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

Background Information: OriginNon-human primates in West-central Africa in

the early 20th century via zoonosisSimian 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 WWIIUnsterile syringes during mass vaccination and

anti-malariaColonization of Africa coincides with emergence

of HIV epidemicSocial changes increased sexual promiscuity,

spread of prostitution, increased syphilis or other genital ulcers

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)

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

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

Background: HIV Mode of InfectionGp 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

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 transcriptionmRNA 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

Background: Acute Myeloid LeukemiaPatient: 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)

Background: Genetic Mutations and HIV ImmunityCCR5 co-receptor CXCR4 co-receptorRequired for macrophage-

tropic HIV variants

Mutated CCR5 gene with 32-nucleotide deletion from coding regionnon-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

Purpose and Goals of the StudyTreat 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

Materials & MethodsCCR5 Genotyping of patient and potential

donorsBigDye Sequencing – improved efficiency over

SangerPolymerase 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

Materials & MethodsImmunospot Assay

Similar to an ELISAWells coated with anti-IFNγ antibodyMonocytes were incubated with CMV or HIV peptidesMonocytes present antigen to T- cells Activated T-cells release IFNγ that binds to anti-IFNγ

antibodyA 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

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

Results – Figure 1Before SCT, the

patient was heterozygous for CCR5

61 days following SCT, patient is homozygous for CCR5Δ32

Complete chimerism was attained

Figure 2 AFollowing 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

Figure 2 BFollowing SCT, the

patient had reduced expression antibodies against HIV-1 polymerase and capsid proteins

Antibodies against envelope proteins not reduced

Figure 3

Figure 4 AIntestinal CD4 T cells do not express CCR5

159 days after SCTIndicates no T cells remaining from before

engraftment

Figure 4 B14.6% of intestinal macrophages express

CCR5 159 after SCTThis is most likely due to macrophages that

have not been broken down and replaced with the new immune systemCould indicate an HIV reservoir

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.

References: BRAGDON v. ABBOTT. The Oyez Project at IIT Chicago-Kent College of Law. 23 January 2013.

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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.