doses can be tested. Other drugs can be provided as a control, but doing so will incur an additional cost.

Other types of cell line (e.g. A549) including primary cells (epithelial or endothelial) can be employed in titer reduction assays using a TCID50 with MTT or CTG or plaque titer as an endpoint.

 

SCREENING FREQUENCY:

• Screening is conducted on a first come, first served basis. 
  Titer Reduction : Every Wednesday

SHIPPING INFORMATION:

• Send by FEDEX or UPS to:

Once you have identified an effective concentration of inhibitor using the HTS and titer reduction assays, one can measure the reduction of virus titer in a time of addition experiment. This will give you information on the antiviral effectiveness of your compound if it is added before infection, or early, middle or late in infection. While the assay is continuing to be refined, addition of compound at 2 hours prior to infection, 2 hours after are a good starting point in a pilot. Advanced studies may measure addition of compound at 2, 4, 6-, 8-, 12- and 24-hours post-infection. 

SCREENING FREQUENCY:

Screening is conducted on a first come, first served basis. 
Time of Addition: Once every 2 weeks

SHIPPING INFORMATION:

• Send by FEDEX or UPS to:

 

As in the titer reduction assay, the chosen cells are grown in 12 to 24-well plates overnight and then infected with virus at a MOI of 0.1 diluted in the appropriate media. 

As with the titer reduction, any cell line can be used that confers infection. We have two human cell lines available; 293T and A549 which harbor the hACE2 receptor. 

Prophylactic addition at 2 hours prior to infection: In three wells (3 biological replicates) two hours prior to infection, a specific amount of compound. i.e. 5 μM, is added to each well.  The cells are washed, the cells are infected for one hour of adsorption at 37C, and then cells are washed with PBS twice and media containing X μM of compound is added to each well.  

Therapeutic addition at 2 hours post-infection: The cells are washed, the cells are infected for one hour of adsorption at 37C, and then cells are washed with PBS twice. At the designated times, in three wells (3 biological replicates) per time point at designated times post infection, a specific amount of compound. i.e. 5 μM, is added to each well.   
After two days, the supernatant is harvested and the amount of virus in each well is measured using a TCID50 or plaque assay. 

The cornerstone service of our facility is the testing of small molecules or vaccines for efficacy in small animal models of bacterial or viral pathogens. Our highly experienced staff each have years of experience in leading and supporting these types of studies for clients from academic, government and industrial sectors. Each study will be supported by extensive documentation in study protocols and reports. The scientific staff at the RBL can provide your team with support from study design to figures for publication to report required for an IND submission.

LC-MS/MS analysis directly in BSL-3 containment is available to examine drug disposition, pharmacokinetics, and exposure response assessments. Triple quadrupole mass spectrometry utilizes select monitoring of single precursor-to-fragment ion transitions specific for drug candidates. This approach allows for high sensitivity, selective, and reproducible quantification of low concentrations of small molecules.

Animal care management and staff are AALAS certified and trained in all aspects of biocontainment safety practices and procedures. The facility features Allentown BCU caging systems and Class II Type A2 biosafety cabinets in all animal holding rooms. We are able to house up to 4,000 mice, 300 rats, and 150 hamsters or guinea pigs under ABSL-2 or -3 containment. RBL staff can be contracted to perform animal work for infectious disease research ranging from weights and daily observations to infections, blood and tissue collection and full necropsy.

 

 

Validated models with mice:

• Influenza BSL-2 strains, HPAIV (intranasal)
• Old World hantaviruses
• Severe acute respiratory syndrome Coronavirus 1 (SARS CoV-1)
• Severe acute respiratory syndrome Coronavirus 2 (SARS CoV-2)
• Middle-East respiratory syndrome Coronavirus (MERS CoV)
• Respiratory Syncytial virus (intranasal)
• Venezuelan equine encephalitis viruses (subcutaneous and intranasal)
• Western equine encephalitis viruses (subcutaneous and intranasal)
• Eastern equine encephalitis viruses (subcutaneous and intranasal)
• Leptospira interrogans; Intraperitoneal, conjunctival, transdermal
• Mycobacterium tuberculosis (intranasal)
• Methicillin-resistant Staphylococcal aureus
• Staphylococcus aureus (laboratory and clinical strains)
• Streptococcus pneumoniae (laboratory and clinical strains)
• Co-infection model with influenza and Streptococcus pneumoniae or Staphylococcus aureus
• Saccharopolyspora rectivirgula
• Other- available on request 

Validated models with ferret:

• Influenza BSL-2 strains (intranasal)
• Influenza HPAI BSL-3 strains (intranasal)
• SARS-CoV-1 and SARS-CoV-2 BSL-3 strains (intranasal) 

**Other small animal models available upon request.

• Xenogen IVIS Spectrum In Vivo Imaging
• CT
• ECHO
• Clinical Signs, Weight, Temperature Measurements
• Clinical Chemistry
• Hematology 

• RNA Isolation/RNA  
• RNA Isolation/qPCRSeq  
• Plaque Assay
• PRNT  
• Cytospin
• Magpix
• LC-MS/MS  
• Pathology  
• Western Blot
• BLAZE
• Cell Sorting and Flow Cytometry
• Immuglobulin Measurements and Subtyping
• Fluorescent Microscopy
• ELISpot
• Protein Target Multiplexing