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.     

Induction and expression of immunity after BCG immunization.

The induction and expression of immunity to Mycobacterium tuberculosis after BCG immunization by intravenous, subcutaneous, and pulmonary routes has been investigated in mice. The speed with which protective immunity was engendered was a function of inoculum size; the immunization route was a less influential factor. Tuberculin hypersensitivity varied both with the inoculim size and immunization route, being least after pulmonary immunization. Once immunity was established, a steady state ensued in which the number of sensitized lymphocytes in the spleen was similar, regardless of the route or dose of vaccination. Actively immunized animals controlled intravenous and subcutaneous challenge infections, regardless of the method of vaccination. However, pulmonary challenge was resisted most efficiently by mice immunized by the pulmonary route. Adoptive immunity was well expressed in the spleen only, but the lungs were no more deficient in this regard than the footpad. It is suggested that enhanced immunity in the lungs depends on a population of resident sensitized lymphocytes.     

Paucibacillary tuberculosis in mice after prior aerosol immunization with Mycobacterium bovis BCG

To develop a murine model of paucibacillary tuberculosis for experimental chemotherapy of latent tuberculosis infection, mice were immunized with viable Mycobacterium bovis BCG by the aerosol or intravenous route and then challenged six weeks later with virulent Mycobacterium tuberculosis. The day after immunization, the counts were 3.71 +/- 0.10 log(10) CFU in the lungs of aerosol-immunized mice and 3.65 +/- 0.11 and 4.93 +/- 0.07 log(10) CFU in the lungs and spleens of intravenously immunized mice, respectively. Six weeks later, the lungs of all BCG-immunized mice had many gross lung lesions and splenomegaly; the counts were 5.97 +/- 0.14 and 3.54 +/- 0.07 log(10) CFU in the lungs and spleens of aerosol-immunized mice, respectively, and 4.36 +/- 0.28 and 5.12 +/- 0.23 log(10) CFU in the lungs and spleens of intravenously immunized mice, respectively. Mice were then aerosol challenged with M. tuberculosis by implanting 2.37 +/- 0.13 log(10) CFU in the lungs. Six weeks after challenge, M. tuberculosis had multiplied so that the counts were 6.41 +/- 0.27 and 4.44 +/- 0.14 log(10) CFU in the lungs and spleens of control mice, respectively. Multiplication of M. tuberculosis was greatly limited in BCG-immunized mice. Six weeks after challenge, the counts were 4.76 +/- 0.24 and 3.73 +/- 0.34 log(10) CFU in the lungs of intravenously immunized and aerosol-immunized mice, respectively. In contrast to intravenously immunized mice, there was no detectable dissemination to the spleen in aerosol-immunized mice. Therefore, immunization of mice with BCG by the aerosol route prior to challenge with a low dose of M. tuberculosis resulted in improved containment of infection and a stable paucibacillary infection. This model may prove to be useful for evaluation of new treatments for latent tuberculosis infection in humans.     

Delayed-type hypersensitivity in mice immunized with Trypanosoma rhodesiense antigens.

When mice were immunized intravenously, subcutaneously, or by the footpad route with formaldehyde-killed Trypanosoma rhodesiense, delayed-type hypersensitivity was elicited by the use of frozen-thawed trypanosomal antigen. The delayed footpad swelling technique was used to measure delayed hypersensitivity. Hypersensitivity induction was dose dependent (greater than or equal to 10(6) formaldehyde-treated T. rhodesiense) and was affected by the route of immunization. The footpad route induced higher levels of hypersensitivity than other routes of immunization. Mice immunized with a single dose of formaldehyde-treated antigen and challenged with live T. rhodesiense did not survive. Yet, mice immunized subcutaneously with formaldehyde-treated antigen and then injected with frozen-thawed antigen and challenged 28 days after immunization survived. The results suggest that T-cell activation, manifested by delayed hypersensitivity responses, was a necessary component in the protective response, perhaps functioning in a helper cell capacity.     

Macrophage activation and resistance to pulmonary tuberculosis.

Mice were vaccinated with 300 micrograms of BCG cell walls (BCG-CW) in oil-in-water emulsion intravenously or with a high or low dose of living BCG by inhalation (BCG-HD or BCG-LD, respectively). The consequences of vaccination were evaluated in terms of the growth of BCG in the lungs and spleen, lung and spleen weight, resistance to intravenous and airborne challenge with Listeria monocytogenes, airborne challenge with virulent Mycobacterium tuberculosis H37Rv, and transfer of adoptive immunity. BCG-CW and BCG-HD mice developed increased lung weight, which was associated with transiet, low-level resistance to airborne L. monocytogenes and initial resistance to airborne H37Rv. Only BCG-CW mice developed splenomegaly, which was accompanied by high resistance to intravenous challenge with L. monocytogenes. The initial resistance of BCG-CW mice to H37Rv was not sustained, whereas that of BCG-HD mice persisted. There was no initial resistance to H37Rv in BCG-LD mice, but immunity was generated later. Overall, BCG-HD mice were most resistant to H37Rv, and BCG-CW and BCG-LD mice were less but equally resistant.     

Induction and suppression of cross-reactive antituberculosis immunity after Mycobacterium lepraemurium infection of mice.

Mice immunized with 10(8) live Mycobacterium lepraemurium in the footpad showed increased resistance to infection with BCG or M. tuberculosis R1Rv. This resistance could be transferred adoptively with lymphoid cells, signifying that the immunity was cross-reactive rather than nonspecific. Adoptive cross-reactive immunity to M. tuberculosis was also conferred by spleen cells from mice immunized with large doses of living or dead M. lepraemurium intravenously, a route of immunization that suppresses the induction of cell-mediated immunity to that organism. The presence of specific suppressor activity was sought in mice immunized intravenously with M. lepraemurium. It was found that mice preimmunized intravenously with living or dead M. lepraemurium and then infected with BCG did not confer levels of adoptive antituberculosis immunity as high as those conferred by mice immunized with BCG alone. Similarly, a mixture of BCG-sensitized and M. lepraemurium-sensitized cells did not convey as much immunity as BCG-sensitized cells alone, signifying suppression of the effector lymphocytes.     

Dichotomy between macrophage activation and degree of protection against Listeria monocytogenes and Toxoplasma gondii in mice stimulated with Corynebacterium parvum.

In vivo and in vitro experiments were conducted to determine the effect of Corynebacterium parvum treatment of mice on resistance of Listeria monocytogenes and Toxoplasma gondii. Intravenous immunization with C. parvum conferred transient protection against intravenous challenge with Listeria or an avirulent strain of Toxoplasma but did not protect against a virulent strain of Toxoplasma. Compared with the level of protection conferred by C. parvum, a higher degree of resistance was noted when mice infected with Listeria or Toxoplasma were challenged with the homologous infecting organism. Peritoneal macrophages from mice immunized intravenously with C. parvum were activated to kill Toxoplasma in vitro. Whereas resistance to challenge in vivo was transient, this population of activated macrophages persisted. Peritoneal macrophages from C. parvum mice also markedly inhibited [3H]thymidine uptake by L cells.     

Acquired immunity to Salmonella typhimurium and delayed (footpad) hypersensitivity in BALB/c mice.

BALB/c mice are extremely susceptible to salmonella infections. Previous reports have suggested that this natural susceptibility is due to a defect in cell-mediated immunity (CMI) which correlates with their inability to develop a delayed (footpad) hypersensitivity reaction to a salmonella extract when immunized with attenuated salmonellae. We have shown that mice thus immunized are in fact highly resistant to superinfecting intravenous challenge with virulent organisms, at a time when the footpad test is still negative. The footpad test becomes positive 2-3 weeks later, after the appearance of CMI, which is already present at 1 week as measured by determining the fate of a superinfecting challenge in the RES. The positive footpad reactions that develop in BALB/c mice--and also in B10, and CBA and (B10XA/J)F1 mice--are transferable to normal recipients by thetasensitive spleen cells. However, although B10 mice give positive delayed hypersensitivity (DH) reactions, they are more susceptible to salmonellae of intermediate virulence than the DH negative BALB/c strain. We have also shown that previous reports which suggested that susceptible mice did not develop immunity when vaccinated with live organisms are probably due to the salmonella strain used for vaccination, which does not establish a carrier state. A strain which does establish a carrier state effectively immunizes the susceptible BALB/c strain against virulent challenge, indicating that natural susceptibility does not preclude the development of acquired immunity to reinfection. X     

Immune responses in BALB/c mice following immunization with aromatic compound or purine-dependent Salmonella typhimurium strains.

Two near isogenic strains of Salmonella typhimurium HWSH, stably mutated in either the aroA gene affecting the biosynthesis of aromatic compounds, or the purA gene affecting the biosynthesis of purines, were administered intravenously as live attenuated vaccines to BALB/c mice. HWSH aroA-immunized mice were well protected against intravenous (i.v.) challenge with wild-type virulent HWSH for at least 10 weeks, whereas HWSH purA-immunized mice were unprotected. Furthermore, HWSH aroA-immunized mice could also control a heterologous challenge with virulent Listeria monocytogenes at 7 and 14 days post-immunization, whereas mice receiving a similar dose of HWSH purA could not. Increasing the i.v. dose of HWSH purA compared to HWSH aroA induced some resistance to L. monocytogenes. Induction of early anti-S. typhimurium resistance by HWSH aroA immunization appeared slightly later than the anti-L. monocytogenes resistance. Mice immunized with either vaccine were able to mount S. typhimurium-specific T-cell proliferative responses and produced anti-S. typhimurium humoral antibodies in their serum. The antibody titre was greater in those mice immunized with the aroA mutant.     

Protective immunization against experimental Bacteroides (Porphyromonas) gingivalis infection.

The effects of immunization in modulating the pathogenesis of Bacteroides (Porphyromonas) gingivalis infection in a murine model system were examined. BALB/c mice were immunized by intraperitoneal injection with B. gingivalis ATCC 53977 (one injection per week for 3 weeks), or with a lithium diiodosalicylate (LIS) extract (one injection per week for 3 weeks), or with lipopolysaccharide (LPS; one intravenous or intraperitoneal injection) from this same strain. Two weeks after the final immunization, the mice were challenged by subcutaneous injection of B. gingivalis ATCC 53977. Mice immunized with bacteria had no secondary lesions and no septicemia, whereas mice immunized with LIS extract had few secondary lesions and no septicemia. Mice immunized with LPS and nonimmunized mice demonstrated secondary abdominal lesions and septicemia after challenge. Bacterial cells and LIS extract, but not LPS, induced serum antibody and antigen reactive lymphocytes, as measured by enzyme-linked immunosorbent assay, immunoblot, Western immunoblot transfer, and in vitro lymphoproliferative responses. The present study suggests that immunization with a LIS extract or whole cells may induce a protective response against experimental B. gingivalis infection.     

Specific and nonspecific resistance in mice immunized with irradiated Myobacterium leprae.

Following subcutaneous inoculation of irradiated Mycobacterium leprae (I-ML) into the left hind footpad of mice, there was increased resistance to Listeria monocytogenes, indicative of macrophage activation, at the immunization site. In spite of the high level of localized macrophage activation which was proportioned to the immunizing dose of I-ML, no such activity could be demonstrated systemically in these mice, as evidenced by the absence of increased resistance to an intravenous challenge with L. monocytogenes. Under these conditions, I-ML-immunized mice were nonetheless resistant to intravenous infection with either M. tuberculosis or M. bovis BCG, and this immunity was transferred to normal recipients using spleen or lymph node cells. Neonatal thymectomy completely abolished the development of antimycobacterial immunity after vaccination with I-ML, but immunity was restored by an intraperitoneal infusion of syngeneic thymocytes. Systemic nonspecific resistance could be generated in I-ML-immunized mice by an intravenous injection of disrupted I-ML. This study reveals that, after subcutaneous vaccination with I-ML, there is local accumulation of activated macrophages at the inoculation site and a widespread distribution of lymphocytes which are sensitized to mycobacterial antigens. Nonspecific resistance is mediated by the former cells and specific antimycobacterial immunity by the latter.     

Induction of protective immunity against a Chlamydia trachomatis genital infection in three genetically distinct strains of mice

To establish the feasibility of inducing a protective immune response against a chlamydial genital infection in animals with different genetic backgrounds, groups of C3H/HeN (H-2k), BALB/c (H-2d) and C57BL/6 (H-2b) mice, were immunized intranasally with elementary bodies (EB) of the Chlamydia trachomatis mouse pneumonitis biovar. Following the intranasal immunization strong Chlamydia-specific humoral and cell-mediated immune (CMI) responses were detected in the three strains of mice. Eight weeks following immunization the animals were challenged with C. trachomatis in the genital tract. Vaginal cultures showed that the three strains of mice immunized with EB were significantly protected in comparison to the sham immunized animals. To determine the ability of this immunization protocol to protect against infertility six weeks after the genital challenge the animals were mated. Mice of the three strains immunized with EB showed significant protection as demonstrated by the number of animals that were fertile, and the number of embryos present in their uterine horns, in comparison to the sham immunized mice.     

Direct correlation between delayed footpad reaction and resistance to local bacterial infection.

The resistance to bacteria was studied at the site of delayed footpad reaction in mice immunized with Listeria monocytogenes. When a challenge injection of listeria was given into the footpad of immune mice, no enhancement of bacterial elimination was observed before the generation of delayed footpad reactivity. After the generation of delayed reactivity, an enhanced elimination of listeria or Salmonella typhimurium was observed only at the site of strongly positive delayed footpad reaction elicited with listerial antigen. Such an enhancement in bacterial elimination was also found at the site of delayed footpad reaction induced by immunization with heterologous erythrocytes. Both delayed footpad reaction and local resistance could be transferred locally by immune spleen cells in a dose-dependent fashion, but were completely abrogated when recipient mice were treated with carrageenan or whole-body X irradiation. Macrophage accumulation was an important factor in the expression of resistance at the reaction site. These results suggest that the delayed footpad reaction contributes to host defense by enhancing the local resistance to bacteria.     

The relative difference in anti-Listeria resistance of C57BL/6 and A/J mice is not eliminated by active immunization or by transfer of Listeria-immune T cells.

In this study, we examined the effects of active and adoptive immunization on the anti-Listeria resistance of innately resistant C57BL/6 and innately susceptible A/J mice. Although active immunization with a sublethal dose of viable Listeria monocytogenes markedly enhanced the anti-Listeria resistance of both C57BL/6 and A/J mice, the 100-fold difference between the two strains in innate anti-Listeria resistance was not diminished. Following immunization with an equivalent sublethal dose (0.1 LD50) of L. monocytogenes, both C57BL/6 and A/J mice generated T cells that could transfer significant and comparable protection to syngeneic recipients that were challenged with up to a 10 LD50 dose of L. monocytogenes. When the absolute number of viable Listeria was compared, however, it was clear that T cells from immunized C57BL/6 mice were capable of transferring protection to syngeneic recipients at Listeria challenge doses that were more than 100-fold greater than could T cells from Listeria-immunized A/J mice. Both active immunization and adoptive transfer of syngeneic Listeria-immune T cells enhanced the accumulation of inflammatory neutrophils and macrophages in C57BL/6 and A/J mice. More inflammatory neutrophils were recovered from actively immunized C57BL/6 than from A/J mice, whereas more inflammatory macrophages were obtained from adoptively immunized C57BL/6 than from A/J mice. These results provide further evidence for the beneficial role of inflammation in genetically determined innate resistance and T-cell mediated resistance to listeriosis. These data also suggest that some mechanism in addition to inflammatory responsiveness may be responsible for limiting the expression of acquired anti-Listeria resistance in genetically susceptible A/J mice.     

Virus strain specificity of challenge immunity to coronavirus

Summary The resistance of immunized mice to challenge with the same or a different strain of mouse hepatitis virus (MHV) was examined as a model of challenge immunity to coronavirus infection. Genetically susceptible BALB/cByJ mice were given an intranasal immunizing infection of respiratory-type MHV-JHM, MHV-S, or enterotropic MHV-Y. Control mice were sham-immunized with sterile tissue culture fluid. Recovered mice were challenged intranasally with MHV-JHM, MHV-S or sterile tissue culture fluid at 30 days after immunization. Resistance to challenge inoculation was evaluated in groups of mice at 4 and 30 days after challenge. At 4 days, the prevalence of MHV lesions in nose and liver was tabulated and MHV titers in liver were determined. At 30 days, the prevalence of residual brainstem spongiform lesions was tabulated and serum antibody to MHV-JHM and MVH-S was quantified by enzyme immunoassay. Mice immunized with MHV-JHM or MHV-S resisted challenge with the MHV homotype, but MHV-S-immunized mice were fully susceptible to challenge with MVH-JHM. Mice immunized with enterotropic MHV-Y were only partially protected against challenge with antigenically related, but biologically different MHV-S. Serum antibody responses to MHV supported these observations. These data indicate that challenge immunity to coronaviruses is strong, but highly virus strain-specific.     

Lymphocyte recruitment and protective efficacy against pulmonary mycobacterial infection are independent of the route of prior Mycobacterium bovis BCG immunization

Mycobacterium tuberculosis infects humans through the lung, and immunity to this chronic infection is mediated primarily by CD4(+) T lymphocytes. Recently we have demonstrated that the recruitment of lymphocytes to the lung during primary aerosol M. tuberculosis infection in mice occurs predominantly through the interaction of alpha(4)beta(1) integrin on CD4(+) T cells and vascular cell adhesion molecule-1 on the pulmonary endothelium. To investigate the effect of route of immunization with Mycobacterium bovis BCG on the pattern of T-cell recruitment to the lung, we have analyzed the differences in expression of integrins on activated memory CD4(+) T cells infiltrating the lung following primary BCG immunization by aerosol, intravenous, and subcutaneous routes and after subsequent aerosol challenge with M. tuberculosis. There were marked differences in the patterns of recruitment of activated CD4(+) T cells to the lung following primary immunization by the three routes. Expansion of CD44(hi) CD62L(low) CD4(+) T cells in the lung occurred following aerosol and intravenous BCG immunizations, and the lymphocyte recruitment was proportional to the pulmonary bacterial load. The majority of infiltrating CD4(+) T cells expressed alpha(4)beta(1) integrin. On subsequent exposure to aerosol BCG rapid expansion of gamma interferon-secreting alpha(4)beta(1)(+) CD4(+) T cells occurred to the same extent in all immunized mice, regardless of the route of immunization. Similar expansion of alpha(4)beta(1)(+) CD4(+) memory T cells occurred following M. tuberculosis challenge. The three routes of BCG immunization resulted in the same level of protection against aerosol M. tuberculosis or BCG challenge in both the lungs and spleen. Therefore, recruitment of effector T lymphocytes and protective efficacy against pulmonary mycobacterial infection are independent of the route of prior BCG immunization.     

Protection Against Meningococcal Challenge Afforded Mice After Immunization with Typhoid-Paratyphoid Vaccine USP

Mice immunized with typhoid-paratyphoid vaccine USP were found to be protected when challenged with serological groups A, B, and C of Neisseria meningitidis. Mice immunized with this vaccine both 14 and 7 days prior to challenge were protected against mean lethal doses (LD₅₀) of 5,220 and 1,151 of groups A and B, respectively, and in two separate experiments they were protected against 3,568 and 268 LD₅₀ of group C. Mice immunized four or more times on successive days prior to challenge were also protected. They demonstrated tolerance and survived a much larger LD₅₀ challenge than mice immunized by any other immunization schedule. Mice immunized once or twice anytime within 7 days prior to challenge were not protected and thus failed to exhibit nonspecific resistance. Serum antibody probably was responsible for the protection afforded mice immunized 14 and 7 days prior to challenge. However, this was not demonstrated conclusively.     

Protective efficacy of H antigen from Histoplasma capsulatum in a murine model of pulmonary histoplasmosis

We previously reported that immunization with H antigen from Histoplasma capsulatum did not protect mice against an intravenous challenge with yeasts. Here, we investigated the utility of H antigen to protect mice in a model of pulmonary histoplasmosis. Mice immunized with H antigen and challenged intranasally 4 weeks postvaccination were protected against sublethal and lethal challenges with H. capsulatum yeasts. If the challenge was performed 3 months after vaccination, there was a reduction in fungal burden following sublethal challenge and a modest delay in mortality in mice given a lethal inoculum. Vaccination was associated with production of gamma interferon, granulocyte-macrophage colony-stimulating factor, interleukin-4, and interleukin-10 by splenocytes. Vaccination with H antigen was not accompanied by a major expansion of CD4(+) or CD8(+) cells in spleens of mice. These results demonstrate that H antigen may be useful as a protective immunogen against pulmonary exposure to H. capsulatum.     

Partial protection against Brucella infection in mice by immunization with nonpathogenic alphaproteobacteria

Previous findings indicate that Brucella antigens and those from nonpathogenic alphaproteobacteria (NPAP) are cross-recognized by the immune system. We hypothesized that immunization with NPAP would protect mice from Brucella infection. Mice were immunized subcutaneously with heat-killed Ochrobactrum anthropi, Sinorhizobium meliloti, Mesorhizobium loti, Agrobacterium tumefaciens, or Brucella melitensis H38 (standard positive control) before intravenous challenge with Brucella abortus 2308. Cross-reacting serum antibodies against Brucella antigens were detected at the moment of challenge in all NPAP-immunized mice. Thirty days after B. abortus challenge, splenic CFU counts were significantly lower in mice immunized with O. anthropi, M. loti, and B. melitensis H38 than in the phosphate-buffered saline controls (protection levels were 0.80, 0.66, and 1.99 log units, respectively). In mice immunized intraperitoneally with cytosoluble extracts from NPAP or Brucella abortus, protection levels were 1.58 for the latter, 0.63 for O. anthropi, and 0.40 for M. loti. To test whether the use of live NPAP would increase protection further, mice were both immunized and challenged by the oral route. Immunization with NPAP induced a significant increase in serum immunoglobulin G (IgG), but not serum or fecal IgA, against Brucella antigens. After challenge, anti-Brucella IgA increased significantly in the sera and feces of mice orally immunized with O. anthropi. For all NPAP, protection levels were higher than those obtained with systemic immunizations but were lower than those obtained by oral immunization with heat-killed B. abortus. These results show that immunization with NPAP, especially O. anthropi, confers partial protection against Brucella challenge. However, such protection is lower than that conferred by immunization with whole Brucella or its cytosoluble fraction.     

Pyelonephritis XIV. Effect of Immunization on Experimental Escherichia coli Pyelonephritis

Mice immunized subcutaneously with heat-killed Escherichia coli were protected from pyelonephritis produced by the intravenous route, but there was little or no protection from ascending infection. No significant protection from ascending or hematogenous pyelonephritis was demonstrated when immunization was accomplished by injecting heat-killed E. coli into the bladder. Heat-killed E. coli injected either subcutaneously or into the bladder protected mice from early endotoxic death after intravenous or bladder challenge.