**Development of Humanized Mice to Study Asexual Blood Stage**  *Plasmodium falciparum* **Infection**

R.K. Tyagi1,2\*, N.K. Garg3**,** T. Sahu4 and P. Prabha5 *1Department of Global Health, College of Public Health, University of South Florida, Tampa, Florida, 2Malaria Vaccine Development Laboratory, Institute Pasteur Paris, 3Shantha Biotechnics Ltd., (A Part of Sanofi Aventis Group), Hyderabad, AP, 4National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD, 5Gene Regulation Laboratory, School Of Biotechnology, Jawaharlal Nehru University, New Delhi, 1,4USA 2France 3,5India* 

#### **1. Introduction**

90 Malaria Parasites

Persson, K.E., Lee, C.T., Marsh, K. & Beeson, J.G. (2006). Development and optimization of

Ridley, R.G. (2002). Medical need, scientific opportunity and the drive for antimalarial

Snow, R.W., Guerra, C.A., Noor, A.M., Myint, H.Y. & Hay, S.I. (2005). The global

Trager, W. & Jensen, J.B. (1997). Continuous culture of *Plasmodium falciparum*: its impact on

Webster, H.K., Boudreau, E.F., Pavanand, K., Yongvanitchit, K. & Pang, L.W. (1985).

Yayon, A., Vande Waa, J.A., Yayon, M., Geary, T.G. & Jensen, J.B. (1983). Stage-dependent

inhibitory antibodies. *J. Clin. Microbiol*., Vol. 44, pp. 1665-1673,

malaria research. *Int. J. Parasitol.*, Vol. 27, pp. 989-1006,

drugs. *Nature*, Vol. 415, pp. 686-693,

pp. 214-217,

642-647,

high-throughput methods to measure *Plasmodium falciparum*-specific growth

distribution of clinical episodes of *Plasmodium falciparum* malaria. *Nature,* Vol. 434,

Antimalarial drug susceptibility testing of *Plasmodium falciparum* in Thailand using a microdilution radioisotope method. *Am. J. Trop. Med. Hyg.*, Vol. 34, pp. 228-235, Willet, G.P. & Canfield, C.J. (1984). *Plasmodium falciparum*: continuous cultivation of

erythrocyte stages in plasma-free culture medium. *Exp. Parasitol*., Vol. 57, pp. 76-80,

effects of chloroquine on *Plasmodium falciparum* in vitro. *J. Protozool.,* Vol. 30, pp.

Infectious diseases continue to heavily burden our global society. Endemic and epidemic malaria results in severe disease in an estimated half-a-billion people each year, and causes over 1.5 million deaths annually. Although progress has been made in the prevention and treatment of *falciparum* malaria infections, more effective, tolerable and affordable therapies are urgently needed. This deadly parasite displays unique human tropism, and the development of novel intervention strategies have been hampered by the lack of robust, cost effective, and predictive animal models that accurately reproduce the hallmark of human infections. While rodents and non-human primates have been employed in biomedical research and drug/vaccine development, they often do not yield reliable preclinical results that translate into effective human treatments. "Humanized" mice have recently emerged as powerful tools in the investigation of human diseases (Legrand et al., 2006; Manz, 2007; Shultz et al., 2007). These are amenable animal models transplanted with various kinds of human cells and tissues (and/or equipped with human transgenes) that may be ideally suited for direct investigation of human infectious agents such as malaria. Despite the challenges, humanized mouse technology has made rapid progress over the last few years, and it is now possible to achieve significant levels of human chimerism in various hosts, organ/tissues, particularly the immune systems, liver and muscles. Such humanized mice provide a new opportunity to perform preclinical studies of intractable human malaria

<sup>\*</sup>Corresponding Author

Asexual Blood Stage Malaria in a Humanized Mouse Model 93

The second breakthrough was the development of immunodeficient non-obese diabetic (NOD)-*scid* mice (Shultz et al., 1995). Crossing the *scid* mutation onto different strain backgrounds led to the observation that NOD-*scid* mice supported higher levels of engraftment with human PBMCs than did any of the other strains that were tested, including C3H/HeJ-*scid* and C57BL/6-*scid* mice (Hesselton et al., 1995). Furthermore, it was observed that NK-cell activity, which is one of the main impediments to the engraftment of human haematopoietic cells (Christianson et al., 1996), was lower in NOD-*scid* mice than in CB17-*scid* mice (Shultz et al., 1995). NOD-*scid* mice also have additional defects in innate immunity that allow higher levels of human PBMC (Hesselton et al., 1995) and HSC (Lowry et al., 1996; Pflumio et al., 1996) engraftment. Incremental improvements in the extent of human-cell engraftment as a result of the development of new genetic variations of NOD*scid* mice occurred over the following 10 years (TIMELINE), but the use of humanized NOD*scid* mice as a model for human immunity remains limited by their relatively short life span, and the residual activity of NK cells and other components of innate immunity, which

The third breakthrough was the humanization of immunodeficient mice homozygous for targeted mutations at the interleukin-2 receptor (IL-2R) γ-chain locus (*Il2rg*; also known as the common cytokine-receptor γ-chain, γc) (Ishikawa et al., 2005; Ito et al., 2002; Shultz et al., 2005;Traggiai et al., 2004). These mice support greatly increased engraftment of human tissues, HSCs and PBMCs compared with all previously developed immunodeficient humanized mouse models. The IL-2R γ-chain is a crucial component of the high-affinity receptors for IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21, and it is required for signaling through these receptors (Sugamura et al., 1996). The absence of the IL-2R γ-chain leads to severe impairments in T- and B-cell development and function, and completely prevents NK-cell

After an obtainment of proliferation of *Theileria sergenti* in scid mouse grafted with bovine-RBC (Tsuji et al., 1992), several studies raised the possibility of obtaining *P. falciparum* in immune deficient mice grafted with huRBCs (Badell et al., 2000; Moore et al., 1995; Tsuji et al., 1995). The researchers used different strategies to improve parasite survival considering the fast clearance of the parasite in few hours after being inoculated. Moore *et al* tried to adapt parasite *in vitro* partially in the serum of scid mice in place of human serum before being inoculated into the mouse (Moore et al., 1995). A concentration of mouse serum higher than 5% was found toxic for the parasite in culture. In this study, the NOD/scid mice were intraperitoneally infected with 1% adapted parasite followed by 1 ml huRBCs injection everyday. The parasite survival lasting up to 10 days was achieved with this protocol. Although splenectomized mice had shown an improvement of parasite growth (>15 days), variations in the parasitaemia from one mouse to another was detected. Another research group (Badell et al., 1995) developed another strategy of *P. falciparum* survival in the immunodeficient mice. They also noted that parasite inoculated through i.p. route into the scid mouse was eliminated in few hours from the circulation. Moreover, mouse serum added in *in vitro* cultures did not show any toxicity to parasite growth and parasite survival was observed even at 10% serum. This experiment further confirmed the important role of

impedes the engraftment of the human lymphoid compartment.

development (Cao et al., 1995; DiSanto et al., 1995; Ohbo et al., 1996).

*P. falciparum* **in humanized mice** 

**3. Optimization of immunosuppression protocols: Engraftment of** 

parasite. However, the relevance of *Plasmodium falciparum*-infected humanized mice for malaria studies has been questioned because of the low percentage of mice in which the parasite develops. Malaria caused by *Plasmodium falciparum* is difficult to model in the laboratory because of the specificity of this parasite for its human host, the complexity of its life cycle and the substantial diversity of parasite strains. Consequently, most experimental in vivo studies on malaria have heavily relied on different combinations of various murine strains and Plasmodium spp. of rodent (Carlton et al., 2001; Hernandez-Valladares et al., 2005) but biological differences between parasite species remain a major limitation. For example, there are many indications that in human cerebral malaria, the preferential sequestration of parasitized erythrocytes in the brain capillaries is the central precipitating step (Medana and Turner, 2006), whereas this phenomenon is much less evident in rodent models (Lou et al., 2001).

Limitations of experimental models have also hampered the evaluation of the impact of new drugs or vaccines prior to clinical trials. During the preclinical screening of a new drug, its activity has to be evaluated first against the development of *P. falciparum* in vitro, then against the infection of a rodent Plasmodium in a mouse model and, finally, against *P. falciparum* infection in a monkey model (Fidock et al., 2004) or in a humanized mouse model.

#### **1.1 Perspectives**

The stability, reproducibility and long-standing tendency of parasitaemia in the developed humanized model based on NOD/SCID/IL2Rγ-null mouse (NSG), a mouse strain with profoundly deficient adaptive and innate immunity (NSG-IV model) might be validated by selecting artesunate resistant mutants of *P. falciparum* through prolonged exposure of the parasite to increasing levels of the drug.

#### **2. The development of humanized mice**

Advances in the ability to generate humanized mice have depended on a systematic progression of genetic modifications to develop immunodeficient host mice. Three main breakthroughs have occurred in this field (TIMELINE). First, the discovery of the *Prkdcscid*  (protein kinase, DNA activated, catalytic polypeptide; severe combined immunodeficiency, abbreviated *scid*) mutation in CB17 mice (Bosma et al., 1983) was soon followed by the observation that human PBMCs (Mosier et al., 1988), fetal haematopoietic tissues (McCune et al., 1988) and HSCs (Lapidot et al., 1992) could engraft in these mice. However, engraftment occurred at only a very low level, and the engrafted human cells failed to generate a functional human immune system. The limitations impeding human-cell engraftment in CB17-*scid* mice include the spontaneous generation of mouse T and B cells during aging (known as leakiness) and high levels of host natural killer (NK)-cell and other innate immune activity, which limit the engraftment of the human hematopoietic compartment (Greiner et al., 1998). The *scid* mutation also results in defective DNA repair and, consequently, an increase in radio sensitivity. Targeted mutations at the recombinationactivating gene 1 (*Rag1*) and *Rag2* loci prevent mature T- and B-cell development in the mice but do not cause leakiness or radio sensitivity. However, these mice retained high levels of NK-cell activity and had limited engraftment of human HSCs (Greiner et al., 1998; Mombaerts et al., 1992; Shinkai et al., 1992).

parasite. However, the relevance of *Plasmodium falciparum*-infected humanized mice for malaria studies has been questioned because of the low percentage of mice in which the parasite develops. Malaria caused by *Plasmodium falciparum* is difficult to model in the laboratory because of the specificity of this parasite for its human host, the complexity of its life cycle and the substantial diversity of parasite strains. Consequently, most experimental in vivo studies on malaria have heavily relied on different combinations of various murine strains and Plasmodium spp. of rodent (Carlton et al., 2001; Hernandez-Valladares et al., 2005) but biological differences between parasite species remain a major limitation. For example, there are many indications that in human cerebral malaria, the preferential sequestration of parasitized erythrocytes in the brain capillaries is the central precipitating step (Medana and Turner, 2006), whereas this phenomenon is much less evident in rodent

Limitations of experimental models have also hampered the evaluation of the impact of new drugs or vaccines prior to clinical trials. During the preclinical screening of a new drug, its activity has to be evaluated first against the development of *P. falciparum* in vitro, then against the infection of a rodent Plasmodium in a mouse model and, finally, against *P. falciparum* infection in a monkey model (Fidock et al., 2004) or in a humanized mouse model.

The stability, reproducibility and long-standing tendency of parasitaemia in the developed humanized model based on NOD/SCID/IL2Rγ-null mouse (NSG), a mouse strain with profoundly deficient adaptive and innate immunity (NSG-IV model) might be validated by selecting artesunate resistant mutants of *P. falciparum* through prolonged exposure of the

Advances in the ability to generate humanized mice have depended on a systematic progression of genetic modifications to develop immunodeficient host mice. Three main breakthroughs have occurred in this field (TIMELINE). First, the discovery of the *Prkdcscid*  (protein kinase, DNA activated, catalytic polypeptide; severe combined immunodeficiency, abbreviated *scid*) mutation in CB17 mice (Bosma et al., 1983) was soon followed by the observation that human PBMCs (Mosier et al., 1988), fetal haematopoietic tissues (McCune et al., 1988) and HSCs (Lapidot et al., 1992) could engraft in these mice. However, engraftment occurred at only a very low level, and the engrafted human cells failed to generate a functional human immune system. The limitations impeding human-cell engraftment in CB17-*scid* mice include the spontaneous generation of mouse T and B cells during aging (known as leakiness) and high levels of host natural killer (NK)-cell and other innate immune activity, which limit the engraftment of the human hematopoietic compartment (Greiner et al., 1998). The *scid* mutation also results in defective DNA repair and, consequently, an increase in radio sensitivity. Targeted mutations at the recombinationactivating gene 1 (*Rag1*) and *Rag2* loci prevent mature T- and B-cell development in the mice but do not cause leakiness or radio sensitivity. However, these mice retained high levels of NK-cell activity and had limited engraftment of human HSCs (Greiner et al., 1998;

models (Lou et al., 2001).

**1.1 Perspectives**

parasite to increasing levels of the drug.

**2. The development of humanized mice** 

Mombaerts et al., 1992; Shinkai et al., 1992).

The second breakthrough was the development of immunodeficient non-obese diabetic (NOD)-*scid* mice (Shultz et al., 1995). Crossing the *scid* mutation onto different strain backgrounds led to the observation that NOD-*scid* mice supported higher levels of engraftment with human PBMCs than did any of the other strains that were tested, including C3H/HeJ-*scid* and C57BL/6-*scid* mice (Hesselton et al., 1995). Furthermore, it was observed that NK-cell activity, which is one of the main impediments to the engraftment of human haematopoietic cells (Christianson et al., 1996), was lower in NOD-*scid* mice than in CB17-*scid* mice (Shultz et al., 1995). NOD-*scid* mice also have additional defects in innate immunity that allow higher levels of human PBMC (Hesselton et al., 1995) and HSC (Lowry et al., 1996; Pflumio et al., 1996) engraftment. Incremental improvements in the extent of human-cell engraftment as a result of the development of new genetic variations of NOD*scid* mice occurred over the following 10 years (TIMELINE), but the use of humanized NOD*scid* mice as a model for human immunity remains limited by their relatively short life span, and the residual activity of NK cells and other components of innate immunity, which impedes the engraftment of the human lymphoid compartment.

The third breakthrough was the humanization of immunodeficient mice homozygous for targeted mutations at the interleukin-2 receptor (IL-2R) γ-chain locus (*Il2rg*; also known as the common cytokine-receptor γ-chain, γc) (Ishikawa et al., 2005; Ito et al., 2002; Shultz et al., 2005;Traggiai et al., 2004). These mice support greatly increased engraftment of human tissues, HSCs and PBMCs compared with all previously developed immunodeficient humanized mouse models. The IL-2R γ-chain is a crucial component of the high-affinity receptors for IL-2, IL-4, IL-7, IL-9, IL-15 and IL-21, and it is required for signaling through these receptors (Sugamura et al., 1996). The absence of the IL-2R γ-chain leads to severe impairments in T- and B-cell development and function, and completely prevents NK-cell development (Cao et al., 1995; DiSanto et al., 1995; Ohbo et al., 1996).

## **3. Optimization of immunosuppression protocols: Engraftment of**  *P. falciparum* **in humanized mice**

After an obtainment of proliferation of *Theileria sergenti* in scid mouse grafted with bovine-RBC (Tsuji et al., 1992), several studies raised the possibility of obtaining *P. falciparum* in immune deficient mice grafted with huRBCs (Badell et al., 2000; Moore et al., 1995; Tsuji et al., 1995). The researchers used different strategies to improve parasite survival considering the fast clearance of the parasite in few hours after being inoculated. Moore *et al* tried to adapt parasite *in vitro* partially in the serum of scid mice in place of human serum before being inoculated into the mouse (Moore et al., 1995). A concentration of mouse serum higher than 5% was found toxic for the parasite in culture. In this study, the NOD/scid mice were intraperitoneally infected with 1% adapted parasite followed by 1 ml huRBCs injection everyday. The parasite survival lasting up to 10 days was achieved with this protocol. Although splenectomized mice had shown an improvement of parasite growth (>15 days), variations in the parasitaemia from one mouse to another was detected. Another research group (Badell et al., 1995) developed another strategy of *P. falciparum* survival in the immunodeficient mice. They also noted that parasite inoculated through i.p. route into the scid mouse was eliminated in few hours from the circulation. Moreover, mouse serum added in *in vitro* cultures did not show any toxicity to parasite growth and parasite survival was observed even at 10% serum. This experiment further confirmed the important role of

Asexual Blood Stage Malaria in a Humanized Mouse Model 95

Time line: Important events in the development of humanized mice

(2002-2005). BALB/c-Rag2-/-, Il2rg-/-, NOD-shiscid Il2rg-/- & NOD/LtSzscid Il2rg-/- mouse models reported for the functional human immune system after human HSC engraftment

**2002**

Preclinical ridge between mice and human

**2003+…** 

NOD-Rag1-/-Prf1- /- mice

NOD-scid β2m mice

**2000**

NOD-Rag1-/ mice

**1997**

A main barrier to achieving an improved, workable *P. falciparum* mouse model is the strong pro-inflammatory effect of the parasite itself. In humans the asexual erythrocytic stages of *P. falciparum* are known to result in a systemic inflammatory process that is responsible for many of the symptoms of the disease. A second significant practical problem with all existing models developed to date is that huRBC are injected by the intra-peritoneal (IP) route, which relies on the successful migration of huRBC into the blood stream across the peritoneum. This is a process that is not properly understood, and, therefore, prevents any rational analysis and further improvement of the model. With these limitations of current models in mind, we thought to improve the *P. falciparum* humanized mouse model, particularly in terms of control of inflammatory reactions, and reproducibility of parasitemia. We decided to address these issues by using the IV route for huRBC and parasite administration, and by investigating other means to increase control over the mouse innate immune response. The use of this IV model led us to identify, among several factors investigated, the effect of aging and that of inosine as significant in reducing inflammatory reactions, and therefore improving *P. falciparum* growth. Moreover, after using various strains of immunodeficient mice, we investigated, as others [14], the value of

(1995-1999). Il2rg-/ targeted t ti

Fig. 1. Chronological events in the development of humanized mouse model

NOD-scid mice with an increased engraftment of human PBMCs & HSCs

**1995**

Description of the CB17 scid mouse strain

**1983** 

Description of the nude mutation

**1966** 

CB17-scid mice engrafted with human fetal tissues, adult blood cells & HSCs

**1988**

Rag1- & Rag2 targeted mutations

**1992**

residual innate immune effectors of mice in the clearance of parasite. The efforts have been made to control innate immune effectors by various means such as irradiation and cyclophosphamide treatment however with limited success. This group was able to establish an immunomodulatory protocol for BXN mouse to deplete innate immune regulatory cells and could achieve an average of 0.1% parasitaemia. This immunomodulatory protocol comprised of an i.p. injection of 0.2 ml of dichloro-methylene-diphosphonate (CL2MDP) loaded liposome once per week to destroy the murine macrophages in conjunction with an administration of an anti-PMN monoclonal antibody at every 5th day to block the polymorphonuclear cells and also the injection of 1 ml of huRBCs & AB+ human serum in 1 ml of 10%-RPMI every day. Tsuji *et al*, used splenectomized scid mice and administered huRBCs by intravenous route from day 1 to 5. As from day 1 to 4 mice received an intraperitoneal injection of 500μl of human serum as well as a subcutaneous injection of an anti-mouse RBC antibody to replace murine RBC by huRBCs. Afterwards, the mouse was reconstituted with the huRBCs followed by the irradiation, and was also injected with 1.6-4% parasitized huRBCs through an intravenous route. By employing this protocol almost complete substitution of murine RBC by huRBCs was achieved (Tsuji et al., 1995). Until recently, immunomodulatory protocol (destroying macrophages with CL2MDP-lip and blocking PMN with an anti-PMN monoclonal antibody) was applied to the NOD/scid mice (Moreno Sabater et al., 2005). Moreover, a comparative study was carried out to show differences in the capabilities of *P. falciparum* survival in BXN and NOD/scid mice using the protocol with two intraperitoneal infections (i.e. primary infection on 3rd day and secondary infection on 18th day) and with the injection of immunosuppressors along with fresh huRBCs at every three days interval. In brief NOD/scid mouse allowed a better development of the parasite for 35 days with 75% infectivity compared to only 8% with BXN mice. In the BXN mouse strain the parasite could grow until day 7 post second infections followed by a gradual decrease in parasitaemia. On the contrary NOD/scid mice showed 0.25% average parasitaemia until day 17 post second infection. Therefore this protocol with NOD/scid mice showed an improvement of parasite survival (Moore et al., 1995). Interestingly, this protocol does not imply either the need of splenectomy or *in vitro* adaptation of the parasite prior to infection. Recently Inigo *et al* (Angulo-Barturen et al., 2008), developed a murine model (NOD/scidβ2m-/-) to study *falciparum*-malaria in non-myelodepleted mice grafted with human erythrocytes with considerable success in terms of parasitaemia and with 100% infectivity. They claimed to generate *in vivo* strains of *P. falciparum* able to grow reproducibly in peripheral blood of humanized mouse with out using immunosuppressors to deplete murine phagocytes. Despite the significant success with this model cumbersome administration of 1ml RBC pellet i.p. everyday and *in vitro*  adaptation of *falciparum* strains prior to infection raise questions over its ideal nature. The same group (Jimenez-Diaz et al., 2009) came up with the same protocol applied to NOD/SCIDIL-2Rγ-/- strain with slight modifications for the survival of the parasite.

Therefore to complement the descriptive analysis in humans by an experimental approach in a model, we decided to perform in *P. falciparum* NOD/SCID model a systematic and stepwise analysis of innate cell responses and inflammation mediators produced in response to the grafting of HuRBC, of *P. falciparum*, as well as to agents employed to control innate defences. The results brought new insights about the role and potency of innate defences against human xenografts, such as HuRBC, and human pathogens, such as *P. falciparum* (Arnold et al.).

residual innate immune effectors of mice in the clearance of parasite. The efforts have been made to control innate immune effectors by various means such as irradiation and cyclophosphamide treatment however with limited success. This group was able to establish an immunomodulatory protocol for BXN mouse to deplete innate immune regulatory cells and could achieve an average of 0.1% parasitaemia. This immunomodulatory protocol comprised of an i.p. injection of 0.2 ml of dichloro-methylene-diphosphonate (CL2MDP) loaded liposome once per week to destroy the murine macrophages in conjunction with an administration of an anti-PMN monoclonal antibody at every 5th day to block the polymorphonuclear cells and also the injection of 1 ml of huRBCs & AB+ human serum in 1 ml of 10%-RPMI every day. Tsuji *et al*, used splenectomized scid mice and administered huRBCs by intravenous route from day 1 to 5. As from day 1 to 4 mice received an intraperitoneal injection of 500μl of human serum as well as a subcutaneous injection of an anti-mouse RBC antibody to replace murine RBC by huRBCs. Afterwards, the mouse was reconstituted with the huRBCs followed by the irradiation, and was also injected with 1.6-4% parasitized huRBCs through an intravenous route. By employing this protocol almost complete substitution of murine RBC by huRBCs was achieved (Tsuji et al., 1995). Until recently, immunomodulatory protocol (destroying macrophages with CL2MDP-lip and blocking PMN with an anti-PMN monoclonal antibody) was applied to the NOD/scid mice (Moreno Sabater et al., 2005). Moreover, a comparative study was carried out to show differences in the capabilities of *P. falciparum* survival in BXN and NOD/scid mice using the protocol with two intraperitoneal infections (i.e. primary infection on 3rd day and secondary infection on 18th day) and with the injection of immunosuppressors along with fresh huRBCs at every three days interval. In brief NOD/scid mouse allowed a better development of the parasite for 35 days with 75% infectivity compared to only 8% with BXN mice. In the BXN mouse strain the parasite could grow until day 7 post second infections followed by a gradual decrease in parasitaemia. On the contrary NOD/scid mice showed 0.25% average parasitaemia until day 17 post second infection. Therefore this protocol with NOD/scid mice showed an improvement of parasite survival (Moore et al., 1995). Interestingly, this protocol does not imply either the need of splenectomy or *in vitro* adaptation of the parasite prior to infection. Recently Inigo *et al* (Angulo-Barturen et al., 2008), developed a murine model (NOD/scidβ2m-/-) to study *falciparum*-malaria in non-myelodepleted mice grafted with human erythrocytes with considerable success in terms of parasitaemia and with 100% infectivity. They claimed to generate *in vivo* strains of *P. falciparum* able to grow reproducibly in peripheral blood of humanized mouse with out using immunosuppressors to deplete murine phagocytes. Despite the significant success with this model cumbersome administration of 1ml RBC pellet i.p. everyday and *in vitro*  adaptation of *falciparum* strains prior to infection raise questions over its ideal nature. The same group (Jimenez-Diaz et al., 2009) came up with the same protocol applied to

NOD/SCIDIL-2Rγ-/- strain with slight modifications for the survival of the parasite.

(Arnold et al.).

Therefore to complement the descriptive analysis in humans by an experimental approach in a model, we decided to perform in *P. falciparum* NOD/SCID model a systematic and stepwise analysis of innate cell responses and inflammation mediators produced in response to the grafting of HuRBC, of *P. falciparum*, as well as to agents employed to control innate defences. The results brought new insights about the role and potency of innate defences against human xenografts, such as HuRBC, and human pathogens, such as *P. falciparum*

Fig. 1. Chronological events in the development of humanized mouse model

A main barrier to achieving an improved, workable *P. falciparum* mouse model is the strong pro-inflammatory effect of the parasite itself. In humans the asexual erythrocytic stages of *P. falciparum* are known to result in a systemic inflammatory process that is responsible for many of the symptoms of the disease. A second significant practical problem with all existing models developed to date is that huRBC are injected by the intra-peritoneal (IP) route, which relies on the successful migration of huRBC into the blood stream across the peritoneum. This is a process that is not properly understood, and, therefore, prevents any rational analysis and further improvement of the model. With these limitations of current models in mind, we thought to improve the *P. falciparum* humanized mouse model, particularly in terms of control of inflammatory reactions, and reproducibility of parasitemia. We decided to address these issues by using the IV route for huRBC and parasite administration, and by investigating other means to increase control over the mouse innate immune response. The use of this IV model led us to identify, among several factors investigated, the effect of aging and that of inosine as significant in reducing inflammatory reactions, and therefore improving *P. falciparum* growth. Moreover, after using various strains of immunodeficient mice, we investigated, as others [14], the value of

Asexual Blood Stage Malaria in a Humanized Mouse Model 97

Numerous attempts have been made to increase the success rate of the grafting of infected RBC. Un-sized dichloromethylene diphosphonate (Cl2-MDP) encapsulated in liposome (clolip) (provided by N. Van Rooijen, Amsterdam, The Netherlands) is injected through intraperitoneal (i.p.) route in order to reduce the number of tissue MP, as described previously (van Rooijen and van Kesteren-Hendrikx, 2003). The anti-PMN monoclonal antibody NIMP-R14 (Lopez et al., 1984) is purified from a hybridoma. Its activity is compared to that of two other anti-PMN monoclonal antibodies: RB6-8C5 (purified from the hybridoma) and 1A8 (BioXcell, Lebanon). The NIMP-R14 monoclonal antibody is used in all the studies, unless specified. Various agents (from Sigma) (Table 1) are used to further reduce innate immunity such as dexamethasone (1-5 mg/kg/day), TGF-β (100 ng - 1 μg/day) (PeproTech, Rocky Hill, NJ), cyclophosphamide (75 mg/kg/day), cisplatinium (1-10 mg/kg/day), and TMβ-1 monoclonal antibody that targets NK cells (1 mg/kg/day).

> **% of sucrose(more than 2 days**

ng/day 17 23.5 7.25 8

µg/day 3 66.6 10 13

50 mg/Kg 12 41.6 6.8 9

mg/Kg 25 56 11.07 19

mg/kg 13 77 8.25 34

µg/day 4 50 2.75 4

0.3 2 100 2 2 1% 2 100 4 4 5% 2 100 5.5 6 7% 2 100 4 4 10% 2 100 5.5 6

mg/Kg 4 100 4.5 5

DSMO 5 % 18 88.8 7.18 12

Splenctomy 5 60 7 10 Cyclophopshamide 75 mg/Kg 7 100 7.6 9

P.Falciparum 7 71.42 11 24

*P.Falciparum* 52 90.24 11.09 34

Trolox 4 mg/Kg 5 60 3.85 6 Anti-NK(TMBβ-1) 1 mg 15 53.3 4.12 8

Bleeding 20 35 7.42 14

Folinic acid mg/Kg 4 100 4.5 5 Coinfection *P.Chabaudi* and *P.Yoellii*, NAC and Vitamine E seem to have a beneficial effect in *P.Falciparum*  survival; however results are very heterogeneous form one mouse to other and from one experiment to other

Table 1. Various immune suppressants to control innate immunity

**Paracitemia length average (days)** 

**Best parasitemia (days)** 

**5. Protocols to control residual innate immunity** 

**5.1.1 Immunomodulatory agents to suppress innate immunity** 

**Expertiments Performed Using I.P. Protocol With Vaious Immunomodulatory Reagent**

**mouse** 

**Protocol tested Dose Nb of** 

100

100

TGFβ

Coinfection *P.Chabaudi*

Coinfection *P.Yoellii*

*P.Falciparum* with various amount

one

NAC <sup>100</sup>

Vitamine E <sup>200</sup>

Futhan <sup>200</sup>

pABA <sup>400</sup>

**5.1 (I.P. Protocol)** 

the NOD/SCID/IL-2Rc-null mouse (NSG mouse) which, due to the knock out of the γ-chain of the IL-2 receptor, has been shown to better tolerate a variety of transplanted human cells (Ito et al., 2002; King et al., 2007; Watanabe et al., 2007). The resulting new IV model based on NSG mice presents several advantages over previously available models. It offers greater reproducibility, with 100% of mice successfully grafted without the need for mouse-adapted parasites, consistent curves of parasitemia, and high levels of infection with up to 40–50% of total erythrocytes infected (Arnold et al., 2010).
