Long-Term Control of HIV by CCR5 Delta32/Delta 32 Stem
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Transcript Long-Term Control of HIV by CCR5 Delta32/Delta 32 Stem
Hutter, G., Nowak, D., Mossner, M., Ganepola, S., Mussig, A., Allers, K., et al (2009). LongTerm 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: 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
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 hostcell membrane
Contains virally encoded
proteins gp120 and gp41
Nucleocapsid contains:
RNA genome
Integrase
Reverse transcriptase
Protease
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
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 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)
Background: Genetic Mutations and HIV
Immunity
CCR5 co-receptor
CXCR4 co-receptor
Required for macrophage-tropic
HIV variants
Required for lymphocyte-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
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 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
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
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
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 1
Before SCT, the
patient was
heterozygous for
CCR5
61 days following
SCT, patient is
homozygous for
CCR5Δ32
Complete
chimerism was
attained
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
Figure 2 B
Following SCT, the
patient had reduced
expression antibodies
against HIV-1
polymerase and capsid
proteins
Antibodies against
envelope proteins not
reduced
Figure 3
Figure 4 A
Intestinal CD4 T cells do not express CCR5 159 days
after SCT
Indicates no T cells remaining from before
engraftment
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
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:
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National Cancer Institute at the National Institute of Health. (2013, 01 24). General information about acute
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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.