Generation of a Nonhuman Primate Model of Severe Combined Immunodeficiency Using Highly Efficient Genome Editing.

Recent advances in genome editing have facilitated the generation of nonhuman primate (NHP) models, with potential to unmask the complex biology of human disease not revealed by rodent models. However, their broader use is hindered by the challenges associated with generation of adult NHP models as well as the cost of their production. Here, we describe the generation of a marmoset model of severe combined immunodeficiency (SCID). This study optimized zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) to target interleukin-2 receptor subunit gamma (IL2RG) in pronuclear stage marmoset embryos. Nine of 21 neonates exhibited mutations in the IL2RG gene, concomitant with immunodeficiency, and three neonates have currently survived from 240 days to 1.8 years. Our approach demonstrates highly efficient production of founder NHP with SCID phenotypes, with promises of multiple pre-clinical and translational applications.

Lymphocytes and macrophages are infected by Theileria equi, but T cells and B cells are not required to establish infection in vivo.

Theileria equi has a biphasic life cycle in horses, with a period of intraleukocyte development followed by patent erythrocytic parasitemia that causes acute and sometimes fatal hemolytic disease. Unlike Theileria spp. that infect cattle (Theileria parva and Theileria annulata), the intraleukocyte stage (schizont) of Theileria equi does not cause uncontrolled host cell proliferation or other significant pathology. Nevertheless, schizont-infected leukocytes are of interest because of their potential to alter host cell function and because immune responses directed against this stage could halt infection and prevent disease. Based on cellular morphology, Theileria equi has been reported to infect lymphocytes in vivo and in vitro, but the specific phenotype of schizont-infected cells has yet to be defined. To resolve this knowledge gap in Theileria equi pathogenesis, peripheral blood mononuclear cells were infected in vitro and the phenotype of infected cells determined using flow cytometry and immunofluorescence microscopy. These experiments demonstrated that the host cell range of Theileria equi was broader than initially reported and included B lymphocytes, T lymphocytes and monocyte/macrophages. To determine if B and T lymphocytes were required to establish infection in vivo, horses affected with severe combined immunodeficiency (SCID), which lack functional B and T lymphocytes, were inoculated with Theileria equi sporozoites. SCID horses developed patent erythrocytic parasitemia, indicating that B and T lymphocytes are not necessary to complete the Theileria equi life cycle in vivo. These findings suggest that the factors mediating Theileria equi leukocyte invasion and intracytoplasmic differentiation are common to several leukocyte subsets and are less restricted than for Theileria annulata and Theileria parva. These data will greatly facilitate future investigation into the relationships between Theileria equi leukocyte tropism and pathogenesis, breed susceptibility, and strain virulence.

Infection of immunodeficient horses with Sarcocystis neurona does not result in neurologic disease.

Equine protozoal myeloencephalitis is a progressive neurologic disease of horses most commonly caused by infection with the apicomplexan parasite Sarcocystis neurona. Factors affecting neuroinvasion and neurovirulence have not been determined. We investigated the pathogenesis of infection with S. neurona in horses with severe combined immune deficiency (SCID). Two immunocompetent (IC) Arabian horses and two Arabian horses with SCID were infected orally with 5 x 10(5) sporocysts of S. neurona. Four IC horses and one SCID horse were infected intravenously (i.v.) with 5 x 10(8) merozoites of the WSU-1 isolate of S. neurona. Despite prolonged parasitemia and persistent infection of visceral tissues (skeletal muscle, cardiac muscle, lung, liver, and spleen) as demonstrated by PCR and culture, SCID horses did not develop neurologic signs after oral or i.v. infection. S. neurona was undetectable in the neuronal tissues of SCID horses by either PCR, immunohistochemistry, or culture. In contrast, although parasitemia was undetectable in orally infected IC horses and of only short duration in i.v. infected IC horses, four of six IC horses developed neurologic signs. S. neurona was detectable by PCR and/or culture of neural tissue but not visceral tissue of IC horses with neurologic disease. Infected SCID horses are unable to clear S. neurona from visceral tissues, but the infection does not result in neurologic signs; in contrast, IC horses rapidly control parasitemia and infection of visceral tissues but frequently experience neuroinvasion and exhibit clinical signs of neurologic disease.

Partial protection of severe combined immunodeficient mice against infection with Babesia microti by in vitro-generated CD4+ T cell clones.

Because CD4+ T cells were considered to be involved in protection against infection with Babesia microti, specific CD4+ T cells were generated in vitro from recovered BALB/c mice and their protective activity was tested in vivo. The cells produced varying amounts of interferon (IFN)-gamma in vitro in response to parasite antigen. In passive transfer experiments, three out of eleven T cell clones tested exerted protective activity in the early phase after infection. However, there seemed to be no correlation between this protection and in vitro IFN-gamma production by the T cell clones. Although the protection was partial and short-lived, the result provided direct evidence that CD4+ T cells play a crucial role in defense against B. microti.

IL-12 protects immunocompetent and immunodeficient neonatal mice against infection with Cryptosporidium parvum.

The protozoan parasite Cryptosporidium parvum (Cp) causes diarrhea that can be acutely severe in immunocompetent persons and can become chronic and life-threatening in immunocompromised individuals. Because studies in mice have implicated IFN-gamma in protection against this parasite, and IL-12 can induce IFN-gamma production, we examined the ability of IL-12 to prevent or cure Cp infection in neonatal BALB/c and SCID mice. Treatment of both immunocompetent and immunodeficient mice with IL-12 before experimental inoculation with Cp oocysts prevented or greatly reduced the severity of infection. Intestinal epithelial cell invasion and/or early intracellular development of Cp was inhibited by exogenous IL-12. The protective effect of IL-12 was completely blocked by anti-IFN-gamma mAb. Established infections were associated with elevated IFN-gamma gene expression and were not ameliorated by IL-12 treatment, even though such treatment further enhanced IFN-gamma gene expression. The severity of Cp infection was, however, exacerbated by treatment with anti-IL-12 Ab. These observations provide the first evidence that treatment with exogenous IL-12 can prevent Cp infection through an IFN-gamma-dependent, specific immune system-independent mechanism, and that endogenous IL-12 production has a role in limiting Cp infection.

The severe combined immunodeficient mouse as a model for Encephalitozoon cuniculi microsporidiosis.

Microsporidia have been recently recognized as opportunistic pathogens in AIDS patients. In attempt to develop an animal model with features similar to the infections observed in the immunodeficient patients, the adult severe combined immunodeficient mice (SCID) were administered both intraperitoneally and perorally by 2 x 10(7) spores of the murine isolate of E. cuniculi. The experimental inoculation caused a severe, fatal disease characterized by the dissemination of microsporidia into the host tissues. The dominant route of E. cuniculi dissemination in the SCID mice was continual direct extension from the site of inoculation to adjacent tissues and organs, terminating in hematogenous spread of infection in the host. The different courses of microsporidiosis in SCID mice relative to the mode of inoculation (i.p. vs. p.o.) was observed. The survival time of i.p. infected SCID mice was 3 weeks--vs. 5 weeks in p.o. infected SCID mice. Experimental microsporidiosis in SCID mice should provide a useful model for studies in microsporidial pathogenesis, mechanisms of resistance, immunotherapy, and in evaluating potential antimicrosporidial agents.

Cryptosporidiosis in adult and neonatal mice with severe combined immunodeficiency.

Cryptosporidium parvum causes protracted diarrhoea in immunodeficient hosts. To characterize the role that T and B lymphocytes play in the eradication of the parasite from the intestinal mucosa, the course of infection in mice with severe combined immunodeficiency (SCID) was studied. Twenty-nine SCID and 26 BALB/c adult mice received 10(6) oocysts intragastrically. The course of infection in the two strains was similar until 2 months after inoculation, when moderate numbers of organisms were identified in the villous and crypt mucosa of the ileum and proximal colon of SCID mice. Three months after inoculation, SCID mice developed wasting and progressive intestinal and biliary tract disease. At 5 months, mortality of 72 and 0 per cent, respectively, was observed in the SCID and BALB/c mice. Twenty-four SCID and 26 BALB/c neonatal mice were also inoculated with C. parvum. Cryptosporidiosis occurred in SCID and BALB/c mice within 2 weeks of inoculation. Subsequently, BALB/c, but not SCID mice, eradicated the parasite from their intestinal mucosa. SCID mice developed progressively severe cryptosporidiosis which killed all animals within 7 weeks. Responses mediated by B or T cells, or both, appeared to play a role in eradicating C. parvum from the intestinal mucosa, since SCID mice were more severely affected than BALB/c mice. The different course of infection in adult and neonatal SCID mice indicated that other age-related factors also played a role in containing C. parvum infection.