Prophylactic immunization against experimental leishmaniasis. III. Protection against fatal Leishmania tropica infection induced by irradiated promastigotes involves Lyt-1+2- T cells that do not mediate cutaneous DTH

Protective immunity against fatal L. tropica infection in genetically vulnerable BALB/c mice can be induced by prophylactic immunization with irradiated promastigotes even when heat-killed. Such immunity is adoptively transferable transiently into intact or durably into sub-lethally irradiated (200 or 550 rad) syngeneic recipients by splenic T but not B cells. The effector T cells are of the Lyt-1+2- phenotype, devoid of demonstrable cytotoxic activity. The immune splenic T cell population expresses specific helper activity for antibody synthesis. A causal role for helper T cells in this capacity, however, seems unlikely, because it was shown in the accompanying paper that antibody does not determine the protective immunity against L. tropica. The immunized donors show no detectable cutaneous DTH or its early memory recall in response to live or killed promastigotes or a soluble L. tropica antigen preparation. Spleen, lymph node, and peritoneal exudate cells from protectively immunized donors similarly fail to transfer DTH locally or systemically. These cells also lack demonstrable suppressive activity against the expression or induction of DTH to L. tropica. Thus, protection against L. tropica induced by prophylactic i.v. immunization with irradiated promastigotes appears to be conferred by Lyt-1+2- T cells that are distinguishable from T cells mediating either both DTH and T help, or cytotoxicity.     

Vaccination against cutaneous leishmaniasis in a murine model. I. Induction of protective immunity with a soluble extract of promastigotes

BALB/c mice can be protected against a fatal Leishmania major infection by immunization with whole radio-attenuated promastigotes; however, neither the antigens responsible for protection nor the protective immunologic mechanisms have been defined. In this study, the ability of promastigote fractions to elicit similar immunity to that obtained with whole organisms, and the immune responses associated with such protection were analyzed. Intraperitoneal immunization with a soluble, membrane-free parasite extract was found to induce protection against L. major challenge equal to that obtained with whole organisms. Induction of immunity (89% protection in seven experiments) was most effective with 100 micrograms of the soluble leishmanial antigen (SLA) and required concomitant injection of the bacterial adjuvant, Corynebacterium parvum (CP), followed by an i.p. boost of SLA alone 1 wk later. Vaccinated animals exhibited Leishmania-specific cell-mediated immunity, as assessed both by lymphocyte transformation and the production of macrophage-activating factors (MAF). In addition, although SLA + CP-immunized mice failed to exhibit delayed-type hypersensitivity (DTH) before challenge, splenic lymphocytes from these mice could transfer a local DTH reaction to naive recipients. Immunization also induced the production of antibodies against two major metabolically labeled proteins of m.w. 30,000 and 53,000, but failed to stimulate a detectable humoral response against promastigote surface antigens. Thus, these experiments demonstrate that vaccine-induced immunity against cutaneous leishmaniasis is strongly associated with the induction of cell-mediated immunity, but does not require the development of an antibody response to promastigote surface antigens. In addition, these studies establish the feasibility of employing soluble, nonmembrane-derived parasite material as a source of protective immunogens.     

Adoptive protection of the Mycobacterium tuberculosis-infected lung. Dissociation between cells that passively transfer protective immunity and those that transfer delayed-type hypersensitivity to tuberculin

Adoptive transfer of protective immunity to an aerogenic infection with the facultative intracellular bacterium Mycobacterium tuberculosis was mediated by a population of T cells acquired in the spleen of donor mice at the height of the primary cell-mediated immune response to an immunizing infection with M. bovis bacillus Calmette-Guerin. Successful adoptive immunotherapy was ablated by prior exposure of immune donor cells to ionizing radiation or by treatment of these cells with antibody raised against the Ly-2 marker. In contrast, however, the capacity of immune donor cells to passively transfer delayed-type hypersensitivity (DTH) responses to tuberculin was unaffected by prior treatment with antibody to Ly-2, but was completely ablated by treatment by antibody to Ly-1. These results indicate, that DTH and protective anti-tuberculous immunity are dissociable phenomena, mediated by separate populations of T lymphocytes.     

Tumor bearer T cells suppress BCG-potentiated antitumor responses. I. Requirements for their effect

Mice bearing established syngeneic tumors fail to reject them when immunized according to protocols based on optimal conditions for BCG potentiation of specific delayed-type hypersensitivity (DTH) and antitumor immunity. Serum factors from mice bearing either the poorly immunogenic mastocytoma, P815 (MA), or the more antigenic sarcoma, Meth A, have been shown to depress both DTH and antitumor immunity. This report demonstrates that lymphoid cells adoptively transferred from these tumor-bearing hosts also can suppress the efferent and afferent phases of DTH to tumor-specific antigens in both BCG-primed and unprimed syngeneic hosts. Suppressor cells (SC) were detected in spleen, thymus, and lymph nodes draining the tumor site, but not in distant superficial lymph nodes. Maximal suppressor activity apeared 6 days after tumor implantation and waned by 18 days. Suppression of the afferent phase of both the BCG-primed and unprimed responses was antigen specific; suppression of the efferent phase of the BCG-primed response was also specific but SC could partially suppress the unprimed responses to sheep red blood cells (SRBC). Amputation of 6-day-old tumors resulted in the disappearance of splenic SC within 2 days but did not affect SC in draining lymph nodes. SC suppressed DTH in a dose-dependent manner but even the highest doses tested did not totally eliminate the response. Depression of the peak DTH reaction was not accompanied by significant abrogation of antitumor activity. If, however, SC were transferred during the ongoing antitumor response, immunity was partially suppressed. Efferentphase SC were sensitive to treatment with anti-Thy 1 sera and complement but were unaffected by B-cell depletion.     

Prophylactic immunization against experimental leishmaniasis. V. Mechanism of the anti-protective blocking effect induced by subcutaneous immunization against Leishmania major infection

The responsiveness of BALB/c mice to protective i.v. immunization with 150,000-rad irradiated or heat-killed Leishmania major promastigotes can be totally suppressed by prior subcutaneous (s.c.) injection of the same "vaccine." Induction of this effect is leishmania specific for although prevention of protection against L. major infection can be obtained with either homologous or Leishmania donovani promastigotes, it does not follow s.c. administration of an immunogenic Trypanosoma cruzi epimastigote preparation. Multiple s.c. injections of irradiated L. major promastigotes do not inhibit the subsequent antibody response of any major isotype to i.v. immunization, but rather induce some priming. The same s.c. injections induced delayed-type hypersensitivity (DTH) reactivity that could be transferred locally or systemically, although it was weaker than in mice with cured infections. Parallel cell-mediated immunity (CMI) responses were also reflected in vitro in specific lymphocyte transformation assays. Despite this evidence of a DTH/helper type of T cell response, transfer of 5 X 10(7) viable T cell-enriched spleen cells from 4 X s.c. immunized donors to normal recipients completely abrogated the protective response to i.v. immunization. Conversely, T cell-depleted (anti-Thy-1.2 + C treated) cells were without effect. The inhibitory T cells were defined by monoclonal antibody pretreatment as possessing an Lyt-1+2-,L3T4+ phenotype. T cells from s.c. immunized donors were also shown, by mixed transfer experiments, to counteract completely the protective effect of T cells from i.v. immunized donors in 550-rad irradiated recipients. They were as potent as suppressor T cells from donors with progressive disease both in this capacity and in abrogating the prophylactic effect of sublethal irradiation itself. The similarities and differences between suppressor and immune effector T cells induced by s.c. or i.v. immunization and those arising in response to leishmanial infection itself are discussed.     

Murine T cell clones specific for Hymenolepis nana: generation and functional analysis in vivo and in vitro.

To examine the role of the T cell in protective immunity to Hymenolepis nana, H. nana-specific clonal lymphocytes were generated from mesenteric lymph nodes of BALB/c mice infected with H. nana, and some of their functions were analyzed in vitro and in vivo. Following limiting dilution techniques, five clones were generated from mesenteric lymph node cell populations. All of these clones expressed the L3T4⁺, Lyt-2.2⁻ phenotype and proliferated in vitro in response to soluble egg antigen of H. nana. Of five clones, three secreted interleukin 2 (IL-2) and interferon-γ (IFN-γ) after stimulation with egg antigen. Furthermore, these three clones conferred local delayed-type hypersensitivity to egg antigen. The remaining two clones produced interleukin 4 (IL-4) in response to egg antigen, and could not mediate local delayed-type hypersensitivity. Adoptive transfer experiments using clonal lymphocytes were also undertaken in an attempt to define cell types involved in protective immunity. Clonal lymphocytes secreting both IL-2 and IFN-γ transferred protective immunity, equivalent to that obtained by non-cultured-sensitized mesenteric lymph node cells. They were effective in very small numbers. However, clonal lymphocytes that secreted IL-4 did not transfer protective immunity. These results suggest that helper T lymphocytes, especially the Th1 subtype, are involved in protective immunity against H. nana.     

Cellular changes in the intestinal lymph of sheep infected with the enteric nematode, Trichostrongylus colubriformis

Some changes produced in the cell populations of intestinal lymph by infection with the enteric nematode, Trichostrongylus colubriformis, were studied in sheep regularly re-infused with all discharged lymph. Lymphocyte traffic through the intestinal lymphatic duct was reduced until day 35 of primary infection, mainly due to the absence of lymphocytes with smaller cell volumes, but was increased two-fold after day 70 and in immune sheep. Antigen-reactive lymphocytes in blood and lymph were assayed by the uptake of 3H-thymidine in cell culture stimulated by extracts from the larvae of T. colubriformis. Cells from the blood and lymph of immune sheep were highly reactive to worm antigen. A relatively smaller reactivity was present in the blood of worm-free sheep and was abolished during the first 12 days of primary infection. Antigen reactive cells were not detected in intestinal lymph until 12 days after primary infection, and in vitro antigen reactivity in intestinal lymph of immune sheep was increased after challenge with infective larvae. Responses to the mitogens, concanvalin A and phytohaemagglutinin, in cultures of cells from both intestinal lymph and blood were depressed on days 7 and 12 of primary infection. It is proposed that the diminished traffic of lymphocytes in intestinal lymph and the reduced numbers of mitogen and nematode antigen-reactive lymphocytes in both blood and intestinal lymph during the early stages of infection with T. colubriformis is closely related to the slow development of protective immunity to this parasite.     

Routes of inoculation and the immune response to a resolving genital flavivirus infection in a novel murine model

The prolonged, abnormal immune response patterns produced by many sexually transmitted viruses have been intensively studied. Because normal antiviral immune responses in the vagina are less well-defined, we developed a resolving murine model using vaginal inoculation with the flavivirus, West Nile virus. Infection resulted in 12% mortality, with sterile protective immunity to vaginal or systemic re-challenge. B-cell numbers increased in the vaginal mucosa from day 1-7 after primary infection, while similar increases in B220(+), CD4(+) and CD8(+) lymphocytes in the draining lymph node were delayed by 48 h. By day 4 postinfection, a MHC-II(+) dendritic cell population became depleted from the stroma and formed aggregates below the basement membrane at points of demonstrable epithelial infection. In contrast, primary systemic or intradermal inoculation resulted in 80-90% mortality, but also conferred protective sterile immunity to vaginal West Nile virus re-challenge. Intravaginal and intradermal immunization elicited comparable, accelerated accumulation of larger B-cell numbers in the mucosa and draining lymph node upon intravaginal re-challenge than systemic immunization. However, accumulation of CD4(+) T cells in both sites in the intradermally immunized group was significantly greater than in intravaginally or systemically immunized mice. Accelerated accumulation of dendritic cells occurred at periodic sub-basement membrane sites in the absence of detectable virus 1 day after vaginal re-challenge, irrespective of the route of immunization. These data illustrate the diversity of possible effective immune responses to West Nile virus in the vaginal mucosa. They show primary vaginal inoculation produces effective immunity to flavivirus infection with lower mortality than other routes and suggest a local role for vaginal mucosal dendritic cells in both primary and secondary responses.     

Separate transfer of mouse protection and delayed-type hypersensitivity with Salmonella typhimurium transfer factor

Delayed-type hypersensitivity (DTH) induced with Salmonella typhimurium transfer factor (TF) contributed to an increase in mean survival days of mice challenged with homologous organisms and afforded only a low level of host protection as determined by survival rate, compared with that obtained by active immunization. TF of other enteric bacteria could transfer DTH which is cross-reactive to salmonella antigen but did not afford host protection. Although TF of Listeria monocytogenes did not transfer the cross-reactive DTH, it could confer the significant increase in mean survival days against the lethal challenge with S. typhimurium. Listerial ribosomal vaccine conferred the high level of mouse protection without inducing DTH to salmonella antigen. The resistance generated upon active immunization with listerial ribosomal vaccine could be enhanced by the injection of S. typhimurium TF to the same level as that obtained after immunization with homologous ribosomal vaccine. Among salmonella TF, there could be no cross-reactive immunity between S. typhimurium and S. choleraesuis, although the cross-reactive DTH was observed. The DTH transfer ability of TF was sensitive to Pronase which could not affect the ability to transfer host immunity, but RNase could abolish the ability to transfer host immunity without impairing DTH transfer activity. These results suggest that in mouse typhoid infection, DTH is not associated with host protection as determined by survival rate.     

Studies on the mechanism of the enhancement of delayed-type hypersensitivity by pertussigen

The potentiation of delayed-type hypersensitivity (DTH) reactions by pertussigen, a protein toxin from Bordetella pertussis, has been studied in adoptive transfer assays. Lymph node or spleen cells from mice treated with or without pertussigen at the time of immunization with protein antigens were transferred to naive, syngeneic recipients that were challenged with antigen. Cells from donors treated with pertussigen had the capacity to transfer vigorous, antigen-specific DTH reactions. Cells from immunized donors not given pertussigen transferred little or no DTH. These results indicate that pertussigen is able to augment DTH reactions by potentiating the antigen reactivity of cell populations in lymphoid organs. The phenotype of the effector cells induced by pertussigen was Thy-1 positive, L3T4 positive, and Ly-2 negative. Cells from mice given pertussigen and an irrelevant antigen had no influence on specific DTH responses, suggesting that pertussigen enhances the activity of the antigen-specific cell type mediating DTH. The effect of pertussigen and of immunization on the lymphocyte subpopulations present in the lymph nodes was studied by analysis of suspensions of lymph node cells by flow cytometry. In immunized and in nonimmune mice, pertussigen increased the ratio of Ly-2-negative:Ly-2-positive T cells, and reduced the overall proportion of B cells. In immunized mice, pertussigen induced a much higher proportion of large dividing cells from 5 days after sensitization onwards. The relevance of these changes in lymphocyte behavior to the development of enhanced and prolonged DTH in mice given pertussigen is discussed.     

Effect of antigen challenge on lymph node lymphocyte adhesion to vascular endothelial cells and the role of VLA-4 in the rat.

Lymphocytes from antigen-stimulated lymph nodes avidly migrate from the blood to cutaneous sites of inflammation such as DTH reactions or contact sensitivity. One of the initial steps in this migration is the adhesion of the lymphocyte to endothelial cells (EC); therefore, the adhesion of lymphocytes from antigen-stimulated lymph nodes to microvascular EC in the rat was examined. Two to five days after subcutaneous immunization with antigen, lymphocytes that adhered to unstimulated and IFN-gamma-, TNF-alpha-, IL-1 alpha-, and LPS-treated EC were increased in the regional lymph nodes. The enhanced adhesion was attributable to low-density lymphoblast-enriched lymph node cells while small high-density lymphocytes displayed little or no increase in their adhesion. Lymphoblast adhesion required the stimulation of the EC with 10 times the concentrations of IFN-gamma and TNF-alpha required for peritoneal exudate lymphocyte adhesion. There was a synergistic increase in the adhesion of the low-density lymphocytes to EC stimulated with combinations of IFN-gamma and TNF-alpha. Antibody to VLA-4 inhibited about 40% of the stimulated adhesion to EC treated with IFN-gamma, TNF-alpha, or LPS. In vivo anti-VLA-4 inhibited lymphoblast migration to IFN-gamma, TNF-alpha, LPS, and DTH reactions by 60%. Thus antigen stimulates the generation of low-density lymphoblasts that have an enhanced adherence to cytokine- and LPS-treated EC through a partially VLA-4-dependent mechanism and the migration of these cells to cutaneous inflammatory reactions is dependent upon VLA-4.     

Effect of a single exposure to ultraviolet radiation on Mycobacterium bovis bacillus Calmette-Guerin infection in mice

BALB/c and C3H mice were exposed on the dorsal skin to 45 kJ/m2 of UVB radiation from FS-40 sunlamps 3 days before infection with 1 x 10(6) live units of Mycobacterium bovis bacillus Calmette-Guérin (BCG) (Tice strain) in the footpad. At regular intervals, groups of mice were tested for a delayed type hypersensitivity (DTH) response to the purified protein derivative (PPD) of tubercle bacilli, and the course of infection was monitored by measuring the size of the infected footpad, enlargement of the draining lymph node, and the number of bacteria in the spleen and lymph node. In both strains the DTH response to PPD was significantly delayed in UV-treated mice compared to unirradiated mice, when tested 21 and 42 days after BCG infection. By day 50, no significant difference was detected in the DTH response between irradiated and unirradiated mice. UV treatment reduced the size of the lymph node draining the site of BCG infection in both strains of mice and the size of the infected footpad in C3H mice but not in BALB/c mice. In both strains of mice the total number of bacteria in the spleen and the draining lymph node increased after UV irradiation. When irradiated 3, 5, 18, or 21 days after BCG infection, BALB/c mice also showed a significant decrease in their DTH response to PPD, indicating that the UV-induced suppression of BCG occurs both at the induction and the elicitation stages of the immune response. Thus, mice exposed to a single dose of UV radiation either before or after BCG infection showed an impaired DTH response to mycobacteria, which was accompanied by an increase in the multiplication of bacteria in the tissues, even though the organisms were introduced at an unirradiated site. These studies demonstrate that a systemic effect of UV irradiation can interfere with the development and expression of immunity to pathogenic bacteria in mice.     

Involvement of the Th1 subset of CD4+ T cells in acquired immunity to mouse infection with Trypanosoma equiperdum.

Heat- or merthiolate-inactivated Trypanosoma equiperdum was administered to recipient mice that were subsequently challenged with viable inocula of the same stabilate. Only mice inoculated with merthiolate-killed parasites were completely protected from a challenge inoculum of 10(3) trypanosomes, an effect that was abolished by prior immunosuppression of mice. Immune sera from protected animals contained high levels of interferon (IFN)-gamma and specific IgG2a antibodies. Spleen cells from these mice produced high amounts of interleukin (IL)-2 and IFN-gamma in vitro in response to specific antigen or concanavalin A, whereas splenocytes from mice receiving heat-killed parasites produced high amounts of IL-6. In contrast, the production of tumor necrosis factor (TNF)-alpha and colony-stimulating activity (CSA) was not significantly different in mice receiving either killed parasite preparation. The protection in immunized mice was associated with the detection of strong delayed-type hypersensitivity (DTH) to T. equiperdum antigens, an effect that could be adoptively transferred onto naive recipients by specifically immune CD4+ lymphocytes. These results suggest that the development of protective immunity in mice to T. equiperdum by our immunization protocol may involve the activity of helper/DTH T cells, particularly those of the Th1 subset.     

Different time course patterns of local expression of delayed-type hypersensitivity to sheep red blood cells in mice.

The delayed-type hypersensitivity (DTH) reaction, a peripheral expression of cell-mediated immunity is still a crucial in vivo immunological test. Nevertheless, the biological significance of its time course remains unclear. Thus, an exhaustive study of DTH was undertaken in mice immunized with increasing doses of sheep red blood cells (SRBC) inoculated intravenously (iv) or subcutaneously. The results showed that overall DTH reactions peaked at 18 hr except in mice iv immunized with the lowest doses (10(5) and 10(6)) and elicited at Day 4. The protracted DTH reaction was shown to be associated with an histological picture of tuberculin-type reaction. A part of the 18-hr DTH reaction is mediated by serum in mice inoculated with large doses of SRBC; nevertheless, numeration by limiting dilution analysis of circulating DTH cells showed that the frequency of these cells correlates with the 18-hr DTH level. The protracted DTH shown at 42 and 48 hr, 4 days after immunization with 10(5) and 10(6) SRBC, could not be transferred in naive recipients with immune spleen cells; it was independent of the antigen life span and did not result from immunization modulation at the bone marrow level on recruitable cells.     

Cross-reactive cellular and humoral immunity to carcinogen-induced chicken fibrosarcomas. I. Protective cross-immunity between transplantable tumor lines

Dimethylbenz[a]anthracene (DMBA)-induced transplantable fibrosarcomas in B2 homozygous chickens (SC line) grow progressively in normal chickens, but are rejected by chickens immunized previously with irradiated tumor cells and Corynebacterium parvum. Tumor-immune chickens resist challenge by the immunizing tumor lines as well as by some, but not all, fibrosarcoma lines. The pattern of cross-reactivity between four DMBA-induced transplantable tumor lines was examined in detail. Ability to reject a tumor challenge correlated very well (p less than 0.001) with the presence of delayed-type hypersensitivity (DTH) to that tumor. Immunization with one of two of the DMBA-induced lines tested also caused rejection of transplantable tumors developed from methylcholanthrene-induced and benzo(a)pyrene-induced primary fibrosarcomas. Although immunization with tumor caused DTH to chicken embryo fibroblasts (CEF), immunization with CEF failed to cause protective immunity or DTH to tumors. Presence of protective immunity, where tested, also correlated with the ability of spleen cells from immune donors to inhibit tumor growth in Winn tests. Humoral immunity exhibited even greater cross-reactivity than did cellular immunity. Distinct patterns of cross-reactivity were nevertheless observed with respect to the serum antibodies as detected in ELISA. Two of these patterns were also observed in several sera from primary tumor-bearing chickens, both including reactivity with CEF. Such reactivity was absent from normal chicken sera.     

Delayed-type hypersensitivity to Babesia microti-infected erythrocytes in mice

Strong delayed-type hypersensitivity (DTH) to Babesia microti was elicited when intraerythrocytic parasites (IEP) were inoculated subcutaneously into the flank of normal mice 6 to 14 days before challenge in the ipsilateral footpad with 10(8) IEP. Intraperitoneal or intravenous administration of antigen did not sensitize mice for DTH. When challenge was given 21 days after immunization, the response was approximately half of the maximum and then rose again slowly over the next 3 weeks to levels that were not significantly different from those maximal values. The response was similar in seven strains of mice, regardless of sex. The response was classified as a true DTH reaction on the basis of kinetics, histology, and the transfer of responsiveness with immune T lymphocytes of the Ly 1+ phenotype, but not with serum. The reaction was specific for IEP since control groups given two injections of red blood cells from uninfected syngeneic mice (NRBC) or one injection of NRBC or sheep red blood cells (SRBC) and one of IEP never developed significant footpad swelling. Freed parasites obtained by osmotic rupture, density gradient sedimentation, and lethally irradiated IEP were also effective for elicitation of DTH. Anti-IEP DTH was expressed in a dose-dependent fashion with 10(6), 10(7), or 10(8) parasites sufficing for immunizing inoculum as long as 10(8) parasites were used as the challenge dose. Mice immunized and challenged with 10(8) lethally irradiated IEP (60 krad, 60Co), were protected against subsequent intraperitoneal challenge with 10(8) viable IEP. If mice were infected intraperitoneally with 10(8) IEP at any time between 21 days before immunization to 2 hr after challenge, their ability to respond to immunization and challenge was profoundly depressed. These data suggest that development of a strong anti-parasite DTH response can occur in parallel with resistance to infection, but is not a rapid sequela of bloodborne infection.     

B Cells Regulate Neutrophilia during Mycobacterium tuberculosis Infection and BCG Vaccination by Modulating the Interleukin-17 Response

We have previously demonstrated that B cells can shape the immune response to Mycobacterium tuberculosis, including the level of neutrophil infiltration and granulomatous inflammation at the site of infection. The present study examined the mechanisms by which B cells regulate the host neutrophilic response upon exposure to mycobacteria and how neutrophilia may influence vaccine efficacy. To address these questions, a murine aerosol infection tuberculosis (TB) model and an intradermal (ID) ear BCG immunization mouse model, involving both the μMT strain and B cell-depleted C57BL/6 mice, were used. IL (interleukin)-17 neutralization and neutrophil depletion experiments using these systems provide evidence that B cells can regulate neutrophilia by modulating the IL-17 response during M. tuberculosis infection and BCG immunization. Exuberant neutrophilia at the site of immunization in B cell-deficient mice adversely affects dendritic cell (DC) migration to the draining lymph nodes and attenuates the development of the vaccine-induced Th1 response. The results suggest that B cells are required for the development of optimal protective anti-TB immunity upon BCG vaccination by regulating the IL-17/neutrophilic response. Administration of sera derived from M. tuberculosis-infected C57BL/6 wild-type mice reverses the lung neutrophilia phenotype in tuberculous μMT mice. Together, these observations provide insight into the mechanisms by which B cells and humoral immunity modulate vaccine-induced Th1 response and regulate neutrophila during M. tuberculosis infection and BCG immunization.     

Induction of partial protection against Leishmania donovani by promastigote antigens in negatively charged liposomes

Negatively charged liposomes, proposed as potential vaccine adjuvants, have been extensively studied in association with various antigens. In the present study, we investigated the adjuvanicity of negatively charged liposomes to enhance the protective immunity of membrane antigens of Leishmania donovani promastigotes (LAg). In comparison to the control mice immunized with phosphate-buffered saline and empty liposomes, immunization with free LAg led to significant levels of protection against infection with virulent promastigotes. Encapsulation of LAg in liposomes also induced effective protection. However, the level of protection by LAg-liposome was not significantly different from that induced by free LAg. Investigation of the immune responses showed, in contrast to free LAg, that immunization with LAg-liposome elicited strong antibody responses. IgG isotype analysis revealed the presence of all 4 isotypes. However, the titer of IgG1 was significantly higher than IgG2a, IgG2b, and IgG3. Following infection, stimulation of IgG and IgG isotypes did not differ in the different immunization groups. Delayed-type hypersensitivity (DTH) analysis after immunization showed significant induction by LAg and LAg-liposomes, in comparison to controls. With infection, again, the level of DTH in all the groups became almost comparable. Stimulation of insufficient cellular response, as reflected by DTH and potentiation of IgG1 over IgG2a, IgG2b, and IgG3 suggest a dominance of Th2 response with this liposome-antigen formulation, resulting in weak protection against visceral leishmaniasis.     

Induction of delayed-type hypersensitivity and antitumor immunity by systemic BCG

Both delayed-type hypersensitivity (DTH) and antitumor resistance induced in mice by intravenous (i.v.) and local injection of highly immunogenic irradiated Meth A cells were potentiated by prior systemic BCG infection. DTH and antitumor immunity were not elicited by i.v. injection of poorly immunogenic irradiated mastocytoma cells, P 815 (MA), but were induced by the local injection of these cells when animals were systemically infected with BCG. The level of the potentiated response corresponded with the dose of immunogen up to an optimum, beyond which additional immunogen was suppressive. At all dose levels the subcutaneous (s.c.) route of immunogen inoculation was more effective than the i.v. route. Significant DTH was first detected 7 days after the local administration of immunogen and was correlated with antitumor immunity. Systemically administered BCG grew mainly in the liver and spleen until the development of maximal tuberculin sensitivity when the number of organisms decreased. However, the small number of mycobacteria that reached the peripheral lymph nodes remained constant after maximal tuberculin sensitivity but failed to augment the cell proliferation that occurred in these lymph nodes as a result of the local inoculation of irradiated tumor cells. Autoradiographs of such nodes revealed proliferation in the thymus-dependent areas whereas nodes from mice immunized with immunogen alone manifested B- as well as T-cell activity. Local immunization in both BCG-infected and uninfected hosts was also associated with a proliferative response in the red pulp of the spleen but the BCG-infected hosts differed conspicuously by virtue of the presence of tubercles and depletion of lymphoid cells from the periarteriolar sheath. Immunity generated by the local administration of immunogen in systemically infected mice was tumor specific and could be adoptively transferred with spleen cells.     

Fusogenic potential of prokaryotic membrane lipids. Implication in vaccine development.

Development of protective immunity against many pathogens, particularly viruses, requires fine orchestration of both humoral- and cell mediated-immunity. The immunization of animals with soluble antigens usually leads to the induction of humoral immune responses. In contrast, the activation of a cell-mediated immune response against exogenous antigens has always been a challenge, requiring special strategies to expose them to the proteasome, a multifunctional protease complex in the cytosol of the target cells. The degradation of the protein by the cytosolic proteolytic system forms a cardinal step for the induction of cytotoxic T lymphocytes (CTLs). In the present study, we report that a potent primary CTL response against a soluble protein, ovalbumin, can be induced in mice by encapsulating it in the liposomes comprised of Escherichia coli membrane lipids. These lipids were shown to induce strong membrane-membrane fusion as evident from resonance energy transfer and content mixing assays. Furthermore, the fusion of these liposomes with living cells (J774 A1) was demonstrated to result in effective transfer of a fluorescent lipid probe to the plasma membrane of the cells. Moreover, ricin A, a protein synthesis inhibitor that does not cross plasma membrane, was demonstrated to gain access to the cytosol when it was encapsulated in these liposomes. Finally, the liposomes were demonstrated to behave like efficient vehicles for the in vivo delivery of the antigens to the target cells resulting in the elicitation of antigen reactive CD8+ T cell responses.