U.S. patent application number 14/887857 was filed with the patent office on 2016-04-21 for recombinant non-human mammalian model for hepatitis infection and immunopathogenesis.
The applicant listed for this patent is The University of North Carolina at Chapel Hill. Invention is credited to Lishan Su, Michael Washburn, Liguo Zhang.
Application Number | 20160106076 14/887857 |
Document ID | / |
Family ID | 40985850 |
Filed Date | 2016-04-21 |
United States Patent
Application |
20160106076 |
Kind Code |
A1 |
Su; Lishan ; et al. |
April 21, 2016 |
RECOMBINANT NON-HUMAN MAMMALIAN MODEL FOR HEPATITIS INFECTION AND
IMMUNOPATHOGENESIS
Abstract
Provided herein is a recombinant non-human mammal having an
immune system including human immune cells and having a liver
including human liver cells, and methods for producing the same.
Also provided are methods of screening a compound for activity in
treating hepatitis, comprising: administering a test compound to a
recombinant non-human mammal as described herein; and then
detecting the presence or absence of said activity in said mammal
(e.g., by biochemical assay), said presence of said activity in
said mammal indicating that said compound has activity in treating
hepatitis. Methods of making fusion cells useful for the production
of human monoclonal antibodies are also provided.
Inventors: |
Su; Lishan; (Chapel Hill,
NC) ; Zhang; Liguo; (Chapel Hill, NC) ;
Washburn; Michael; (Durham, NC) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The University of North Carolina at Chapel Hill |
Chapel Hill |
NC |
US |
|
|
Family ID: |
40985850 |
Appl. No.: |
14/887857 |
Filed: |
October 20, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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12918676 |
Sep 15, 2010 |
9173383 |
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PCT/US09/01081 |
Feb 20, 2009 |
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14887857 |
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61030328 |
Feb 21, 2008 |
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Current U.S.
Class: |
435/5 ; 435/451;
800/9 |
Current CPC
Class: |
A01K 2227/105 20130101;
C12N 15/02 20130101; A01K 2207/12 20130101; A01K 2267/0337
20130101; A01K 2217/206 20130101; A01K 67/0275 20130101; A01K
2217/052 20130101; A01K 2217/30 20130101; A01K 2207/15 20130101;
G01N 33/5088 20130101; A01K 67/0271 20130101; C12N 9/6475 20130101;
C12N 15/8509 20130101; A01K 2217/15 20130101 |
International
Class: |
A01K 67/027 20060101
A01K067/027; C12N 15/02 20060101 C12N015/02; G01N 33/50 20060101
G01N033/50 |
Goverment Interests
STATEMENT OF GOVERNMENT SUPPORT
[0002] This invention was made with Government support under grants
R21-CA99939 and RO1-AI41356 from the National Institutes of Health.
The United States Government has certain rights to this invention.
Claims
1. A recombinant non-human mammal comprising: (a) an immune system
comprising: human T cells, human B cells, human natural killer
cells, human monocytes and macrophages and human dendritic cells,
so that said mammal expresses a human immune system phenotype; and
(b) a liver comprising human hepatocytes, so that said mammal
expresses a human liver phenotype.
2. The mammal of claim 1, wherein said human liver cells comprise
at least 20% by volume of said liver of said mammal.
3. The mammal of claim 1, wherein said mammal is a Rag2-gammaC
double knockout mammal.
4. The mammal of claim 1, wherein said mammal comprises non-human
cells that contain a liver-specific inducible promoter operatively
associated with a nucleic acid encoding a product toxic to said
non-human cells.
5. The mammal of claim 4, wherein said liver-specific inducible
promoter comprises a FKBP inducible promoter and said nucleic acid
encoding a product toxic to said non-human cells comprises a
Caspase8 gene.
6. The mammal of claim 1, wherein said mammal is infected with a
virus.
7. The mammal of claim 1, wherein said mammal is infected with
HIV-1 virus, a hepatitis virus, or both.
8. The mammal of claim 7, wherein said hepatitis virus is Hepatitis
B virus (HBV) or Hepatitis C virus (HCV).
9. The mammal of claim 1, wherein said mammal is a mouse.
10. A method of screening a compound for activity in treating
hepatitis, comprising: administering a test compound to the
recombinant non-human mammal of claim 1; and then detecting the
presence or absence of said activity in said mammal, said presence
of said activity in said mammal indicating that said compound has
activity in treating hepatitis.
11. The method of claim 10, wherein said detecting step is carried
out by a biochemical assay.
12. A method of making a non-human transgenic mammal comprising an
immune system, said immune system comprising: human T cells, human
B cells, human natural killer cells, human monocytes and
macrophages and human dendritic cells, so that said mammal
expresses a human immune system phenotype; and a liver comprising
human hepatocytes, so that said mammal expresses a human liver
phenotype; said method comprising the steps of: (a) providing a
BalbC/Rag2.sup.-/-.gamma..sub.c.sup.-/- double knockout transgenic
mammal; (b) transplanting human CD34+ hematopoietic stem cells into
said double knockout transgenic mammal, wherein said stem cells
differentiate into human T cells, human B cells, human natural
killer cells, human monocytes and macrophages and human dendritic
cells in said transgenic mammal; and (c) transplanting human liver
cells into said double knockout transgenic mammal, wherein said
liver cells form human hepatocytes in said liver of said transgenic
mammal.
13. The method of claim 12, wherein said human CD34+ hematopoietic
stem cells and said human liver cells are autogeneic with respect
to each other.
14. The method of claim 12, wherein said human liver cells comprise
human parenchyma hepatoblasts.
15. The method of claim 12, wherein said human CD34+ hematopoietic
stem cells and said human liver cells are transplanted
simultaneously.
16. The method of claim 12, wherein said transplanting steps are
carried out when said transgenic mammal is from 0 to 10 days
old.
17. The method of claim 12, wherein said transplanting steps are
carried out when said transgenic mammal is from 1-3 days old.
18. The method of claim 12, wherein said transgenic mammal further
comprises an Alb-FKBP-Casp8 transgene.
19. The method of claim 12, further comprising the step of
administering a c-Met agonist selective for human c-Met to said
transgenic animal.
20. The method of claim 19, wherein said c-Met agonist is an
agonistic antibody against human c-Met.
21. The method of claim 19, wherein said administering step is
carried out by injecting an anti-C-met antibody.
22. A method of making fusion cells useful for the production of
human monoclonal antibodies, said method comprising: isolating a
human antibody-secreting B lymphocyte from a recombinant non-human
mammal of claim 1; and then fusing said antibody-secreting B
lymphocyte with immortal cells to form said fusion cells.
23. The method of claim 22, wherein said immortal cell is a human
or mouse myeloma cell.
24. The method of claim 22, wherein said antibody-secreting B
lymphocyte is isolated from a spleen or lymph node of said
recombinant non-human mammal.
25. The method of claim 22, wherein said mammal is infected with a
virus.
26. The method of claim 22, wherein said mammal is infected with
HIV-1 virus, a hepatitis virus, or both.
27. The method of claim 26, wherein said hepatitis virus is
Hepatitis B virus (HBV) or Hepatitis C virus (HCV).
Description
RELATED APPLICATIONS
[0001] This application is a continuation of and claims priority to
U.S. patent application Ser. No. 12/918,676, filed Sep. 15, 2010,
now U.S. Pat. No. 9,173,383, which is a 35 U.S.C. .sctn.371
national phase entry of PCT Application PCT/2009/001081, filed Feb.
20, 2009, and published in English on Aug. 27, 2009, as
International Publication No. WO 2009/105244, and which claims the
benefit of U.S. Provisional Patent Application Ser. No. 61/030,328,
filed Feb. 21, 2008, the disclosure of each of which is
incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
[0003] The present invention concerns transgenic non-human animals
and methods of making and using the same.
BACKGROUND OF THE INVENTION
[0004] Liver disease induced by hepatitis C virus (HCV) and
hepatitis B virus (HBV) is a global health problem. The World
Health Organization estimates that 350-400 million people are
chronically infected, and about one million die annually due to
chronic hepatitis, cirrhosis or liver cancer. Another 200 million
people are infected by HCV, of whom 70%-85% will become chronically
infected. HBV and HCV infection is the leading cause of liver
disease in Asia. In Western countries, HCV infection is the leading
indication for liver transplantation and a major cause of liver
cancer.
[0005] The liver is a unique organ for immune responses and
viruses/tumors (for review, see Crispe, I. N. 2003. Hepatic T cells
and liver tolerance. Nat Rev Immunol 3:51-62). The liver
intrinsically dampens the immune responses to foreign antigens
filtering through it from the intestines. An allogeneic liver
transplant is often accepted with minimal or no immune suppression.
Tumors with tumor-specific antigens can metastasize to and survive
in the liver in immuno-competent patients. Infection of hepatocytes
by viruses such as HCV often leads to specific immune tolerance to
the virus, and to chronic or persistent infection (Bowen et al.
2005. Adaptive immune responses in acute and chronic hepatitis C
virus infection. Nature 436:946-52; Grakoui et al. 2003. HCV
persistence and immune evasion in the absence of memory T cell
help. Science 302:659-62).
[0006] Although the liver may provide an immunologically privileged
site for infections, most infections in the liver, such as HAY and
MHV, and HBV in adults, are effectively cleared and accompanied
with lasting protective immunity. It is unusual that greater than
80% of HCV infection in immuno-competent hosts leads to persistent
infection. HCV-encoded factors and/or unique host cell tropism may
contribute to the efficient immune evasion and HCV persistence.
[0007] The liver consists of unique subsets of antigen presenting
cells and lymphocytes. In addition to dendritic cells, a large
number of liver macrophages (or Kupffer cells) and sinusoidal
endothelial cells in the liver also have efficient phagocytosis
activity and express various levels of MHC and T cell costimulatory
molecules. However, they often show suboptimal T cell activation
activity in vivo (Everett et al. 2003. Kupffer cells: another
player in liver tolerance induction. Liver Transpl 9:498-9; Parker
et al. 2005. Liver immunobiology. Toxicol Pathol 33:52-62;
Racanelli et al. 2006. The liver as an immunological organ.
Hepatology 43:S54-S62; Sun et al. 2003. Hepatic allograft-derived
Kupffer cells regulate T cell response in rats. Liver Transpl
9:489-97; Wiegard et al. 2005. Murine liver antigen presenting
cells control suppressor activity of CD4+CD25+ regulatory T cells.
Hepatology 42:193-9). The liver also contains lymphoid cells with
unique features. Up to 25% of lymphoid cells belong to the NKT cell
population that expresses TCR as well as NK markers. Their function
in the liver is not clear, but they have been implicated in
clearing infections in the liver (Behar et al. 1999. Susceptibility
of mice deficient in CD1D or TAP1 to infection with Mycobacterium
tuberculosis. J Exp Med 189:1973-80; Skold et al. 2003. Role of
CD1d-restricted NKT cells in microbial immunity. Infect Immun
71:5447-55).
[0008] HCV/HBV coinfection with the HIV-1 virus, which is highly
prevalent among intravenous drug users, leads to accelerated liver
disease progression (Bani-Sadr et al. 2006. Hepatic steatosis in
HIV-HCV coinfected patients: analysis of risk factors. Aids
20:525-31; Brau, N. 2003. Update on chronic hepatitis C in
HIV/HCV-coinfected patients: viral interactions and therapy. Aids
17:2279-90; Sabin et al. 2004. HIV/HCV coinfection, HAART, and
liver-related mortality. Lancet 364:757-8; author reply 758). Liver
failure is increasingly affecting HIV-1/HCV-coinfected patients, as
their AIDS-free survival is being prolonged by highly active
antiretroviral therapy (HAART).
[0009] The available treatment for HCV infection is far from
optimal, and HIV-1/HCV-coinfected patients show even worse
responses to pegylated interferon plus rivabirin than
HCV-monoinfected patients (Sola et al. 2006. Poor response to
hepatitis C virus (HCV) therapy in HIV- and HCV-coinfected patients
is not due to lower adherence to treatment. AIDS Res Hum
Retroviruses 22:393-400). There is a great need for alternative
treatment options for hepatitis infection.
[0010] A relevant small animal model for research on HCV/HBV
infection and pathogenesis is therefore needed. However, HCV fails
to infect murine cells due to blocks at multiple steps of the HCV
life cycle. HCV and HBV can only infect, establish chronic
infection and to lead to liver pathogenesis in humans. Only a
reduced chronic infection and immuno-pathogenesis are observed in
chimpanzees, which provides the only current non-human animal model
for HCV infection (Pietschmann et al. 2003. Tissue culture and
animal models for hepatitis C virus. Clin Liver Dis 7:23-43).
[0011] The Alb-uPA transgenic mouse, developed in 1990 by Heckel et
al. (1990. Neonatal bleeding in transgenic mice expressing
urokinase-type plasminogen activator. Cell 62:447-56) to study
plasminogen hyperactivation and therapeutic protocols to prevent
bleeding, contains a tandem repeat of four murine uPA genes under
the control of an albumin promoter. The transgene overexpression
results in profound hypo-fibrinogenemia and accelerated hepatocyte
death. Homozygous animals can be rescued by transplantation of
murine or human hepatocytes, which undergo rapid proliferation to
replace the dying hepatocytes (Mercer et al. 2001. Hepatitis C
virus replication in mice with chimeric human livers. Nat Med
7:927-33; Meuleman et al. 2005. Morphological and biochemical
characterization of a human liver in a uPA-SCID mouse chimera.
Hepatology 41:847-56; Meuleman et al. 2006. Immune suppression
uncovers endogenous cytopathic effects of the hepatitis B virus. J
Virol 80:2797-807). Transplanted human hepatocytes can be infected
with HBV and HCV (Mercer et al. 2001. Hepatitis C virus replication
in mice with chimeric human livers. Nat Med 7:927-33; Meuleman et
al. 2006. Immune suppression uncovers endogenous cytopathic effects
of the hepatitis B virus. J Virol 80:2797-807).
[0012] A molecularly cloned, cell culture-produced hepatitis C
virus (HCVcc) genome has been recently shown to support efficient
replication in vitro (Blight et al. 2000. Efficient initiation of
HCV RNA replication in cell culture. Science 290:1972-4; Lindenbach
et al. 2005. Complete replication of hepatitis C virus in cell
culture. Science 309:623-6) and in vivo (Lindenbach et al. 2006.
Cell culture-grown hepatitis C virus is infectious in vivo and can
be recultured in vitro. Proc Natl Acad Sci USA 103:3805-9). The
HCVcc is infectious in uPA-SCID mice reconstituted with human
hepatocytes, and infection can be serially passaged to a naive
animal.
[0013] Infectivity of HCV can be studied in the uPA-SCID mice
transplanted with human hepatocytes (Kneteman et al. 2006. Anti-HCV
therapies in chimeric scid-Alb/uPA mice parallel outcomes in human
clinical application. Hepatology 43:1346-53; Lindenbach et al.
2005. Complete replication of hepatitis C virus in cell culture.
Science 309:623-6; Mercer et al. 2001. Hepatitis C virus
replication in mice with chimeric human livers. Nat Med 7:927-33;
Meuleman et al. 2005. Morphological and biochemical
characterization of a human liver in a uPA-SCID mouse chimera.
Hepatology 41:847-56). However, immuno-pathogenesis cannot because
uPA mice have no immune system. In addition, the uPA-SCID mouse is
very sick and not suitable for many studies.
[0014] The RagFah.gamma.C TKO mouse also allows efficient
engraftment of human hepatocytes in a uPA transgene-dependent
fashion (Azuma et al. 2007. Robust expansion of human hepatocytes
in Fah(-/-)/Rag2(-/-)/I12rg(-/-) mice. Nat Biotechnol 25:903-10).
In the B6 Rag/.gamma.C DKO background, the fumarylacetoacetate
hydrolase (Fah) mutation is crossed to generate the RagFah.gamma.C
triple KO mice. After pretreatment with a urokinase-expressing
adenovirus, these animals could be highly engrafted with human
hepatocytes. However, due to lack of a functional immune system
(which is not suitable for human immune system development), it is
not possible to study HCV/HBV immunopathogenesis in these
uPA-SCID/-TKO models.
[0015] Thus, a mouse model having both a functional human immune
system and a human liver is needed to study HCV/HBV infection,
immune responses and pathogenesis.
[0016] Two human-mouse chimera models with human lymphoid organs
implanted in immunodeficiency mice have been constructed to study
HIV-1 infection in vivo. The hu-PBL-SCID mouse is limited due to
its lack of human hemato-lymphoid organs and its selective
engraftment of xeno-reactive human T cells (Mosier et al. 1988.
Transfer of a functional human immune system to mice with severe
combined immunodeficiency. Nature 335:256-9; Mosier et al. 1991.
Human immunodeficiency virus infection of human-PBL-SCID mice.
Science 251:791-4; Tary-Lehmann et al. 1994. Anti-SCID mouse
reactivity shapes the human CD4+ T cell repertoire in hu-PBL-SCID
chimeras. J Exp Med 180:1817-27). The SCID-hu Thy/Liv mouse has an
intact human thymus organ, which allows investigation of HIV-1
pathogenesis in the thymus (McCune et al. 1991. The SCID-hu mouse:
a small animal model for HIV infection and pathogenesis. Annu Rev
Immunol 9:399-429; McCune et al. 1988. The SCID-hu mouse: murine
model for the analysis of human hematolymphoid differentiation and
function. Science 241:1632-9; Su et al. 1995. HIV-1-induced
thymocyte depletion is associated with indirect cytopathogenicity
and infection of progenitor cells in vivo. Immunity 2:25-36).
However, no human B or myeloid cells and very low levels of human T
cells are detected in the peripheral organs or blood. Therefore, no
significant primary human immune responses are observed in the
model.
[0017] A more relevant in vivo non-human animal model that allows
hepatitis infection as well as hepatitis and HIV co-infection is,
therefore, needed.
SUMMARY OF THE INVENTION
[0018] Provided herein is a recombinant non-human mammal (e.g., a
mouse) comprising: (a) an immune system comprising, consisting of,
or consisting essentially of: human T cells, human B cells, human
natural killer cells, human monocytes and macrophages and human
dendritic cells, so that the mammal expresses a human immune system
phenotype; and (b) a liver comprising, consisting of, or consisting
essentially of human hepatocytes, so that the mammal expresses a
human liver phenotype. In some embodiments, the human liver cells
comprise at least 20, 30, 40 or 50% of said liver of said mammal
(by weight, by volume and/or by number of cells) (measured, e.g.,
by human albumin staining). In some embodiments, the mammal is a
Rag2-gammaC double knockout mammal. In some embodiments, the mammal
comprises non-human cells that contain a liver-specific promoter
(e.g., an albumin promoter) operatively associated with a nucleic
acid encoding a product with inducible toxicity to said non-human
cells (e.g., FKBP-Caspase 8). In some embodiments, the mammal is
infected with a virus, e.g., HIV-1 virus, a hepatitis virus (e.g.,
HBV or HCV), or both.
[0019] Also provided herein are methods of screening a compound for
activity in treating hepatitis, comprising: administering a test
compound to a recombinant non-human mammal as described herein; and
then detecting the presence or absence of said activity in said
mammal (e.g., by biochemical assay), said presence of said activity
in said mammal indicating that said compound has activity in
treating hepatitis.
[0020] Further provided are methods of making a non-human
transgenic mammal comprising an immune system, said immune system
comprising: human T cells, human B cells, human natural killer
cells, human monocytes and macrophages and human dendritic cells,
so that the mammal expresses a human immune system phenotype; and a
liver comprising human hepatocytes, so that the mammal expresses a
human liver phenotype. The methods comprise the steps of: a)
providing a BalbC/Rag2.sup.-/-.gamma..sub.c.sup.-/- double knockout
transgenic mammal (optionally further comprising an Alb-FKBP-Casp8
transgene); b) transplanting human CD34+ hematopoietic stem cells
into said double knockout transgenic mammal, wherein said stem
cells differentiate into human T cells, human B cells, human
natural killer cells, and human dendritic cells in said transgenic
mammal; and c) transplanting human liver cells into said double
knockout transgenic mammal, wherein said liver cells form human
hepatocytes in said liver of said transgenic mammal. In some
embodiments, the human CD34+ hematopoietic stem cells and the human
liver cells are autogeneic. In some embodiments, the human liver
cells comprise human parenchyma hepatoblasts. In some embodiments,
the human CD34+ hematopoietic stem cells and the human liver cells
are transplanted simultaneously. In some embodiments, the
transplanting steps are carried out when the transgenic mammal is
from 0 to 10 days old. In some embodiments, the methods further
comprise the step of administering (e.g., by injection) a c-Met
agonist (e.g., an anti-C-met antibody) selective for human c-Met to
said transgenic animal.
[0021] Methods of making fusion cells useful for the production of
human monoclonal antibodies are also provided, said method
comprising: isolating a human antibody-secreting B lymphocyte from
a recombinant non-human mammal (e.g., from a spleen or lymph node),
said mammal comprising: (a) an immune system comprising: human T
cells, human B cells, human natural killer cells, human monocytes
and macrophages and human dendritic cells, so that said mammal
expresses a human immune system phenotype; and (b) a liver
comprising human hepatocytes, so that said mammal expresses a human
liver phenotype; and fusing said antibody-secreting B lymphocyte
with immortal cells (e.g., human or mouse myeloma cells) to form
said fusion cells. In some embodiments, the mammal is infected with
a virus, e.g., HIV-1 virus, a hepatitis virus (e.g., HBV or HCV),
or both.
[0022] Also provided is the use of a non-human transgenic mammal,
cell or cell culture as described herein for the preparation of a
composition or medicament for carrying out a method of treatment as
described herein, or for making an article of manufacture as
described herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] FIGS. 1A-1H. Long-term, stable human hemato-lymphopoiesis in
HSC-DKO Mice. Human fetal liver CD34+ cells were injected
intra-hepatically in newborn (1-3 days old) DKO mice. At 12-50
weeks post transplant, the human CD45.sup.+, murine CD45.sup.-
cells in PBMC, lymph node (LN), spleen, bone marrow and thymus were
analyzed by multi-color FACS. FIG. 1A: Human CD45 reconstitution
(12-16 weeks old) in 5 cohorts of DKO-hu HSC mice from 5
independent donor fetal livers. Shown is average % human CD45+
cells in PB of reconstituted DKO-hu HSC mice. Error bars indicate
standard errors of each cohort (n=numbers of mice/cohort). FIG. 1B:
Human cells reconstitution in PBMC of the transplanted mice.
Individual bars represent percentage of human CD45+ cells of 12
mice from the same cohort (14 weeks post transplant). The darker
inside bars indicate the portion of human CD3+CD4+ cells. FIG. 1C:
Total number of splenocytes and thymocytes from a typical DKO-hu
cohort (n=12) is compared to wild type and DKO mice. Error bars are
standard deviations. FIG. 1D: Stable reconstitution of naive and
resting human T cells. Human T cells from a DKO-hu mouse at 50
weeks are analyzed for expression of CD45RO and CD69. FIG. 1E/F:
Human CD4+ T cells purified from DKO-hu mice or from human PBMC are
stimulated with various doses of anti-CD3 mAb with (solid squares)
or without (open squares) anti-CD28 mAb. Human T proliferation is
measured by 3-H thymidine incorporation for 16 hr after 3 days post
co-culture. FIG. 1G: Tolerization of human T cells to both BalbC
host cells and to the donor human cells in DKO-hu HSC mice.
Splenocytes from DKO-hu mouse cohort A (A1 and A2), cohort B (B)
and untransplanted DKO mouse (DKO) are prepared and mixed in
culture in triplicates at 2.times.10e5 total cells/well (in mixed
cultures, 1.times.10e5 cells per donor cells are used). Similar
human cells (about 50%) are engrafted in A1, A2 and B DKO-hu mice.
Only A1/B and A2/B co-culture show significant proliferation
(p<0.05) in the MLR assay. FIG. 1H: DKO-hu mice are vaccinated
with Ova protein and Ova-specific T cell recall response is
measured at 3 weeks post vaccination. Splenocytes from vaccinated
or control "cohort-mate" mice are compared for response to Ova
protein challenge in vitro. Human T cell proliferation is measured
as above.
[0024] FIGS. 2A-2H. HIV-1 Replication and Pathogenesis in DKO-hu
HSC mice. DKO-hu HSC mice were infected with HIV-R3A or JRCSF (5 ng
p24/mouse). DKO mice or mock infected DKO-hu mice are used as
controls. For R3A infection, plasma samples were collected at 1, 2,
3, 4 and 12 weeks post infection and HIV genome copy numbers were
determined with the Roche Amplicor HIV-1 Monitor Kit (FIG. 2A).
(FIG. 2B) FACS analysis of human CD45, CD3, CD4 and CD8 cells from
blood samples are performed and relative CD4 depletion is shown (as
% CD3+CD4+ or CD4/CD8 ratio). Open symbols are mock controls and
solid symbols are HIV-infected mice. (FIG. 2C/D) JRCSF infection is
similarly analyzed at 1, 2, 4, 6, and 18 wpi. Shown are data from
four infected DKO-hu mice and two mock control mice (FIG. 2D).
(FIG. 2E-H) Spleen (FIG. 2E/F) or mLN (FIG. 2E/F) samples from
DKO-hu mice infected with R3A (FIG. 2E/G, R3A-2 wpi, 3 mocks and 4
R3A-infected mice) or JRCSF (FIG. 2F/H, JRCSF-4 wpi, 4 mocks and 5
JRCSF-infected mice) are summarized for relative CD4 and CD8 in
human leukocytes. Error bars are SE. *, p<0.05; **,
p<0.01.
[0025] FIGS. 3A-3F. Reconstitution of human leukocytes (CD45+) and
hepatocytes (Alb+). (FIG. 3A-B): Both T cells and myeloid cells
were present in the reconstituted liver. Leukocytes from DKO-hu
HSC/Hep mice were isolated. FIG. 3A: human CD45+CD3- cells were
analyzed for CD4 and CD11c expression. All myeloid cell types
(monocytes and macrophage/Kupfer cells; myeloid DC and PDC-CD123+,
not shown) and B cells are present. FIG. 3B: human CD45+CD3+ cells
were analyzed for CD4 and CD8 expression. (FIG. 3C-E) Liver
sections from DKO-hu HSC/Hep mice at 5 weeks post transplant were
stained with anti-human albumin and DAPI. Human albumin+ cells are
detected in the liver parenchyma (FIG. 3C) or around the central
vein (FIG. 3D). FIG. 3E: no primary antibody control. Human
Albumin+ cells exist in the liver parenchyma. All slides are
counter-stained with DAPI for DNA (blue). FIG. 3F: When the human
albumin levels in the blood were measured in a representative
cohort (n=7), a steady level of human albumin (150-350 ng/ml) was
detected from 5-15 weeks post transplant. Pre-transplant sera were
used as background (Non-tran).
[0026] FIGS. 4A-4C. Generation of the FKBP-Casp8 fusion gene with
inducible cell killing activity. (FIG. 4A) Inducible activation of
Caspase 8 through dimerization. The chemical dimerizer AP20187
causes dimerization/activation of Caspase 8 through interaction of
adjacent FKBP binding sites. M, myristoylation signal; FKBP, FK506
binding domain; caspase, human activated caspase 8 (fragment
Ser217-Aps479. ref) was cloned into the pC4M-Fv2E vector (Ariad
Pharmaceuticals) to express the FKBP2-Casp8 fusion protein whose
activation is induced by dimerization with AP20187. (FIG. 4B-C)
Dose-dependent induction of apoptosis in cells transfected with
FKBP-Caspase 8. 293T cells were co-transfected with plasmids
expressing eGFP alone, or with CMV-promoter driven FKBP-Casp8.
Transfected cells were cultured for 30 hours and then various
amounts of AP20187 dimerizer was added. The cells were then
cultured for 24 hours, harvested, stained with 7AAD, and analyzed
by FACS for GFP and 7AAD. (FIG. 4B) GFP+ (transfected) cells were
gated and the percentage of dead cells (7AAD+) was analyzed. (FIG.
4C) GFP-(untransfected bystander) cells were similarly analyzed. At
least 3 independent experiments are repeated.
[0027] FIGS. 5A-5C. Generation of Alb-FKBP-Casp8 transgenic DKO
(AFC8/DKO) mice. (FIG. 5A) The Alb-FKBP-Casp8 transgene structure
and AFC8/DKO-hu HSC/Hep mice: the FKBP2Casp8 fusion gene was cloned
into the liver specific transgenic construct with the Albumin
promoter (see Heckel et al. 1990. Neonatal bleeding in transgenic
mice expressing urokinase-type plasminogen activator. Cell
62:447-56). (FIG. 5B) Hepatocyte-specific apoptosis with expression
from the albumin promoter/enhancer. Alb-FKBP-Casp8 functions in
HepG2 but not 293T cells. As described in FIG. 5B, the FKBP-Casp8
gene controlled by CMV enhancer or the hepatocyte-specific albumin
promoter was co-transfected with GFP-expressing plasmid in 293T and
HepG2 cells. Transfected cells were cultured for 30 hours and
AP20187 dimerizer (2 nM) was added to the culture medium. The cells
were then cultured for 24 hours, harvested, stained with 7AAD, and
analyzed for GFP and 7AAD. GFP+ cells were analyzed for 7AAD uptake
(dead cells). (FIG. 5C) Generation of Alb-FKBP-Casp8/DKO transgenic
mice. Standard transgenic mouse procedure was used to inject the
transgene construct into fertilized DKQ embryos. In the initial
screening of 11 mice from two injections, one transgenic founder
was identified. PCR is run using primers that amplify the
transgenic construct or the mouse endogenous p18 gene (see Kovalev
et al. 2001. An important role of CDK inhibitor p18(INK4c) in
modulating antigen receptor-mediated T cell proliferation. J
Immunol 167:3285-92). 300 fg of transgene plasmid DNA, 100 ng of
mouse genomic DNA, a mixture of 300 fg plasmid+100 ng mouse DNA,
Water+PCR mixture, and DNA from a transgenic founder mouse was
shown.
[0028] FIG. 6. Spleen tissue of DKO-hu HSC mice at 20.times. and
40.times., Mock versus vaccinated with HBsAg (the surface antigen
of HBV).
[0029] FIG. 7. ELISPOT assay of splenocytes from mock or immunized
DKO-hu mice shows that the HBV vaccine induced IgM+ B cells, but
not IgG+ B cells. However, IgG+ B cell induction is achieved with
anti-CD3/CD28 mAb (T activation) in vitro.
[0030] FIG. 8. Antigen-Fe fusion proteins give enhanced
antigen-specific IgG induction in vivo.
[0031] FIGS. 9A-9C. Dimer injection in AFC8/DKO mice leads to
transient mouse liver damage and enhanced human hepatocyte
engraftment (50-100.times.). AFC8/DKO transgenic mice were treated
with AP20187 (5 .mu.g/g). FIG. 9A. Plasma ALT levels were measured
at 24 hr and 72 hr (and 3-6 days, not shown) post treatment. A
second injection of AP20187 6 days after 1st injection again leads
to elevated ALT levels. Two non-TG DKO mice were used as controls
(dashed line, background). FIG. 9B. Livers were harvested at 24 h
after the 2nd injection and H&E stained. Accumulation of fat
droplets (arrows) was observed in TG (but not in Non-TG) livers. V,
vein. FIG. 9C. Enhanced human albumin levels in AP20187-treated
AFC8-DKO-hu mice. AFC8/DKO or DKO mice were treated with AP20187 (5
ug/g) one hour prior to intra-splenic injection of human
hepatocytes (2.times.10e6/mouse). Sera were collected at 80 days
post transfer and human albumin levels were determined by ELISA.
Thus, one injection of dimerizers significantly enhanced human
hepatocyte engraftment (100.times.).
[0032] FIGS. 10A-10D. HBV infection of DKO-hu HSC/Hep mice led to
elevated liver inflammation and long term HBV infection. DKO-hu
HSC/Hep mice were infected with HBV (patient serum, 1.times.10e9
HBV genomes/mouse) or mock. At 37 wpi, two mock and two
HBV-infected mice were terminated. FIG. 10A. HBV-infected DKO-hu
HSC/Hep mice had enlarged livers with 2-3.times. more nucleated
cells. Error bars indicate SD and p value is shown. FIG. 10B.
H&E staining of mock and HBV-infected livers. Elevated
infiltration of leukocytes and vacuoles (arrows) were detected in
the HBV-infected liver. FIG. 10C. HBV genome DNA in the infected
mouse liver. Liver DNA (5 ng) was used to amplify HBV genome (core
sequences) by nested PCR (HBV core sequences). 1. mock liver DNA;
2. HBV-infected liver DNA; 3. 3,000 HBV genomes+mock liver DNA; and
4. 300,000 HBV genomes+mock liver DNA. FIG. 10D. Detection of HBV+
cells with the Anti-HBV core antibody in the HBV infected DKO-hu
liver. Liver section from the infected mouse was stained with
anti-HBV core antibody, and HBV+ cells are indicated by the arrows.
Isotype controls or mock liver sections show no signals (not
shown).
[0033] FIGS. 11A-11C. HCV infection in DKO-hu HSC/Hep mice is
associated with elevated levels of T cell activation and liver
pathology. DKO-hu HSC/Hep mice were infected with HCV (patient
serum, 1.times.10e7 HCV genomes/mouse) or mock. At 25 wpi, two mock
and one HCV-infected mice were analyzed. FIG. 11A. HCV genome RNA
in the infected mouse blood was detected (2.times.10e5 copies/ml).
Mock infected mice showed background level and patient HCV stock
was also used as a control. FIG. 11B. HCV-infected DKO-hu HSC/Hep
mice had higher levels of activated CD4 or CD8 T cells (% HLA-DR+).
FIG. 11C. H&E and anti-human albumin staining of HCV-infected
livers. Human albumin+ hepatocytes and vacuoles were detected in
the HCV-infected liver.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0034] The present invention is explained in greater detail below.
The disclosures of all United States patent references cited herein
are to be incorporated by reference to the extent they are
consistent with the disclosure herein.
[0035] As used herein in the description of the invention and the
appended claims, the singular forms "a," "an" and "the" are
intended to include the plural forms as well, unless the context
clearly indicates otherwise. Furthermore, the terms "about" and
"approximately" as used herein when referring to a measurable value
such as an amount of a compound, dose, time, temperature, and the
like, is meant to encompass variations of 20%, 10%, 5%, 1%, 0.5%,
or even 0.1% of the specified amount. Also, as used herein,
"and/or" or "/" refers to and encompasses any and all possible
combinations of one or more of the associated listed items, as well
as the lack of combinations when interpreted in the alternative
("or").
[0036] An improved non-human animal model of hepatitis infection is
provided herein. The model has well-studied hemato-lymphoid cells
and liver target cells which are human, and HCV can establish
infection and lead to T cell tolerance with similar features of T
cell responses as in HCV infected human or chimps. In some
embodiments, the non-human animal is genetically inbred,
inexpensive and can be manipulated by genetic, immunological and
pharmacological means.
[0037] The model is also useful for studying HCV/HBV and HIV
co-infection and for identifying or confirming therapeutic
strategies for controlling hepatitis diseases. The model is further
useful for studying human liver development/regeneration,
identifying and screening compounds, and determining the
pharmacokinetics of preclinical drugs.
[0038] Besides infection and immuno-pathogenesis, the model is
useful to study human liver stem cells, hepatocyte
development/liver regeneration and autoimmune hepatitis. First,
different subsets of human hepatocyte progenitors can be tested.
Second, the non-human animal is efficiently reconstituted with a
functional human immune system with T, B and myeloid cells in
lymphoid organs including liver. These human immune cells are
present as normal resting cells and respond to antigenic
stimulation in vitro or in vivo (Gimeno et al. 2004. Monitoring the
effect of gene silencing by RNA interference in human CD34+ cells
injected into newborn RAG2-/- gammac-/- mice: functional
inactivation of p53 in developing T cells. Blood 104:3886-93;
Traggiai et al. 2004. Development of a human adaptive immune system
in cord blood cell-transplanted mice. Science 304:104-7; and FIG.
3). The model is further useful for human liver regeneration,
immuno-pathogenesis and immune-based therapeutics. With the model
including a human liver, drug liver toxicity, metabolism, and
pharmacokinetics may also be tested.
[0039] "DKO-hu HSC" as used herein refers to the RAG2-/-
.gamma.C-/- double knockout non-human mammal (with the suicidal
transgene) comprising a human immune system formed from human
hematopoietic stem cell administration.
[0040] "DKO-hu HSC/Hep" as used herein refers to the DKO-hu HSC
non-human mammal (with the suicidal transgene) further comprising
human liver cells (e.g., human hepatocytes). "Hepatitis" is an
inflammation of the liver characterized by the presence of
inflammatory cells in the liver. Acute hepatitis lasts for less
than 6 months, while chronic hepatitis lasts 6 months or longer.
Causes of hepatitis include, but are not limited to, certain
viruses, toxins (e.g., alcohol), infections elsewhere in the body,
an autoimmune response, metabolic disease, etc. Hepatitis caused by
viruses include, but are not limited to, Hepatitis A, Hepatitis B,
Hepatitis C, Hepatitis D (as a co-infection with Hepatitis B
virus), Hepatitis E, Hepatitis F, Hepatitis G, Herpes simplex,
Cytomegalovirus, Epstein-Barr, yellow fever, adenovirus, etc.
[0041] "Hepatitis C virus" or "HCV" is the virus that causes
Hepatitis C. Acute Hepatitis C symptoms, if present, are mild, and
therefore the infection is often not diagnosed at this early stage.
About 20-30% of people infected with HCV will clear the virus from
their systems during the acute phase (i.e., the first 6 months
after infection). The remaining 70-80% will develop a chronic
infection (i.e., lasting 6 months or longer). Symptoms during the
chronic phase are normally also mild, until significant scarring of
the liver has occurred. Once chronic, there is little chance of
clearing the virus without medical treatment. Co-infection of
Hepatitis C with HIV, which is also a blood-borne virus, is common
in the United States, particularly among intravenous drug
users.
[0042] "Hepatitis B virus" or "HBV" is the virus that causes
Hepatitis B. Children are more susceptible than adults to develop a
chronic HBV infection. While more than 95% of adults are able to
clear the virus without medical treatment, 70% of children ages
1-6, and only 5% of newborns who acquire HBV from their mothers can
clear the infection without treatment. Those who do not clear the
virus become chronic carriers.
[0043] "HIV" is the human immunodeficiency virus. It is a
retrovirus that can lead to acquired immune deficiency syndrome
(AIDS), a condition in which the immune system is compromised and
the body is susceptible to opportunistic diseases that can be
life-threatening. HIV primarily infects immune system cells, and
leads to a decrease in the number of CD4+ T cells, which are
important for cell-mediated immunity. The most common strain of HIV
is HIV-1. A second strain, HIV-2, is also known.
[0044] "Transplanting," "engrafting" or "grafting" is the placement
of cells or tissue originating from one animal (e.g., a human) into
another (e.g., a mouse). In some embodiments cells are autogeneic
(i.e., from the same individual animal or subject), isogeneic
(i.e., a genetically identical but different animal or subject,
e.g., from an identical twin, also known as syngeneic), allogeneic
(i.e., from a non-genetically identical member of the same species)
or xenogeneic (i.e., from a member of a different species, also
known as xenografting). In some embodiments, stem/progenitor cells
are engrafted into the non-human animal.
[0045] "Infectivity" is the characteristic of an infectious agent
(i.e., an agent that causes infection) that enables it to enter,
survive and multiply in a suitable host. Similarly, an "infection"
is the invasion by and multiplication of one or more pathogenic
microorganisms (e.g., viruses, bacteria, etc.) in a bodily part or
tissue, which may produce subsequent tissue injury and progress to
overt disease, or the pathological state resulting from having been
infected by such microorganisms.
[0046] "Immunopathogenesis" includes the cellular and mechanistic
events underlying the typical course of development of an infection
or disease that involves the immune response or the products of an
immune response. In some embodiments, immunopathogenesis includes a
dysfunction of the immune response (e.g., HIV infection lowers T
cell count).
[0047] A "human immune system phenotype" is an immune system of a
non-human animal that includes all or essentially all human cells
of the hematopoietic lineages, including adaptive immune system
cells such as T cells, B cells, dendritic cells, natural killer
(NK) cells, monocytes and macrophages, etc.
[0048] "Human" cells include, but are not limited to, cells that
are directly isolated from human tissue as well as cells derived
from human cells, e.g., stem cells (such as hematopoietic stem
cells) differentiated into immune system cells (e.g., in a
non-human host animal, in vitro, etc.).
[0049] "Hematopoietic stem cells" are stem cells typically found in
bone marrow, cord blood, fetal liver and/or mobilized peripheral
blood that can differentiate into all types of blood cells,
including myeloid and lymphoid lineages. In some embodiments,
hematopoietic stem cells are directly delivered (e.g., by direct
injection) into the liver of the animal (e.g., newborn mice).
[0050] The "adaptive immune system" includes immune cells that are
able to remember and recognize antigens or pathogens that had
previously evoked an immune response, and are thereby able to mount
a stronger response upon subsequent exposures, giving rise to
"immunity" to a pathogen.
[0051] A "lymphocyte" is a type of white blood cell typically found
in vertebrates. Large granular lymphocytes include the natural
killer (NK) cells, which are involved in innate immunity. Small
lymphocytes include the T cells and B cells, which are involved in
adaptive immunity. T cells (e.g., helper T cells, cytotoxic T
cells) are typically involved in the cell-mediated immune response,
while B cells are typically involved in the humoral immune
response. T cells and B cells typically recognize non-self antigens
presented on the surface of cells and elicit an immune response
tailored to the non-self antigens. After activation, T cells and B
cells typically leave behind memory cells that can elicit a
stronger response if the antigens are again detected.
[0052] A "human liver phenotype" is a liver organ of a non-human
animal that includes human liver cells. "Human liver cells" are
cells that normally form the liver organ in humans, and include,
but are not limited to, human hepatoblasts, hepatocytes, hepatic
stellate cells, Kupffer cells, sinusoidal endothelial cells,
lymphoid cells, etc.
[0053] "Hepatocytes" are the primary cells of the liver organ.
Human liver cells may also include stem/progenitor cells of the
liver (e.g., hepatocyte progenitor cell). "Hepatoblasts" are fetal
liver stem-progenitor cells.
[0054] In some embodiments, human liver cells (e.g., hepatoblasts)
are directly delivered (e.g., by direct injection) into the liver
of the animal (e.g., newborn mice). Other methods for the
introduction of human liver cells into a non-human animal are known
in the art. See, e.g., U.S. Pat. No. 7,161,057 to Kneteman et al.,
which discusses infusing hepatocytes into the spleen of the
animal.
[0055] In some embodiments, the liver organ of the non-human animal
comprises at least 10, 20, 30, 40, or 50% of one or more types of
human liver/leukocyte cells (e.g., hepatocytes) by weight, by
volume and/or by cell count. In some embodiments, the liver organ
comprises at least 60, 70 or 80% of human liver/leukocyte cells by
weight, by volume or by cell count.
[0056] "Isolated" signifies that the cells are placed into
conditions other than their natural environment. However, the term
"isolated" does not preclude the later use of these cells
thereafter in combinations or mixtures with other cells.
[0057] "Non-human animals" of the present invention are, in
general, mammals including primates, such as monkeys, more
preferably rodents, and are more particularly mice and rats.
Animals may be male or female, and may be of any age including
adult. In some embodiments animals are laboratory animals (e.g.,
non-human primates, rodents, dogs, pigs, birds, etc.). In some
embodiments animals are mammalian laboratory animals.
[0058] A "recombinant" or "transgenic" non-human animal refers to a
non-human animal that has a genome or genetic material that is
augmented or altered in some fashion with a construct comprising a
recombinant nucleic acid (e.g., a "transgene") that is introduced
into one or more of the somatic and/or germ cells of the mammal.
The nucleic acid or portions thereof may be, for example, of the
same species (homologous) or of another species (heterologous) with
respect to the host mammal.
[0059] A "recombinant" nucleic acid refers to a nucleic acid that
has been manipulated in vitro, for example, by molecular biology
techniques as described herein and as known in the art.
[0060] A "knockout" of a target gene refers to an alteration in a
host cell genome that results in altered expression of the target
gene (typically a reduction in expression), e.g., by introduction
of a mutation into a coding or noncoding region of the target gene,
which mutation alters expression of the target gene. Mammals may be
heterozygous or homozygous with respect to the mutation or
insertion that causes the knockout.
[0061] "Wild type" gene or protein sequences of a given species are
those DNA or protein sequences that are generally accepted in the
art as being the most highly conserved within or across
species.
[0062] By the term "express" or "expression" of a nucleic acid
coding sequence, it is meant that the sequence is transcribed, and
optionally, translated. Transcription can be measured by any means
well known by those of skill in the art, e.g., measuring the
relative levels of mRNA expression (e.g., with a northern blot,
quantitative PCR, etc.). Typically, expression of a coding region
will result in production of the encoded protein or polypeptide
(measured by, e.g., western blot).
[0063] The production of transgenic animals is known and can be
carried out in accordance with known techniques or variations
thereof which will be apparent to those skilled in the art, for
example, as disclosed in: U.S. Pat. No. 7,022,893 to Takeda et al.
and U.S. Pat. No. 6,218,595 to Giros et al., as well as U.S. Pat.
No. 6,344,596 to W. Velander et al. (American Red Cross); U.S. Pat.
No. 6,339,183 to T. T. Sun (New York University); U.S. Pat. No.
6,331,658 to D. Cooper and E. Koren; U.S. Pat. No. 6,255,554 to H.
Lubon et al. (American National Red Cross; Virginia Polytechnic
Institute); U.S. Pat. No. 6,204,431 to P. Prieto et al. (Abbott
Laboratories); U.S. Pat. No. 6,166,288 to L. Diamond et al.
(Nextran Inc., Princeton, N.J.); U.S. Pat. No. 5,959,171 to J. M.
Hyttinin et al. (Pharming BV); U.S. Pat. No. 5,880,327 to H. Lubon
et al. (American Red Cross); U.S. Pat. No. 5,639,457 to G. Brem;
U.S. Pat. No. 5,639,940 to I. Garner et al. (Pharmaceutical
Proteins Ltd.; Zymogenetics Inc); U.S. Pat. No. 5,589,604 to W.
Drohan et al. (American Red Cross); U.S. Pat. No. 5,602,306 to
Townes et al. (UAB Research Foundation); U.S. Pat. No. 4,736,866 to
Leder and Stewart (Harvard); and U.S. Pat. No. 4,873,316 to Meade
and Lonberg (Biogen).
[0064] Human immune system. Preferably, the non-human animal has an
immune system comprising human immune cells. The transplantation of
human CD34+ cells into SCID or NOD/SCID mice leads to the
generation of mainly human myeloid and B cells in the mouse bone
marrow, but inefficient peripheral engraftment of human cells,
especially human T cells (Lapidot et al. 1992. Cytokine stimulation
of multilineage hematopoiesis from immature human cells engrafted
in SCID mice. Science 255:1137-41; Larochelle et al. 1996.
Identification of primitive human hematopoietic cells capable of
repopulating NQD/SCID mouse bone marrow: implications for gene
therapy. Nat Med 2:1329-37). More recently, a mouse model with a
functional human immune system has been reported. The Rag2-.gamma.C
double knockout (DKO) mouse lacks T, B and NK cells, and serves as
better hosts for engraftment of human cells/tissues. Therefore, in
preferred embodiments the non-human animal is a Rag2-.gamma.C
double knockout (DKO) transgenic non-human animal.
[0065] In some embodiments, cord blood CD34+ human HSC are injected
directly into the liver of newborn DKO animals (see Traggiai et al.
2004. Development of a human adaptive immune system in cord blood
cell-transplanted mice. Science 304:104-7). The new born liver
environment appears to support efficient human HSC engraftment and
reconstitution of the animal with a functional human immune system
in central and peripheral lymphoid organs.
[0066] Remarkably, long term human T cell development occurs
efficiently in the mouse DKO thymus, and normal human T, B, NK and
dendritic cells are readily detected in peripheral lymphoid tissues
such as spleen, lymph nodes (LN) and peripheral blood (PB).
Importantly, de novo human B and T cell responses are elicited in
the hu-HSC-DKO mouse by standard immunization (human TT-specific
IgG induction) or infection with the human tumor virus EBV
(expansion of EBV-specific CD8 T cells).
[0067] Both CCR5 and CXCR4 are expressed on human immature and
mature T cells (Zhang et al. 2007. HIV-1 infection and pathogenesis
in a novel humanized mouse model. Blood 109:2978-81). DKO-hu HSC
mice allow efficient HIV-1 infection with high plasma viremia. High
levels of productive infection occur in the thymus, spleen and
lymph nodes. Human CD4+ T cells are gradually depleted by HIV-1 in
a dose-dependent manner. In addition, HIV-1 infection persists in
infected DKO-hu HSC mice for at least 19 weeks, with infectious
HIV-1 in lymphoid tissues. Thus, the DKO-hu HSC mouse can serve as
a relevant in vivo model to investigate mechanisms of HIV-1
infection and immuno-pathogenesis.
[0068] Combined with engrafted human liver cells, the HSC-DKO mouse
as described herein can serve as a model to investigate mechanisms
of HCV immuno-pathogenesis and how coinfection with HIV-1 may
affect HCV replication and/or pathogenesis.
[0069] Human liver cells. In preferred embodiments, the non-human
mammal comprises human liver cells. Human liver cells may be
introduced into the non-human mammal by the procedures provided
herein or by procedures known in the art (see, e.g., U.S. Pat. No.
7,161,057 to Kneteman, incorporated by reference herein).
[0070] In some embodiments, human hepatoblasts/progenitors are
isolated from human fetal liver tissues. Hepatocytes (or parenchyma
cells) may be isolated from livers by collagenase digestion as
described (Meuleman et al. 2005. Morphological and biochemical
characterization of a human liver in a uPA-SCID mouse chimera.
Hepatology 41:847-56; Schmelzer et al. 2006. The phenotypes of
pluripotent human hepatic progenitors. Stem Cells 24:1852-8).
EpCAM, a transmembrane glycoprotein, has been shown to mark human
hepatic stem or progenitor cells as it is expressed by hepatic
progenitors but not hepatocytes (de Boer et al. 1999. Expression of
Ep-CAM in normal, regenerating, metaplastic, and neoplastic liver.
J Pathol 188:201-6). Transplantation of EpCAM+ cells into the liver
of mice gives rise to human liver tissue expressing human-liver
specific proteins (de Boer et al. 1999. Expression of Ep-CAM in
normal, regenerating, metaplastic, and neoplastic liver. J Pathol
188:201-6; Schmelzer et al. 2006. The phenotypes of pluripotent
human hepatic progenitors. Stem Cells 24:1852-8).
[0071] In some embodiments, approximately one million (10.sup.6)
CD34+ HSC cells are co-transferred with approximately one million
(10.sup.6) parenchyma cells (comprising human hepatoblasts and/or
hepatic stem cells) (105 EpCAM+ hepatoblasts) into the liver of 1-
to 3-day-old DKO or AFK8/DKO mice previously irradiated at 400 rad
(sublethal). In some embodiments, cells are co-injected in the
liver of newborn DKO mice.
[0072] Antagonistic antibody against c-met. To improve human
hepatocyte growth, in some embodiments an agonistic antibody
against human c-Met (c-Met mAb, mouse IgG1) is used that activates
human but not murine c-Met as reported (Ohashi et al. 2000.
Sustained survival of human hepatocytes in mice: A model for in
vivo infection with human hepatitis B and hepatitis delta viruses.
Nat Med 6:327-31).
[0073] DKO-hu HSC/Hep mice in AFC8/DKO mice. In another embodiment,
DKO-hu HSC/Hep mice are generated in an AFC8/DKO background
(FKBP-Casp8 gene under control of the albumin promoter, see Heckel
et al. 1990. Neonatal bleeding in transgenic mice expressing
urokinase-type plasminogen activator. Cell 62:447-56) (FIG.
6&7). Transfer of adult hepatocytes into uPA-SCID mice has led
to efficient engraftment of human hepatocytes (70%) in the chimeric
liver (Mercer et al. 2001. Hepatitis C virus replication in mice
with chimeric human livers. Nat Med 7:927-33; Meuleman et al. 2005.
Morphological and biochemical characterization of a human liver in
a uPA-SCID mouse chimera. Hepatology 41:847-56). However, the
uPA-SCID or uPA-TKO mouse (Azuma et al. 2007. Robust expansion of
human hepatocytes in Fah(-/-)/Rag2(-/-)/I12rg(-/-) mice. Nat
Biotechnol 25:903-10) has no immune system, is difficult to breed
and not optimal for most studies. Hepatocytes of the AFC8/DKO mouse
can be inducibly depleted with the FKBP dimerizer ligand AP20187
(Burnett et al. 2004. Conditional macrophage ablation in transgenic
mice expressing a Fas-based suicide gene. J Leukoc Biol 75:612-23;
Pajvani et al. 2005. Fat apoptosis through targeted activation of
caspase 8: a new mouse model of inducible and reversible
lipoatrophy. Nat Med 11:797-803).
[0074] Methods of screening compounds. The present invention also
provides methods of screening a compound for activity in treating
hepatitis and/or HIV infection. In some embodiments the method
comprises administering a test compound to a mammal as described
herein, and then detecting the presence or absence of activity in
treating and/or preventing hepatitis and/or HIV infection in the
mammal. The administering step may be carried out by any suitable
technique depending upon the particular compound, including
parenteral injection, oral administration, inhalation
administration, transdermal administration, etc.
[0075] "Treat" refers to any type of treatment that imparts a
benefit to a subject, e.g., a subject afflicted with or at risk for
developing a disease or condition (e.g., a liver infection and/or
HIV/AIDS, etc.). Treating includes actions taken and actions
refrained from being taken for the purpose of improving the
condition of the subject (e.g., the relief of one or more
symptoms), delay in the onset or progression of the disease,
disease prevention (e.g., immunization), etc.
[0076] Production of monoclonal antibodies. Monoclonal antibodies
can be produced in a hybridoma cell line formed according to
well-known technique of Kohler and Milstein, (1975) Nature
265:495-97, using a human immune cell isolated from the spleen of a
non-human mammal with a human immune system/human liver phenotype
for the fusion. For example, human spleen cells are isolated from a
non-human animal having a human immune system. The spleen cells are
then immortalized by fusing them with myeloma cells or with
lymphoma cells, typically in the presence of polyethylene glycol,
to produce hybridoma cells. The hybridoma cells are then grown in a
suitable medium and the supernatant screened for monoclonal
antibodies having the desired specificity. Monoclonal Fab fragments
can be produced in bacterial cells such as E. coli by recombinant
techniques known to those skilled in the art. See, e.g., W. Huse,
(1989) Science 246:1275-81.
[0077] In some embodiments of the present invention, vaccination of
the DKO-hu HSC with a human HBV vaccine induces IgM-producing human
B cells, but very low levels of human IgG+ B cells. Human IgG+ B
cells can be increased by activating human T cells in the spleen in
vitro e.g., with anti-CD3/CD28 mAb. Further, immunization of the
non-human animal with a specific protein may induce
antigen-specific human IgG producing B cells. In some embodiments,
antigen-specific human IgG induction can be enhanced in vivo by
using the fusion protein with the antigen and the Fc domain of
human IgG.
[0078] The present invention is explained in greater detail in the
following non-limiting Examples.
EXAMPLE 1
[0079] Stable reconstitution of DKO mice with CD34+ HSC (DKO-hu HSC
mice>1 yr). Human fetal liver derived CD34+ HSC
(0.5-1.times.10.sup.6/mouse) were transplanted into newborn DKO
mice intra-hepatically. Functional lymphoid organs are formed in
the DKO-HSC mice as reported (see Baenziger et al. 2006.
Disseminated and sustained HIV infection in CD34+ cord blood
cell-transplanted Rag2-/-gamma c-/- mice. Proc Natl Acad Sci U.S.A.
103:15951-6; Traggiai et al. 2004. Development of a human adaptive
immune system in cord blood cell-transplanted mice. Science
304:104-7; Zhang et al. 2007. HIV-1 infection and pathogenesis in a
novel humanized mouse model. Blood 109:2978-81). Experiments showed
that >95% of the transplanted DKO mice have stable human cell
engraftment with human CD45+ cells in the blood for at least 50
weeks (FIG. 1A; Zhang et al. 2007. HIV-1 infection and pathogenesis
in a novel humanized mouse model. Blood 109:2978-81; and data not
shown). T, B, monocytes, mDC and PDC are stably reconstituted (FIG.
1B and data not shown). About 30.times.10.sup.6 total splenocytes
(40% of wild type mice) and 20.times.10.sup.6 thymocytes (20% of
wild type mice) are generated in DKO-hu mice (FIG. 1C). Most CD4
and CD8 T cells express a resting naive phenotype (CD45RO-CD69- and
CD62L+CCR7+, FIG. 1D and data not shown). When their proliferation
response to TCR stimulation is compared to human CD4 T cells (FIG.
1F), DKO-hu derived human CD4 T cells (FIG. 1E) show identical
response to different doses of anti-CD3 mAb and to CD28
co-stimulation.
[0080] Human T cells are negatively selected by both mouse MHC/APC
and human MHC/APC because both human and mouse antigen presenting
cells (APC) are detected in the mouse thymus (see Traggiai et al.
2004. Development of a human adaptive immune system in cord blood
cell-transplanted mice. Science 304:104-7). Thus, human T cells
developed in the DKO-hu HSC mouse are tolerized to both BalbC mouse
cells and to human cells from "cohort-mate" DKO-hu mice of the same
donor ("inbred/syngeneic" hu-mice) shown by lack of mixed
lymphocyte response (MLR) between splenocytes from DKO, A1 and B
DKO-hu mice, and between "syngeneic" A1 and A2 DKO-hu mice.
However, either A1 or A2 cells react strongly with the "allogeneic"
cells from the cohort B DKO-hu mice (FIG. 1G). In addition,
immunization of DKO-hu mice with Ova protein induces Ova-specific
human T and B cell responses (FIG. 1H and data not shown).
Therefore, normal human T and B cells are generated in the DKO-hu
mouse.
EXAMPLE 2
[0081] HIV-1 infection and pathogenesis in DKO-hu HSC mice. Both
CCR5 and CXCR4 are expressed on human immature and mature T cells
in DKO-hu mice. DKO-hu HSC mice allow efficient HIV-1 infection
with high plasma viremia. High levels of productive infection occur
in the thymus, spleen and lymph nodes. Interestingly, both CD45RO+
(memory/effector) and CD45RO- (naive) CD4T cells are productively
infected as stained for HIV gag p24. Human CD4+ T cells are rapidly
depleted by a pathogenic HIV-1-R3A (FIG. 2A/B). In addition, HIV-1
infection persists in infected DKO-hu HSC mice for at least 19
weeks, with infectious HIV-1 in lymphoid tissues. DKO-hu mice were
also infected with the less pathogenic HIV-1 isolate JRCSF
(CCR5-tropic). High levels of HIV replication were detected at 1 to
18 weeks post infection (FIG. 2C). CD4+ T cells in the blood were
only slowly decreased but maintained at steady levels for 13-128
weeks (FIG. 2D). When lymphoid organs were analyzed from R3A-(2
wpi) or JRCSF-(4 wpi) samples, R3A infection almost completely
depleted human CD4+ T cells in the spleen and dramatically depleted
CD4 T cells in mLN, whereas JRCSF infection did not significantly
deplete human CD4 T cells in the spleen or mLN (FIG. 2E-H).
Interestingly, there is an increase in the CD8 levels in the mLN
for both infections.
[0082] At 22 wpi with JRCSF, HIV infection is associated with an
enlarged spleen and activated HLA-DR+ T cells in DKO-hu mice. CD4
depletion is also observed in lymph node organs and the remaining T
cells show activated phenotypes. Therefore, HIV infection leads to
immuno-pathogenesis, correlated with hyper-immune activation and
inflammation. Indeed, inflammatory cytokines are induced by HIV
infection in plasma as well as in lymphoid tissues (data not
shown).
[0083] Thus, the DKO-hu HSC mouse can serve as a relevant in vivo
model to investigate mechanisms of HIV-1 infection and
immuno-pathogenesis. HIV-R3A can be used to study acute HIV
infection with a rapid CD4 depletion, and JRCSF can be used to
study acute HIV infection, immuno-response and chronic HIV
infection and immunopathogenesis.
EXAMPLE 3
[0084] Development of the DKO-hu HSC/Hep mouse. To develop the
DKO-hu HSC/Hep mice, we isolated human CD34+ HSC and human
hepatoblasts/progenitors from human fetal liver tissues.
Hepatocytes (or parenchyma cells) are isolated from livers by
collagenase digestion as described (Meuleman et al. 2005.
Morphological and biochemical characterization of a human liver in
a uPA-SCID mouse chimera. Hepatology 41:847-56; Schmelzer et al.
2006. The phenotypes of pluripotent human hepatic progenitors. Stem
Cells 24:1852-8). EpCAM, a transmembrane glycoprotein, has been
shown to mark human hepatic stem or progenitor cells as it is
expressed by hepatic progenitors but not hepatocytes (de Boer et
al. 1999. Expression of Ep-CAM in normal, regenerating,
metaplastic, and neoplastic liver. J Pathol 188:201-6).
Transplantation of EpCAM+ cells into the liver of mice gave rise to
human liver tissue expressing human-liver specific proteins (de
Boer et al. 1999. Expression of Ep-CAM in normal, regenerating,
metaplastic, and neoplastic liver. J Pathol 188:201-6; Schmelzer et
al. 2006. The phenotypes of pluripotent human hepatic progenitors.
Stem Cells 24:1852-8).
[0085] One million (1.times.10e6) CD34+ HSC cells are
co-transferred with 1.times.10e6 parenchyma cells (10e5 EpCAM+
hepatoblasts) into the liver of 1- to 3-day-old DKO or AFK8/DKO
mice previously irradiated at 400 rad (sublethal). 20-30 DKO-hu
mice are generated from each fetal liver tissue donor.
[0086] Human T/B/DC cells are analyzed by FACS at different time
points after HSC transplant as previously reported (FIG. 1A-1H;
Meissner et al. 2004. Characterization of a thymus-tropic HIV-1
isolate from a rapid progressor: role of the envelope. Virology
328:74-88; Su et al. 1995. HIV-1-induced thymocyte depletion is
associated with indirect cytopathogenicity and infection of
progenitor cells in vivo. Immunity 2:25-36). Human cells (CD45+)
are analyzed for CD4, CD8, CD25 (Treg), HLA-DR (activated T cells),
CD27/CD45RO (naive and memory T cell subsets), CD19 (B cells),
CD11c (mDC) and CD123 (PDC). Antibodies with appropriate labels are
purchased from BD-Pharmingen.
[0087] When the parenchyma cell suspensions prepared from fetal
livers were analyzed, 12% of liver cell suspensions from fetal
livers are EpCAM+ cells, of which more than 80% are hepatoblasts
and hepatic stem cells (Schmelzer et al. 2006. The phenotypes of
pluripotent human hepatic progenitors. Stem Cells 24:1852-8). Thus,
the parenchyma cells prepared from fetal livers are enriched with
human hepatoblasts.
[0088] These cells are co-injected in the liver of newborn DKO
mice. As shown above, human CD34+ HSC reconstituted the human blood
system with a functional human immune system. Regarding the
hepatocyte reconstitution, human liver stem/progenitor cells have
been documented in both CD34+ cells (Dan et al. 2006. Isolation of
multipotent progenitor cells from human fetal liver capable of
differentiating into liver and mesenchymal lineages. Proc Natl Acad
Sci USA 103:9912-7) and in hepatocyte-like parenchyma EpCAM+ cells
(Schmelzer et al. 2006. The phenotypes of pluripotent human hepatic
progenitors. Stem Cells 24:1852-8). These progenitor cells injected
in the liver give rise to human hepatocytes in the chimeric
mouse.
[0089] When CD34+ cells were co-transplanted with parenchyma
hepatoblasts, significant and stable human albumin in the chimeric
mouse blood was detected (FIG. 3A-3F). The engraftment of human
blood cells was monitored by FACS for human leukocytes (>20% in
total PBMC). Human albumin in the plasma of mice was measured
(100-500 ng/ml) by ELISA assay (FIG. 3F) and, for human
hepatocytes, by IF staining of human Alb+ cells in the liver (FIG.
3C-E).
EXAMPLE 4
[0090] Multiple types of human cells are developed in the liver of
DKO-hu HSC/Hep mice. When liver leukocytes from the DKO-hu HSC
mouse were isolated, 80% showed human CD45+ expression. Both
lymphoid cells and myeloid cells were present in the reconstituted
liver (FIG. 3A-B). Immuno-staining of liver section also
demonstrates reconstitution of human hepatocytes (Albumin+, FIG.
3C-E). Therefore, human hepatocytes as well as human T/B/myeloid
cells are present in the chimeric liver.
EXAMPLE 5
[0091] FKBP-Casp8 transgenic mice with inducible death of liver
cells. The DKO-hu HSC/Hep mouse model was improved by 1) boosting
human hepatocyte cell growth with anti-human c-Met mAb, and 2) by
selective ablation of murine hepatocytes (AFC8/DKO-hu HSC/Hep
mice).
[0092] The newborn BalbC/Rag2.sup.-/-.gamma..sub.c.sup.-/- DKO
mouse is currently the most permissive mouse model for the
engraftment of human tissues, allowing long-term development of
human primary and secondary immune organs. When human liver
stem/progenitor cells (hepatoblasts) are transplanted into newborn
(1-3 days) DKO mice, human liver cells are also detected in the
chimeric mice. However, the levels of human hepatocyte engraftment
is relatively low. Therefore, to enhance human liver cell
engraftment in the DKO-hu mouse, murine hepatocyte death is induced
after human liver cell transfer.
[0093] The FKBP-Caspase8 fusion gene with inducible cell killing
activity was constructed (FIG. 4A-4C; and Chang et al. 2003.
Activation of procaspases by FK506 binding protein-mediated
oligomerization. Sci STKE 2003:PL1). Addition of FKBP dimerizer
AP20187 to cells transfected with the FKBP-Casp8 and/or GFP led to
death of GFP+/FKBP-Casp8+ cells, but not GFP- bystander cells, in a
dose-dependent fashion (FIG. 4B/C). The drug had no detectable
effect on cells transfected with only GFP expressing gene.
Therefore, FKBP-Casp8 activation by AP20187 kills target cells
expressing the gene but not bystander cells, an important point for
depleting specific target cells in vivo.
[0094] DKO female mice are super-ovulated and fertilized eggs are
isolated by standard procedures in the UNC transgenic core
facility. The Alb-FKBP-Casp8 transgene is injected into fertilized
DKO eggs and implanted into surrogate mother mice. Screening for
the Alb-FKBP-Casp8 transgene is done with PCR (FIG. 5C; Kovalev et
al. 2001. An important role of CDK inhibitor p18(INK4c) in
modulating antigen receptor-mediated T cell proliferation. J
Immunol 167:3285-92). FKBP-Casp8/DKO transgenic founders are
confirmed by Southern blot and its expression confirmed by
western/IF (anti-human Caspase 8 mAb) of liver tissues. Spleen,
kidney, heart and thymus are used as control tissues. After
establishing the AFC8/DKO founder mice that express the fusion
protein in the liver, we test the dose and duration of AP20187 to
induce hepatocyte apoptosis in the mouse. For peritoneal
injections, AP20187 (Ariad Pharmaceuticals) is prepared (1 mg/ml in
a solution consisting of 4% ethanol, 10% PEG-400, and 1.7% Tween).
The AP20187 dose is adjusted to deliver 0.25, 1, 4 and 10 mg/kg
(3-5 mice/dose). This dose range of AP20187 is effective in
inducing Capsase8 activation and target cell depletion. No toxicity
is observed in mice because AP20187 is engineered for in vivo
purposes and does not interact with endogenous FKBP (Burnett et al.
2004. Conditional macrophage ablation in transgenic mice expressing
a Fas-based suicide gene. J Leukoc Biol 75:612-23; Pajvani et al.
2005. Fat apoptosis through targeted activation of caspase 8: a new
mouse model of inducible and reversible lipoatrophy. Nat Med
11:797-803).
[0095] The AFC8/DKO mouse was used to construct AFC8/DKO-hu HSC/Hep
mice as described above. At 3-8 weeks post transplant of human
cells, AP20187 is administered to induce death of murine
hepatocytes. Hepatocyte depletion is monitored by measuring serum
ALT level and increased human hepatocytes by blood levels of human
albumin at 1, 2, 4, 6 and 8 weeks post drug treatment. At 2, 4 and
8 weeks post induction, we harvest the chimeric liver of selected
AFC8/DKO-hu HSC/Hep mice to monitor murine hepatocyte death
(apoptosis markers) and human hepatocytes (human albumin+ cells),
and expression of other human liver-specific genes listed
above.
[0096] For peritoneal injections, AP20187 (Ariad Pharmaceuticals)
is prepared (1 mg/ml in a solution consisting of 4% ethanol, 10%
PEG-400, and 1.7% Tween). The AP20187 dose is adjusted to deliver
0.25, 1, 4 and 10 mg/kg (3-5 mice/dose). This dose range of AP20187
is effective in inducing Capsase8 activation and target cell
depletion. No toxicity is observed in mice because AP20187 is
engineered for in vivo purposes and does not interact with
endogenous FKBP (Burnett et al. 2004. Conditional macrophage
ablation in transgenic mice expressing a Fas-based suicide gene. J
Leukoc Biol 75:612-23; Pajvani et al. 2005. Fat apoptosis through
targeted activation of caspase 8: a new mouse model of inducible
and reversible lipoatrophy. Nat Med 11:797-803).
[0097] When expressed from the liver-specific albumin promoter in
the transgenic construct (FIG. 5A; and Heckel et al. 1990. Neonatal
bleeding in transgenic mice expressing urokinase-type plasminogen
activator. Cell 62:447-56), FKBP-Casp8 only kills HepG2 cells, but
not 293T cells, in a dimer-dependent fashion (FIG. 5B). This
confirms the hepatocyte-specific expression of the transgene. DKO
transgenic mice have been generated with the Alb-FKBP-Casp8
construct (FIG. 5C).
[0098] Human hepatocyte level and functions are closely monitored
by measuring human albumin expression. Expression of human
liver-specific marker genes including human albumin,
.alpha.-fetoprotein (AFP, highly expressed in fetal liver but not
adult liver), CytochromeP450/CYP3A4 and CYP1A2 are measured by
TaqMan RT-PCR and by detecting protein expression by IF, as
reported (Azuma et al. 2007. Robust expansion of human hepatocytes
in Fah(-/-)/Rag2(-/-)/I12rg(-/-) mice. Nat Biotechnol 25:903-10;
Meuleman et al. 2005. Morphological and biochemical
characterization of a human liver in a uPA-SCID mouse chimera.
Hepatology 41:847-56).
[0099] Dimer injection in AFC8/DKO mice led to transient mouse
liver damage and enhanced human hepatocyte engraftment
(50-100.times.) (FIG. 9A-9C). AFC8/DKO transgenic mice were treated
with AP20187 (5 .mu.g/g). Plasma ALT levels were measured at 24 hr
and 72 hr (and 3-6 days, not shown) post treatment (FIG. 9A). A
second injection of AP20187 6 days after 1st injection again led to
elevated ALT levels. Two non-TG DKO mice were used as controls
(dashed line, background). Livers were harvested at 24 h after the
2nd injection and H&E stained (FIG. 9B). Accumulation of fat
droplets (arrows) was observed in TG (but not in Non-TG) livers. V,
vein. Enhanced human albumin levels were found in AP20187-treated
AFC8-DKO-hu mice (FIG. 9C). AFC8/DKO or DKO mice were treated with
AP20187 (5 .mu.g/g) one hour prior to intra-splenic injection of
human hepatocytes (2.times.10e6/mouse). Sera were collected at 80
days post transfer and human albumin levels were determined by
ELISA. Thus, one injection of dimerizers significantly enhanced
human hepatocyte engraftment (100.times.).
[0100] The improved DKO-hu HSC/Hep mouse is used for HBV, HCV
and/or HIV infection.
EXAMPLE 6
[0101] Agonistic antibody against human c-Met. Hepatocyte Growth
Factor (HGF) binds to c-Met (the HGF receptor) and is essential in
the development and regeneration of the liver. To improve human
hepatocyte growth, an agonistic antibody against human c-Met is
used (c-Met mAb, mouse IgG1) that activates human but not murine
c-Met as reported (Ohashi et al. 2000. Sustained survival of human
hepatocytes in mice: A model for in vivo infection with human
hepatitis B and hepatitis delta viruses. Nat Med 6:327-31).
[0102] In each cohort, 50% of the DKO-HSC/Hep mice are injected
i.v. with the anti-C-Met mAb 3D1 (Genentech, South San Francisco,
Calif.), at 50 .mu.g/mouse weekly from 2-8 weeks post transplant
(Ohashi et al. 2000. Sustained survival of human hepatocytes in
mice: A model for in vivo infection with human hepatitis B and
hepatitis delta viruses. Nat Med 6:327-31). Mouse IgG1 is used for
controls. Human albumin levels are measured weekly in blood before,
during and after treatment, and human hepatocytes are measured by
IF of liver sections. Proliferation of human hepatocytes (Alb+) is
analyzed by Ki67 staining or by in vivo BrDU pulse labeling
followed by FACS or IF (Kovalev et al. 2001. An important role of
CDK inhibitor p18(INK4c) in modulating antigen receptor-mediated T
cell proliferation. J Immunol 167:3285-92). 3-4 cohorts are tested
to monitor the anti-c-Met effect on human hepatocyte
engraftment.
EXAMPLE 7
[0103] Production of Antigen-specific Human IgG in DKO-hu HSC Mice.
Vaccination of the DKO-hu HSC mouse with a human HBV vaccine
induced only IgM-producing human B cells but very low levels of
human IgG+ B cells (FIG. 6). Human IgG+ B cells were increased by
activating human T cells in the spleen in vitro with anti-CD3/CD28
mAb (FIG. 7). Similarly, immunization with the chicken ovalbumin
protein induced very low levels of Ova-specific human IgG (FIG. 8).
Ova-specific human IgG induction is enhanced by using the fusion
protein between the same chicken ovalbumin protein with the Fc
domain of human IgG (FIG. 8).
EXAMPLE 8
[0104] HBV infection of DKO-hu HSC/Hep mice leads to elevated liver
inflammation and long term HBV infection. DKO-hu HSC/Hep mice were
infected with HBV (patient serum, 1.times.10e9 HBV genomes/mouse)
or mock. At 37 wpi, two mock and two HBV-infected mice were
terminated.
[0105] HBV-infected DKO-hu HSC/Hep mice had enlarged livers with
2-3.times. more nucleated cells (FIG. 10A), and elevated
infiltration of leukocytes and vacuoles (arrows) were detected in
the HBV-infected liver (FIG. 10B). The HBV genome DNA was detected
in the infected mouse liver (FIG. 10C), and HBV+ cells were also
detected with the Anti-HBV core antibody (FIG. 10D).
EXAMPLE 9
[0106] HCV infection in DKO-hu HSC/Hep mice is associated with
elevated levels of T cell activation and liver pathology. DKO-hu
HSC/Hep mice were infected with HCV (patient serum, 1.times.10e7
HCV genomes/mouse) or mock. At 25 wpi, two mock and one
HCV-infected mice were analyzed. A. HCV genome RNA in the infected
mouse blood was detected (2.times.10e5 copies/ml). Mock infected
mice showed background level and patient HCV stock was also used as
a control. B. HCV-infected DKO-hu HSC/Hep mice had higher levels of
activated CD4 or CD8 T cells (% HLA-DR+). C. H&E and anti-human
albumin staining of HCV-infected livers. Human albumin+ hepatocytes
and vacuoles were detected in the HCV-infected liver. HCV+
hepatocytes in the liver tissue are also measured.
[0107] HCV genomes were detected in the infected DKO-hu mice at 25
wpi, but not in mock infected DKO-hu mice or HCV-infected DKO mice
(FIG. 11A and data not shown). In addition, elevated levels of
activated human T cells were detected (FIG. 11B), as well as liver
pathology characteristic of virus-induced hepatitis/liver diseases
(FIG. 11C).
* * * * *