Protection of mice against brucellosis by intranasal immunization with Brucella melitensis lipopolysaccharide as a noncovalent complex with Neisseria meningitidis group B outer membrane protein

Intranasal immunization of mice with purified Brucella melitensis lipopolysaccharide (LPS) as a noncovalent complex with Neisseria meningitidis group B outer membrane protein (GBOMP) elicited a high-titer anti-LPS systemic antibody response and a significant mucosal antibody response. The anti-LPS immunoglobulin G (IgG) antibody was predominantly of the IgG1 subtype, although there was some response of the IgG2a, IgG2b, and IgG3 subtypes. The antibody titer remained high for 16 weeks postimmunization. Immunized mice and sham-immunized control mice were challenged intranasally with 10(4) CFU of virulent B. melitensis strain 16 M 4 weeks after the second dose of vaccine. The numbers of bacteria in lungs, livers, and spleens at 3 days, 9 days, and 8 weeks postchallenge were determined. Bacteria were found in lungs of all mice on day 3, but there was no disseminated infection of liver or spleen. By day 9, 40% of the mice had infected spleens and livers. At 8 weeks postchallenge, spleens of 25 of 62 immunized mice were infected, compared to 61 of 62 control mice (P < 0.0001). The livers of 12 of 43 immunized mice were infected, compared to 22 of 36 control mice (P = 0.005). In contrast, the lungs of 26 of 46 immunized mice were still infected, compared to 27 of 44 control mice. The numbers of bacterial CFU in lungs of immunized and control animals were identical. These studies show that intranasal immunization with B. melitensis LPS-GBOMP subunit vaccine significantly protects mice against intranasal challenge with virulent B. melitensis. Vaccination reduces bacterial dissemination to spleen and liver but has no effect on the course of lung infection.     

Vaccine and serum-mediated protection against brucella infection of mouse placenta

Pregnant mice inoculated i.p. or i.v. with virulent Brucella abortus Strain 544 displayed strong infection, evidenced by brucella enumeration in placentas and dams' spleens. Vaccination 1 month before pregnancy decreased frequency of placental colonization and number of brucella per infected placenta and per spleen. Protein-bound cell wall peptidoglycan (PG) fractions extracted from brucella protected mice as well as H38 killed and B19 attenuated living vaccines. The lipopolysaccharide (LPS) fraction was comparatively less active. Immune serum injected i.v., either 24 h before or just after i.v. challenge of 15-day pregnant mice effectively transferred resistance to placental colonization. Anti LPS, anti PG and anti killed brucella sera protected mice as well as vaccination one month before pregnancy. The immunity transferred persisted for at least 3 days.     

Vaccine and serum-mediated protection against brucella infection of mouse placenta.

Pregnant mice inoculated i.p. or i.v. with virulent Brucella abortus Strain 544 displayed strong infection, evidenced by brucella enumeration in placentas and dams'' spleens. Vaccination 1 month before pregnancy decreased frequency of placental colonization and number of brucella per infected placenta and per spleen. Protein-bound cell wall peptidoglycan (PG) fractions extracted from brucella protected mice as well as H38 killed and B19 attenuated living vaccines. The lipopolysaccharide (LPS) fraction was comparatively less active. Immune serum injected i.v., either 24 h before or just after i.v. challenge of 15-day pregnant mice effectively transferred resistance to placental colonization. Anti LPS, anti PG and anti killed brucella sera protected mice as well as vaccination one month before pregnancy. The immunity transferred persisted for at least 3 days.     

Schistosoma japonicum: the modulation of lung granuloma and inhibition of egg maturation in mice by human sera

Human sera taken from patients with chronic schistosomiasis japonica have been demonstrated to have two effects on mice. Sera from those patients reduced the size of granuloma in mice sensitised for accelerated granuloma formation to eggs entrapped in the lungs of mice injected with the sera shortly before and at day 2 after intravenous egg challenge. The sera with this effect on the mouse lung granuloma models caused large segmented precipitates in the optimised circumoval precipitin test (COPT). Such sera also reduced the rate at which eggs matured in the liver and intestines of mice infected with S. japonicum. The results strongly support our postulate that a major cause of granuloma modulation in cases of chronic schistosomiasis japonica is antiembryonation immunity and that mice provide useful models for the analysis of our postulate. Identification of egg antigens responsible for the anti-embryonation effect should facilitate progress towards the development of a vaccine against granulomatous disease.     

Comparison of protective efficacy of subcutaneous versus intranasal immunization of mice with a Brucella melitensis lipopolysaccharide subunit vaccine

Groups of mice were immunized either subcutaneously or intranasally with purified Brucella melitensis lipopolysaccharide (LPS) or with LPS as a noncovalent complex with Neisseria meningitidis group B outer membrane protein (LPS-GBOMP). Control mice were inoculated with sterile saline. Two doses of vaccine were given 4 weeks apart. Mice were challenged intranasally with virulent B. melitensis strain 16M 4 weeks after the second dose of vaccine. Sera, spleens, lungs, and livers of mice were harvested 8 weeks after challenge. The bacterial loads in the organs were determined by culture on brucella agar plates. Protective efficacy was determined by comparing the clearance of bacteria from organs of immunized mice with the clearance of bacteria from organs of control mice. At 8 weeks postchallenge there was significant protection from disseminated infection of spleens and livers of mice intranasally immunized with either vaccine compared to infection of control mice (P < 0.01). There was no significant difference in clearance of bacteria from the lungs of immunized mice and control mice. However, mice immunized subcutaneously with either LPS or LPS-GBOMP vaccine showed significant protection against infection of the spleen (P < 0.001), liver (P < 0.001), and lungs (P < 0.05). These results show that intranasal immunization of mice with either vaccine provided significant protection against disseminated infection of the spleen and liver but subcutaneous immunization of mice with the vaccines conferred significant protection against infection of the spleen, liver, and lungs.     

Characteristics of Rickettsia mooseri infection of normal and immune mice.

Rickettsia mooseri infection initiated by subcutaneous injection has been studied in BALB/c mice with the objective of developing a model for the study of immune mechanisms. Characterization of infection included the following: measurement of the replication, dissemination, and clearance of rickettsiae; measurement of correlates of the immune response, including humoral antibody, hypersensitivity to subcutaneously inoculated rickettsial antigen, and activation of nonspecific macrophage microbicidal capacity; and measurement of resistance to a second homologous challenge. Local infection at the site of subcutaneous injection progressed through day 5 and was controlled by day 7. Systemic infection as determined by the presence of rickettsiae in spleen was first detected on day 7 and progressed through day 14; however, rickettsiae persisted in this organ at reduced numbers through at least day 28. Control of the local infection at the site of subcutaneous injection occurred at about the time humoral antibodies and hypersensitivity reactions to subcutaneously injected rickettsial antigens became demonstrable and was paralleled by a capacity to resist homologous subcutaneous challenge at a site distant from that of the primary infection. Systemic infection progressed in spite of this acquired immune capacity and was controlled in the spleen in parallel with the development of enhanced macrophage microbicidal capacity in the liver. The results show that an acquired immunity is capable of restricting rickettsial growth at subcutaneous sites at a time when rickettsiae are increasing in titer in deep organs.     

Antibody and delayed-type hypersensitivity responses to Ochrobactrum anthropi cytosolic and outer membrane antigens in infections by smooth and rough Brucella spp.

Immunological cross-reactions between Brucella spp. and Ochrobactrum anthropi were investigated in animals and humans naturally infected by Brucella spp. and in experimentally infected rams (Brucella ovis infected), rabbits (Brucella melitensis infected), and mice (B. melitensis and Brucella abortus infected). In the animals tested, O. anthropi cytosolic proteins evoked a delayed-type hypersensitivity reaction of a frequency and intensity similar to that observed with B. melitensis brucellin. O. anthropi cytosolic proteins also reacted in gel precipitation tests with antibodies in sera from Brucella natural hosts with a frequency similar to that observed with B. melitensis proteins, and absorption experiments and immunoblotting showed antibodies to both Brucella-specific proteins and proteins common to Brucella and O. anthropi. No antibodies to O. anthropi cytosolic proteins were detected in the sera of Brucella-free hosts. Immunoblotting with sera of Brucella-infected sheep and goats showed immunoglobulin G (IgG) to Brucella group 3 outer membrane proteins and to O. anthropi proteins of similar molecular weight. No IgG to the O-specific polysaccharide of O. anthropi lipopolysaccharide was detected in the sera of Brucella-infected hosts. The sera of sheep, goats, and rabbits infected with B. melitensis contained IgG to O. anthropi rough lipopolysaccharide and lipid A, and B. ovis and O. anthropi rough lipopolysaccharides showed equal reactivities with IgG in the sera of B. ovis-infected rams. The findings show that the immunoresponse of Brucella-infected hosts to protein antigens is not necessarily specific for brucellae and suggest that the presence of O. anthropi or some related bacteria explains the previously described reactivities to Brucella rough lipopolysaccharide and outer membrane proteins in healthy animals.     

Endogenous gamma interferon mediates resistance to Brucella abortus infection.

Depletion of endogenous gamma interferon (IFN-gamma) with anti-IFN-gamma monoclonal antibody resulted in increased numbers of Brucella abortus in the spleen and liver of infected CBA mice. This increase was accompanied by a decrease in splenomegaly and a lower proportion of macrophages in the spleen. Furthermore, treatment of recipient mice with anti-IFN-gamma antibody blocked the adoptive transfer of resistance with immune T cells. Together, the results indicated that endogenous IFN-gamma plays an important role in mediating resistance to primary and secondary Brucella infection.     

Protective effect of Plantago major L. Pectin polysaccharide against systemic Streptococcus pneumoniae infection in mice

The antibacterial effect of a soluble pectin polysaccharide, PMII, isolated from the leaves of Plantago major, was examined in inbred NIH/OlaHsd and Fox Chase SCID mice experimentally infected with Streptococcus pneumoniae serotype 6B. Serotype 6B is known to give a more protracted infection when injected intraperitoneally into susceptible mice than more virulent serotypes like type 4. PMII was administered i.p. either once 3 days before challenge or once to thrice from 3 to 48 h after challenge. The number of bacteria in blood and the mouse survival rate were recorded. Pre-challenge administration of PMII and also lipopolysaccharide (LPS), included as a control, gave a dose-dependent protective effect against S. pneumoniae type 6B infection. However, injection of PMII after establishment of the infection in NIH/OlaHsd mice had no effect. The data demonstrate that, firstly, the polysaccharide fraction PMII from P. major protects against pneumococcal infection in mice when administered systemically prechallenge, and secondly that the protective effect is owing to stimulation of the innate and not the adaptive immune system.     

Oral vaccination with Brucella melitensis WR201 protects mice against intranasal challenge with virulent Brucella melitensis 16M

Human brucellosis can be acquired from infected animal tissues by ingestion, inhalation, or contamination of conjunctiva or traumatized skin by infected animal products. In addition, Brucella is recognized as a biowarfare threat agent. Although a vaccine to protect humans from natural or deliberate infection could be useful, vaccines presently used in animals are unsuitable for human use. We tested orally administered live, attenuated, purine auxotrophic B. melitensis WR201 bacteria for their ability to elicit cellular and humoral immune responses and to protect mice against intranasal challenge with B. melitensis 16M bacteria. Immunized mice made serum antibody to lipopolysaccharide and non-O-polysaccharide antigens. Splenocytes from immunized animals released interleukin-2 and gamma interferon when grown in cultures with Brucella antigens. Immunization led to protection from disseminated infection and enhanced clearance of the challenge inoculum from the lungs. Optimal protection required administration of live bacteria, was related to immunizing dose, and was enhanced by booster immunization. These results establish the usefulness of oral vaccination against respiratory challenge with virulent Brucella and suggest that WR201 should be further investigated as a vaccine to prevent human brucellosis.     

Effect of tilorone on susceptibility of mice to primary or secondary infection with Listeria monocytogenes.

Subcutaneous administration of the drug tilorone did not increase the susceptibility of CD-1 male mice to intravenous or intraperitoneal infection with an alpha-toxin-deficient mutant of the Smith diffuse strain strain of Staphylococcus aureus. In contrast, a single injection of this drug greatly increased the susceptibility of mice to an intravenous or an intraperitoneal infection with Listeria monocytogenes, an intracellular parasite. The effects of tilorone, which were maximal when the drug was administered on the day of the listerial challenge or one day thereafter, were reflected by relatively lower mean lethal dose values and enhanced proliferation of Listeria in the spleen and liver. The ability of tilorone, administered at the time of listerial challenge, to decrease the resistance against the challenge was partially abrogated by previous administration of the same drug. Tilorone, administered at the time of immunization with a very small number of viable Listeria, greatly enhanced the potency of the immunization. On the other hand, tilorone, administered to Listeria-immunized mice near to the time of a listerial challenge, slightly reduced the protective effects of the prior immunization.     

Studies on the immune protection to murine experimental brucellosis conferred by Brucella fractions. I. Positive role of immune serum.

Mouse inoculation with three different phenol-insoluble fractions extracted from Brucella melitensis (fractions 'PI', '4A' and '5') induces an acceleration of the blood clearance of i.v. inoculated live Brucella and a diminution of the rate of multiplication of the injected bacteria in the spleen. Preincubation of the challenge inoculum in immune serum or i.p. injections of immune serum confer a good specific protection to non-immunized hosts. The results observed with fractionated sera suggest that, not only antibodies, but also other serum constituents may participate in the protective activity of immune sera. This discussed in terms of the respective importance of humoral and cellular immunity to Brucella and of the choice of the best preparations for human or animal vaccination.     

A 'safe-site' for Salmonella typhimurium is within splenic cells during the early phase of infection in mice

Salmonella typhimurium infection in mice is focused on the spleen and liver, and prolonged infection can lead to sepsis and death. After intravenous infection with a moderate dose of S. typhimurium, the few bacteria that survive in the spleen and liver grow in a 'safe-site' where they are protected from immune destruction. In this study, we demonstrated that the lack of killing of resident salmonella in the spleen and liver was not because the salmonella were transformed within the host and became resistant to killing, or because the infected mice lost the ability to kill salmonella. We showed that the salmonella were within an intracellular 'safe-site' that protected them from killing. Brief treatment of salmonella-infected mice with gentamicin reduced the numbers of salmonella in the blood but had no effect on the numbers in the liver and spleen, suggesting an intracellular location of the salmonella. After dissociation of spleen cells from recently infected mice, 60% of the salmonella remained cell associated. These cell-associated salmonella, unlike cell-free salmonella, were resistant to killing by gentamicin. The cell-associated salmonella were rendered susceptible to gentamicin after sonication, providing confirmation of their previous intracellular location.     

Course of infection with the emergent pathogen Brucella microti in immunocompromised mice

A new Brucella species, Brucella microti, has been isolated from wild rodents and found to be pathogenic in mice. The biological relevance of this new mouse pathogen is clear, as it allows us to study Brucella infection in a species-specific model. The course of infection in wild-type (wt) and immunodeficient mice that lack B (Jh), T and B (SCID), or T, B, and NK (SCID.Beige) cells was analyzed over 3 weeks. wt mice completely cleared bacteria from the liver and spleen after that time. However, SCID mice showed a much higher bacterial load in the spleen and liver than wt and Jh mice after 1 week and maintained the same level during the next 2 weeks. All mice tested survived for the 3 weeks. In contrast, the bacterial levels in mice that lacked NK cell activity progressively increased and these mice succumbed to infection after 16 to 18 days. Histopathology analysis of infected mice showed extensive areas of necrotic tissue and thrombosis in liver after 1 week in all infected SCID.Beige mice but were not seen in either SCID or wt animals. These processes were dramatically increased after 21 days, corresponding with the death of SCID.Beige animals. Our results indicate that T and/or B cells are required for the control of infection with the mouse pathogen Brucella microti in liver and spleen but that NK cells are crucial for survival in the absence of B and T cells. In addition, they suggest that controlled granuloma formation is critical to clear this type of infection in wt mice.     

Restoration of immunological responses by THF, a thymic hormone, in mice infected with murine cytomegalovirus (MCMV).

Cytomegalovirus causes T cell immune impairment in infected mice, reflected by a decreased response to the T cell mitogens PHA and Con A, and reduction of Con A-induced IL-2 secretion. Concomitantly, a marked increase in the spleen weight of infected mice was found. Histological examination of the liver revealed focal hepatitis. These signs of infection lasted from day 5 to day 14 after infection when mice slowly started to recover. Systemic treatment of MCMV-infected mice with thymic humoral factor (THF) resulted in a reconstitution of the mitogenic responses, IL-2 secretion, normalization of spleen weight and recovery of liver inflammation. Unlike other thymic hormones, THF did not affect interferon synthesis and NK cytotoxicity in infected mice. It is concluded that THF restores immune competence of mice immunosuppressed as a result of MCMV infection, through modulation of the T cell compartment.     

Prophylaxis or treatment of experimental brucellosis with interleukin-1.

Intravenously injected recombinant human interleukin-1 alpha (IL-1 alpha) given to mice 4 h before infection with Brucella abortus 19 depressed the growth of bacteria in the spleen and liver. However, the same dose (10(5) U) or a 10-fold higher dose was not able to decrease numbers of bacteria when given to chronically infected mice. IL-1 injected into normal mice induced a dramatic increase 2 h later in colony-stimulating activity in serum, measured by bone marrow proliferation, and in colony-stimulating factor 1, measured by radioimmunoassay. Colony-stimulating factor levels declined but remained higher than normal for at least 12 h. The early peak stimulation was not observed in chronically infected mice, but the more prolonged elevation was. As a result of IL-1 treatment, the number of colony-forming cells, especially in the spleen, was increased in normal and acutely or chronically infected mice. Myeloperoxidase staining of newly formed monocytes and polymorphonuclear cells in the spleen revealed an increase in the number of these cells in normal and acutely infected mice as a result of IL-1 treatment, but there was no increase in the already high numbers in chronically infected mice. The relationship between these observations and the basis of chronic infection are discussed.     

Virulence criteria for Brucella abortus strains as determined by interferon regulatory factor 1-deficient mice

Interferon regulatory factor 1-deficient (IRF-1(-/-)) mice infected with virulent Brucella abortus 2308 at 5 x 10(5) CFU developed acute hepatitis similar to many natural hosts but, unlike natural hosts, IRF-1(-/-) mice were unable to resolve infection and died. In contrast, IRF-1(-/-) mice survived when infected at 5 x 10(5) CFU with several attenuated Brucella strains (S19, RB51, cbp, and cyd). The survival of infected IRF-1(-/-) mice is likely a function of the level of virulence of each Brucella strain and the extent of retained immunity. Further, these findings suggest that adaptive immunity may be important to the survival of IRF-1(-/-) mice since attenuated Brucella strains can protect IRF-1(-/-) mice against lethal challenge with virulent Brucella: Using the IRF-1(-/-) mouse model, the following set of criteria were identified to define Brucella virulence: (i) the day of death for 50% of mice infected with 5 x 10(5)CFU of Brucella, (ii) the extent of liver toxicity, and (iii) the minimum immunizing dose of Brucella to protect against challenge with virulent S2308. Thus, IRF-1(-/-) mice are important to determining the level of Brucella virulence, to evaluating Brucella mutants for attenuation, and to investigating adaptive immunity in brucellosis.     

Protective Effect of β-Glucan Against Mycobacterium bovis, BCG Infection in BALB/c Mice

β-1,3-Glucan is a potent stimulator of macrophage functions and has a protective effect against a range of infections in rodent models. We examined whether the agent could also protect against the intracellular Mycobacterium bovis , bacillus Calmette–Guérin (BCG) infection in mice. BCG-susceptible BALB/c mice were injected intravenously (i.v.) with β-glucan or vehicle 3 days before, or with β-glucan 7 days after i.v. challenge with live BCG bacilli. The animals were killed 4 or 8 weeks later, their organs were homogenized and applied to object slides and stained with auramin for counting of bacilli, or seeded onto agar in Petri dishes. Mice treated with β-glucan both pre- and postchallenge had significantly lower numbers of BCG bacilli and BCG colony-forming units in spleen homogenates compared with controls 4 weeks after challenge. A similar, but not statistically significant, tendency was observed in spleen homogenates from mice killed 8 weeks after challenge. In homogenates of liver and lungs there were similar findings, but less pronounced. There was a dose-dependent effect of β-glucan injected before BCG challenge on the number of BCG bacilli found in spleen and liver homogenates. In addition, antibody cross-reactivity was demonstrated between M. tuberculosis cell wall and β-glucan. The results suggest that β-glucan has a protective effect against M. bovis , BCG infection in susceptible mice.     

Transfer of immunity against lethal murine Francisella infection by specific antibody depends on host gamma interferon and T cells.

Both serum and spleen cells from mice immune to Francisella tularensis transfer protection to naive recipients. Here we characterize the mechanism of protection induced by transfer of immune mouse serum (IMS). IMS obtained 4 weeks after intradermal infection with 10(3) bacteria of the live vaccine strain (LVS) contained high levels of immunoglobulin G2 (IgG2a) and IgM (end point titers, 1:16,600 and 1:7,200, respectively) and little IgG1, IgG2b, or IgG3. LVS-specific antibodies were detected 5 days after intradermal infection, and reached peak levels by 2 weeks postinfection. Only sera obtained 10 days or more after sublethal infection, when IgG titers peaked, transferred protection against a challenge of 100 50% lethal doses (LD50s). Purified high-titer IgG anti-LVS antibody but not IgM anti-LVS antibody was responsible for transfer of protection against an intraperitoneal challenge of up to 3,000 LD50s. IMS had no direct toxic effects on LVS and did not affect uptake or growth of bacteria in association with peritoneal cells. One day after LVS infection, liver, spleen, and lung tissue from mice treated with IMS contained 1 to 2 log units fewer bacteria than did tissue from mice treated with normal mouse serum or phosphate-buffered saline. Between 2 and 4 days after infection, however, bacterial growth rates in tissues were similar in both serum-protected mice and unprotected mice. Bacterial burdens in IMS-treated, LVS-infected mice declined in infected tissues after day 5, whereas control animals died. This lag phase suggested that development of a host response was involved in complete bacterial clearance. In fact, transfer of IMS into normal recipients that were simultaneously treated with anti-gamma interferon and challenged with LVS did not protect mice from death. Further, transfer of IMS into athymic nu/nu mice did not protect against LVS challenge; protection was, however, reconstituted by transfer of normal T cells into nu/nu mice. Thus, "passive" transfer of protection against LVS with specific antibody is not passive but depends on a host T-cell response to promote clearance of systemic infection and protection against lethal disease.     

Effects of active and passive immunization in the Mycoplasma arthritidis infection of rats

Viable, heat-inactivated, formalin-inactivated and sonicated Mycoplasma (M.) arthritidis antigens as well as immune sera against M. arthritidis and spleen cells from Lewis rats recovered from M. arthritidis infection were injected into naive Lewis rats prior to a challenge infection with 10(7) cfu of M. arthritidis and tested for their protective effects. Viable mycoplasmas induced arthritis combined with the production of high titers of antibodies against M. arthritidis and resistance to a second infection. The application of the inactivated M. arthritidis-antigens in emulsion of incomplete Freund's Adjuvant (ICFA) to naive rats, which also induced a strong antibody production, as well as the inoculation of reconvalescent serum from rats infected with M. arthritidis and hyperimmune serum against M. arthritidis from rabbits and mice protected rats also from an outbreak of arthritis after challenge. The injection of sonicated M. arthritidis antigen without ICFA which failed to induce the production of antibodies and the transfer of spleen cells from rats recovered from M. arthritidis infection had no protective function. The investigations showed, that antibodies play an important role in the prevention of M. arthritidis infections in Lewis rats.