Lessons Learned from the Humanized Mouse

Use of humanized mouse model as a translational medicine tool
Past successes:
1. Pathogenesis studies
2. Drug screening
3. Gene therapy
Future aims:
1. Gene therapy
2. Vaccine development
3. Immune reconstitution/enhancement
4. Viral transmission
5. Viral latency/ eradication
6. Organ regeneration/replacement
7. Novel therapeutic development

SCID, NSG
Rag2-/-
Black 6 SCID

• First humanized mouse used in HIV studies.
• Involves injecting human Peripheral Blood
Lymphocytes into the peritoneum of SCID mice. Cells are typically removed from mice by intraperitoneal lavage.
• Key Strength: can assess effects rapidly and directly on mature human blood cells in vivo.
• Key Weaknesses: limited number of time points, short experimental duration (<3-4 weeks).

Typical Experiment: hu-PBL SCID Mouse Model
7 -14 days 7 -21 days
Typically there are 6-30 mice per experiment. The limiting factor in these studies is the number of human cells. You need >2 x 10 7 human cells/mouse.
Lavage and
Assessment:
Real Time
Quantitative PCR

Analysis of radio-labeled HIV specific antibody
213 Bismuth
HIV Infected
213 Bismuth
HIV Infected

213 Bismuth
213 Bismuth

Experimental Results
HIV DNA Viral Load
120
100
80
60
40
20
0
Saline n = 5 mice/group
Cold antigp41 non-specific
50 uCi anti-gp41
50 uCi non-specific
100 uCi
Treatment anti-gp41
100 uCi non-specific
200 uCi anti-gp41
200 uCi
Dadachova et al. PLoS One 2012

Bismuth213- labeled neutralizing anti-gp41 monoclonal antibody targets and eliminates HIV infected cells in vivo.
Platelet counts and Pathology similar in all experimental conditions:
Low toxicity of radioactive compound
Eliminates productively infected cells.
Decreases Viral DNA load.

• Developed at Stanford, the SCID-human fetal thymus and liver model.
• Involves transplanting human fetal thymus and liver in SCID mice. Cells or HIV is then injected into tissue. Tissue is obtained by biopsy.
• Key Strengths: can assess viral infection of human T cells in human tissue in vivo, system to study T cell development.
• Key Weaknesses: surgery required, technically complex.

• Established in the late 1980 ’ s/early 1990 ’ s as a model system to study HIV pathogenesis in vivo.
• Played a key role in studies on:
– HIV Pathogenesis
– Gene Therapy
– HIV Latency
– Embryonic Stem Cell Development
– Engineering T cell Immunity

How does HIV infection perturb T cell development?

Thymopoiesis
Thymus
CD4 SP
Quiescent
Peripheral
Circulation
CD4 + /
CD45RA +
CD4
-
/ CD8
-
CD4
+
/ CD8
+
Transcriptionally Active
CD8 SP
Quiescent
CD8 + /
CD45RA +

The SCID-hu mouse model
Human fetal thymus
Human fetal liver
Thy/Liv implant
3-4 months
SCID-hu mouse
CD8

Typical Experiment: hu-PBL SCID Mouse Model
Biopsy and
Assessment
SCID-hu
3-22 weeks
SCID-hu
Tissue processing
Assay
1. PCR
2. Flow cytometry
Experiments typically consist of 6-30 mice, and have 3 time points. The limiting factor is the amount of fetal tissue.

Uninfected
HIV infected

HIV infection causes loss of immature thymocytes
What viral factors are involved in this process?

HIV Reporter Virus muHSA (CD24)

HSA Expression in Thymocytes
Jamieson et al.

Do high levels of HIV destroy the ability of thymic stroma to direct T cell differentiation?

Why is reconstitution of thymocytes transient?

Viral / Thymocyte Dynamics Following Antiretroviral Therapy

• Reconstitution of thymopoiesis is transient following HAART.
• The transiency is caused by breakthrough in viral replication to antiretroviral treatment.
• The SCID-hu thy/liv model is highly useful in examining HIV infection in the context of developing T cells.

Modeling HIV Latency

HIV-1
Thy/Liv
Implant
CD4 SP /HSA
-
4-6 weeks
Biopsy + Protease Inhibitor
CD8 SP /HSA
-

Latent HIV in Thymocytes from SCID-hu Mice
Day 3
Day 0
99% <1%
21%
MFI: 510
79%
CD45
+ Protease Inhibitor CD24
99% <1%
5%
MFI: 225
CD8
CD45
95%

The Search for Agents That Activate Latent HIV
Prostratin
Phorbol ester
Used in tea in Samoa to treat various illnesses
Activates latent virus without inducing T cell replication
Further testing is required to define effects on immune system
IL-7
Naturally occuring cytokine
Induces some cell proliferation, but phenotype is maintained
Potently induces expression of latent HIV
Further development is required

Anti-gp120
Anti-HIV Immunotoxin gp120
Infected Cell
3B3:N31H/Q100eY(dsFv)-PE
McHugh et al.; 2002
Pseudomonas
Exotoxin

Elimination of Latent HIV

I.
Immunotoxins can be used to kill cells induced to express previously latent virus
II.
Pre-treatment with IL-7 or with prostratin plus immunotoxin results in a decrease in rescuable latent virus upon subsequent co-stimulation.
III. These agents may prove useful as adjunctive therapeutics to purge the latent
HIV reservoir

Model #3:
The Non-obese diabetic (NOD), SCID, IL-2 receptor γ knockout (NSG), humanized bone marrow, fetal liver and thymus (BLT) mouse model
The NSG-BLT model
• Recently developed model, pioneered by J. Victor Garcia in Texas.
• Involves transplanting human fetal thymus and liver in NSG mice, the irradiating them and the injecting human stem cells intraveneously, which allow them to become engrafted in mouse bone marrow. Mice become engrafted with multiple human cell types that arise from stem cells within 6-8 weeks.
• HIV is then injected. HIV replication and peripheral blood cells are monitored following bleeding of the mice. Tissue is obtained by biopsy and/ or sacrificing mice.

Model #3:
The Non-obese diabetic, SCID, IL-2 receptor γ knockout (NSG), humanized bone marrow, fetal liver and thymus (BLT) mouse model
• Key Strengths: can assess viral infection of multiple types of human cells in vivo, slow and steady rate of T cell depletion and viral replication (mimics natural history in humans), easy to manipulate, develop immune responses.
• Key Weaknesses: surgery required, technically complex, immuodeficient status of mice make them highly susceptible to graft versus host disease, lower experimental numbers.

Humanized Mouse Model of HIV Infection:
The NSG-BLT Model
1. Implant fetal thymus and liver tissue.
Infect with
HIV-1 liv thy
NSG
3 weeks
Irradiate
6-12 weeks
NSG
4. Analyze human cell reconstitution
3. i.v. Inject
2. Sort CD34+ Stem Cells
CD34+
CD34+
CD34+
CD34+
CD34+
5. Analyze effects of infection
Each experiment typically contains 6-15 mice. Human cell reconstitution frequency is far lower than SCID –hu.

Multlineage Hematopoesis in
Humanized mice
Myeloid stem cell
CD34+
BM Stem cell
Lymphoid stem cell
Erythroid progenitor
Megakaryoblast
Eosinophil progenitor
Basophil progenitor
Myelomonocytic progenitor
B progenitor
T progenitor
Red blood cells
Megakaryocyte
Platelets
Monocyte
DP Thymocyte
NK Cell
Eosinophil Basophil Neutrophil Macrophage B cell
CD8 +
T cell
CD4 +
T cell

Multilineage Hematopoiesis in NSG-LTL mice
%CD45+ mean=53% ± 29% range 19%-80% n=12

HIV Infection of NSG-BLT mice
Mice were either
1. Untreated
2. HIV infected
3. HIV infected but treated with pre-exposure prophylaxis of emtricitabine (FTC)/ tenofovir disoproxil fumarate (TDF)
Conclusion: Pre-exposure prophylaxis
Prevents infection in this model
Denton et al.,PLoS Med. 2008 Jan 15;5(1)

Targeting immune responses could augment rejection of
Infectious agents (chronic viruses) or tumors in certain individuals or disease states
HIV disease: weakened immune system, viral drift
Cancer: Escape of tumor cells from immune surveillance
Transgenic mouse models suggest that introduction of antigen receptors into stem cells can result in functional effector cells targeting the antigen
Can this type of approach be done in humans?
Can we enhance immune capabilities in humans?

Killing of HIV
Infected Cells
HIV Infected
Cells
Stem Cell
Mature
T-Cells
HIV-Specific
T Cell
Expansion of
HIV-Specific
Cells
Incomplete
Clearance of
HIV Infected
Cells
Thymus Periphery

T Cell Recognition Of Virally-Infected Cell

Step 1:
Strategy for Cloning HIV-Specific T Cell Receptors
HIV Infected
Individual
T cell
T cell
T cell
T cell
T cell
T cell
T cell
T cell
T cell
T cell
T cell
T cell
T cell
T cell
T cell
T cell
1) Purify T Cells
2) Culture in Presence of
Known HIV peptide
TCR

TCR

IRES eGFP
3) Molecularly Clone
HIV-Specific
TCR

ES or iPS Cells
Myeloid stem cell
BM Stem cell
Class I Restricted
TCR Gene
Lymphoid stem cell
Erythroid progenitor
Megakaryoblast
Eosinophil progenitor
Basophil progenitor
Myelomonocytic progenitor
B progenitor
T progenitor
Red blood cells
Megakaryocyte
Platelets
Monocyte
DP Thymocyte
NK Cell
Eosinophil Basophil Neutrophil Macrophage B cell
CD8 +
T cell
CD4 +
T cell

HIV Gag SL9-Specific T Cell Receptor
Restricted to HLA-A2.01

Fetal Liver
ESC
ESC
ESC
ESC
ESC
2. Transduce with
Anti-HIV TCR
(SL9 Peptide Specific)
CD34+
CD34+
CD34+
CD34+
CD34+
1. Sort CD34+
Irradiate
SCID-hu
HLA-A2.1+
3-12 weeks
3. Analyze
TCR
Expression
CD8
Kitchen et al, PLoS One, 2009

HIV-TCR Transduced Thymocytes
Week 4
MHC Tetramers

Antigen Responsiveness of
Transgenic T Cells

HLA-A*0201+
Tissue
Humanized Mouse Model of HIV Infection:
The NSG-CTL Model
Fetal Liver
1. Sort CD34+
CD34+
CD34+
4. Thaw and Transduce with
Anti-HIV TCR
Or Control TCR
CD34+
Infect with
HIV-1
NL4-3HSA-HA
CD34+
CD34+
CD34+
CD34+
CD34+
2a. Viably freeze fraction
Irradiate
2. Transduce with
Anti-HIV TCR or
Control TCR
NSG
3 weeks
NSG
5. Tail Vein Inject
6-12 weeks
6. Analyze
TCR
Expression/Fu nction
3. Combine with fetal thymus tissue and liver stroma, implant under kidney capsule

HIV-Specific TCR expressing cells are found in multiple organs in NSG-LTL humanized mice
10
5
10
4
10
3
10
2
0
0
Bone Marrow
10
2
10
3
0.75%
SL9 Tetramer
10
4
10
5
10
4
10
3
10
5
10
2
0
0 10
2
Thymus
10
3
1.26%
10
5
10
4
10
3
10
4
10
5
10
2
0
0 10
2
Spleen
10
3
1.47%
10
5
10
4
10
3
10
4
10
5
10
2
0
0 10
2
Liver
1.31%
10
5
10
4
10
3
Peripheral Blood
10
3
10
4
10
5
10
2
0
0 10
2
10
3
1.28%
10
4
10
5

Suppression of HIV Replication by HIV-specific TCR
20
15
10
5
35
30
25 p=0.43
0
SL9-TCR Control Uninfected
TCR
Week 2 p=0.004
SL9-TCR Control
TCR
Uninfected
Week 6

Suppression of CD4 Depletion by HIV-specific TCR
100
90
80
70
60
50
40
30
20
10
0 p=0.20
p=0.88
p=0.19
SL9-TCR Control
TCR
Uninfected p=0.29
p=0.05
p=0.01
SL9-TCR Control
TCR
Uninfected

HIV-Specific TCR suppression of plasma vRNA in vivo p=0.05
1000000
100000
10000
1000
100
10
1 p=0.02
SL9-TCR Control
TCR
Week 2
SL9-TCR Control
TCR
Week 6

HIV-specific TCR does not drive short-term viral evolution
AMINO ACID ALIGNMENT
I NPUT VI RUS: SLYNTVATL
Co nt r o l TCR- CONT AI NI NG M SLYNTVATL
HI V SL- 9 TCR- CONTAI NI NG M SLYNTVATL

Immune Correlates of Anti-Viral Efficacy
A.
Uninfected Infected
4.04
SL-9 Tetramer
12.7
57.2
16.8
7.91
25.8
C.
16
14
12
10
8
6
4
2
0
0
Week 6 p=0.044
50 100 150 200 250 300 350 400 450
Copies vRNA /ml at Week 6
B.
0
0
10.7
53.8
3.57
19.3
CCR7
6
5
4
3
2
1
Week -2 p=0.0292
50 100 150 200 250 300 350 400 450
Copies vRNA/ml at Week 6
D.
18
16
14
12
10
8
6
4
2
0
Week -2 Week 4 Week 6

Stem cell
TCR

TCR

TCR

TCR

TCR

TCR

TCR

TCR

T cell
Viral Vectors
Containing Cloned
TCRs
Virus
Infected cells
T cell
T cell
T cell
T cell

Human blood-forming stem cells can be genetically modified with an HIV- specific T cell receptor and mature into functional CD8+ T cells in vivo in humanized mice.
HIV specific TCR lowers viral replication in vivo in humanized mice.

• There are a variety of different type of mutations that produce immunodeficient mice.
• Immunodeficient mice allow human cell engraftment.
• The type of mouse and the system of humanization has to be carefully considered depending on the study.
• The interest in these models has significantly expanded with the development of new strains and better human cell engraftment and function.