We provide the HIV/AIDS research community with distinct biological research products not readily available from other sources. 

The Biological Products Core makes these products available to Frederick National Laboratory for Cancer Research, intramural, and extramural investigators at no cost. Extramural laboratories must establish a Material Transfer Agreement between their requesting institution and the National Cancer Institute prior to receiving products. 

Key among these research products are purified retrovirus preparations, such as HIV and simian immunodeficiency virus (SIV). These can be provided in infectious, chemically inactivated, and genetically replication-incompetent forms.   

We also produce and purify control microvesicle preparations, produce transfection-derived infectious stocks, produce and purify monoclonal antibodies, and produce HIV p24ca antigen capture immunoassay key reagents developed by the AIDS and Cancer Virus Program. 

Purified preparations of native virions crucial to HIV/AIDS mitigation and basic research 

Early in the HIV/AIDS pandemic, our laboratory provided the large quantity of purified HIV that others needed to develop initial immunoassays for screening donated blood for the presence of antibodies against HIV so contaminated units would not be used for transfusion. Recently, our purified virus preparations enabled a wide range of basic research, including demonstration that cell-free HIV virions cross the blood-brain barrier in vivo, elucidation of the structure of an entire HIV RNA genome at single-nucleotide resolution, determination of the complete glycan composition of virion-derived retroviral SU protein, and determination of the in situ 3D structure of retroviral heterotrimeric envelope protein by cryo-EM.   

Establishing the biochemical and biophysical properties of native viral proteins and the viral genome is a key component of elucidating the basic biology of HIV. Information about the nature of the HIV envelope protein provides a framework for identifying potential viral envelope protein targets for antibody-mediated viral neutralization, offers detailed information about how the SU protein glycan shield contributes to shielding the virus from antibody-mediated neutralization, and provides a basis for the development of recombinant proteins for use as experimental vaccine immunogens or in structural studies. 

Collaboration Opportunities

We provide several services for investigators, including the production and purification of virus materials, development of infected cell lines, and provisioning of key reagents. Email Julian Bess for requests.

Extramural laboratories can obtain our research products through a Material Transfer Agreement between the requesting institution and the National Cancer Institute. Visit the NCI Technology Transfer Center for more information.

Incubator and roller rack used to propagate cell cultures in roller bottles
Large quantity production

HIV, SIV with high SU protein content

We were the first laboratory to derive a cell line that produced HIV with high SU protein content. Analyzed purified virus from the cell line produced the first cryo-EM images of the trimeric envelope protein spikes. Since then, we’ve developed additional cell lines and also limiting dilution clones that produce either HIV or SIV with high SU protein content. Our program environment makes us well-positioned to perform this key scope of work as we collaborate with other sections and cores within the AIDS and Cancer Virus Program for FACS analysis of the cell lines, biochemical analysis of the purified virus preparations, and provirus sequencing.
What is this? Incubator and roller rack used to propagate cell cultures in roller bottles
FPLC system used for the purification of monoclonal antibodies and cytokines

Development of clonal cell lines producing replication-incompetent HIV

Many laboratories are either not permitted to work with infectious virus or have equipment that can't be used to analyze infectious virus safely. To address these problems for the research community, we used CRISPR/Cas-9 gene-editing methods to knock out the proviral integrase gene in one of our clonal cell lines expressing HIV. After editing, we derived several limiting dilution clones in which the proviral integrase gene was essentially eliminated while progeny virus yield and SU protein content remained high. Virions from these cell lines were demonstrated to be replication incompetent, and we’ve begun producing purified, concentrated virus preparations from them for use by the research community.
What is this? FPLC system used for the purification of monoclonal antibodies and cytokines

Our capabilities and specializations

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Production and purification of HIV, SIV, or control microvesicles 

Continuous T-cell lines used to derive virus are typically cultured in T-flasks or roller bottles. The cells are removed by centrifugation, and the virus-containing supernatant is filtered through a 0.45 µm membrane. Virus is purified from the filtrate by centrifugation through a 20% sucrose pad. This method provides virus of at least equivalent purity to that purified by sucrose density gradient centrifugation. Control microvesicles are prepared using the same method but omitting the filtration step. 

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  • Batch sizes of 30 mL to 6 L 

  • Have in infectious (non-treated); chemically inactivated (typically Aldrithiol-2 treatment); or, in one case, genetically replication-incompetent (provirus integrase gene knockout) form 

  • Virus preparations provide substantial added value due to extensive analyses performed on each lot 

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Development of productively infected cell lines or cell clones 

The quality of our purified virus preparations is principally due to the utilization of selected cell lines derived by our laboratory. Development of these cell lines is a laborious, time-consuming process. 

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  • Usually infect a SUP-T1-based cell line with a virus infectious stock or infectious molecular clone (preferred) and derive a stable, productively infected cell line 

  • Prepare limiting dilution clone(s), if needed 

  • Prepare viably frozen cell stocks 

  • Test the cell line(s) for contamination by adventitious agents: bacteria, mold, or mycoplasma 

  • Produce and analyze purified virus preparation(s) to determine virus yield and biochemical phenotype 

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Provisioning of key reagents required for our in-house-developed HIV-1 p24CA antigen capture assay 

We provide qualified key reagents prepared in our laboratory and instructions describing how they can be used by recipient laboratories to set up their own assays to determine the concentration of HIV p24 in their samples. The concentration of p24 is typically used as a surrogate for the concentration of HIV present while noting that low-speed culture supernatants can contain non-virion-associated p24 in addition to virion-associated p24. 

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  • Capture mAb: Purified mouse anti-HIV p24 

  • Primary Ab: Rabbit anti-HIV p24 polyclonal antibody 

  • Standard: Detergent-disrupted HIV preparation with known p24 concentration