Cellular Immune Responses in Guinea Pigs Immunized with Cell Walls of Histoplasma capsulatum Prepared by Several Different Procedures

Since guinea pigs immunized with water-washed cell walls of Histoplasma capsulatum developed cellular immune responses detectable with cytoplasmic substances, attempts were made to determine whether cytoplasmic contamination of the walls was responsible for the induction of the immune response. Cell walls were treated by several procedures designed to remove possible contamination, namely, extraction with lipid solvents, incubation with proteolytic enzymes, and washing with sodium dodecyl sulfate, and each of the treated preparations was compared with water-washed walls for its ability to induce cellular responses demonstrable with cytoplasmic substances. For comparison, wall glycoprotein was also used as a test antigen. Immune responses were assessed by gross and histological examinations of skin test sites and by assays for the production of migration inhibition factor. A portion of the material inducing the response detectable with cytoplasmic substances was apparently removed or altered by each of the purifying procedures. The cellular immune responses to wall glycoprotein were also altered, however, indicating that more than the mere removal of cytoplasmic substances had occurred. On the basis of the data collected from each of the cellular assays involving wall glycoprotein as the test antigen, the hypothesis is proposed that sodium dodecyl sulfate altered or removed protein from the wall and thus augmented its ability to induce a more intense immediate-type hypersensitivity, whereas incubation with Pronase altered the walls in such a way as to shift the balance toward a more intense delayed-type hypersensitivity. The latter effect was probably due to the removal of carbohydrate from the wall by glucanase or to mannosidase contaminating the Pronase preparation.     

Serological and Cellular Immune Activity of Peptidoglucomannan Fractions of Candida albicans Cell Walls

A two-stage extraction of isolated cell walls of C. albicans resulted in 45% solubilization into antigens of high molecular weight leaving a wall residue which also had antigenic properties. Ice-cold dilute alkali removed 25% of the defatted cell walls. The extract was nondialyzable, had a glucose-to-mannose ratio of 2:3 and an amino acid content of 7.32%, and was designated peptidoglucomannan (PGM). An additional 26% of the walls resistant to stage I were solubilized by sonic treatment yielding a fraction having a glucose-to-mannose ratio of 6:1, termed soluble mannoglucan (sMG). The residue after extraction and sonic treatment contained 10.9% mannose, which was the insoluble mannoglucan. The gel permeation behavior of PGM and sMG on BioGel A5M was similar; each contained two components, one estimated to exceed 5 x 10(6) molecular weight and a second smaller species. The soluble cell wall fractions were active in immunodiffusion and carried antigenic group specificity. Immunoelectrophoresis of PGM, sMG, and mannan revealed some heterogeneity. The insoluble mannoglucan had agglutinating activity. A distinctive immunodiffusion pattern of cell wall antigens was formed with the serum of a leukemic patient with candidiasis. All three cell wall antigens and mannan elicited delayed-type hypersensitivity as measured by skin-test and specific inhibition of macrophage migration. A dose of 25 mug of PGM was sufficient to inhibit 89.9% migration in the peritoneal exudates of guinea pigs immunized with cell walls, and 10 mug of PGM inhibited 91.7% migration in guinea pigs immunized with insoluble mannoglucan.     

Cellular Immune Activity of a Galactomannan-Protein Complex from Mycelia of Histoplasma capsulatum

A galactomannan-protein complex was extracted from defatted mycelium of Histoplasma capsulatum with 0.25 N alkali at 25 C. It accounted for 7.5% of the cell dry weight in nondialyzable form and had a galactose-mannose ratio of 2:5. The complex containing 68% protein was separated into polysaccharide and glycoprotein components by ion-exchange chromatography. The galactomannan formed a single precipitate in immunodiffusion, sharing a common antigen with the glycoprotein which contained two precipitating antigens. The glycoprotein elicited delayed-type hypersensitivity in guinea pigs as measured by skin-test and macrophage migration inhibitory factor assay, with 5 mug resulting in 86% specific inhibition. The galactomannan was skin-test negative, but 10 mug resulted in 80% specific inhibition of migration.     

Immunotherapy of a guinea pig hepatoma with mycobacterial vaccines: comparison of BCG cell walls and cell wall skeletons.

BCG cell wall skeletons (SK) derived from BCG cell walls (CW) by treatment with proteolytic enzymes and organic solvents were tested for their potency to cause regression of a transplanted guinea pig hepatoma. On a weight basic, SK were as effective as CW in causing tumor regression, and they, as well as purified protein derivative of mycobacteria, provoked delayed cutaneous hypersensitivity reactions in animals immunized with CW or with SK. On a weight basis, CW were more active than SK in eliciting delayed cutaneous hypersensitivity in sensitized guinea pigs whether the animals were immunized with CW or with SK. In unimmunized animals the inflammatory response to intradermally administered CW was greater than that evoked by SK. CW and SK provoked delayed cutaneous hypersensitivity reactions of similar strength in animals immunized with living BCG. This study provided no compelling reasons for using SK instead of CW in clinical trials of cancer treatment by mycobacterial vaccines.     

Isolation of a purified skin test antigen from Blastomyces dermatitidis yeast-phase cell wall.

Preparative polyacrylamide gel electrophoresis was used to isolate individual components of an alkaline-soluble-water-soluble fraction of the cell wall of Blastomyces dermatitidis yeast phase. One component isolated demonstrated exceptional specificity and reactivity when tested on guinea pigs infected with B. dermatitidis. This component displayed no cross-reactivity when tested on animals infected with Histoplasma capsulatum. The significance of isolation of a purified, specific antigen is discussed.     

Acute Cavitary Histoplasmosis in Rhesus Monkeys: Influence of Immunological Status

Male rhesus monkeys were randomized into groups according to body weight, immunized with different Histoplasma capsulatum antigens, and two weeks later were infected intratracheally with 10(8)H. capsulatum yeast cells. Complement fixation antibody titers, skin tests, and chest X rays were performed at weekly intervals from immunization until autopsy, at which time the spleens were cultured and the lungs and other organs were dissected. Pulmonary cavities were found in 33% of the animals, and extrapulmonary dissemination was present in 85% of the animals. Delayed hypersensitivity and circulating antibody activity was detected in all animals at some time during the experimental period; however, animals which developed pulmonary cavities had a longer period before circulating antibodies were detected than animals which did not develop pulmonary cavities. Delayed hypersensitivity developed at approximately the same time in both cavitary and noncavitary animals. Early appearance of delayed hypersensitivity was associated with reduced amounts of extrapulmonary dissemination, in that animals with a later onset of skin test reactivity had more H. capsulatum cultured from the spleen. There was no correlation between the onset or titers of circulating antibodies and the spleen culture results.     

Immunobiological activity of cell wall antigens of Staphylococcus aureus

Guinea pigs sensitized with washed, Formalin-killed cells of Staphylococcus aureus, strains 263 or Copenhagen, were skin-tested with various antigens from these strains including washed viable and heat-killed whole cells, cell walls, the peptidoglycan complexes of the walls, teichoic acid, teichoic acid-peptidoglycan fragments, and peptidoglycan fragments. In nonsensitized control animals, all antigens but teichoic acid elicited acute inflammatory reactions which decreased in size after 10 hr. In animals sensitized with the Copenhagen strain, the reactions to all antigens but teichoic acid and peptidoglycan fragments from either strain remained erythematous and indurated for at least 30 hr and were interpreted as hypersensitivity of the delayed type. Responses in animals sensitized with strain 263 generally resembled those in controls, although in some experiments there was evidence of hypersensitivity.     

Immunization with extracellular proteins of Mycobacterium tuberculosis induces cell-mediated immune responses and substantial protective immunity in a guinea pig model of pulmonary tuberculosis.

We have studied the capacity of a selected fraction of Mycobacterium tuberculosis extracellular proteins (EP) released into broth culture by mid-logarithmic-growth-phase organisms to induce cell-mediated immune responses and protective immunity in a guinea pig model of pulmonary tuberculosis. Guinea pigs infected with M. tuberculosis by aerosol but not uninfected control guinea pigs exhibit strong cell-mediated immune responses to EP, manifest by dose-dependent cutaneous delayed-type hypersensitivity and splenic lymphocyte proliferation. Guinea pigs immunized subcutaneously with EP but not sham-immunized control guinea pigs also develop strong cell-mediated immune responses to EP, manifest by dose-dependent cutaneous delayed-type hypersensitivity and splenic lymphocyte proliferation. EP is nonlethal and nontoxic to guinea pigs upon subcutaneous immunization. Guinea pigs immunized with EP and then challenged with aerosolized M. tuberculosis exhibit protective immunity. In five independent experiments, EP-immunized guinea pigs were consistently protected against clinical illness, including weight loss. Compared with EP-immunized guinea pigs, sham-immunized control guinea pigs lost 12.9 +/- 2.0% (mean +/- SE) of their total weight. EP-immunized guinea pigs also had a 10-fold reduction in viable M. tuberculosis bacilli in their lungs and spleens (P = 0.004 and 0.001, respectively) compared with sham-immunized control animals. In the two experiments in which some guinea pigs died after aerosol challenge, EP-immunized animals were protected from death. Whereas all 12 (100%) EP-immunized guinea pigs survived challenge with aerosolized M. tuberculosis, only 6 of 12 (50%) sham-immunized control guinea pigs survived challenge (P = 0.007, Fisher exact test). This study demonstrates that actively growing M. tuberculosis cells release immunoprotective molecules extracellularly, that a subunit vaccine against tuberculosis is feasible, and that extracellular molecules of M. tuberculosis are potential candidates for a subunit vaccine.     

Immunotherapy of guinea pigs with a transplanted hepatoma: comparison of intralesionally administered killed BCG cells and BCG cell walls.

Heat-killed whole BCG cells (KC) and BCG cell walls (CW) were each tested in emulsified form for their potency to cause regression of a transplanted guinea pig hepatoma. On a weight basis, KC were at least as effective as CW in causing tumor regression and elimination of microscopic lymph node metastasis, and they, as well as purified protein derivative of mycobacteria, provoked delayed cutaneous hypersensitivity reactions in animals immunized with CW or with KC. On a weight basis, KC were as active as CW in eliciting delayed cutaneous hypersensitivity in sensitized guinea pigs whether the animals were immunized with CW or with KC. In unimmunized animals the inflammatory response to intradermally administered KC was similar to that induced by CW. Because KC are easier to prepare than CW, it is suggested that whole killed BCG might be used instead of CW in clinical trials of cancer treatment requiring administration of nonliving mycobacteria.     

In vivo and in vitro cell-mediated immune responses to a cell wall antigen of Blastomyces dermatitidis.

An alkali-soluble, water-soluble cell wall fraction of Blastomyces dermatitidis, designated B-ASWS, was evaluated as an antigen for detecting in vivo (skin tests) and in vitro migration inhibition factor (MIF) production and lymphocyte transformation (LT) responses in Blastomyces-infected guinea pigs. The biological activity of B-ASWS was compared with that of blastomycin KCB-26. The superiority of B-ASWS, in terms of its sensitivity and specificity, was evident in in vivo and in vitro assays. Skin tests responses were obtained in 21 of the 24 Blastomyces-infected guinea pigs, whereas only one of the 14 Histoplasma-infected guinea pigs were significantly greater than those obtained using cell populations from Histoplasma-infected or noninfected guinea pigs. The con-MIF and LT in peritoneal exudate cells and lymph node cells of homologuosly infected animals. In each biological system, the response of the Blastomyces-infected guinea pigs were significantly greater than those obtained using cell populations from Histoplasma-infected or non-infected guinea pigs. The contrasting efficacy of B-ASWS as compared with blastomycin KCB-26, suggests that the cell wall antigen will be a useful tool for detecting cell-mediated immune responses in blastomycosis.     

Protective efficacy of a 62-kilodalton antigen, HIS-62, from the cell wall and cell membrane of Histoplasma capsulatum yeast cells.

We reported previously that a detergent extract of the cell wall and cell membrane of Histoplasma capsulatum yeast cells contains antigens recognized by T cells. In T-cell immunoblot analysis, a region encompassing 62 kDa was stimulatory for an H. capsulatum-reactive T-cell line and T-cell clones derived from C57BL/6 mice. In this study, we isolated a 62-kDa band, termed HIS-62, from electrophoresed cell wall and cell membrane of H. capsulatum yeast cells and examined its antigenicity and immunogenicity. C57BL/6, BALB/c, and CBA/J mice that were immunized with viable H. capsulatum yeast cells mounted a delayed-type hypersensitivity response to HIS-62 that was stronger than that of normal controls. Spleen cells from each strain of mouse immunized with viable yeast cells proliferated vigorously in response to HIS-62; conversely, splenocytes from control animals did not recognize this antigen. A T-cell line and 5 of 5 T-cell clones from C57BL/6 mice, 10 of 15 BALB/c T-cell hybridomas, and 8 of 12 CBA/J T-cell hybridomas recognized HIS-62. A cutaneous delayed-type hypersensitivity response to the antigen was apparent in each strain of mouse that was injected with 80 micrograms of HIS-62 mixed with Freund adjuvant. In addition, spleen cells from HIS-62-immunized mice proliferated in vitro in response to this antigen. Vaccination of each strain of mouse with 80 micrograms of HIS-62 conferred protection against a lethal intravenous challenge with H. capsulatum yeast cells. Thus, HIS-62 appears to be an important target of the cellular immune response to H. capsulatum and induces a protective immune response in mice.     

Influenza in ferrets and guinea pigs: effect on cell-mediated immunity.

Two different animal models were studied to determine whether localized upper respiratory tract viral infection was associated with suppression of systemic cell-mediated immunity. During influenza infection in ferrets, there was no significant decrease in lymphocyte responsiveness to phytohemagglutinin (PHA). Guinea pigs given influenza showed no significant change in their response to PHA or to picryl human serum albumin (picHSA), to which they had been immunized previously. Delayed hypersensitivity skin test responses to picHSA in guinea pigs remained intact during infection. No change in the percentage of circulating T lymphocytes was detected during influenza infection. Transfer of immunity to nonsensitized recipient guinea pigs from picHSA-sensitized guinea pigs was accomplished during influenza infection. Lack of a suppressive effect on systemic cell-mediated immunity after influenza challenge in these two animal models of mild influenza confirmed previous findings in humans with mild influenza infection.     

Protective immunity in murine histoplasmosis: functional comparison of adoptively transferred T-cell clones and splenic T cells.

In this study, I examined whether a murine T-cell line and three clones that recognize Histoplasma capsulatum antigens in vitro could confer protection in vivo against a challenge of Histoplasma yeasts. C57BL/6 mice were each inoculated with 5 X 10(4) yeasts intravenously; 1 h later, 5 X 10(6) or 2 X 10(7) resting T cells were inoculated intravenously. At week 1 of infection, the T-cell line and all clones failed to reduce the number of H. capsulatum CFU in the spleens of mice compared with numbers in infected controls. Administration of recombinant interleukin 2 or cyclophosphamide to infected mice did not potentiate the functional activity in vivo of either the T-cell line or the clones. In contrast, inoculation with 2 X 10(7) CD4+ but not CD8+ cells isolated from the spleens of mice immunized with 10(6) viable yeast cells sharply diminished the number of CFU in the spleens of infected animals. Moreover, splenic CD4+ cells from immune mice transferred a delayed-type hypersensitivity response, whereas the T-cell line and clones did not. Injection Injection of an equal number of cloned T cells and CD8+ splenocytes from immune mice did not transfer resistance to infected mice. Additional studies were undertaken to determine if the ineffectiveness of cloned T cells was associated with a failure to migrate to and survive within spleens of infected mice. B6.PL Thy-1a/Cy mice, which are genetically identical to C57BL/6 mice except that T cells of the former bear Thy-1.1 rather than Thy-1.2, were inoculated with Histoplasma yeasts and then injected with immune CD4+ splenocytes or a T-cell clone. At days 1 and 7 of infection, virtually no Thy-1.2+ cells were detected in the spleens of infected mice given cloned T cells. However, the spleens of animals inoculated with immune CD4+ cells contained a small but significant (P less than 0.01) proportion of Thy-1.2+ cells at both day 1 and day 7 postinoculation of H. capsulatum. Thus, the failure of T-cell clones to transfer protection against H. capsulatum may be explained by defective trafficking or poor survival in vivo or both.     

Influenza virus infection of the guinea pig: Immune response and resistance

Guinea pigs were inoculated by intranasal inoculation with unadapted, influenza virus A/England/42/72, and virus was recovered from nasal washings between 3 and 10 days post-inoculation. Infected animals did not exhibit a febrile response to infection, did not produce local antibody and produced only relatively low levels of serum antibody. However, they developed delayed-type hypersensitivity to influenza virus, demonstrable by both skin tests and macrophage migration inhibition tests, which was similar to that of man. The relevance of the influenza virus specific delayed hypersensitivity in immunity to infection was examined in this model. Guinea pigs previously infected with virus or passively immunized with hyperimmune serum were relatively resistant to reinfection with influenza virus A/England/42/72. Inoculation of guinea pigs with spleen cells from immune donor animals, together with or without immune serum, did not give or enhance resistance to challenge virus infection. The results do not suggest a role for delayed hypersensitivity response in immunity to influenza virus infection.     

Isolation of Skin Test-Active Preparations from Yeast-Phase Cells of Blastomyces dermatitidis

Cell wall and cytoplasmic fractions were isolated from mechanically disrupted yeast-phase cells of Blastomyces dermatitidis in an effort to obtain a reliable skin-test antigen. The biological activities of these fractions were compared with those of two blastomycin preparations. The cytoplasmic antigens exhibited a low index of specificity yet exceeded the specificities of the blastomycins. The skin test-active component(s) of the cytoplasmic material had a molecular weight between 10,000 and 30,000 and could easily be concentrated by ultrafiltration on a PM-10 membrane. Unlike the cytoplasmic antigens and blastomycins, an alkali-soluble, water-soluble cell wall antigen effectively distinguished guinea pigs that were sensitized to B. dermatitidis from those sensitized to Histoplasma capsulatum. The biological activity of the yeast-phase antigen could be quantitated, on a weight basis, after purification by ultrafiltration (PM-10 membrane).     

Skin Testing of Guinea Pigs and Footpad Testing of Mice With a New Antigen for Detecting Delayed Hypersensitivity to Cryptococcus neoformans

This study was undertaken to evaluate the potential of a cryptococcal culture filtrate antigen, cryptococcin C184, for detecting delayed hypersensitivity in Cryptococcus neoformans-injected animals. The antigen was tested on guinea pigs which had received saline or C. neoformans and on animals sensitized to Histoplasma capsulatum, Blastomyces dermatitidis, Candida albicans, or Sporothrix schenckii. A delayed-type hypersensitivity response was elicited by cryptococcin C184 in C. neoformans-injected guinea pigs, whereas no indurations or erythemas were seen at 48 h after skin testing of saline controls or heterologously sensitized guinea pigs. Besides being specific for Cryptococcus, the antigen showed a high degree of sensitivity and was reproducible. Footpad tests were conducted with the antigen on mice which had previously received either 10(5) viable C. neoformans cells or saline. Delayed hypersensitivity was indicated in the C. neoformans-injected mice by the increase in thickness of antigen-injected footpads when compared with the saline-injected footpads. In control mice, antigen- and saline-injected footpads were comparable in thickness 24 h after injection. Mice sensitized to B. dermatitidis were footpad tested with C184, and no cross-reactivity was demonstrated.     

Intradermal and oral immunization with recombinant Mycobacterium bovis BCG expressing the simian immunodeficiency virus Gag protein induces long-lasting, antigen-specific immune responses in guinea pigs

To develop a new recombinant BCG (rBCG) vaccine, we constructed rBCG that expresses the full-length Gag protein of simian immunodeficiency virus (rBCG-SIVGag) at a level of 0.5 ng/mg after 3 weeks of bacterial cell culture. Intradermal (i.d.) inoculation of guinea pigs with 0.1 mg of rBCG-SIVGag resulted in the induction of delayed-type hypersensitivity (DTH) responses to both purified protein derivative (PPD) of tuberculin and SIV Gag p27 protein; responses that were maintained for the duration of the 50-week study. In contrast, guinea pigs orally vaccinated with 160 mg of the same antigen exhibited a long-lasting DTH response to the SIV Gag p27 protein, but mounted no response to PPD. Proliferative responses to SIV Gag p27 and PPD antigens were detected in both i.d. and orally immunized animals; however, the levels of PPD-specific responses were significantly higher in guinea pigs immunized by the i.d. than the oral route. A significant increase in the level of PPD- and SIV Gag p27-specific IFNgamma mRNA expression was also detected in both immunization groups receiving rBCG-SIVGag. In addition, both i.d. and oral immunization with rBCG-SIVGag induced PPD- and SIV Gag p27-specific serum IgG responses. Insertion of the SIV gag gene into BCG did not appear to change the ability of rBCG-immunized animals to elicit PPD-specific immune responses. These results indicate that rBCG-SIVGag has the ability to effectively induce long-lasting, cell-mediated and humoral immunity against both viral and bacterial antigens in guinea pigs, suggesting that rBCG-Gag has the potential to elicit immunities specific not only for tuberculosis but also for HIV at human doses.     

Cell-Mediated Immunity in Guinea Pig and Man to a Salmonella typhi Glycoprotein Complex Assessed by Migration Inhibition Methods

A Salmonella typhi glycoprotein preparation in concentrations of 5 to 10 _μg/ml strongly inhibited the migration of peritoneal exudate cells (PEC), obtained from guinea pigs immunized with S. typhi vaccine in Freund's complete adjuvant (FCA) PEC from animals injected with saline in FCA were also inhibited but to a lesser extent, whereas cells from guinea pigs that had received only S. typhi vaccine or normal guinea pigs showed variable and weaker inhibition. PEC from all guinea pigs receiving FCA were strongly inhibited by 5 to 15 _μg PPD/ml. These PPD concentrations had no inhibitory effect on exudate cells from guinea pigs receiving S. typhi vaccine only. The S.typhi glycoprotein concentration (5 and 10 _μg/ml) primarily used in the migration inhibition studies had negligible inhibitory effect on the phytohemagglutinin (PHA)-induced DNA, RNA, and protein syntheses in guinea pig lymph node cells. In the human system only blood leukocytes of donors who had received their third or fourth booster of heat-killed S. typhi vaccine within the latest 2 years were inhibited in their in vitro migration by the S. typhi glycoprotein complex.     

Cell-mediated immunity in experimental Nocardia asteroides infection.

Experimental mycetoma-like lesions developed in guinea pigs after subcutaneous injection of Nocardia asteroides. Although delayed hypersensitivity appeared earlier, increased macrophage migration inhibition and microbicidal activity appeared after 7 weeks. When the lesions healed, high cell-mediated immunity was present. Cell-mediated immunity was transferred to normal recipient guinea pigs from healed donor guinea pigs by spleen cell transfer. Recipient guinea pigs showed marked protection against challenge with N. asteroides.     

Comparative adjuvant activities of Legionella pneumophila and Mycobacterium tuberculosis

Adjuvant activity of heat-killed Legionella pneumophila was demonstrated and compared with that of inactivated Mycobacterium tuberculosis H37Rv. The two species of bacteria were suspended separately in oil and Arlacel A. Bovine serum albumin (BSA) in saline was then emulsified within the respective adjuvants and injected intradermally into guinea pigs. Antibodies to the BSA antigen in the sera of the animals were quantitated with the kinetic-dependent enzyme-linked immunosorbent assay (k-Elisa). Guinea pigs immunized with BSA in adjuvant with killed L. pneumophila produced high titers of anti-BSA antibody, which, on the average, were nearly as high as in those immunized with BSA in complete Freund's adjuvant with M. tuberculosis H37Rv, and which were much greater than in others immunized with incomplete adjuvant, lacking bacteria. Moreover, with a polypeptide hapten, the L. pneumophila evoked as much or more antibody in rabbits as the mycobacterium adjuvant. The effect of the legionella adjuvant upon the cellular immune response was examined using skin tests. For this purpose guinea pigs were immunized with picryl-guinea pig albumin in these adjuvants. 6 weeks later, they were skin-tested with that antigen. They showed reactions which appeared to have immediate as well as delayed components when examined grossly and histologically. Others, immunized with incomplete adjuvant, did not exhibit delayed reactions. Accordingly, heat-killed L. pneumophila acts as a potent adjuvant. Under the circumstances of these experiments, it was as effective as heat-killed M. tuberculosis.