AIDS VACCINE RESEARCH LABORATORY David Watkins, P.I.

• Home
• About the Lab
• Research
• Lab Members
• Publications
• Services
• Links

• Research Interests
• Immunology
• Molecular
• Protein
• Virology

Virology Core

Thomas Friedrich, Ph.D. – Assistant Scientist.  Dr. Friedrich is responsible for running the Virology Core Facility at WNPRC.  In this capacity he oversees production of SIVmac239, as well as related SIV viruses, titering virus and providing virus to all projects in the lab.  Gemma May, B.S. prepares samples for challenge experiments and runs qPCR for viral loads or all users of the Virology Core Facility services.  She reports data back to the users and enters it into the lab database

Our Virology Core is responsible for three distinct services:

1. the production and quality assurance of high-titer SIV stocks;
2. conducting virus detection and assays for clinical and tissue culture samples;
3. challenge models

1. Stock Production
In the calendar year 2004, we produced four large-scale, high-titer SIV stocks.  Two of these stocks were produced onsite, and two were made at the New England Primate Research Center in collaboration with Dr. Ronald Desrosiers (Friedrich 2004).  Clonal virus stocks are prepared by transfection of adherent cell lines with plasmid DNA containing the proviral sequence.  Supernatants harvested from transfected cells may contain enough virus to use as a laboratory stock, but typically such supernatants are used to seed infection of primary lymphocytes or lymphocyte-derived, immortalized cells (such as CEMx174) so that the virus stock can be expanded.  Lymphocyte-expanded virus stocks made in our laboratory usually reach titers of about 100,000 TCID₅₀, equivalent to 100 – 300 ng p27 antigen or 1 – 5 billion vRNA copy equivalents, per milliliter culture fluid.  All coding regions of each virus stock are bulk sequenced to ensure that no mutations have been introduced during the culture period.  The yield from such preparations can be modulated to suit the needs of the experiment, but we have produced between 100 and 800 vials containing 0.5 ml high-titer virus stock, which is archived in the vapor phase of liquid nitrogen.  The stocks we have made this year are as follows, and together comprise over 1,100 frozen aliquots:
   -SIV239 wild type, grown on CEMx174 cells
   -3xSIV, a genetically engineered escape mutant variant, grown on CEMx174 cells
   -SIV239 wild type, grown on primary lymphocytes at NEPRC
   -SIV239 wild type, product of 293T cell transfection performed at NEPRC
  -Stocks of SIV251, SIV239∆nef,  SIVsmE543, and SIVsmE660  will be available from Dr. Desrosiers.

2. Virus quantitation
Virus quantitation assays performed by Virology Services include standard TCID₅₀ assays and p27 antigen ELISAs, but our main focus is on ultrasensitive quantitative RT-PCR-based detection of virus in plasma and tissue culture samples.  We have dramatically improved the sensitivity of our QRT-PCR assay over the past year, and now routinely achieve a dynamic range of greater than 7 orders of magnitude, with a sensitivity threshold of about 5 vRNA copies/ml sample.  We amplify and detect a conserved region of gag, using an approach adapted in consultation with Michael Piatak and Jeffery Lifson at the National Cancer Institute (Lifson 2001) (Piatak et al., MS in prep).  Currently, we use primers specific for the SIV239 sequence, but the assay has been modified by Lifson et al. for the detection multiple SIV strains, including SIV251 and SIVsmE660.  Our assay is performed on the Roche LightCycler.

3. Challenge model
We will use Indian rhesus macaques for our vaccine and challenge experiments.  We have recently established a low-dose weekly repeated i.r. challenge model that required low doses (30- 500 TCID₅₀) of SIVmac239 (McDermott 2004).  The lower dose challenges are thought to be a more physiologically appropriate model for mucosal HIV transmission.