The Tissue Analysis and Retroviral Protein Chemistry Core provides state-of-the-art tissue analysis capabilities, including immunofluorescence, single- and multi-label immunohistochemistry, singleplex and multiplex in situ hybridization, ultra-high-plex spatial stain, quantitative image analysis, laser-capture microdissection, antibody conjugation, and characterization. 

The issue is in the tissue 

HIV infection and persistence occurs in the tissues. Therefore, localizing, identifying, and quantifying the virus present within tissues is key to better understanding the infection and developing appropriate interventions.

The core supports investigators within the AIDS and Cancer Virus Program and both intramural and extramural collaborating investigators who are working on studies relevant to major questions in HIV/AIDS research and in which tissue-based analyses provide a critical enabling aspect of the research. 

HIV and more 

Adapting workflow and assays to meet the needs of principal investigators, we provide expertise for analysis of specimens from human and animal model studies, with emphasis on specimens from nonhuman primates infected with a simian immunodeficiency virus (SIV).

Studies supported by the core are aimed at better understanding HIV/SIV mucosal transmission, reservoir biology, pathogenesis, and therapeutic intervention strategies.

The core adapted standard operating procedures developed for HIV/SIV to other infectious agents, such as SARS-CoV-2, Zika, MNPXV, and gammaherpesviruses.  

Collaboration Opportunities

We work with investigators through various partnership mechanisms, including Material Transfer Agreements (MTAs), FNL Cooperative Research and Development Agreements (FNL CRADAs), Technical Service Agreements (TSAs), and Collaboration Agreements. 

Contact Claire Deleage to inquire about and engage the core’s services. 

RNAscope+ FUSION
Work in progress

In depth characterization of infected cells and cellular reservoirs in tissues

In pursuing an HIV cure, the characterization of cells harboring viral RNA (vRNA) and viral DNA (vDNA) is fundamental. We are currently combining two state-of-the-art assays, RNAscope and Pheno-Cycler Fusion, to allow characterization of the cellular reservoir in any tissues while preserving the microenvironment of the organ.
What is this? Merging technologies. RNAscope+ FUSION
Information
HIV viral persistence

CTLA-4+PD-1− memory CD4+ T cells previously unrecognized component of SIV, HIV

CTLA-4 cells predict size of viral reservoir during ART and may represent a therapeutic target for functional HIV-1 cure.
Donor aspect of sexual transmission

Macrophages: Major population of infected cells in semen

High levels of virus and infected cells in macaque semen during primary infection may contribute to SIV transmission.
Human skin biopsy showing MPXV in red, DC-SIGN in blue and Cytokeratin in green in a hair follicle.
RNASCOPE

Prolonged Mpox disease in people with advanced HIV calls for aggressive treatment and immune optimization

Immunofluorescence of skin biopsies demonstrated a dense immune infiltrate predominantly composed of myeloid and CD8+ T cells, with a strong type I interferon local response. RNAscope detected large amounts of MPXV virus in epithelial cells (epithelium and hair follicles) and dendritic cells.
Read the study
What is this? Human skin biopsy showing MPXV in red, DC-SIGN in blue and Cytokeratin in green in a hair follicle.
Information

Our capabilities and specializations

LN of acutely infected RM
LN of acutely infected RM: RNAscope combined with immunofluorescence (vRNA [red], CD4 [green], myeloid cells [blue]).

RNA/DNAscope  

Highly sensitive, next-generation in situ hybridization strategies are designed to detect viral and cellular RNA and DNA. Extremely sensitive (down to 2 copies) and remarkably specific, the approach uses powerful Z pairs technology to initiate amplification of the signal. 

  • Enables detection of single virions, productively infected cells, and vDNA+ cells in situ. Gives location of the cells harboring vRNA and/or vDNA in tissues 

  • Combined with IFA, enables phenotyping of cells harboring viral genome 

  • Due to high sensitivity, can assess effect of interventions on active replication and/or reservoir population 

Pheno-Cycler Fusion assay on acutely infected RM- representative image of lymph node with 26 different cell markers on one tissue section.
Pheno-Cycler Fusion assay on acutely infected RM: representative image of lymph node with 26 different cell markers on one tissue section. 

Pheno-cycler fusion 

A new ultra-high-plex spatial phenotyping assay is designed to stain for up to 50 markers on a formalin-fixed and paraffin-embedded (FFPE) tissue section. The approach uses barcoded antibody, allowing for ultra-high-plex spatial staining. 

  • Spatial phenotyping enables investigation of impact of infection or any intervention on entire microenvironment of tissues 

  • Tool evaluates state of activation, inflammation, and functionality of each cell within a tissue without disrupting spatial relationships of cells within tissues 

  • Unique tools to investigate cell neighborhood and relationships 

LCM area cut
Laser-capture microdissection of an area cut.

Laser-capture microdissection 

LCM is a method to capture individual cells or areas within FFPE or frozen tissues, then extract RNA or DNA for further investigations.   

  • Capture specific cells of interest (infected cells, CD4+ T cells, myeloid cells) or cut specific area of tissue (B-cell follicles in a lymph node or lymphoid aggregates in the GI tract) 

  • Extract RNA or DNA, allowing many different downstream applications, including proportion of population of variants, RNA transcript profiling, and cDNA library generation