Einstein-Montefiore CFAR Virology Core Director: Dr. Ganjam V. Kalpana, Ph.D

Einstein-Montefiore CFAR

Virology Core

Director: Dr. Ganjam V. Kalpana, Ph.D.

Overarching Goal

• To provide CFAR Investigators with the appropriate support, training and access for the most relevant and cutting-edge approaches to rapidly and efficiently support their virology research programs.

Virology Core Aims• Providing the Infrastructure, reagents, assays and technologies to

enable basic and translational researchers to Investigate HIV Infection, pathogenesis and therapy;

• Performing cellular and molecular assays characterizing HIV-lnfection to support clinical and translational investigators engaged in patient-based studies of HIV/AIDS patients; and

• Providing training to investigators In a wide range of cellular and molecular assays used in the study of HIV pathogenesis.

• Maintaining a central repository of molecular clones of infectious HIV, viral isolates, reporter HIV clones and HIV reporter cells lines as well as HIV-based expression vectors.

Virology Core Training ProgramsMeasurement of p24 antigen by ELISA Co-culture, quantification and sequence analysis of HIV

from PBMCs Determining the viral titers, infectious units (i.u.) and

m.o.i. of HIV using reporter cell linesReal-time PCR for quantification of HIV RNA and

cellular gene expressionApplication of advanced virological techniques to

analyze all stages of HIV-1 replication-including entry, reverse transcription, nuclear localization, integration, transcription, assembly and particle production.

Virological Core Services• HIV culture and isolation • HIV p24 antigen capture assay • Repository of titered primary HIV isolates • HIV-1 neutralization assays • Quantitative HIV-1 DNA and RNA measurements• HIV and cellular RNA quantification by real-time qPCR• Determination of viral tropism. • Reporter virus entry assay. • HIV sequence analysis. • Patient sample processing and storage.• Immunosorting of control and HIV-infected cellular

subpopulations. • Measurement of serum HCV antibody and plasma HCV virus

levels.  

Molecular Biological Core Services• Routine

o Custom vector and plasmid design and construction o Provision and expression of reporter and full-length molecular

clones of viruses of many cladeso Site-directed mutagenesiso Large-scale expression of recombinant HIV, human or other

pathogen proteins

• Advanced  o Lentiviral vector construction and preparation of virus

particles o RNA interference analysis o Genetic and biochemical systems to study protein-protein

interactions o Chromatin immunoprecipitation (ChIP) techniques o FRET-based BLam assay (beta-Lactamase based assay) o Heteroduplex mobility assay (HMA)

• The core (in collaboration with ATRP program) purchased a new Applied Biosystem qPCR machine to facilitate the demand for use of this assay in studying HIV-1 infection.

• Core is developing methods to carry out microRNA analysis in HIV-1 infected samples.

• In collaboration with Epigenomics and Genomics Center, the core is planning to optimize the methods for RNA/microRNA-seq, ChIP-seq and Affymatrix techniques. Consultation for the developing and carrying out these techniques will be provided for CFAR users.

Expansion of Core Activities and Innovation

Expansion of Core Activities and Innovation

M200spSample preperation

station

M2000rtRT-PCR machine

In collaboration with Bronx WHIS (PI: Dr. Anastos) and the Einstein’s Microbicide program (PI: Dr. Herold), CFAR has

purchased an Abbott m2000 machine

Expansion of Core Activities and Innovation

• Abbot m2000 Provides automation for sample preparation and real time PCR.

• Ideal for using a variety of clinical samples and allows the use of bar coded tubes, creates an efficient workflow, minimizes errors and contamination.

• Barcoded Laboratory Tubes, Precision Pipetting-no manual mixing or manipulation, Open Mode-Flexible protocol for various sample types and volumes

• Efficient Sample Extraction--Flexible throughput options of 24 to 96 samples—allows isolation of nucleic acids, RNA or DNA from variety of samples (serum, plasma) based on the magnetic bead method.

• Samples are prepared automatically and loaded onto 96 well plates and sealed for transfer to PCR machine.

Expansion of Core Activities and Innovation --- continued

• Abbot m2000 currently being used for HIV-1 viral load assays (range 40 to 10,000,000 copies/mL).

• Can be used for other clinical applications.Other viral loads—HCV, HBV

Detection of cellular transcripts-biomarkers, depending on the availability of the kit to use in the Abbott machine.

Expansion of Core Activities and Innovation --- continued

Technical Innovations—Alpha Technology

AlphaLISA-instead of p24 ELISAVarious enzymatic- Kinase AssaysProtein-protein interactions

A reduced number of assay steps---no washing steps.

Automated---good for adopting to HTS Versatile—measure large biomolecules (up to 2000 kDa) or weak interactions (mM).

Proposed Activities forNext Project Period

• The Core will expand its sequencing activities for molecular characterization of clinical isolates

• Expansion of virological support for WIHS studies

• Expansion of support to carry out epigenetic (methylation and ChIP assays) and microRNA analysis during HIV-1 pathogenesis

• Expand service to include support of protein production

Accomplishments

• Over the past 5 years, we have contributed to data in 31 publications

• In the past year we have supported studies by 32 NlH-funded investigators.

• We have expanded the Core services In response to the needs of Investigators including developing highly cost-effective lab-developed assays.