Patent Abstract:
provided is a transgenic animal model for testing immunogenicity and protective efficacy of human vaccines and the method for generating such a multitransgenic animal . also disclosed are methods for screening compositions for human vaccine development . more specifically , a mouse model capable of expressing human leukocyte antigen dr4 , and human costimulatory molecules upon infusion of human hla - matched hematopoietic stem cells , which can develop into a functional human immune system is provided .

Detailed Description:
unless defined otherwise , all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which this invention belongs . although any methods , devices and materials similar or equivalent to those described herein can be used in the practice or testing of the invention , the preferred methods , devices and materials are now described . all publications mentioned herein are incorporated herein by reference for the purpose of describing and disclosing , for example , the cell lines , constructs , and methodologies that are described in the publications which might be used in connection with the presently described invention . the publications discussed above and throughout the text are provided solely for their disclosure prior to the filing date of the present application . nothing herein is to be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior invention . the term “ transgene ” refers to the genetic material which has been or is about to be artificially inserted into the genome of an animal , particularly a mammal and more particularly a mammalian cell of a living animal . “ transgenic animal ” refers to a non - human animal , usually a mammal , having a non - endogenous ( i . e ., heterologous ) nucleic acid sequence present as an extrachromosomal element in a portion of its cells or stably integrated into its germ line dna ( i . e ., in the genomic sequence of most or all of its cells ). heterologous nucleic acid is introduced into the germ line of such transgenic animals by genetic manipulation of , for example , embryos or embryonic stem cells of the host animal . “ humanized mouse ” refers to immunocomprimised mice engrafted with human haematopoietic stem cells or tissues , or mice that transgenically express human genes . “ inbreeding ” refers the mating of closely related individuals or of individuals having closely similar genetic constitutions . “ backcross ” refers to mating the crossbred offspring of a two - way crossback to one of the parent breeds . one aspect of the instant invention is a transgenic animal model whose genome comprising a nucleic acid construct comprising as least one transgene linked to a promoter effective for expression of human leukocyte antigen dr4 and / or human costimulatory molecules ( cd80 ), which develops a functional human immune system upon infusion of human hla - matched hematopoietic stem cells . in one embodiment , one human gene was inserted into the genome of a genetically - altered mouse already containing a human gene , thereby producing a multi - transgenic mouse . another aspect of the instant invention is directed to a method for producing a multi - transgenic animal in general . typically , transgenic mice are generated by microinjecting a foreign gene into fertilized eggs isolated from a normal , nontransgenic mouse . in the instant invention , it has been shown that it is possible to create a mouse expressing additional human transgenes by starting with a mouse that is already transgenic . that is , single - cell embryos ( fertilized eggs ) from an existing transgenic mouse have been harvested and additional transgene dna fragments have been microinjected into the cells . the results shown herein demonstrate that existing fertilized eggs from transgenic mice can withstand the microinjection process to successfully produce a multi - transgenic mouse . two mouse strains were genetically modified to sustain the development of human hematopoietic system upon infusion of human stem cells . these strains are : strain # 1 : nod . rag1ko . il2rγcko mice expressing hla - dr4 , thereafter referred as drag mice . strain # 2 : hla - dr4 tg , hcd80 tg , rag1ko , il2rγko ( nod background ), thereafter referred as drag80 mice . other mouse strains obtained during from process for generation of above strains are : strain # 3 : hla - dr4 tg , abb ko , rag2 ko ( c57bl / 6 background ). strain # 4 : hla - dr4 tg , hcd80 tg , abb ko , rag2 ko ( c57bl / 6 background ) c57bl / 6 mice expressing transgenically hla - dr4 ( hla dr * 0401 / i - e d ) molecules under mouse i - e d promoter ( line 4149 ) ( taconic farms , inc ., new york ), and rag2 ko c57bl / 6 mice ( taconic farms , inc ., new york ) are used as parental ( p ) mice to generate f1 mice that are heterozygous for all target genes of the parent mouse ( hla - dr4 , abb ko , and rag2 ) ( fig1 ). f1 mice are then intercrossed to generate f2 mice that were screened for expression of hla - dr4 tg and ko mutations for rag2 and abb loci . screening of mice was performed by facs analysis using specific antibodies and confirmed by pcr . as shown in fig1 , panel b , f2 mice that expressing hla - dr4 molecules , and ko for abb and rag2 genes were selected . this novel mouse strain f2 ( hla - dr4 tg , abb ko , rag2ko in c57bl / 6 background ) is currently maintained at the wrair vetmed facility . step 2 . generation of hla - dr4 tg , hcd80 tg , abb ko , rag2 ko mice in c57bl / 6 background ( m3 ) the c57bl / 6 mouse expressing transgenically hla - dr4 and hcd80 molecules , and showing knock out mutation of the abb , rag2 loci ( m3 ) is generated by microinjection of rip - hcd8 dna into fertilized eggs of f2 mice ( hla - dr4 tg , abb ko , rag2 ko in c57bl / 6 background ) ( fig2 ). genetic construction of rip - hcd80 : the gene encoding for human cd80 ( hcd80 ) was cloned by rt - pcr from total rna extracted from boleth ( human b lymphoblastoma ) cells . the genomic region of rat insulin promoter ( rip ) was cloned by pcr from dna extracted of splenic cells of rip - ha tg mice ( a gift from dr . harald von boehmer at inserm , france ) ( 11 ). primers used for cloning of hcd80 gene and rip contained restriction sequences that allowed the assembling of rip and hcd80 in frame . to assess the structural integrity and the ability of the chimeric rip - hcd80 genetic construct to express hcd80 protein , the rip - hcd80 genetic construct was cloned in a pcdna3 vector , and used to transfect btc - 6 mouse insulinoma cells . fig2 a demonstrated hcd80 expression on transfected cells . generation of hla - dr4 tg , hcd80 tg , abb ko , rag2 ko mice : the rip - hcd80 construct was introduced into fertilized eggs of hla - dr4 tg , abb ko , rag2 ko mice ( f2 ). the developed embryos were implanted in uterus of surrogate female dams , using standard procedures at the mouse genetics core , mount sinai school of medicine , new york , n . y . progeny was screened by pcr using specific primers for the rip - cd80 genetic construct ( fig2 b ). expression of hcd80 in pancreatic beta - cells from transgenic littermates was assessed by immunohistochemistry using anti - hcd80 abs . the results were shown in fig2 c , right panel demonstrated expression of hcd80 tg in pancreatic beta - cells . this new mouse strain m3 ( hla - dr4 tg , hcd80 tg , abb ko , rag2 ko ) is currently maintained at wrair vetmed facility . step 3 . generation of drag mice ( hla - dr4 tg , rag1ko , il2γ ko in nod background ) and drag80 mice ( hla - dr4 tg , hcd80 , rag1ko , il2rγko in nod background ) the drag and drag80 mouse strain were generated by inbreeding the hla - dr4 and hcd80 tg from the f2 / m3 mouse strain into the nod / rag1 background ( fig3 a ), followed by introduction of the il2rγ ko mutation into their genetic background ( fig3 b ) fig3 a is a schematic representation showing the inbreeding the hcd80 tg into the nod background . c57bl / 6 mice expressing transgenically hla - dr4 molecules ( f2 ) or c57bl / 6 mice expressing transgenically hla - dr4 and hcd80 molecules , and with knock out mutation at abb , and rag2 loci were backcrossed with rag1ko mice in nod background ( the jackson laboratories , bar harbor , me .) for twelve generations . litters from each generation ( f1 - f12 ) were screened for mice were screened for hla - dr4 using specific primer sets ( seq id nos . 1 - 4 ) or hcd80 tgs , and rag1 ko mutations . rag1 ko litters were identified by the absence of cd3 + t cells upon staining of peripheral blood cells with anti - mouse cd3 ( beckton dickinson ™, san jose ) the level of inbreeding of f12 mice in the nod background was 100 %, as determined by microsatellite analysis ( mappairs , diversified biopharma solutions inc ., loma linda , calif .) ( fig3 a , lower panel ). f12 mice showed complete microsatellite identity with nod for the 36 microsatellite regions . next , f12 : hla - dr4 tg , hcd80 tg , rag1 ko ( nod ) and f12 : hla - dr4 tg , rag1 ko ( nod ) male mice were crossed with il2rγko , rag1ko ( nod ) female mice ( the jackson laboratories , bar harbor , me .) ( fig3 b ) and the male progeny was screened for hla - dr4 transgene ( the il2rγ gene is located in the x chromosome ). positive males were crossed with nod . rag1 ko . il2rγko females and all progeny were screened via pcr for hla - dr4 and hcd80 tgs and il2rγko mutations ( fig3 b ). mice having the genotype hla - dr4 tg , rag1ko , il2rγ ko ( strain # 1 , drag ), and mice having the genotype hla - dr4 tg , hcd80 tg , rag1ko , il2rγko ( strain # 2 , drag80 ) were selected . these new mouse strains are currently maintained at the wrair vetmed facility . the newly - developed humanized mice drag ( hla - dr4 tg , rag1 ko , il2rγko and drag80 ( hla - dr4 tg , hcd80 , rag1ko , il2r ko drag80 ) are superior than the current humanized mouse strains ( nod - scid ko ; nod - scid ko , il2rγ ko ; nod - rag1 ko , nod - rag1 ko il2rγ ko ; balb / c - rag1ko ; and balb / c - rag1 ko , il2r il2rγ ko ). they are capable of : 1 ) providing high level of human t cell reconstitution and frequencies of cd4 t and cd8 t cell subsets ( fig4 ) 2 ) allowing development of human regulatory cd4 + foxp3 + t cells ( fig6 ) 3 ) allowing development of serum levels of human igm and igg comparable to human blood ( fig6 c , fig7 ) 4 ) allowing development of human igg1 , igg2 , igg3 , igg4 subclasses ( fig8 ) and 5 ) providing the ability to elicit specific igg antibodies upon vaccination ( fig9 ) to test the functionality of the new strain of transgenic mouse , mice were infused with human hematopoietic stem cells ( hsc ). umbilical cord blood ( ucb ) were purchased from allcells ( emeryville , calif .) and promocell ( heidlbery , germany ). screening were carried out using drb1 * 04 ssp unitray ( invitrogen ®, san diego , calif .). hla - drb1 * 04 - positive cord bloods were enriched for cd 34 + stem cells using easy sep , human progenitor cell enrichment kit ( stemcell technologies , vancouver , bc ). prior to enrichment , the frequency of cd 34 + cells in ucbs were 1 . 0 ± 5 % and after enrichment 62 . 0 ± 8 . 6 %. 4 - 6 week old mice were irradiated at ( 350 - 450 rads ), and injected intravenously with 40 , 000 - 80 , 000 human hematopoietic stem cells . the mice tested were drag mice ( hla - dr4 tg , rag1ko , il2rγ ko in nod background ). as control , rag1ko , il2rg ko littermates ( negative for hla - dr4 and hcd80 tgs ) were used . the control mice are identical to the commercial strain nod - rag1 ko , il2rγ ko . their use allowed a strict comparison between the control and the test mouse models . previous studies demonstrated no significant difference between nod - rag1 ko , il2r littermates and the other commercial strains in terms of development of a human immune system ( 3 , 4 ). higher level of human t cell reconstitution and frequencies of cd4 t and cd8 t cell subsets in drag mice as compared to control mice . pre - t cells derived from differentiation of hematopoietic stem cells in the bone marrow migrate to the thymus to undergo either positive ( survival ) or negative ( deletion ) selection upon recognition of self - peptides presented by thymic stromal cells in the context of mhc molecules . this is a physiological process aimed at preventing autoimmunity , by means of elimination of t cells that can react against self - antigens . t cell precursors in thymus that are positively selected receive survival signals that enable them to migrate and repopulate peripheral lymphoid organs ( 12 ). current humanized mouse models do not express human mhc ( hla ) molecules and consequently the differentiation of t cells is thought to occur extra - thymically . extra - thymic differentiation of human t cells in the current humanized mouse models may thus account for poor human t cell development and function , as it has been widely reported ( 2 - 9 ). transgenic mouse described in the application ( drag mice ) express transgenically hla - dr4 molecules and consequently they are expected to allow thymic differentiation of t cell precursors derived from hla - matched hhscs . to test this hypothesis , groups of drag mice and control mice were injected with human hsc of hla - dr4 haplotype , and they were followed for development of human t cells in peripheral blood by facs . blood drawn for the tail vein was collected with heparin - coated capillary tube . erythrocytes were lysed using ack lysis buffer and white blook cells were incubated with fcblock and strined with anti - human cd3 , cd4 , cd8 and cd19 abs ( bd ® biosciences ) and washed with pbs / 1 % bsa / 0 . 1 % na azide and analyzed by bacs on mononuclear fsc / ssc scatter . human peripheral blood from adult healthy volunteers was used as controls and was collected and treated as above . the analyses were performed at various time points upon human hsc infusion . to assess the function of human t cells developed by drag , groups of mice ( both drag and control ) were euthanized and splenic t cells adjusted to 3 × 105 from mice or pbmcs ( 3 × 10 5 ) were stimulated with anti - human cd3 / cd28 - coated magnetic beads . stimulation with cd3 / cd28 antibodies is a common approach to test selectively the function of t cells . cytokines in supernantants were measured by luminex ( invitrogen ® ca ). spleen cells adjusted to 3 × 10 6 cells / ml were depleted of cd4 t cells , cd8 t cells , or both cell subsets with antibody - coated magnetic beads ( dynabeads , invitrogen ). the efficiency of cell depletion was 0 . 95 % as measured by facs using cd3 , cd4 , and cd8 abs . equal volumes ( 0 . 1 ml ) of cell depleted cell suspensions or corresponding volumes of positively isolated cells on the magnetic beads were stimulated with pma / iomycin for 24 h and cytokine secretion in cell culture supernatants were measured by ( invitrogen ® ca . as illustrated in fig4 , panel a , facs analysis of splenic , thymic , and bone marrow cells from naive ( non - hsc infused ) drag and control mice ( n = 3 ) stained with hla - dr antibodies . groups of drag ( n = 15 ) and control ( n = 11 ) mice that were infused intravenously with 80 , 000 enriched - hsc ( cd34 +) cells from umbilical cord blood of hla - dr4 (* 0401 ) newborns were examined in peripheral blood by facs using anti - human cd3 , cd4 , and cd8 . panel b shows percentage of human t cell reconstituted mice at various time points post - infusion of hsc . fisher exact test indicated that the rate of human t - cell reconstitution in drag mice ( 14 out of 15 ) was significantly superior to that in control mice ( 4 out of 11 ) ( p = 0 . 0025 ). in addition , mann - whitney test indicated that the frequency of human t cells in blood of drag mice was significantly higher than that in control mice ( p = 0 . 0044 ). the cut - off for positive human cd3 + t cells was calculated as three times the standard deviation over the background levels of cells from naive ( non - hsc infused ) drag mice that were stained with anti - human cd3 ( 0 . 17 %). ranges are indicated over the plots . thus , the results demonstrated that expression of hla - cd4 molecules in drag mice promoted development of human t cells . panel d shows frequency of human cd4 and cd8 t cells in blood of t cell reconstituted drag mice ( n = 14 ) and control mice ( n = 4 ) at 25 weeks post - infusion of hsc ( upper two panels ). lower histograms shows frequencies of cd4 and cd8 t cells in human blood ( n = 2 ). p values were calculated using the mann - whitney test . t cell - reconstituted drag ( n = 14 ) and control mice ( n = 4 ) developed both cd4 and cd8 t cells in blood , but the frequency of cd4 t cells was significantly higher in drag mice ( fig4 d , p = 0 . 0032 ). of note , the frequency of blood circulating cd4 t cells in drag mice was comparable to that of human volunteers ( fig4 d , upper and lower panels ). on the other hand , the frequency of human cd8 t cells in blood of both groups of mice was similar ( fig4 d , p = 0 . 36 ) and at the same time lower than that in blood of human volunteers . t cell - reconstituted drag ( n = 14 ) and control mice ( n = 4 ) developed both cd4 and cd8 t cells in blood , but the frequency of cd4 t cells was significantly higher in drag mice ( fig4 d , p = 0 . 0032 ). of note , the frequency of blood circulating cd4 t cells in drag mice was comparable to that of human volunteers ( fig4 d , upper and lower panels ). on the other hand , the frequency of human cd8 t cells in blood of both groups of mice was similar ( fig4 d , p = 0 . 36 ) and at the same time lower than that in blood of human volunteers . as illustrated in fig5 , the t cells from drag mice , but not t cells from control mice , responded vigorously to cd3 / cd28 stimulation , as measured by the level of cytokines secreted in cell culture supernatants . the lack of t cell response in control mice is in agreement with previous studies indicating that t cells from stem cell - reconstituted nod - scid respond poorly to cd3 / cd28 stimulation in vitro ( 3 ). on the other hand , t cell response of drag mice was similar to that of t cells from human blood . this result demonstrated that t cells from drag mice are fully functional , whereas the t cells from control mice are impaired in function . regulatory t cells , particularly those from the cd4 + foxp3 + subset ( tregs ), are an important compartment of the immune system , whose function is to maintain self - tolerance in periphery and to down - regulate aggressive immune responses to pathogens once the infection has been cleared . development of tregs by the current humanized mouse model has not been reported in the literature . however , as illustrated in fig6 , at 6 months after infusion of hsc , a much higher frequency ( p = 0 . 021 ) of human cd4 + foxp3 + tregs was detected in spleens of drag mice as compared to control mice . thus , our results indicated that a major regulatory cell compartment ( cd4 + foxp3 + tregs ) was also reconstituted in drag mice upon infusion of hla - dr - matched hsc . dendritic cells ( dcs ) are critical components of the innate immune system due to their potent ability to process antigen and present immunogenic peptides to t cells for activation . the frequency of dcs in human spleen has been estimated as 2 . 3 % of the mononuclear population [ as illustrated in table 2 , both drag and control mice developed human dcs ( cd 11c + cd 19 −). the frequency of human dcs in spleens of drag mice ( 2 . 9 %) was slightly higher than that in control mice ( 1 . 5 %), though it did not reach statistical significance . natural killer ( nk ) cells are characterized by expression of cd 16 , an immunoglobulin fc receptor that allows them to eliminate target cells through a process known as “ antibodydependent - cell - cytotoxicity ” or adcc . the frequency of nk cells in human spleen has been estimated as 15 %. as shown in table 2 , the numbers of human nk cells in both drag and control mice were insignificant ( 0 . 05 % vs . 0 . 07 %), which indicated that expression of hla - dr4 did not improve development of human nk cells . in aggregate , these results demonstrated that expression of hla - dr4 molecules in drag mice favored engraftment of hsc in bone marrow , homing and development of bone - marrow derived human t cell precursors in thymus , and proficient repopulation of peripheral lymphoid organs with mature cd4 and cd8 t cells . expression of hla - dr4 molecules however did not improve development of human nk cells . the longitudinal facs analysis in blood of drag and control mice using anti - human cd 19 revealed that the rate of human b - cell reconstitution was similar between the two strains . thus , 14 out of 15 ( 93 . 3 %) drag mice and 9 out of 11 ( 81 . 8 %) control mice were reconstituted with human b ( cd 19 + ) cells by 10 weeks after hsc infusion ( fig7 a ). although not statistically significant ( p = 0 . 31 ), 25 weeks after infusion of hsc the overall frequency of blood circulating human b cells in drag mice was slightly lower than in control mice ( fig7 b ). noteworthy , spleens of drag mice contained similar numbers of human b cells as compared to control mice ( b cell frequency : 47 . 9 %, range [ 45 . 9 - 50 ] vs . 60 % [ 55 - 65 . 9 ]; total number of b cells / spleen : 5 . 5610661 . 95 vs . 6 . 35610662 . 75 , respectively ). this indicated that expression of hla - dr4 molecules in drag mice did not improve significantly the reconstitution with human b cells in the peripheral blood on in the spleen as compared to control mice . the function of b cells is to secrete antibodies ( immunoglobulins ) that are mainly found in blood and body fluids . the function of immunoglobulins is to counterattack pathogens such as viruses , bacteria , and parasites as well as to control the homeostatic growth of commensal bacteria in the intestinal tract ( reviewed in 14 ). in mammalians there are five classes of immunoglobulins , namely igd , igm , igg , iga , and ige . in physiological conditions , igd is found only on the surface of b cells . upon antigen encountering , b cells undergo a rearrangement at the dna level to switch antibody class from igd to a secretory form of igm . fully functional b cells then undergo a further dna rearrangement to switch class from igm to either igg , iga , or ige . the igg class is by far the most abundant immunoglobulin in blood . thus , we investigated the kinetics of human immunoglobulin reconstitution in blood of drag mice . as illustrated in fig7 c , the levels of human igm in plasma from b cell reconstituted drag mice ( n = 14 ) were significantly higher than those from reconstituted control mice ( n = 9 ) ( p = 0 . 0002 ). in addition , the drag mice were able to reconstitute plasma igg whilst control mice failed to do so ( fig7 d ). the results indicated that the mechanism of immunoglobulin class switch was preserved in drag mouse . the human identity of ig heavy chain and light chains in blood of drag mice was confirmed by immunoelectrophoresis ( fig8 a ). human igg class comprises four different subclasses that differ in structure and biological functions , namely ( i ) ability to cross placenta , ( ii ) ability to activate the complement cascade and ( iii ) affinity binding to fc receptors expressed on phagocytic cells . as illustrated in fig8 c , drag mice reconstituted plasma levels of all human igg subclasses , with igg2 as the most prevalent followed by igg1 , igg3 and igg4 , whereas in the human blood the most prevalent subclass is igg1 , followed by igg2 , igg3 and igg4 . in addition , 12 out of 14 ( 85 . 7 %) drag mice reconstituted plasma levels of human iga ( fig8 d ) and 7 out of 14 ( 50 %) reconstituted plasma levels of human ige ( fig8 e ). in contrast , control mice did not show detectable levels of human iga or ige ( fig8 d & amp ; e ). the results demonstrated that human b cells developed by drag were functional as they secreted all human immunoglobulin classes , whereas those from control mice were impaired in their ability to undergo class switching and secreted only igm . a major function of the immune system is to protect against infections by eliciting specific antibodies that bind to and eliminate pathogens . tetanus toxoid , a formalin - detoxified form of clostridium tetani toxin adjuvanted in alumn , is a licensed human vaccine that induces neutralizing anti - tetanus toxin ( tt ) igg antibodies . we investigated whether human b cells from drag mice could elicit specific humoral responses upon immunization with tt vaccine . groups of hsc - infused drag and control mice were immunized with 1 flocculation unit of tt vaccine ( sanofi pasteur ) by the intramuscular route , and the titers of anti - tt igg abs were measured by elisa 14 days later . as illustrated in fig9 , immunized - drag mice were able to elicit anti - tt igg antibodies , whereas the control mice failed to do so . these results clearly indicated that human b cells developed by drag mice are fully functional . in addition , the results demonstrated that the “ human immune system ” developed by drag mice is superior to that developed by blt mice , currently referred as the golden standard for humanized mice , since the blt mice cannot elicit specific igg antibodies upon tt immunization . experiments similar to those contained in example 2 are conducted to test the functionality of drag80 . 1 shultz , et al . humanized mice in translational biomedical research , nat . rev . immunol . 7 ( 2007 ) 2 heuts et al . alloreactivity but failure to reject islet transplants by humanized balb / c rag2gc mice . scand j . immunol . 71 : 83 - 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