Protection of cattle from bovine herpesvirus type I (BHV-1) infection by immunization with individual viral glycoproteins

The major glycoproteins gI, gIII, and gIV of bovine herpesvirus-1 (BHV-1) were found to induce high levels of antibody in cattle which could neutralize virus and participate in antibody-dependent cell cytotoxicity of BHV-1-infected cells. Immunized animals were fully protected from disease, using a BHV-1/Pasteurella haemolytica aerosol challenge model but not from infection with the virus. Thus, virus could still replicate in the nasal passages of immunized animals, although to a lesser extent than in placebo-treated animals or animals immunized with a commercial killed whole virus vaccine. Systemic spread of the virus in immunized animals did not appear to occur since there was not a dramatic alteration of leukocyte function following challenge. These results suggest that any one of the three major BHV-1 glycoproteins may be useful as a subunit vaccine either individually or in combination.     

Immunomodulation of Japanese encephalitis vaccine through CpG oligodeoxynucleotides in mice

Synthetic oligodeoxynucleotides (ODN) containing unmethylated cytosine guanine (CpG) dinucleotides motifs act as immune adjuvant and provide means of modulation to immune responses when co-delivered with antigens. They stimulate both innate and adaptive immune responses and induce T helper 1 (Th1) immune responses. We investigated the immunomodulation of Japanese encephalitis (JE) vaccine using CpG ODN as an adjuvant. Mice were immunized with one dose of JE vaccine 0.1 ml with different concentrations (10, 25 and 100 microg) of CpG ODN. The serum antibody level and cytokines were evaluated and compared with mice immunized with two doses of JE vaccine alone. Our studies revealed that anti-JE antibody level in mice immunized with single dose of 0.1 ml JE vaccine and 100 microg CpG ODN were almost equal to mice immunized with two doses of JE vaccine alone. Furthermore, CpG ODN enhanced the production of TNF-alpha and Th1-mediated cytokines, including IFN-gamma and IL-2 compared with JE vaccine alone. In addition, absence of any significant changes in biochemical, haematological and histological studies suggest that CpG ODN are safe adjuvants for JE vaccine. Therefore, it is inferred that CpG ODN are effective and improve the efficacy of JE vaccine.     

Anti-idiotypic antibodies induce neutralizing antibodies to bovine herpesvirus 1.

A neutralizing murine monoclonal antibody (mAb) of the IgG2a isotype (MM-113), specific for bovine herpesvirus 1 (BHV-1) glycoprotein gIV, was used to develop anti-idiotypic antibodies (anti-Id) in a calf. The bovine anti-Id were isolated from the serum of the immunized calf by affinity chromatography on an MM-113-Sepharose column, followed by repeated adsorption on a murine IgG2a column. The anti-Id thus obtained specifically reacted with MM-113, but not with isotype-matched controls. They also inhibited the binding of MM-113 to BHV-1 in a concentration-dependent manner. Mice immunized with the anti-Id produced neutralizing antibodies to BHV-1. The anti-Id bound to cells permissive to BHV-1 in a cell-binding radioimmunoassay (RIA).     

Modulation of immune responses to bovine herpesvirus-1 in cattle by immunization with a DNA vaccine encoding glycoprotein D as a fusion protein with bovine CD154

The objective of this study was to determine whether a DNA vaccine encoding bovine CD154 linked to a truncated version of bovine herpesvirus-1 (BHV-1) glycoprotein D (tgD-CD154) induces enhanced tgD-specific immune responses in cattle. In vitro characterization demonstrated that tgD and tgD-CD154 both bind to cultured bovine B cells, whereas only tgD-CD154 induces interleukin-4-dependent proliferation, suggesting that tgD-CD154 specifically binds the CD40 receptor and induces receptor signalling. Calves were immunized with plasmid encoding either tgD or tgD-CD154 by intradermal injection with a needle-free device. After two immunizations, tgD-specific immune responses were observed in both vaccinated groups and after challenge with BHV-1 these responses further increased. Animals immunized with plasmid encoding tgD-CD154 had significantly higher tgD-specific serum titres of immunoglobulins G and A but significantly lower numbers of tgD-specific interferon-gamma-secreting cells than animals immunized with plasmid encoding tgD after BHV-1 challenge. This suggests that the expression of an antigen as a chimeric protein with CD154 can qualitatively alter immune responses in cattle. Since we previously showed that plasmid encoding tgD-CD154 induces significantly enhanced secondary tgD-specific antibody responses in sheep, there appear to be interspecies differences in the immune responses induced by tgD-CD154, which suggests that both proteins in the chimeric molecule may influence protein targeting and the induction of an immune response.     

Induction of mucosal immune responses by administration of liposome-antigen formulations and interleukin-12.

We examined the effect of interleukin-12 (IL-12) on the induction of mucosal immune responses following intranasal immunization with liposome-antigen formulations. We assessed the immune response to two recombinant glycoproteins (gD and gB) from bovine herpesvirus type 1 (BHV-1). Positively charged liposomes induced significantly higher gD-specific IgA titers than did immunization with antigen alone. This liposome formulation was selected to further assess the ability of IL-12 to influence mucosal immune responses. Intranasal immunization with IL-12 gD-liposome formulations did not alter the induction of mucosal immune responses. However, a significant increase in anti-gD antibody responses was induced in serum after intranasal immunization with IL-12 gD-liposome when compared with animals immunized with gD-liposomes. Mucosal antibody responses induced by a subcutaneous priming followed by an intranasal boost were significantly higher than those induced by two intranasal immunizations with the same IL-12 liposome-gD formulations. Furthermore, this immunization protocol resulted in the induction of high levels of interferon-gamma (IFN-gamma) in the lungs of subcutaneously primed mice. These findings indicate that the immunomodulatory effects of IL-12 influenced immune responses to a vaccine antigen when delivered intranasally and that these responses can be further enhanced by subcutaneous priming.     

Identification of different target glycoproteins for bovine herpes virus type 1-specific cytotoxic T lymphocytes depending on the method of in vitro stimulation.

Vaccinia virus recombinants expressing the three major bovine herpes virus-1 (BHV-1) glycoproteins gI, gIII and gIV were used to identify the major target antigens for BHV-1-specific CTL isolated from immune cattle. Peripheral blood mononuclear cells (PBMC) expanded in vitro in the presence of interleukin-2 (IL-2) and lysed both gIII- and gIV-infected target cells. Secondary in vitro stimulation of PBMC was also performed in the presence of either fixed BHV-1-infected autologous fibroblasts or ultraviolet (UV)-inactivated virus. Both methods of antigen presentation allowed the proliferation of BHV-1-specific CTL but the target glycoprotein for these CTL differed depending on the method of stimulation. Vaccinia-gIV-infected targets were lysed predominantly when PBMC were stimulated by fixed infected fibroblasts, whilst PBMC stimulated by UV-inactivated virus lysed mostly vaccinia-gIII-infected targets. This observation could be explained by a different processing pathway of BHV-1 antigens in each cell type involved.     

Age- and dose-interval-dependent antibody responses to inactivated poliovirus vaccine

Antibody responses were studied in five groups of children immunized with different three-dose schedules of inactivated poliovirus vaccine (IPV). The age of the child at the first dose (1-4 months) and the interval between the first and second doses (2-4 months) influenced the initial responses in a serotype-dependent manner. All the groups attained sufficient antibody level after three doses but the third dose given at 18 months resulted in higher persisting antibody levels than that given at 12 months. The highest persisting antibody titers against PV1 and PV2 (but not against PV3) at the age of 3 years were measured in the control group immunized with the current schedule (P < 0.001) in which the first dose is given at 6 months. The level of maternal antibodies present at the time of the first dose correlated negatively with the antibody titers as late as at 3 years of age. It is concluded that three doses of IPV given in widely variable schedules may result in satisfactory immune responses in children but, for optimal results, very early onset of the program and short dosage intervals should be avoided.     

Biolistic-mediated gene transfer using the bovine herpesvirus-1 glycoprotein D is an effective delivery system to induce neutralizing antibodies in its natural host

A genetic vaccine consisting of the bovine herpesvirus-1 (BHV-1) glycoprotein D (gD) gene was constructed and administered to cattle using the biolistic (gene-gun) process. Results were compared to standard intramuscular injection of an inactivated whole BHV-1 commercial vaccine. Cattle genetically immunized by the gene-gun-delivered gD subunit vaccine developed high titers of IgG antibodies specific to gD demonstrating that this immunization method is a potent humoral response inducer. Further, gene-gun vaccinated cattle produced high neutralizing antibody titers to BHV-1 similar to levels induced in the commercial vaccine immunized animals. Additionally, cellular immunity was measured by an increased level of IFN-gamma mRNA detected in PBMC of cattle immunized with the gD gene or with the commercial vaccine, whereas augmented levels of IL-4 were not detected following vaccination. Because of its simplicity and effectiveness in inducing an immune response in cattle similar to a commercial vaccine, gene-gun delivery of a subunit BHV-1 gD vaccine would be a viable alternative to current immunization protocols.     

BHV-1-Specific CD4+, CD8+, and gammadelta T cells in calves vaccinated with one dose of a modified live BHV-1 vaccine

Expression of the high-affinity interleukin 2 receptor alpha chain (CD25) was used to monitor antigen-specific activation of T lymphocyte subsets (CD4+, CD8+, and gammadelta T cells) from cattle immunized with modified live bovine herpesvirus-1 (BHV-1). Calves seronegative for BHV-1 were either vaccinated with one dose of modified live vaccine containing BHV-1 or not vaccinated to serve as negative controls. Two animals vaccinated 7 and 5 weeks before the start of the experiment with two doses of modified live vaccine containing BHV-1 served as positive controls. Blood samples were taken from the nonvaccinate group, the positive control group, and the vaccinate group at 0, 21, 35, 60, and 90 days postinoculation (PI). Isolated peripheral blood mononuclear cells from immunized and control animals were incubated for 5 days with and without live BHV-1 ISU99. Compared to the nonvaccinates, a significant (p < 0.05) increase in expression of CD25 by CD4+, CD8+, and gammadelta T lymphocytes from the vaccinate group was detected following in vitro exposure to live BHV-1 after vaccination. This is apparently the first report using live BHV-1 to stimulate lymphocytes in vitro and showing CD8+ T cell activation. Peripheral blood from the positive control animals was depleted of CD4+, CD8+, or gammadelta T lymphocytes prior to incubation with BHV-1 to assess bystander activation in the CD25 expression assay. When incubated with live BHV-1, depletion of CD4+ T cells depressed the expression of CD25 by CD8+ T cells, but not gammadelta T cells. Depleting CD8+ or gammadelta T cells prior to in vitro culture with BHV-1 did not affect the expression of CD25 by the remaining T lymphocyte subsets. Vaccinates were protected from challenge with virulent BHV-1 at 110 days postvaccination compared to nonvaccinates. Expression of CD25 appears to be a useful marker for evaluating induction of antigen-specific T lymphocyte subset responses following vaccination.     

Monoclonal antibodies that distinguish between encephalitogenic bovine herpesvirus type 1.3 and respiratory bovine herpesvirus type 1.1.

Seven mouse hybridoma cell lines producing monoclonal antibodies (MAb) against an encephalitogenic strain of bovine herpesvirus type 1.3 (BHV-1.3) were established. The clones producing MAb were selected to be specific for BHV-1.3 by enzyme-linked immunosorbent assay. Only L1B neutralized virus without complement. With the addition of complement, five of the MAb neutralized BHV-1.3 but not the respiratory strain BHV-1.1. The anti-BHV-1.3-specific MAb Q10B, L6G, and L1B precipitated glycoproteins from BHV-1.3 that were analogous to the gI, GIII, and gIV glycoproteins of BHV-1.1, respectively. The other four MAb precipitated unknown proteins. None of the anti-BHV-1.3 MAb precipitated BHV-1.1 glycoproteins. The majority of the anti-BHV-1.3 MAb did not react with BHV-1.1 by immunoblotting, but O7E (unknown protein pattern by radioimmunoprecipitation) was reactive with five proteins (M(r)s of 33,000, 43,000, 70,000, 141,000, and 190,000) of BHV-1.3 and with a different pattern of proteins of BHV-1.1 (M(r)s of 30,000, 38,000, 83,000 and 144,000). Two of the MAb, L6G and O7E, conjugated with peroxidase were found to be useful for detecting BHV-1.3 antigen by immunochemistry in Formalin-fixed brain tissue from experimentally infected calves.     

Cloning and sequencing of truncated gIV glycoprotein gene of an Indian isolate of bovine herpesvirus 1

Bovine herpesvirus 1 (BHV-1) has been reported from the Indian subcontinent in late 70's. In order to identify the origin of an Indian isolate of BHV-1 (IBR/H 167 VS) and its molecular relationship to known strains of BHV-1, a 680 bp region of the glycoprotein gene gIV was amplified by polymerase chain reaction (PCR), cloned and sequenced. Comparison of this sequences with the corresponding one of an European strain of BHV-1 (Cooper) revealed more than 99% nucleotide (nt) homology. We conclude that the Indian isolate under study does not differ from the Cooper strain regarding the gIV gene.     

Intercellular trafficking of the major tegument protein VP22 of bovine herpesvirus-1 and its application to improve a DNA vaccine

Summary. Intercellular spread of bovine herpesvirus-1 (BHV-1) VP22 was demonstrated in living COS-7 cells transfected with a plasmid expressing VP22-YFP (yellow fluorescence protein) and CFP (cyan fluorescence protein) bicistronically. The intercellular trafficking property of VP22 was localized to the C-terminal portion of the molecule (amino acids 121–258; VP22-C). Plasmids encoding a truncated form of BHV-1 glycoprotein D (tgD) fused to VP22, VP22-C, or the N-terminal portion of VP22 (amino acids 1–120; VP22-N) were constructed. Mice immunized with plasmid encoding tgD-VP22 or tgD-VP22-C developed stronger immune responses when compared to animals immunized with plasmid encoding tgD or tgD fused to tgD-VP22-N.     

Adaptive immune responses fail to provide protection against genital HSV-2 infection in the absence of IL-15

IL-15 plays a crucial role in innate defense against viral infections. The role of IL-15 in the generation and function of adaptive immunity, following mucosal immunization, against genital HSV-2 has not been studied. Here, we report that immunized IL-15(-/-) mice were able to generate antibody and T cell-mediated immune responses against HSV-2, comparable to those seen in immunized B6 mice. However, immunized IL-15(-/-) mice were not protected against subsequent HSV-2 challenge, compared to B6 immunized mice, even with a ten times lower challenge dose. We then examined if the adaptive immune responses generated in the absence of IL-15 could provide protection against HSV-2 in an IL-15-positive environment. Adoptive transfer of lymphocytes from immunized IL-15(-/-) to naive mice were able to provide protection against HSV-2 challenge similar to protection with immunized cells from control mice. This suggests that the adaptive immune responses raised in the absence of IL-15 are functional in vivo. Reconstitution of the innate components, particularly IL-15, NK cells and NK cell-derived IFN-gamma, in immunized IL-15(-/-) mice restored their protective adaptive immunity against subsequent genital HSV-2 challenge. Our results clearly suggest that innate antiviral activity of IL-15 is necessary for protective adaptive immunity against genital HSV-2 infection.     

Induction of specific transplantation immunity by oral immunization with allogeneic cells

Oral administration of antigen has been shown to be effective for both positive and negative modulation of immune responses. In the present study we characterized changes in the reactivity of the immune system after oral immunization with allogeneic spleen cells. Mice were orally immunized for 10 consecutive days with fresh allogeneic spleen cells, and the phenotype, proliferative response, cytotoxic activity and cytokine production profile of recipient spleen cells were assessed 1 or 7 days after the last immunization dose. Although no significant changes in the proportion of CD4+, CD8+ or CD25+ cells were observed in the spleen of orally immunized mice, significant activation of alloreactivity in spleen cells was found. Cells from orally immunized mice exhibited enhanced proliferation and cytotoxic activity after stimulation with specific allogeneic cells in vitro, and produced considerably higher concentrations of interferon-gamma (IFN-gamma) and significantly less interleukin (IL)-4 than did cells from control mice. The production of IL-2 was essentially unchanged and that of IL-10 was only slightly increased. The systemic allosensitization induced by oral immunization was demonstrated in vivo by increased resistance to the growth of allogeneic tumours induced by subcutaneous inoculation of high doses of tumour cells. In addition, orthotopic corneal allografts in orally immunized recipients were rejected more rapidly (in a second-set manner) than in control, untreated recipients. These data show that oral immunization with allogeneic cells modulates individual components of the immune response and that specific transplantation immunity, rather than tolerance, is induced in the treated recipients.     

Monoclonal antibody analysis of bovine herpesvirus-1 glycoprotein antigenic areas relevant to natural infection

Neutralizing antigenic areas on the glycoproteins of bovine herpesvirus-1 (BHV-1) were identified by reciprocal competition radioimmunoassays using monoclonal antibodies. Three interrelated and two independent antigenic areas were identified on the 77-kDa (K) gIV envelope glycoprotein. Antigenic analysis of this protein has not been previously described. Four interrelated and one independent antigenic areas were found on the 97K gIII envelope glycoprotein. A third group of monoclonal antibodies reacting in Western blot with the 74K subunit of gI, a 130K disulfide-linked 74K/55K heterodimer, revealed four interrelated antigenic areas. All of the antigenic areas on all three glycoproteins were reactive with neutralizing monoclonal antibodies and all were targets for antibody-complement lysis. However, antibodies against gIV were the most efficient at neutralizing the virus and rendering infected cells susceptible to antibody-complement lysis. Convalescent sera from experimentally infected calves were used in a competitive radioimmunoassay to confirm that each antigenic area on the gI, gIII, or gIV glycoproteins was a target for bovine antibodies during primary infection with BHV-1.     

Intranasal immunization with a colloid-formulated bacterial extract induces an acute inflammatory response in the lungs and elicits specific immune responses

Nonspecific stimulation of lung defenses by repeated oral administration of immunomodulators, such as bacterial extracts, has shown potential for the prevention of respiratory tract infections. Here, we show that intranasal (i.n.) immunization with a bacterial extract formulated as a colloid induces an acute inflammatory response in the lungs characterized by increased production of CCL and CXCL chemokines and a major influx of dendritic cells (DCs) and neutrophils, with a higher proportion of DCs showing an activated phenotype (high CD80/CD86 expression). Cytokine levels measured in bronchoalveolar-lavage samples showed a small increase in the production of tumor necrosis factor alpha and similar levels of the other cytokines measured (interleukin 10 [IL-10], IL-12, and gamma interferon [IFN-gamma]) in immunized mice compared with control mice. However, the recall response of primed animals after antigenic challenge induced increased expression of IL-12 and IFN-gamma mRNAs in lung homogenates. Overall, all these effects were not due to the lipopolysaccharide content in the bacterial extract. Furthermore, we found that three i.n. doses administered 2 to 3 weeks apart were enough to elicit long-lasting specific serum immunoglobulin G (IgG) and secretory IgA antibody responses. Assessment of IgG subclasses showed a balanced pattern of IgG1-IgG2a responses. The serum total IgE concentrations were also elevated in immunized mice 2 weeks after the third dose, but they significantly decreased soon afterwards. Our results suggest that simple formulations of bacterial extracts administered i.n. are highly immunogenic, eliciting local and systemic immune responses, and may serve as the basis for cost-effective immunotherapies for the prevention and treatment of respiratory infections.     

Oral Tolerisation to Pyruvate Dehydrogenase Complex as a Potential Therapy for Primary Biliary Cirrhosis

Primary biliary cirrhosis (PBC) is characterised by immune-mediated damage to the intra-hepatic biliary epithelial cells (BEC). Immuno-modulatory/suppressive therapy represents, therefore, a logical approach to treatment in this disease. Conventional immuno-suppressive and immuno-modulatory agents suffer from the breadth of their action and/or excessive side effects. Autoreactive responses to pyruvate dehydrogenase complex (PDC) have been extensively characterised in PBC, and implicated in target cell damage. The aim of the current study was to study the potential efficacy of an antigen specific approach (oral tolerisation with autoantigen) to modulation of anti-PDC immune responses characteristic of PBC, utilising a mouse model of PDC immuno-reactivity. Groups of SJL/J mice were orally dosed with PDC alone, dosed with carrier only (saline) but systemically sensitised with PDC in adjuvant, or orally dosed with PDC at high (5 mg) or low (0.01 mg) dose and systemically sensitised with PDC. Oral dosing with PDC in isolation had no adverse effects on the animals and did not prime anti-PDC responses at doses below 1 mg. Pre-dosing with PDC at both high and low doses was effective at skewing the phenotype of the T-cell response to PDC induced by subsequent sensitisation away from the disease associated Th-1 phenotype (IL-2 and IFN- &#110 secreting) and towards a theoretically protective Th-2 phenotype (IL-4 secreting) in a majority of, but not all, animals. No augmentation of Th-1 response was seen in any animal. Although the effects on liver histology remain to established oral tolerisation with PDC holds promise as a novel, antigen specific approach to therapy in PBC.     

Desensitization of delayed-type hypersensitivity in mice: possible involvement of interleukin 2-dependent regulatory mechanisms in desensitized mice

The systemic injection of high doses of antigen into a previously immunized animal results in a state of transient anergy with respect to cell-mediated immune responses. This phenomenon is known as desensitization. The results presented here demonstrated that serum interleukin 2 (IL-2) activity was found transiently in desensitized mice at early stage (3 hr after the challenge). Subsequently, these mice could not develop in vivo (footpad swelling) and in vitro (lymphocyte proliferation) manifestations of cell-mediated immune responses 1 day after the challenge. Antigen-nonspecific and specific suppression of IL-2 production was observed in desensitized mice. The serum from 3 hr-desensitized mice containing endogenous IL-2 activity showed a marked suppressive effect on IL-2 production. Exposure of lymph node cells to IL-2 was capable of inhibiting IL-2 production in vitro. Additionally, in vivo administration of exogenous IL-2 into preimmunized mice led to the failure of development of footpad responses to antigen. These results suggest that IL-2-dependent regulatory mechanisms of T cell-mediated immune responses play an important role in the immunosuppression of desensitized mice.     

Maternal tolerance is not critically dependent on interleukin-4

Pregnant animals can generate and maintain immune responses to fetal antigens. This however, does not usually lead to fetal loss. At least two types of immune response are recognized. T helper type 1 (Th1) responses support the generation of cellular cytotoxicity. In contrast, Th2-type responses support the production of non-cytotoxic antibody and suppress the Th1-type. One attempt to explain why the fetus is not generally rejected has been to suggest that during pregnancy Th2-type responses are dominant. These responses rely heavily on interleukin-4 (IL-4) for both functions. This work focuses on maternal immunity to the male antigen H-Y, which is expressed in male fetuses. When injected with male spleen cells, female mice of certain strains mount a cytotoxic immune response to H-Y. However, pregnant females immunized in this way do not deliver litters with fewer males. To help delineate the possible role of IL-4 in such maternal tolerance, female mice genetically deficient in IL-4 were studied. The results show that: (1) deficiency in maternal IL-4 does not affect fertility, (2) deficiency in IL-4 is not associated with selective loss of male offspring in unimmunized mice, (3) pregnancy does not obliterate anti-H-Y reactivity in immunized mice and (4) maternal immunity to H-Y in the absence of IL-4 does not result in loss of male offspring. The results suggest that IL-4-dependent Th2-type responses are not critical to maternal tolerance. Other cytokines must be examined for their role in this phenomenon.     

Essential pathogenic role of endogenous IL-18 in murine diabetes induced by multiple low doses of streptozotocin. Prevention of hyperglycemia and insulitis by a recombinant IL-18-binding protein: Fc construct

IL-18 is a cytokine structurally and functionally related to IL-1 that, in synergy with IL-12, stimulates the synthesis of IFN-gamma from T lymphocytes and natural killer cells. Because IFN-gamma plays a key pathogenic role in the development of murine immunoinflammatory diabetes induced by multiple low doses of streptozotocin (STZ) we investigated the effect of negating the actions of endogenous IL-18 in this model by administering recombinant IL-18-binding protein:Fc (IL-18 bp:Fc). C57BL/6 mice were injected once daily with 40 mg/kg STZ for 5 consecutive days, day 0 being the first day of STZ challenge. Relative to control animals treated in parallel with either PBS or human IgG, mice treated from day -3 to day 7 with daily doses of 150 microg of IL-18 bp:Fc exhibited lower incidence of diabetes and milder insulitis. In contrast, mice that were treated with IL-18 bp:Fc from day 7 to day 14 exhibited clinical and histological signs of STZ-induced diabetes similar to those of control mice treated with IgG. The protective effect of IL-18 bp:Fc was accompanied by modified ex vivo immune responses, in that spleen cells and peritoneal macrophages contained fewer IFN-gamma secreting cells and released lower amounts of nitrite (an index of nitric oxide production) and IL-1beta. We conclude that intact IL-18 function is essential for the full diabetogenic effect of low dose STZ in C57BL/6 mice.