Long-Term Control of HIV by CCR5 Delta32/Delta 32 Stem-Cell Transplantation

1. Long-Term Control of HIV by CCR5 Delta32/Delta 32 Stem-Cell Transplantation Hutter, G., Nowak, D., Mossner, M., Ganepola, S., Mussig, A., Allers, K., et al (2009). Long-Term Control of HIV by CCR5 Delta32/Delta32 Stem-Cell Transplantation. The New England Journal of Medicine, 360;7 692-698. Presented by Nathaniel Dusto & Katie Plunkett

2. Background Information: Origin • Non-human primates in West-central Africa in the early 20th century via zoonosis • Simian immunodeficiency disease (SIV) undergoes several mutations  HIV if several rapid successive transmissions • Humans involved in bushmeat activities acquire SIV • Unsafe medical practices in Africa following WWII • Unsterile syringes during mass vaccination and anti-malaria • Colonization of Africa coincides with emergence of HIV epidemic • Social changes increased sexual promiscuity, spread of prostitution, increased syphilis or other genital ulcers

3. Background Information: HIV in the U.S. • 1969 HIV introduced by single Haitian immigrant • Throughout 1970’s-80’s misdiagnosed as Kaposi’s sarcoma, pneumocystis pneumonia, etc. • 1981- diagnosed as HIV/AIDS • HIV precursor to AIDS- CD4+ T cell count below 200 cells per µL • 1998 Bragdon v. Abbott U.S. Supreme Court - infection with HIV constitutes a disability (Americans with Disabilities Act 1990)

4. Background: HIV in the U.S. Pandemic- as of 2010 approximately 34 million people infected worldwide 2010- African Americans have highest proportion of AIDS diagnoses in all regions except the West, where Caucasians account for the highest proportion of diagnoses. Most common in urban populations

5. Background: HIV Viron • RNA retrovirus • Targets CD4+ helper T lymphocytes, macrophages and dendritic cells • Two strains: HIV-1 & HIV-2 • Transmitted via blood, semen, vaginal secretions, and breast milk • Surrounded by lipid based envelope derived from the host-cell membrane • Contains virally encoded proteins gp120 and gp41 • Nucleocapsid contains: • RNA genome • Integrase • Reverse transcriptase • Protease

6. Background: HIV Mode of Infection • Gp 120 envelope extracellular glycoprotein binds host CD4-surface protein and CCR5 or CXCR4 co-receptors • Gp 41 envelope transcellular glycoprotein inserts hydrophobic terminus into host cell membrane • Viron fuses with host cell releasing contents of nucleocapsid

7. Background: Intracellular Mode of Infection • Reverse transcriptase copies viral RNA into ds cDNA • Integrase cleaves 3’ ends of host DNA and interates cDNA into genome • Host cell undergoes transcriptionmRNA leaves nucleus • Viral mRNA is translated and Protease cleaves these proteins, which are then reconstructed • Envelope proteins travel to host cell plasma membrane • Viral genome and other proteins form nucleocapsid • New virus particles bud from cell  exocytose

8. Background: Acute Myeloid Leukemia • Patient: 40 year old, Caucasian male • M4 variant: Cancer of myeloblasts and monoblasts which are progenitor cells to granulocytes and agranulocytes • Symptoms: fever, fatigue, and easy bruising or bleeding • Non-functional cells build-up in the bone marrow and blood  infection, anemia, and hemorrhaging • Four standard treatments : chemotherapy, radiation therapy, stem cell transplant, and other drug therapies (all-trans retinoic acid)

9. Background: Genetic Mutations and HIV Immunity CCR5 co-receptor CXCR4 co-receptor • Required for macrophage-tropic HIV variants • Mutated CCR5 gene with 32-nucleotide deletion from coding regionnon-functional protein • CCR5-delta 32 only present in Caucasians • 10% heterozygous • 1% homozygous • Required for lymphocyte-tropic HIV variants • Infect and destroy activated CD4 T cells • CD4 T cell count less than 200 cells/mm3 or less than 15% indicates disease has progressed to AIDS

10. Purpose and Goals of the Study • Treat 40-year old Caucasian man with newly diagnosed acute myeloid leukemia and pre-existing 10 year HIV-1 infection • Utilize allogenic stem-cell transplantation (SCT) from HLA-matched donor to treat leukemia • Select for SCT donor with homozygosity for CCR5-delta 32 genetic deletion variant • Demonstrate role of CCR5 in HIV-1 infection • Analyze stem cell transplantation as a treatment for HIV-1 infection

11. Materials & Methods • CCR5 Genotyping of patient and potential donors • BigDye Sequencing – improved efficiency over Sanger • Polymerase Chain Reaction (PCR) • Amplify CCR5 DNA from peripheral-blood monocytes, Electrophoresis, Luminescent staining • Amplify HIV-1 RNA in peripheral blood of the patient before and after chimerism

12. Materials & Methods • Immunospot Assay • Similar to an ELISA • Wells coated with anti-IFNγ antibody • Monocytes were incubated with CMV or HIV peptides • Monocytes present antigen to T- cells • Activated T-cells release IFNγ that binds to anti-IFNγ antibody • A biotinylated IFNγ-recognizing antibody was then added, and luminescence was measured. Each IFNγ antibody complex is one spot • Spots produced in antigen-stimulated wells is normalized to controls to account for non-specific IFNγ release

13. Materials and Methods • Immunoblotting • Wells plated with HIV-1 envelope, polymerase, capsid, and HIV-2 envelope proteins • Levels of antibodies against these antigens were labeled and quantified • Flow Cytometry • Mucosal cells from rectal biopsy stimulated to produce CCR5 by phytohemaglutinin • CD3, CD4, CCR5, CD11c, and CD163 all tagged with different colors • Cells focused into a stream and passed through several lasers • Characteristic light scattering and alterations in light wavelength allow identification and quantification of target molecules • Cells expressing sufficient levels of CD3 and CD4 are determined to be T cells, and this population was then analyzed for CCR5 expression • Cells expressing sufficient levels of CD4 were then analyzed for CD163 and CD11c, identifying them as macrophages • These cells were then analyzed for CCR5 expression

14. Results – Figure 1 • Before SCT, the patient was heterozygous for CCR5 • 61 days following SCT, patient is homozygous for CCR5Δ32 • Complete chimerism was attained

15. Figure 2 A • Following SCT, The patients T cells have lost HIV-1 specific reactivity • This is not due to an ablated immune system however, because CMV specific T cells are present

16. Figure 2 B • Following SCT, the patient had reduced expression antibodies against HIV-1 polymerase and capsid proteins • Antibodies against envelope proteins not reduced

17. Figure 3

18. Figure 4 A • Intestinal CD4 T cells do not express CCR5 159 days after SCT • Indicates no T cells remaining from before engraftment

19. Figure 4 B • 14.6% of intestinal macrophages express CCR5 159 after SCT • This is most likely due to macrophages that have not been broken down and replaced with the new immune system Could indicate an HIV reservoir

20. Conclusions & Significance: • The role of CCR5 co-receptor is vital to maintaining HIV infection and disease progression. • Based on overwhelmingly positive results of this case study, further investigation of CCR5 targeted HIV treatments should be explored.

21. References: • BRAGDON v. ABBOTT. The Oyez Project at IIT Chicago-Kent College of Law. 23 January 2013. <http://www.oyez.org/cases/1990-1999/1997/1997_97_156>. • CDC. HIV Surveillance Report, 2010; vol 22. http://www.cdc.gov/hiv/resources/factsheets/geo-bibliography.htm Published March 2012. Accessed May 25, 2012. • Parham, P. (2009). The immune system. (3rd ed.). New York, NY: Garland Science, Taylor & Francis Group, LLC, an informa business. • National Cancer Institute at the National Institute of Health. (2013, 01 24). General information about acute myeloid leukemia. Retrieved from http://www.cancer.gov/cancertopics/pdq/treatment/adultAML/Patient/page1 • Ganepola, S., Gentilini, C., Hilbers, U., Lange, T., Rieger, K., Hofmann, J., Maier, M., Liebert, U. G., Niederwieser, D., Englemann, E., Heilbronn, R., Thiel, E., Uharek, L. (2007). Patients at high risk for CMV infection and disease show delayed CD8 + T-cell immune recovery after allogenic stem cell transplantation. Bone Marrow Transplantation, 39, 293-299. • Blau, I. W., Schmidt-Hieber, M., Leschinger, N., Goldner, H., Knauf, W., Hopfenmuller, W., Thiel, E., Blau, O. (2007). Engraftment kinetics and hematopoietic chimerism after reduced-intensity conditioning with fludarabine and treosulfan before allogenic stem cell transplantation. Annals of Hematology, 86, 583-589. doi 10.1007/s00277-007-0294-6. • Rowlings, P. A., Przepiorka, D., Klein, J. P., Gale, R. P., Passweg, J. R., Henslee-Downey, P. J., Cahn, J., Calderwood, S., Gratwohl, A., Socie, G., Abecasis, M. M. (1997). IBMTR Severity Index for grading acute graft-versus-host disease: retrospective comparison with Glucksberg grade. British Journal of Haematology, 97, 855-864. • Freed, E. O. (1998). HIV-1 Gag Proteins: Diverse Functions in the Virus Life Cycle. Virology, 251, 1-15. • Hutter, G., Nowak, D., Mossner, M., Ganepola, S., Mussig, A., Allers, K., Schneider, T., Hofmann, J., Kucherer, C., Blau, O., Blau, I. W., Hofmann, W. K., Thiel, E. (2009). Long-Term Control of HIV by CCR5 Delta32/Delta32 Stem-Cell Transplantation. The New England Journal of Medicine, 360;7 692-698.