Preventing the Spread of HIV by Vector-Encoded Antibody

Preventing the Spread of
HIV by Vector-Encoded
Antibody
Cancer Research in the 21st Century
David Baltimore
UCL Cancer
Institute
Dedication
K/RITH
Opening
18Oct,
Sept2012
07
11
David Baltimore
A 30-Year-Old Challenge: Making A
Vaccine Against HIV
Because the virus takes years, if ever, to elicit
from a patient’s body antibodies that might
stop the infection– and then it is too late—
and T cells do not sufficiently control the virus,
making a vaccine has been a daunting task.
The Big Advance
• Even though they do not protect the person from
whom they were isolated, broadly neutralizing antiHIV human monoclonal Abs have been found,
isolated and studied in detail
– Some are very broad and quite potent and more
coming weekly
• So the issue becomes, how do we build on this
demonstration that the human repertoire can make
a potentially prophylactic antibody?
• Remember the antibodies:
Broadly Neutralizing Antibodies Against Env
PG9, PG16, PGT121,
PGT128 (Variable Loops
1,2,3)
b12, VRC01, NIH45-46
(CD4 Binding Site)
2G12
(Glycanbinding)
2F5, 4E10
(Membrane
Proximal)
Antibodies from the VRC (Nabel), IAVI (Burton), Rockefeller (Nussenzweig)
How can we use these Abs to provide
anti-HIV prophylaxis?
• We could try to elicit them
• We could make them in large amounts and
inject them
• We could use viral vectors expressing the
appropriate genes to program the body to make
the antibodies
• We call this VIP (Vectored ImmunoProphylaxis)
AAV Mediated Delivery of Neutralizing
Antibodies as Prophylaxis Against HIV:
Vectored ImmunoProphylaxis (VIP)
Alex Balazs
Engineering Ab Production using AAV8
o Positive Characteristics of
Adeno-Associated Virus
o Non-pathogenic in humans
o Non-integrating DNA virus
20nm
giving long-lived expression
following a single administration
o Excellent expression ability in vivo in animals
and suggestive data in humans
o Grown to high titer by transient transfection
o Negative characteristic: very small (5 kB)
HJ Nam et al.
Our AAV Transfer Vector
2-5x
Luciferase
CASI
SV40pA
WPRE
10x
oPromoter
oPolyadenylation Signal
oAdditional Post-transcriptional regulatory elements (WPRE)
ITR
CASI Prom.
IgG1 HC
2A
IgG1 LC
AAV IgG Expression Vector - 4.5 kb
WPRE
SV40pA
ITR
AAV2/8-Mediated Luciferase Expression
Intramuscular Injection
Intravenous Injection
107
108
109
Photons/sec/cm2
1010
AAV2/8 Mediates Long-Term Gene Expression in
Immunodefficient (Rag2/γc-/-) Mice
107
108
109
Photons/sec/cm2
1010
NOD/SCID/γ-/- HuPBMC Model as a Model of CD4 Depletion
During HIV Infection
Transfer Human PBMCs
2 Wks
Challenge with HIV
Sample Weekly
Experimental Design to Test
Antibodies In Vivo
• Administer antibody expression vectors or control
• Humanize immunodeficient mice by engrafting with PBMCs
• Challenge with HIV
• Quantify CD4/CD8 ratios over time
Inject
AAV
D-35
Inject
HuPBMC
HIV
Challenge
IV or IP
-21
0
Expand PBMC 13 Days
D-33
-21
Blood Sampling
7
14
21
28 35 42 49 56
b12 Expressed from CASI Driven AAV Vector Protects Animals
from CD4 Loss Following Intraperitoneal HIV Challenge
Histology of NL4-3 Challenged Humanized Mice
Luciferase
B12
o No evidence of p24+ cells in spleens of mice treated with B12
The Recently Identified VRC01 Antibody Neutralizes a Broad Array of
HIV Strains
Engrafted Human CD4 Cells are Protected by VRC01
N=8-12
Determination of the Minimum Protective
VRC01 Dose In Vivo
• 8.3 µg/mL of VRC01 was sufficient to protect
• 1.6 µg/mL of VRC01 was not sufficient to protect
• The intravenous challenge dose represents
approximately 1x108 physical particles of CXCR4tropic NL4-3
Open Questions
• Will VIP work against R5 virus?
• Will VIP work against founder strains?
• Will VIP work against a mucosal challenge?
VRC01 Protects Against Infection by an R5 Transmitted
Founder HIV Strain (REJO.c)
REJO.c
NL43
Luciferase
b12
VRC01
To examine protection against
mucosal infection we went to a
different model, the BLT mouse
[Mouse with a fetal human liver/
thymus graft populated with
CD34+ hematopoietic stem cells]
Comparison of NSG-HuPBMC and BLT Mouse Models
NSG-HuPBMC
BLT
~ 8 Weeks
~ 24 Weeks
Engrafted lineages
T-Cells
All Lineages
Host tissue engraftment
Limited
Extensive
Non-functional
Some function
Thousands
20-30 Maximum
Difficulty of production
Low
High
Cost per animal
Low
High
Duration of engraftment
Immune Function
Potential group size from single donor
Can VIP Protect a Model of Human-to-Human Transmission?
• Create BLT mice (Dong Song An, UCLA)
• Administer vector intramuscularly
• Challenge intravaginally with HIV repetitively
• Sample repetitively
Surgical
Implantation of
Bone-LiverThymus to
produce BLT
Animal
Inject
AAV
-70
-28
Repetitive “Low-Dose” Intravaginal Challenge with 50ng JR-CSF
Harvest
Tissue
0 7 14 21 28 35 42 49 56 63 70 77 84 91 98105
Blood Sampling for 15 Weeks
VRC01 Antibody is Detected in Vaginal Secretions
Serum
Detection Limit = 70ng/mL
Vaginal Wash
Detection Limit = 1.3ng/mL
Can this level of antibody protect mice from mucosal
challenge?
VRC01 Protects Against Loss of CD4 Cells in BLT Mice
Luciferase
VRC01
VRC01 Protects Against Mucosal HIV Transmission
Splenic
Lymphocytes
Vaginal
Lamina
Propria
Lymphocytes
Plasma
Viral Load
Detection Limit = 200 Copies/mL
VRC01 Reduces Risk of HIV Transmission Through Mucosal Route
Detection Limit = 1500/mL
VRC01 Reduces Risk of HIV
Transmission Through
Mucosal Route
Can VIP Protect Against a Transmitted Founder Strain?
• Create BLT mice
• Administer vector expressing VRC07G54W antibody
• Challenge intravaginally with a Transmitted Founder
Strain of HIV (REJO.c)
Surgical
Implantation of
Bone-LiverThymus to
produce BLT Inject
AAV
Animal
-70
-28
Repetitive “Low-Dose” Intravaginal Challenge with REJO.c
0
Harvest
Tissue
7 14 21 28 35 42 49 56 63 70 77 84 81 88 95 102109116 123130
Blood Sampling for 20 Weeks
VRC07G54W Antibody is Detected in Vaginal Secretions
Serum
Detection Limit = 70ng/mL
Vaginal Wash
Detection Limit = 1.3ng/mL
VRC07G54W Protects Against Loss of CD4 Cells in BLT
Mice
Luciferase
VRC07G54W
VRC01 Protects Against Mucosal HIV Transmission
Gut IntraSplenic
Epithelial
Lymphocytes Lymphocytes
Gut Lamina
Propria
Lymphocytes
Vaginal
Lamina
Propria
Lymphocytes
VIP-Treated Mice Show No Detectable HIV Despite Extensive Mucosal
Challenge
Plasma
Viral Load
Detection Limit = 200 Copies /mL
Conclusions
• Vectored Immunoprophylaxis is capable of protecting
animals from X4 and R5 strains, including a
transmitted molecular founder strain.
• This approach protects humanized mice from
repeated muscosal challenge.
Transitioning to Clinical Development
• Today we know enough to be confident that if
humans act like mice we can protect humans
against HIV infection
• To test human responses we have teamed up
with the Gary Nabel and his team at the VRC at
NIH to run clinical trials
• First need to ask if humans make prophylactic
levels of Ab
• Need to carefully investigate safety (therapeutic
antibodies have proved quite safe)
VIP
• VIP was developed to prevent HIV infection
and does this effectively in humanized mice
• VIP could have wider applicability, for instance
in preventing the spread of influenza virus
• It is relatively cheap and quite stable making it
appropriate for use in the developing world as
well as stock-piling against natural or
bioterrorist-mediated viral diseases
People Who Did This Work
• Alex Balazs with the help of Joyce Chen,
Christin Hoag and Stella Ouyang
• Lili Yang, project manager of Engineering
Immunity Program
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