Previous immunization of mice with herpes simplex virus type-1 strain MP protects against secondary corneal infection

Herpes simplex virus (HSV)-induced ocular disease is occurring in epidemic proportions throughout the world, and is the number one cause of unilateral corneal blindness in all developed countries. We have found, in a mouse model of herpes simplex keratitis (HSK), that products encoded by the Igh-1 locus on chromosome 12 exert a profound influence on the immune/inflammatory response in the cornea after HSV inoculation in the cornea. Thus, mice with Igh-1c or Igh-1d phenotype routinely develop extreme keratopathy and loss of corneal clarity after HSV encounter in the eye, while congenic strains expressing other Igh-1 phenotypes develop substantially less keratopathy. We examined the effect of previous subcutaneous immunization with the mutant, less virulent, MP strain of HSV on the development of keratitis and encephalitis after secondary corneal inoculation with strains MP, mP, F, and KOS. A/J mice (Igh-1c), 5-6 weeks old, were injected sc with live HSV-1 strain MP. Controls were injected with culture media without virus. Three weeks later both immunized and control nonimmunized animals were challenged in the cornea with HSV-1, strains MP, mP, F, and KOS. The animals were clinically scored for keratitis and encephalitis at regular intervals for 21 days following corneal challenge. None of the immunized animals challenged in the cornea with strain MP, 5 X 10(4) plaque-forming units (PFU), developed clinical signs of encephalitis compared to 86% of unimmunized controls. Of the immunized animals challenged in the cornea with strain MP, 5 X 10(4) PFU, only 18% developed a mild keratitis, while 96% of unimmunized controls developed severe keratitis. Mice immunized subcutaneously with MP and subsequently challenged corneally with other HSV-1 strains (mP, F, or KOS) were also protected from development of severe keratopathy.     

Strain variations in the murine cellular immune response to the phenolic glycolipid I antigen of Mycobacterium leprae.

The cellular immune response to the Mycobacterium leprae-specific phenolic glycolipid I was examined in inbred mice immunized with M. leprae by in vivo delayed cutaneous hypersensitivity and in vitro lymphocyte proliferation. Whereas all mouse strains responded to M.leprae-induced delayed-type hypersensitivity and lymphocyte proliferation, only BALB.K was responsive in both assays to the glycolipid. Responsiveness was determined in part by non-H-2 genes, while the influence of H-2 genes was not apparent. Among congenic BALB/c mice differing only at Igh-C allotype loci, variations in responsiveness were found in both delayed-type hypersensitivity and lymphocytes proliferation assays, indicating a possible role for Igh-C loci-linked genes. Unresponsiveness in the lymphocyte proliferation assay to the glycolipid was inherited as a dominant trait in one set of responder X nonresponder F1 progeny. We conclude that after immunization with M. leprae organisms, the cell-mediated responses to the glycolipid, endowed with a single carbohydrate epitope, are under polygenic control, predominantly non-H-2-linked genes.     

Pathogenesis of latent herpes simplex virus infection of the trigeminal ganglion in guinea pigs: effects of age, passive immunization, and hydrocortisone.

Latent herpes simplex virus (HSV) infection of the trigeminal ganglion, after corneal inoculation of virus, was investigated in guinea pigs. The effects of several factors on the establishment of ganglionic latency were investigated. Latently infected guinea pigs were clinically normal, and virus was isolated from the trigeminal ganglia by co-cultivation. It was found that newborn guinea pigs were significantly more susceptible than adult animals to the development of latent HSV infection of the trigeminal ganglion. The susceptibility of newborn guinea pigs was very much decreased, however, if they received passive immunization with immune serum or if they were born of actively immunized mothers. On the other hand, the susceptibility of adult animals, usually somewhat resistant to the development of latent HSV ganglionic infection, was markedly increased by the parenteral administration of hydrocortisone.     

Immunological studies of hsv-infection of resistant and susceptible inbred strains of mice

DBA/2 mice were found to be quite susceptible to infection with herpes simplex virus (HSV) while C57BL/6 mice and F1 hybrids between the 2 strains were relatively resistant. This difference was most marked after ip infection, but could also be demonstrated after intracerebral, intravenous or subcutaneous infection. In both strains the LD50 was considerably higher after ip infection than after iv infection, and a dose of X-irradiation was required to kill the mice by sc infection. A/J and BALB/c mice were equally susceptible after ip infection but differed significantly after iv infection. C57BL/6 were made susceptible to ip infection by immunosuppression with antilymphocyte serum or cyclophosphamide. LPS, when given simultaneously with HSV also markedly increased the susceptibility of C57BL/6 mice. Susceptible DBA/2 mice which surrvived a low dose of HSV ip were not immune but C57BL/6 mice surviving a high dose were immune against rechallenge. Both strains of mice could be protected by an apathogenic, tissue-culture-attenuated strain of HSV against infection with the virulent strain. They could also be protected by iv injection of a sublethal dose against a lethal ip infection.     

Chronic progressive deficits in neuron size, density and number in the trigeminal ganglia of mice latently infected with herpes simplex virus

Numerous epidemiological studies have proposed a link between herpes simplex virus (HSV) infection and several common chronic neuropsychiatric and neurodegenerative diseases. Experimental HSV infection of mice can lead to chronic behavioral and neurological deficits and chronic pain. While neuron injury and loss are well-documented consequences of the acute phase of infection, the pathologic consequences of latent HSV infection are poorly understood. To determine whether latent HSV infection can cause neuronal injury in mice, trigeminal ganglia (TG) derived from adult BALB/c mice 1, 12 and 31 weeks after corneal HSV type 1 (HSV-1) inoculation were analyzed for evidence of productive or latent HSV-1 infection, inflammation and changes in neuron size, density and number. We found that latent HSV-1 infection between 12 and 31 weeks after corneal virus inoculation was associated with inflammation and progressive deficits in mean neuron diameter, neuronal nucleus diameter, neuron density and neuron number in the TG relative to mock-infected controls. The extent of neuronal injury during latent infection correlated with the extent of inflammation. These studies demonstrate that latent HSV infection is associated with progressive neuronal pathology and may lead to a better understanding of the role of HSV infections in chronic neurological diseases.     

The mitotic activity of, and antibody production by, sensitized lymphocytes quantified after transfer into irradiated recipients.

Adult thymectomized, irradiated CBA mice were reconstituted with bone marrow and thymus grafts derived from congenic strains differing in immunoglobulin allotype. After challenge with heterologous erythrocytes the allotype of the antibody secreted by the chimaeric spleen cells was the same as that of the bone marrow donor. When mixtures of immune and non-immune cells from the two congenic strains were transferred to irradiated recipients, a preponderance of the immune-donor cells was found dividing in the recipients' spleen 3 days after challenge with the immunizing antigen, and the allotype of the antibody-producing cells 7 days after challenge was almost entirely of immune-donor derivation. Further quantitative studies on the number of mitotically reactive cells observed after immunization with heterologous erythrocytes indicate that after primary and secondary challenge the mitotic activity of these cells is increased respectively four and eight times relative to that found in unsensitized cells.     

Herpetic keratitis in athymic (nude) mice.

The inflammatory response to herpes simplex virus infection of the cornea was studied in athymic nude (nu/nu) and heterozygote (nu/+) BALB/c mice. Although athymic mice were highly susceptible to HSV infection and died 13 to 17 days after corneal inoculation, they failed to develop necrotizing keratitis of the cornea. Heterozygote mice survived the initial virual infection, but many of these mice developed necrotizing keratitis and permanent corneal scarring. Light and electron microscopy showed numerous inflammatory cells (polymorphonuclear leukocytes and lymphocytes) in the corneas of heterozygote mice, but not in the athymic mice. These studies suggest that the immune system plays a dual role in herpes simplex virus infection of the cornea: protection against dissemination of the virus and immunopathogenesis of necrotizing keratitis in the cornea.     

Induction of natural killer cells by herpes-simplex virus type 2 in resistant and sensitive inbred mouse strains

Infection of mice with herpes simplex virus type 2 (HSV 2) stimulated natural killer (NK) cells in a variety of inbred mouse strains including athymic nude mice. Essentially all mouse strains tested exhibited high NK activity on day four after virus inoculation. Assayed 24 hours after infection, SWR/J, AKR/J, SJL/J and C57B1/10J mice were low or negative for these non-virus-specific cytotoxic responses. Whereas the first two mouse strains were most sensitive to the lethal effects of HSV 2, the latter two were highly resistant. Three lines with intermediate susceptibility and three highly resistant strains were all efficient with regard to early NK-cell.     

Effect of Ly 5 allotype on in vitro immune responses.

We have tested in vitro immune responses of several kinds to determine if Ly 5 allotype influences reactivity of murine splenocytes in processes thought to involve the T200 glycoprotein. Matings were established among C57BL/6J (B6) (Ly 5.1) and C57BL/6-Ly 5.2 (B6-Ly 5.2) congenic mice (both H-2b) to obtain sibling mice segregating for alloalleles of the Ly 5 system. F2 progeny of the three Ly 5 genotypes were tested for antibody-dependent cell-mediated cytotoxicity (ADCC), proliferation in allogeneic mixed lymphocyte culture (MLC), mitogen responsiveness, natural killer (NK) cell activity, and cytotoxic T-lymphocyte (CTL)-mediated cytotoxic activity. We observed that in Ly 5 segregant mice, higher CTL activity was associated with the Ly 5.2 type. No allotype effect was observed in MLC, mitogen responses, and NK cell-mediated cytolysis. In the parental and F1 animals, mice carrying the Ly 5.2 allele had significantly higher ADCC levels, though this effect was not seen in the segregants. Our results indicate that Ly 5 or some closely linked gene or genes influence CTL activity of murine splenocytes in vitro.     

Resistance ranking of some common inbred mouse strains to Mycobacterium tuberculosis and relationship to major histocompatibility complex haplotype and Nramp1 genotype.

Six common inbred strains of mice and their F1 hybrids were examined for resistance to infection with the H37Rv strain of Mycobacterium tuberculosis. According to survival times after inoculation of 10(5) CFU intravenously (i.v.), the mice could be classified as being either highly susceptible (CBA, DBA/2, C3H, 129/SvJ) or highly resistant (BALB/c and C57BL/6). F1 hybrids of susceptible and resistant strains were resistant. Although an examination of a limited number of H-2 congenic strains showed that the H-2k haplotype could confer susceptibility on a resistant strain, it was evident that non-major histocompatibility complex (MHC) genes were much more important. Resistant strains all possessed the susceptibility allele of the anti-microbial resistance gene, Nramp1. Results obtained with selected strains infected with 10(2) CFU of M. tuberculosis by aerosol agreed with the results obtained with mice infected i.v. The size of the bacterial inoculum was important in distinguishing between resistant and susceptible strains, in that a 10(7) inoculum overcame the resistance advantage of one strain over another.     

Role for cell-mediated immunity in the resistance of mice to subcutaneous herpes simplex virus infection.

The role of cell-mediated immunity in the resistance of young adult mice to subcutaneous herpes simplex virus (HSV) type I infection was studied in mice receiving immunosuppressive doses of antilymphocyte sera (ALS) or antithymocyte sera (ATS). The effectiveness of these treatments to reduce cell-mediated responses was measured by their ability to prolong the life of allografts transplanted to ALS- or ATS-treated mice. It was found that subcutaneous infection of these mice with HSV resulted in spread of virus from the site of inoculation to the central nervous system. Neutralizing antibody could not be detected in the sera of ALS- or ATS-treated mice after HSV inoculation. Passive transfer of neutralizing antibody to ATS-treated mice did not restore resistance to subcutaneous HSV infection. However, adoptive transfer of HSV-sensitized spleen cells did provide significant protection against infection unless the spleen cells were treated with ATS prior to transfer. These experiments suggest that lymphocytes are involved in a cell-mediated response to subcutaneous HSV infection and demonstrate the importance of a noncompromised immune response in controlling spread of HSV from localized areas of infection.     

H2 complex controls CD4/CD8 ratio, recurrent responsiveness to repeated stimulations, and resistance to activation-induced apoptosis during T cell response to mycobacterial antigens

Genetic variation in the major histocompatibility complex (MHC) influences susceptibility and immune responses to Mycobacterium tuberculosis in mice and humans, but connections among the severity of tuberculosis (TB), dynamic changes in T cell responses to mycobacteria, and MHC genetic polymorphisms are poorly characterized. The overall effect of the MHC genes on TB susceptibility and cellular responses to mycobacteria is moderate; thus, such studies provide reliable results only if congenic mouse strains bearing a variety of H2 haplotypes on an identical genetic background are analyzed. Using a panel of H2-congenic strains on the B10 background, we demonstrate that T cells from mice of three different strains, which are resistant to TB infection, readily respond by proliferation to repeated stimulations with mycobacterial sonicate, whereas T cells from three susceptible mouse strains die after the second stimulation with antigen. This difference is specific, as T cells from TB-susceptible and -resistant mouse strains do not differ in response to irrelevant antigens. The CD4/CD8 ratio in immune lymph nodes correlates strongly and inversely with TB susceptibility, being significantly lower in resistant mice as a result of an increased content of CD8+ cells. These differences between the two sets of mouse strains correlate with an elevated level of activation-induced T cell apoptosis in TB-susceptible mice and a higher proportion of activated CD44+ CD62 ligand- T cells in TB-resistant mice. These results may shed some light on the nature of the cellular basis of MHC-linked differences in susceptibility to TB.     

Resistance and susceptibility of mice to bacterial infection: genetics of listeriosis.

A survey of various strains of mice showed distinct differences in resistance or susceptibility to Listeria monocytogenes. C57B1, related sublines, NZB, and SJL were resistant to Listeria, whereas BALB/c, CBA, A, DBA/1, C3H, LP.RIII, 129, and WB were susceptible. The gene(s) responsible for resistance and susceptibility to Listeria were studied in detail. C57BL6/6, B10.D2, and B10.A mice were 100 times more resistant than were BALB/c, CBA, and A. Resistance of the (C57B1/6 X BALB/C)F1 was intermediate between the two parents, suggesting partial penetration of a dominant gene. Backcross studies in which the (C57B1/6 X BALB/c)F1 were crossed with the susceptible BALB/c parent suggested that a single gene or group of linked genes were the major determinant of resistance, although the possibility that other genes exerted a modifying influence was not excluded. By using the backcross and various congenic and recombinant mice, linkage of the genes involved to the H-1, H-2, H-3, H-4, H-7, or H-8 loci, to the immunoglobulin allotype, to the Thy-1 gene, to the Hc gene specifying C5, or to coat color genes (B, c) was excluded. There was no difference in the response of males and females. In all studies, the powerful overriding influence of the C57B1 genome was evident.     

The Ity/Lsh/Bcg gene significantly affects mouse resistance to Mycobacterium lepraemurium.

Mouse resistance to infection with Mycobacterium lepraemurium was measured by counting the total number of intact acid-fast bacilli in the spleen 8 weeks after i.v. injection of a standard inoculation. The effect of Ityr on resistance to M. lepraemurium was confirmed and the results extended to two Ityr strains of mice, A and C57L, not previously tested. Resistance to M. lepraemurium was also examined in the F1, backcross and F2 generations of BALB/c X CBA crosses, and in the congenic strain B10.LLshr that is Ityr. In all experiments the results were consistent with the view that resistance to M. lepraemurium is significantly affected by a gene close to or identical to the Ity/Lsh/Bcg gene on mouse chromosome 1. Sex had a marked effect on resistance to M. lepraemurium, so that the males of some genetically resistant strains were almost as susceptible as some genetically susceptible females.     

Immunological consequences of innate resistance and susceptibility to BCG

The immunological consequences of genetically controlled innate resistance and susceptibility to Mycobacterium bovis (BCG) infection in mice were investigated. The susceptible (BcgS) BALB/c and the congenic resistant BALB/c.Bcgr mouse strains were employed to test for differences in the specific immune response to BCG. Antigen-specific lymphocyte proliferation and production of interleukin 2 (IL-2) to purified protein derivative (PPD) in vitro were measured following infection of congenic mice with low (10(4)) and high (10(6)) doses of BCG. The lymphocyte subsets were identified by testing the ability of spleen cells to respond after separation of T and B cells and after cytotoxic depletion of T cell subsets. In addition, fluorescence activated cell sorter (FACS) analysis with anti-T and -B cell monoclonal reagents was used to enumerate lymphocyte populations in BCG-infected spleens. The results indicated that the innately resistant mice displayed antigen-specific T helper cell function three weeks following the injection of BCG. The susceptible animals were found to be T cell-unresponsive since they lacked both proliferative and IL-2 secreting specific T cells. No evidence for suppression of IL-2 production or proliferation was detected in BALB/c (susceptible) spleen cultures in cell mixing experiments. The results provide evidence for a regulatory role of the Bcg gene on the generation of lymphocyte responses to BCG.     

Genetic control of antibody responses induced by recombinant Mycobacterium bovis BCG expressing a foreign antigen.

Recombinant Mycobacterium bovis BCG expressing foreign antigens represents a promising candidate for the development of future vaccines and was shown in several experimental models to induce protective immunity against bacterial or parasitic infections. Innate resistance to BCG infection is under genetic control and could modify the immune responses induced against an antigen delivered by such engineered microorganisms. To investigate this question, we analyzed the immune responses of various inbred strains of mice to recombinant BCG expressing beta-galactosidase. These experiments demonstrated that BALB/c mice developed strong antibody responses against BCG expressing beta-galactosidase under the control of two different promoters. In contrast, C57BL/6, C3H, and CBA mice produced high anti-beta-galactosidase antibody titers only when immunized with recombinant BCG expressing beta-galactosidase under the control of the pblaF* promoter, which induced the production of high levels of this antigen. This difference in mouse responsiveness to recombinant BCG was not due to innate resistance to BCG infection, since similar immune responses were induced in Ity(r) and Ity(s) congenic strains of mice. In contrast, the analysis of anti-beta-galactosidase antibody responses of H-2 congenic mice in two different genetic backgrounds demonstrated that H-2 genes are involved in the immune responsiveness to beta-galactosidase delivered by recombinant BCG. Together, these results demonstrate that immune responses to an antigen delivered by recombinant BCG are under complex genetic influences which could play a crucial role in the efficiency of future recombinant BCG vaccines.     

Visceral leishmaniasis in congenic mice of susceptible and resistant phenotypes: immunosuppression by adherent spleen cells.

Visceral leishmaniasis is one of several parasitic diseases of humans characterized by immune suppression. A murine model of disseminated leishmaniasis utilizing inbred strains of specific genetic constitution was used to study the mechanisms of immunosuppression elicited during the course of infection. Resistant (Lshr) and susceptible (Lshs) strains of mice were challenged with amastigotes of Leishmania donovani and evaluated as to immune status at intervals between 2 and 40 weeks after challenge. The proliferative responses of splenic lymphocytes to T-cell mitogens, a B-cell mitogen, and parasite antigens were measured to evaluate the relative immune status of parasitized mice and noninfected control mice. Lymphocytes from resistant C3Heb/FeJ (C3H) mice responded normally to concanavalin A and phytohemagglutinin throughout the course of infection. Parasite antigen responses appeared 2 weeks after challenge of C3H mice and remained vigorous for periods up to 6 months. In contrast, immune suppression during infection was profound in both the curing (C57B1/10) and noncuring (B10.D2) phenotypes of Lshs congenic mice. Both Lshs strains developed severe infection as evidenced by high parasite burdens in the liver and spleen 4 to 5 weeks after challenge; splenic lymphocytes taken from these mice between 2 and 8 weeks became increasingly unresponsive to the T-cell mitogens as well as to parasite antigens. The noncuring B10.D2 mice which suffered chronic infection continued to be suppressed for as long as 40 weeks. C57B1/10 (curing) mice, in contrast, cleared infection between 12 and 16 weeks. After spontaneous recovery or elimination of parasites by antimonial drug therapy, the response of spleen cells to T-cell mitogens or parasite antigens were restored to normal. The spleen cells from the Lshs strains of mice obtained during the height of infection suppressed the proliferative responses of spleen cells from their uninfected counterparts upon cocultivation in vitro. Removal of adherent cells from the suppressive spleen cell populations restored normal mitogen responses. On the basis of adherence characteristics, phagocytosis, and morphology, the suppressor was identified as a macrophage population which appears to be responsible for a nonspecific immunosuppression of Lshs mice with significant parasite burdens of L. donovani.     

In-Vivo Modulation of Macrophage Functions by Herpes Simplex Virus Type 2 in Resistant and Sensitive Inbred Mouse Strains

Intra-peritoneal (i.p.) infection of mice with herpes simplex virus type 2 (HSV 2) attracted macrophages into the peritoneum. Macrophages from moderately and highly HSV 2 resistant mouse strains expressed elevated phagocytosis activity 24 hours after injection. Stimulation of phagocytosis in low resistant strains was generally less effective or absent. This was, in some experiments, due to the fact that macrophages were already highly activated before the experimental infection. I.p. infection also caused HSV replication in the adherent peritoneal exudate cell (PEC) population. The capacity of macrophages supporting HSV 2 replication was low in three of four resistant mouse strains and high in all moderately and highly susceptible and in one of the resistant (SJL) strains when determined 24 hours after infection. Four different F1 hybrids between resistant and susceptible strains exhibited significantly lower yields of virus-producing macrophages than the HSV-sensitive parent. One hybrid between two HSV-susceptible lines restricted virus replication in the PEC population better than both parental strains.     

Cholera toxin as a mucosal adjuvant: effects of H-2 major histocompatibility complex and lps genes.

In previous studies we found that cholera toxin (CT) can act as a mucosal adjuvant; i.e., it can stimulate an intestinal secretory immunoglobulin A (S-IgA) response to an unrelated protein antigen when both are fed together to mice. The purpose of this study was to determine whether the mucosal adjuvanticity of CT is restricted by either H-2 major histocompatibility complex or lps genes by using congenic inbred strains that differ at only a single genetic locus. Groups of five mice each were fed saline, CT (10 micrograms), keyhole limpet hemocyanin (KLH) (5 mg), or both CT and KLH on four different days, and samples of intestinal secretions and plasma were obtained 1 week after the last feeding. In the mice fed both CT and KLH, the intestinal S-IgA anti-KLH response was higher in H-2b congenic strains than in H-2k congenic strains, and in addition there was a highly significant positive correlation between the intestinal S-IgA anti-KLH and S-IgA anti-CT responses in the intestinal secretions of individual mice. Similarly, in the lps congenic strains, mice of the endotoxin-responsive strain that were fed both CT and KLH had substantially higher S-IgA and plasma IgG responses to KLH than did mice of the endotoxin-unresponsive strain. The effect of CT on the induction of oral tolerance to KLH in the H-2 congenic strains was also examined. In contrast to the results above, the abrogation of oral tolerance to KLH by CT occurred in all strains regardless of H-2 haplotype. Similarly, the adjuvant effect of CT on plasma IgG anti-KLH responses after both were given together intraperitoneally was not restricted by H-2. I conclude that the mucosal adjuvanticity of CT is influenced by both the H-2 and lps genetic loci and that it appears to depend on a vigorous mucosal immune response to CT itself.