Embryonic stem cell-derived neuronally committed precursor cells with reduced teratoma formation after transplantation into the lesioned adult mouse brain

The therapeutic potential of embryonic stem (ES) cells in neurodegenerative disorders has been widely recognized, and methods are being developed to optimize culture conditions for enriching the cells of interest and to improve graft stability and safety after transplantation. Whereas teratoma formation rarely occurs in xenogeneic transplantation paradigms of ES cell-derived neural progeny, more than 70% of mice that received murine ES cell-derived neural precursor cells develop teratomas, thus posing a major safety problem for allogeneic and syngeneic transplantation paradigms. Here we introduce a new differentiation protocol based on the generation of substrate-adherent ES cell-derived neural aggregates (SENAs) that consist predominantly of neuronally committed precursor cells. Purified SENAs that were differentiated into immature but postmitotic neurons did not form tumors up to four months after syngeneic transplantation into the acutely degenerated striatum and showed robust survival.     

Future strategies for tolerance induction: a comparative study between hematopoietic stem cells and macrophages

So far, clinical benefit of hematopoietic stem cell induced donor-specific tolerance across major histocompatibility complex (MHC) barriers was hampered by either graft rejection or graft-versus-host disease. An alternative approach focuses on the use of donor-derived cells that bear an inherent mechanism to circumvent allospecific rejection upon injection into non-immunosuppressed hosts. Using a myeloablative conditioning model in the rat, full donor chimeric recipients were generated and their potential to induce long-term cardiac allograft survival was compared with the fate of grafts transferred to non-immunosuppressed host rats pretreated with donor-macrophages derived from the peritoneal cavity in the LEW to DA inbred strain combination. The induction of full multilineage long-term donor-chimerism (> 150 days) after initial host conditioning using two doses of cyclophosphamide and one shot of busulphan prevented acute graft rejection, whereas non-chimeric animals experienced acute and complete rejection. Conversely, vigorous T-cell depletion is required to protect conditioned animals from lethal graft-versus-host disease. Instead, the use of donor intraperitoneal macrophages achieved a state of transient chimerism and subsequent long-term graft survival in fully immunocompetent rats without the need of lethal conditioning regimens. In conclusion, the complex immunologic interactions, observed after allogeneic bone marrow transplantation as a means to induce donor chimerism, and subsequent long-term graft acceptance can be avoided if appropriate cell populations can be identified that, by themselves, induce a transient state of donor chimerism prevailing long enough to deviate allospecific immune responses, as outlined in this study.     

Spatial and temporal kinetics of teratoma formation from murine embryonic stem cell transplantation

Pluripotent embryonic stem (ES) cells have the potential to form teratomas composed of derivatives from all three germ layers in animal models. This tumorigenic potential prevents clinical translation of ES cell research. In order to understand the biology and physiology of teratoma formation, we investigated the influence of undifferentiated ES cell number, migration, and long-term follow up after transplantation. Murine ES cells were stably transduced with a self-inactivating (SIN) lentiviral vector with a constitutive ubiquitin promoter driving a double-fusion (DF) reporter gene that consists of firefly luciferase and enhanced green fluorescent protein (Fluc-eGFP). To assess effects of cell numbers, varying numbers of ES-DF cells (1, 10, 100, 1,000, and 10,000) were injected subcutaneously into the dorsal regions of adult nude mice. To assess cell migration, 1 x 10(6) ES-DF cells were injected intramyocardially into adult Sv129 mice, and leakage to other extracardiac sites was monitored. To assess effects of long-term engraftment, 1 x 10(4) ES-DF cells were injected intramyocardially into adult nude rats, and cell survival response was monitored for 10 months. Our results show that ES-DF cells caused extracardiac teratoma in both immunocompetent and immunodeficient hosts; the lowest number of undifferentiated ES cells capable of causing teratoma was 500-1,000; and long-term engraftment could be shown for >300 days. Collectively, these results illustrate the potent tumorigenic potential of ES cells, which presents an enormous obstacle for future clinical studies.     

Application of chimerism-based drug-induced tolerance to rat into mouse xenotransplantation

The current critical shortage of human donor organs has stimulated the feasibility of the xenogenic transplantation, such as swine to primate. We have previously reported the induction of donor-specific tolerance in MHC-disparated recipient mice by using our cyclophosphamide (CP)-induced tolerance conditioning. In this study, we examined the efficacy of our CP-induced tolerance conditioning in xenogenic transplantation model. F344 rats and B10 mice were used as donors and recipients. Recipient mice were treated with donor spleen cells, CP, Busulfan and bone marrow cells, with or without prior NK-cell depletion. Donor mixed chimerism, and the presence of donor reactive T-cell population were analysed by flow cytometry. The survival of the donor skin grafts were observed after the conditioning. Donor mixed chimerism was temporary induced but terminated at 10 weeks after treatments. Donor-specific prolongation of the skin graft survival was observed after the treatments, however, grafts were rejected in the long term. NK-cell depletion, prior to the treatments, did not affect the levels of the mixed chimerism or graft prolongation. The donor-reactive recipient T-cell population was remained the same level as the untreated mice, suggesting the failure of the induction of the central T-cell tolerance. Thus, partial efficacy of our CP-induced tolerance treatments in the rat to mice xenotransplantation was observed. Our results suggested that the additional treatments were required to establish the stable xenogenic tolerance.     

Localized bone marrow transplantation leads to skin allograft acceptance in nonmyeloablated recipients: comparison of intra-bone marrow and isolated limb perfusion.

It has been shown that engraftment of allogeneic bone marrow cells (BMC) induces tolerance to antigen-matched organs, and infusion of a megadose of cells improves the success of engraftment of T-cell-depleted BMC. This study explores intra-bone marrow injection (IB) and isolated limb perfusion (IL) as means of localized bone marrow transplantation (BMT) and assesses their tolerogenic effect. Intravenous (i.v.), IB, and IL infusion of syngeneic and allogeneic whole BMC rescued 90%-100% of myeloablated recipients. Tracing of PKH-labeled cells revealed early systemic dissipation after IB injection, indicating that it was equivalent to i.v. transplantation. In contrast, IL perfusion led to initial localization of donor BMC. BALB/c recipients conditioned with 70 microg/g busulfan had 58% +/- 5% and 44% +/- 4% donor lymphocytes at 4 weeks after i.v. and IL infusion, respectively, of 10(7) whole BMC from B10 donors. This suggests that cells migrated out of the IL femur and seeded other bones. All recipients accepted donor-matched skin grafts and acutely rejected third party grafts. T-cell depletion lowered the engraftment efficiency of i.v.-BMT by 35% (p < 0.001 versus whole BMC), but not when infused IL (p < 0.001). It is concluded that IL-BMT is a procedure for initial localization of donor cells, which is as efficient as i.v.- and IB-BMT in rescue of myeloablated mice, induction of hemopoietic chimerism, and donor-specific immune nonresponsiveness to secondary skin grafts without myeloablative conditioning. The megadose effect achieved by inoculation of a small hemopoietic space improved engraftment of T-cell-depleted BMC. This approach may have clinical applications.     

Human embryonic stem cells are prone to generate primitive, undifferentiated tumors in engrafted human fetal tissues in severe combined immunodeficient mice

Embryonic stem (ES) cells are uniquely endowed with the capacity of self-renewal and the potential to give rise to all possible cell types. Their differentiation potential has raised hope that these cells could be used as a renewable source for cell transplantation in severe degenerative diseases. However, progress in this direction is still limited. Using two human embryonic stem (ES) cell lines, H1 and HSF-6, and three types of human fetal tissues--thymus, lung and pancreas-we investigated whether engrafted human fetal tissues in severe combined immunodeficient mice (SCID) mice could provide a physiologically-relevant microenvironment for human ES cells to differentiate into mature cells of corresponding tissues. Surprisingly, we observed an aggressive growth of tumors when human ES cells were injected into engrafted human fetal tissues in SCID mice. These tumors displayed histological characteristics of primitive, undifferentiated tumors rather than differentiated teratomas. Additionally, these tumors exhibited a normal karyotype and did not express the characteristic antigens of embryonic carcinomas. We also found differences among human fetal tissue types in their abilities to support the growth of these primitive tumors. Our study supports and validates a previously reported phenomenon in mouse that tumorigenesis of ES cells is host dependent. Our study is also the first report to demonstrate that human ES cells are prone to generate primitive, undifferentiated tumors in human fetal tissue grafts in SCID mice and raises a potential safety concern for using human ES cell-derived cell products in humans.     

Efficient generation of transgenic BALB/c mice using BALB/c embryonic stem cells

BALB/c is one of the most widely used and best characterized mouse strains in immunology. For various applications, it is necessary to generate BALB/c transgenic mice. However, using the conventional microinjection technique it is extremely inefficient to produce transgenic BALB/c mice since the one-cell stage BALB/c embryos are highly vulnerable to pronuclear DNA microinjection. To overcome this problem, we have investigated the generation of Egr-1 (early growth response gene) transgenic mice via the transfection of BALB/c embryonic stem cells. Transfectants carrying Egr-1 constructs comprising either the immunoglobulin heavy chain or the MHC class II promoter/enhancer system were injected into C57BL/6 host blastocysts resulting in chimeric mice. For both type of expression vectors, transgenic offspring of the germline chimeras expressed recombinant Egr-1 in lymphoid tissues containing B cells. This demonstrates the successful generation of Egr-1 transgenic BALB/c mice using transfected ES cell.     

Double negative Treg cells promote nonmyeloablative bone marrow chimerism by inducing T-cell clonal deletion and suppressing NK cell function

The establishment of immune tolerance and prevention of chronic rejection remain major goals in clinical transplantation. In bone marrow (BM) transplantation, T cells and NK cells play important roles for graft rejection. In addition, graft-versus-host-disease (GVHD) remains a major obstacle for BM transplantation. In this study, we aimed to establish mixed chimerism in an irradiation-free condition. Our data indicate that adoptive transfer of donor-derived T-cell receptor (TCR) αβ(+) CD3(+) CD4(-) CD8(-) NK1.1(-) (double negative, DN) Treg cells prior to C57BL/6 to BALB/c BM transplantation, in combination with cyclophosphamide, induced a stable-mixed chimerism and acceptance of C57BL/6 skin allografts but rejection of third-party C3H (H-2k) skin grafts. Adoptive transfer of CD4(+) and CD8(+) T cells, but not DN Treg cells, induced GVHD in this regimen. The recipient T-cell alloreactive responsiveness was reduced in the DN Treg cell-treated group and clonal deletions of TCRVβ2, 7, 8.1/2, and 8.3 were observed in both CD4(+) and CD8(+) T cells. Furthermore, DN Treg-cell treatment suppressed NK cell-mediated BM rejection in a perforin-dependent manner. Taken together, our results suggest that adoptive transfer of DN Treg cells can control both adoptive and innate immunities and promote stable-mixed chimerism and donor-specific tolerance in the irradiation-free regimen.     

The major histocompatibility complex (MHC) of the secondary transplant tissue donor influences the cross-reactivity of alloreactive memory cells

Memory cells are currently thought to be a major barrier to tolerance induction in transplantation. However, whether alloreactive memory cells resulting from a primary transplant have cross‐reactivity in a second transplant is unclear. Here, we used skin transplantation from BALB/c mice donors to presensitize C₅₇BL/6 (B6) mice. One month later, several strains of mice (including BALB/c, DBA/2, NOD, C3H and B6 mice) were chosen as donors to construct a memory model of heterotopic cardiac transplantation. The higher degree of major histocompatibility complex (MHC) mismatch to sensitizing MHC resulted in longer median survival times (MSTs, BALB/c 3.63 days versus C3H 6.08 days). After 3.5 days of cardiac transplantation, compared with the BALB/c and DBA/2 groups, in the groups of NOD and C3H, the infiltration of inflammatory cells in the grafts, the proportion and proliferation of memory cells in spleens and the function of allogeneic antibodies decreased significantly. The varying degrees of MHC mismatch between the primary and secondary donors influenced the intensity of alloreactive memory cell function, the higher degree of MHC mismatch resulted in better tolerance during secondary transplantation, and these may be related to the changed activation, proliferation and function of the alloreactive memory cells.     

Sustained macroscopic engraftment of cynomolgus embryonic stem cells in xenogeneic large animals after in utero transplantation

Because embryonic stem (ES) cells are able to proliferate indefinitely and differentiate into any type of cell, they have the potential for providing an inexhaustible supply of transplantable cells or tissues. However, methods for the in vitro differentiation of human ES cells are still quite limited. One possible strategy would be to generate differentiated cells in vivo. In view of future clinical application, we investigated the possibility of using xenogeneic large animals for this purpose. We transplanted nonhuman primate cynomolgus ES cells into fetal sheep at 43-67 gestational days (full term 147 days, n=15). After birth, cynomolgus tissues, which were mature teratomas, had been engrafted in sheep when more than 1 x 10(6) ES cells were transplanted at <50 gestational days. Despite the sustained engraftment, both cellular and humoral immune responses against the ES cells were detected, and additional transplantation was not successful in the animals. At 2 weeks post-transplantation, the ES cell progeny proliferated when transplanted at 48 (<50) gestational days, whereas they were cleared away when transplanted at 60 (>50) gestational days. These results support the rapid development of the xenogeneic immunological barrier in fetal sheep after 50 gestational days. Notably, a large number of Foxp3(+) regulatory T cells were present around the ES cell progeny, but macrophages were absent when the transplant was conducted at <50 gestational days, implying that regulatory T cells and premature innate immunity might have contributed to the sustained engraftment. In conclusion, long-term macroscopic engraftment of primate ES cells in sheep is feasible despite the xenogeneic immunological barrier.     

Response of genetically susceptible and resistant mice to intranasal inoculation with mouse hepatitis virus JHM

Mouse hepatitis virus (MHV)-JHM infection was studied in genetically susceptible (BALB/cByJ) and resistant (SJL/J) mice following intranasal inoculation at 1, 3, 6 or 12 wk of age. Markers of infection included histology, immunohistochemistry, virus quantification and virus serology. All BALB mice developed severe disseminated disease with high mortality due to encephalitis and hepatitis. Peak MHV titers appeared in brain, liver, spleen and intestine on days 3 or 5. Age at inoculation did not influence virus titers in brain, spleen or intestine, but virus titers in liver were inversely proportional to age at inoculation. In 6-wk-old BALB mice, virus was cleared from spleen, intestine and liver by day 30 and from brain by day 60. In intestine, MHV was localized to lymphoid tissue, without fecal excretion. SJL mice of all ages developed remarkably milder disease with low mortality occurring only among mice inoculated at 1 wk of age. SJL mice inoculated at 1 wk had disseminated infection at day 3, but lesions and antigen were cleared from most organs by day 5. Mice inoculated at 3 and 6 wk of age had minimal or no involvement of peripheral organs, and mice inoculated at 12 wk of age had infections restricted to the nose. At day 5, MHV titers in brain, liver, spleen and intestine were significantly lower or undetectable in SJL mice of all ages compared to age-matched BALB mice. In 6-wk-old mice, MHV was cleared from all organs by day 10. Serum antibody titers to MHV were many-fold higher in BALB mice, compared to SJL mice, which mounted only a modest response.     

Successful pancreatic allografts in combination with bone marrow transplantation in mice.

We have established a new method for pancreatic allografts in mice by combining pancreatic transplantation with allogeneic bone marrow transplantation. In this approach, we first transplanted bone marrow to induce tolerance to both donor-type and host-type major histocompatibility complex (MHC) determinants. Pancreatic tissue from the same mouse strain as bone marrow donor was then grafted under the renal capsule. Acceptance of the grafts was confirmed by histopathological and immunohistochemical techniques. BALB/c mice reconstituted with C57BL/6J bone marrow cells accepted pancreatic tissue from both bone marrow donor (C57BL/6J)-type and host (BALB/c)-type mice. An immunohistochemical study revealed the presence of functional islets under the renal capsules. Assays for both mixed lymphocyte reaction (MLR) and induction of cytotoxic T lymphocytes indicated that the newly developed T cells are tolerant of both donor (stem cell)-type and host-type MHC determinants. By contrast, the T cells of these chimeras showed a significant responsiveness to third party MHC determinants. These findings suggest that pancreatic allografts combined with bone marrow transplantation may become a viable strategy for the treatment of patients with diabetes or patients who have undergone pancreatectomy.     

小鼠胚胎干细胞植入大鼠脑内分化的研究

观察小鼠胚胎干细胞植入大鼠隔区和海马内之后的分化状况。以 SD大鼠为宿主 ,将胚胎干细胞移植入宿主隔区和海马内 ,移植后在 l、2、3、4和 8周取脑 ,冰冻切片 ,进行 Nissl染色和 M6、NSE、GFAP免疫组织化学反应。胚胎干细胞移植入大鼠隔区和海马之后 ,从第 2周开始表达 M6、GFAP、NSE等抗原 ,持续至第 4周 ,主要位于移植区内 ,较少迁移。小鼠胚胎干细胞移植入大鼠隔区和海马内之后 ,分化为神经元和神经胶质细胞     

Generation of hematopoietic humanized mice in the newborn BALB/c-Rag2null Il2rγnull mouse model: a multivariable optimization approach

Hematopoietic humanized mice generated via transplantation of human hematopoietic stem cells (hHSCs) into immunodeficient mice are a valuable tool for studying development and function of the human immune system. This study was performed to generate a protocol that improves development and quality of humanized mice in the BALB/c-Rag2(null)Il2rγ(null) strain, testing route of injection, in vitro culture and freezing of hHSCs, types of cytokines in the culture, and co-injection of lineage-depleted CD34(-) cells. Specific hHSC culturing conditions and the addition of support cells were found to increase the frequency, and human hematopoietic chimerism, of humanized mice. The optimized protocol resulted in BALB/c-Rag2(null)Il2rγ(null) humanized mice displaying more consistent human hematopoietic and lymphoid engraftment. Thus, hematopoietic humanized mice generated on a BALB/c immunodeficient background represent a useful model to study the human immune system.     

Genetic influences on latent inhibition

The present study examined the development of latent inhibition in a number of inbred strains of mice. C57BL/6J, DBA/2J, C3H/Ibg, BALB/cByJ, A/J, CBA/J, 129/SvevTac, 129/SvJ, and AKR/J mice were screened for the development of latent inhibition. The latent inhibition paradigm involved 1 day of either 40 preexposures to the conditioned stimulus (CS) or no preexposure. On the following training day, the CS was twice paired with a shock unconditioned stimulus (US). On a subsequent test day, the strength of the CS-US association was measured. Mice preexposed to the CS should show a weaker CS-US association, which would reflect development of latent inhibition. Significant between-strain differences existed. The 129/Svev, C57BL/6, BALB/cByJ, AKR, and DBA/2 mice developed latent inhibition, but 129/SvJ, CBA, A, and C3H mice did not. Thus, genetic variance contributes to variability in the development of latent inhibition.     

Role of Regulatory T Cells in Transferable Immunological Tolerance to Bone Marrow Donor in Murine Mixed Chimerism Model

Constructing a bone marrow chimera prior to graft transplantation can induce donor-specific immune tolerance. Mixed chimerism containing hematopoietic cells of both recipient- and donor-origin has advantages attributed from low dose of total body irradiation. In this study, we explored the mechanism of mixed chimerism supplemented with depletion of Natural Killer cells. Mixed chimerism with C57BL/6 bone marrow cells was induced in recipient BALB/c mice which were given 450 cGy of γ-ray irradiation (n = 16). As revealed by reduced proliferation and cytokine production in mixed leukocyte reaction and ELISpot assay (24.6 vs 265.5), the allo-immune response to bone marrow donor was reduced. Furthermore, the induction of transferable immunological tolerance was confirmed by adoptive transfer and subsequent acceptance of C57BL/6 skin graft (n = 4). CD4⁺FoxP3⁺ regulatory T cells were increased in the recipient compartment of the mixed chimera (19.2% → 33.8%). This suggests that regulatory T cells may be therapeutically used for the induction of graft-specific tolerance by mixed chimerism.     

Determinants of skeletal muscle contributions from circulating cells, bone marrow cells, and hematopoietic stem cells

To investigate the factors that regulate incorporation into uninjured or damaged skeletal muscle of donor markers derived from unfractionated bone marrow (BM) cells or from highly purified c-kit+ Thy1.1lo Lin- Sca-1+ hematopoietic stem cells (HSCs), we evaluated myofiber chimerism of multiple muscle groups in irradiated and transplanted recipient mice and in unirradiated parabiotic animals. Uninjured panniculus carnosus, diaphragm, and abdominal muscles infrequently incorporated donor markers into myofibers in a subset of animals after either BM or HSC transplantation; however, acute muscle injury was essential to elicit contributions to triceps surae (TS) and tibialis anterior muscles. The low level of incorporation of donor marker-expressing myofibers could not be enhanced either by transplantation into newborn recipients or by induced migration of HSCs into the periphery. Analysis of muscle chimerism in unirradiated animals joined surgically by parabiosis revealed that contributions of circulating cells to myofibers in the TS were injury dependent and that at least some circulating cells with the potential to contribute to regenerating muscle derive from BM, suggesting that hematoablative preconditioning is not required for such contributions. In all cases tested, donor-derived myofibers expressed both donor-specific and host-specific markers, suggesting that they arise by low-level fusion into skeletal muscle of cells that can include the progeny of HSCs. It is not yet clear whether such events represent a normal myogenic pathway or a pathological response to muscle damage.     

The Susceptibility to Cytotoxic T Lymphocyte Mediated Lysis of Chemically Induced Sarcomas from Immunodeficient and Normal Mice

A panel of sarcomas induced with 3-methylcholanthrene in normal and immunodeficient mice was studied for their capacity to present antigen by the endogenous, MHC class I restricted pathway. Lymphocytic choriomeningitis virus was used to infect cultured tumour cells, and the infected cells were tested for susceptibility to cytolysis by virus specific cytotoxic T cells. Tumour cells originating from tumours induced in immunocompetent C.B.-17 mice presented virus antigen more efficiently than tumour cells from immunodeficient SCID mice. No significant difference in virus antigen presentation was found between tumours from nude and nu/+ BALB/c mice. The sensitivity of target cells from the individual tumours to cytotoxic T lymphocyte (CTL) mediated lysis correlated negatively with their sensitivity to natural killer (NK) cell mediated lysis. There was a positive correlation between the sensitivity to CTL mediated lysis and surface expression of the MHC class I molecule L^d of the tumour cells. Tumour cells incapable of in vitro presentation of viral antigen to specific cytotoxic T cells originated from tumours known from previous experiments to be readily accepted after transplantation to immunocompetent, histocompatible recipients.     

Gut-homing CD4+ T cell receptor αβ+ T cells in the pathogenesis of murine inflammatory bowel disease

We studied which T cell subsets from the gut-associated lymphoid tissue (GALT) can migrate out of the gut mucosa and repopulate GALT compartments of an immunodeficient (semi)syngeneic host. Many distinct lymphocyte subsets were found in GALT of immunocompetent H-2^d (BALB/c, BALB/c^dm2, C. B-17+/+) mice. No antigen receptor-expressing lymphoid cells were found in GALT of congenic C. B-17 scid/scid (scid) mice. The heterotopic transplantation of a full-thickness gut wall graft from the ileum or colon of immunocompetent (C. B-17+/+, BALB/c^dm2) donor mice onto immunodeficient scid mice selectively reconstituted a CD3⁺ T cell receptor αβ⁺ CD4⁺ T cell subset. CD4⁺ cells of this subset expressed the surface phenotype of mucosa-seeking, memory T cells. In the immunodeficient scid host, this gut-derived CD4⁺ T cell subset was found in spleen, peritoneal cavity, mesenteric lymph nodes (LN), epithelial layer and lamina propria of the small and large intestine, but not in peripheral LN. Scid mice heterotopically transplanted with gut from a congenic, immunocompetent donor developed clinical and histological signs of inflammatory bowel disease (IBD). Hence, the selective repopulation of GALT compartments with CD4⁺ T cells from normal GALT plays an essential role in the pathogenesis of IBD in an immunodeficient host.     

Duration of challenge immunity to coronavirus JHM in mice

Summary The duration of challenge resistance in mice immunized with mouse hepatitis virus (MHV) strain JHM was examined as a model of immunity to corona-virus infection. Genetically susceptible BALB/cByJ mice were immunized by intranasal (i.n.) or per os (p.o.) inoculation with MHV-JHM or sterile tissue culture fluid (sham) then challenged i.n. with MHV-JHM or sterile tissue culture fluid 1, 6, or 12 months later. Four days after challenge, virus in nasal turbinates and liver was quantified, and prevalance of microscopic lesions in liver and gut-associated lymphoid tissue was tabulated as indices of challenge resistance. MHV-immunized and challenged groups were compared to sham-immunized and challenged groups. Mice immunized by i.n. inoculation were strongly resistant to challenge at 1, 6, and 12 months. Mice immunized by p.o. inoculation were resistant at 1 month, but became partially susceptible to reinfection at 6 and 12 months, based upon all indices. These data indicate that, depending upon route of immunization, mice can become susceptible to reinfection with the same coronavirus strain over time.