Specificity of Acquired Resistance Produced by Immunization with Mycobacterial Cells and Mycobacterial Fractions

Mice were immunized intraperitoneally with 5.0 mg of living and 5.0 mg of heat-killed H37Ra cells of the attenuated strain Mycobacterium tuberculosis and challenged intraperitoneally with Listeria monocytogenes and Klebsiella pneumoniae. The period of protection provided by the living and heat-killed H37Ra cells against both heterologous infections was the same. When mice were immunized intraperitoneally with graded doses of living and heat-killed H37Ra and challenged intraperitoneally with listeria or klebsiella, the lowest immunizing dose providing protection against both klebsiella and listeria challenge was the same for living and heat-killed cells. Living and heat-killed cells also immunized equally effectively when the routes of immunization and challenge were different. Mice also were immunized intraperitoneally with mycobacterial ribosomal fraction, mycobacterial cell walls, and several nonspecific agents (Escherichia coli endotoxin, mineral oil emulsion, and Freund's incomplete adjuvant). The mice were challenged intraperitoneally with listeria or klebsiella at varying times after immunization. The mycobacterial components and all the nonspecific agents provided transitory protection lasting no longer than 4 days after immunization. Only the mycobacterial cell walls and the endotoxin provided protection against listeria challenge. It was concluded that the protection provided by the mycobacterial ribosomal fraction is specific for tuberculosis infection, since this fraction provided no protection against listeria infection and only transitory protection against klebsiella. It was also concluded that the mycobacterial component providing protection against heterologous infections is heat stable and probably is found in the cell wall.     

Specificity of Acquired Resistance Produced by Immunization with Listeria monocytogenes and Listeria Fractions

Mice were immunized with 1.0 mg of an attenuated strain of Listeria monocytogenes to determine the period of protection afforded by this strain when the mice were challenged intravenously with 5 MLD of listeria. Protection appeared 2 days after immunization and was still apparent 4 weeks after immunization. If the challenge dose was decreased to 1 MLD, protection was apparent at 10 weeks. Mice immunized with a comparable dose of mycobacterial cells and challenged intravenously with 1 MLD of listeria showed no protection at 10 weeks. The magnitude of the immune response to listeria challenge was not increased in mice immunized with the same virulent strain as that used for challenge. It was also found that resistance to listeria challenge appeared early after listeria immunization if the immunizing dose was large. As the immunizing dose was decreased and the challenge dose increased, resistance appeared later. Listeria killed by heat or ultraviolet irradiation, living but nonmultiplying streptomycin-dependent listeria, or listeria ribosomal fraction gave no protection against listeria challenge. The magnitude of the immune responses after listeria immunization to listeria challenge and to mycobacteria challenge were compared. It was found that protection after listeria challenge was of longer duration. In addition, a 100-fold larger vaccinating dose was required to give comparable protection against tuberculous infection.     

Quantifying translocation of Listeria monocytogenes in rats by using urinary nitric oxide-derived metabolites

The urinary nitric oxide metabolites NO(2)(-) and NO(3)(-) (summed as NO(x)) are a noninvasive, quantitative biomarker of translocation of salmonella from the intestinal lumen to systemic organs. Listeria monocytogenes is a food-borne gram-positive pathogen that can also cross the intestinal epithelium. In this study, we tested the efficacy of urinary NO(x) as a marker of listeria translocation. Rats (eight per group) were orally infected with increasing doses of L. monocytogenes; control rats received heat-killed listeria. The kinetics of urinary NO(x) and population levels of listeria in feces were determined for 7 days. Another group of rats was killed 1 day after infection to verify translocation by culturing viable listeria from systemic organs. Oral administration of increasing doses of L. monocytogenes resulted in a time- and dose-dependent increase in urinary NO(x) excretion. Translocation was a prerequisite for inducing a NO(x) response, since heat-killed L. monocytogenes did not elevate NO(x) excretion in urine. Fecal counts of listeria also showed dose and time dependency. Moreover, the number of viable L. monocytogenes cells in mesenteric lymph nodes also increased in a dose-dependent manner and correlated with urinary NO(x). In conclusion, urinary NO(x) is a quantitative, noninvasive biomarker of listeria translocation.     

ISA 61 VG佐剂增强单核细胞增生李斯特氏菌灭活疫苗的保护性免疫应答

单核细胞增生李斯特氏菌(Listeria monocytogenes,Lm)是重要的人兽共患李斯特氏菌病的致病菌,疫苗免疫是预防该病原菌感染的有效手段之一。本研究研制了添加矿物油佐剂MontanideTM ISA61VG的新型灭活细菌疫苗,并对其安全性和免疫应答特性进行了研究。结果表明,ISA 61 VG佐剂疫苗具有较好的安全性;诱导小鼠产生的抗李斯特氏菌溶血素O抗体滴度以及IgG2a/IgG1比值显著高于无佐剂免疫组;在致死剂量Lm攻毒下,能对小鼠提供100%的免疫保护。因此,ISA 61VG佐剂能显著增强灭活疫苗诱导宿主产生体液免疫和细胞免疫应答的能力,从而提高灭活疫苗的保护性免疫应答作用,是预防人和动物Lm感染的潜在疫苗候选株。     

Quantifying Translocation of Listeria monocytogenes in Rats by Using Urinary Nitric Oxide-Derived Metabolites

The urinary nitric oxide metabolites NO₂⁻ and NO₃⁻ (summed as NO_x) are a noninvasive, quantitative biomarker of translocation of salmonella from the intestinal lumen to systemic organs. Listeria monocytogenes is a food-borne gram-positive pathogen that can also cross the intestinal epithelium. In this study, we tested the efficacy of urinary NO_x as a marker of listeria translocation. Rats (eight per group) were orally infected with increasing doses of L. monocytogenes; control rats received heat-killed listeria. The kinetics of urinary NO_x and population levels of listeria in feces were determined for 7 days. Another group of rats was killed 1 day after infection to verify translocation by culturing viable listeria from systemic organs. Oral administration of increasing doses of L. monocytogenes resulted in a time- and dose-dependent increase in urinary NO_x excretion. Translocation was a prerequisite for inducing a NO_x response, since heat-killed L. monocytogenes did not elevate NO_x excretion in urine. Fecal counts of listeria also showed dose and time dependency. Moreover, the number of viable L. monocytogenes cells in mesenteric lymph nodes also increased in a dose-dependent manner and correlated with urinary NO_x. In conclusion, urinary NO_x is a quantitative, noninvasive biomarker of listeria translocation.     

Multifunctional, high-level cytokine-producing Th1 cells in the lung, but not spleen, correlate with protection against Mycobacterium tuberculosis aerosol challenge in mice

Boosting bacillus Calmette-Guérin (BCG)-primed mice with a recombinant adenovirus expressing Mycobacterium tuberculosis Ag 85A by different administration routes has very different effects on protection against aerosol challenge with M. tuberculosis. Mice boosted intradermally make very strong splenic CD4 and CD8 Th1 cytokine responses to Ag 85A, but show no change in lung mycobacterial burden over BCG primed animals. In contrast, intranasally boosted mice show greatly reduced mycobacterial burden and make a much weaker splenic response but a very strong lung CD4 and CD8 response to Ag 85A and an increased response to purified protein derivative. This effect is associated with the presence in the lung of multifunctional T cells, with high median fluorescence intensity and integrated median fluorescence intensity for IFN-gamma, IL-2, and TNF. In contrast, mice immunized with BCG alone have few Ag-specific cells in the lung and a low proportion of multifunctional cells, although individual cells have high median fluorescence intensity. Successful immunization regimes appear to induce Ag-specific cells with abundant intracellular cytokine staining.     

Anti-bacterial immunity to Listeria monocytogenes in allogeneic bone marrow chimera in mice

Protection and delayed-type hypersensitivity (DTH) to the facultative intracellular bacterium Listeria monocytogenes (L.m.) were studied in allogeneic and syngeneic bone marrow chimeras. Lethally irradiated AKR (H-2k) mice were successfully reconstituted with marrow cells from C57BL/10 (B10) (H-2b), B10 H-2-recombinant strains or syngeneic mice. Irradiated AKR mice reconstituted with marrow cells from H-2-compatible B10.BR mice, [BR----AKR], as well as syngeneic marrow cells, [AKR----AKR], showed a normal level of responsiveness to the challenge stimulation with the listeria antigens when DTH was evaluated by footpad reactions. These mice also showed vigorous activities in acquired resistance to the L.m. By contrast, chimeric mice that had total or partial histoincompatibility at the H-2 determinants between donor and recipient, [B10----AKR], [B10.AQR----AKR], [B10.A(4R)----AKR], or [B10.A(5R)----AKR], were almost completely unresponsive in DTH and antibacterial immunity. However, when [B10----AKR] H-2-incompatible chimeras had been immunized with killed L.m. before challenge with live L.m., these mice manifested considerable DTH and resistance to L.m. These observations suggest that compatibility at the entire MHC between donor and recipient is required for bone marrow chimeras to be able to manifest DTH and protection against L.m. after a short-term immunization schedule. However, this requirement is overcome by a preceding or more prolonged period of immunization with L.m. antigens. These antigens, together with marrow-derived antigen-presenting cells, can then stimulate and expand cell populations that are restricted to the MHC (H-2) products of the donor type.     

Flt3 ligand pretreatment promotes protective immunity to Listeria monocytogenes

Flt3 ligand (Flt3L) plays a critical role in the proliferation, differentiation and survival of haematopoietic progenitor cells. Its potential use in a clinical setting has been suggested. Here, we report that mice administered Flt3L displayed a nine-fold increase in size of their hepatic non-parenchymal cell population and an approximate 365-fold increase in number of mature dendritic cells within their livers. Such mice exhibited an elevated resistance to secondary infections with Listeria monocytogenes, an intracellular bacterial pathogen. More than 2.0 log(10)fewer listeriae were recovered in the livers of Flt3L-treated, than untreated, mice on day 2 following secondary challenge. Importantly, Flt3L-pretreated mice immunized with an avirulent (listeriolysin O-negative) strain of Listeria harbored significantly fewer ( approximately 1.5 log(10)) organisms in their spleens and livers than did control mice immunized with listeriolysin O-negative listeriae and challenged with a lethal dose of bacteria. The latter finding supports a potential role for Flt3L in strategies to develop vaccines to intracellular pathogens.     

Induction of a cellular immune response to a foreign antigen by a recombinant Listeria monocytogenes vaccine.

A recombinant strain of Listeria monocytogenes that stably and constitutively expresses Escherichia coli beta-galactosidase was used as a live vaccine vector. BALB/c mice were immunized orally or parenterally with the recombinant L. monocytogenes, and their cellular and humoral immune responses to beta-galactosidase were measured. Spleen cells taken 1 week after oral inoculation or 5 weeks after oral or parenteral inoculation (with a boost at 4 weeks) showed beta-galactosidase-specific CTL responses. The CTL line derived from mice immunized i.p. was also shown to be class I restricted and Thy-1.2+, CD8+, and TCR alpha beta+. All mice immunized with the recombinant L. monocytogenes had positive delayed-type hypersensitivity responses to heat-killed L. monocytogenes, but only 15% had a positive delayed-type hypersensitivity reaction to beta-galactosidase. Individual serum samples from mice immunized i.p. or i.v. were tested for antibody to beta-galactosidase. Approximately 11% had low positive titers for beta-galactosidase antibodies. These results demonstrate that both oral and parenteral immunization with recombinant L. monocytogenes results in a cellular immune response to the foreign protein, which is primarily a specific CD8+ CTL response.     

Delayed hypersensitivity in murine salmonellosis: specificity of footpad reaction in mice infected with rough mutants of Salmonella typhimurium

Delayed type (footpad) hypersensitivity (DTH) in BALB/c mice immunized with rough mutant strains of Salmonella typhimurium LT2 was examined. Injection of live organisms of an Rb mutant TV148 strain induced DTH in mice, while injection of the heat-killed organisms did not. The mice immunized with live organisms of the Ra, Rb, Rc, Rd, and Re mutant strains showed positive footpad reactions to the heat-killed cell antigen of LT2 (wild type) strain. The mice immunized with the Rb mutant strain also showed positive footpad swellings in response to heat-killed cell antigens of S. paratyphi A, S. paratyphi B, S. typhi, S. enteritidis, and S. cholerae-suis. Furthermore, positive reactions to antigens of Escherichia coli and Shigella flexneri were seen in the TV148-immunized mice, but the mice did not respond to heat-killed organisms of Pseudomonas aeruginosa or Staphylococcus aureus. The cross-reactive footpad reaction to E. coli could be transferred adoptively with T cells prepared from the spleens of TV148-immunized mice into syngeneic recipients. These results suggest that the cross-reactive DTH antigen(s) is widely distributed among related organisms such as Shigella and Escherichia.     

Enhanced autoimmune arthritis in IFN-gamma receptor-deficient mice is conditioned by mycobacteria in Freund's adjuvant and by increased expansion of Mac-1+ myeloid cells.

Induction of experimental autoimmune diseases often relies on immunization with the organ-specific autoantigens in CFA, which contains heat-killed mycobacteria. In several of these models, including collagen-induced arthritis, endogenous IFN-gamma acts as a disease-limiting factor in the pathogenesis of the disease. Here we show that in collagen-induced arthritis the protective effect of IFN-gamma depends on the presence of mycobacteria in the adjuvant. Omission of mycobacteria inverts the role of endogenous IFN-gamma to a disease-promoting factor. Thus, the mycobacterial component of CFA opens a pathway by which endogenous IFN-gamma exerts a protective effect that supersedes its otherwise disease-promoting effect. Extramedullary hemopoiesis and expansion of the Mac-1+ cell population accompanied the accelerated and more severe disease course in the IFN-gamma receptor knockout mice immunized with CFA. Treatment of such mice with Abs against the myelopoietic cytokines IL-6 or IL-12 inhibited both disease development and the expansion of the Mac-1+ population. We postulate that mycobacteria in CFA stimulate the expansion of the Mac-1+ cell population by a hemopoietic process that is restrained by endogenous IFN-gamma. These results have important implications for the validity of animal models of autoimmunity to study the pathogenesis and to evaluate cytokine-based therapy of autoimmune diseases.     

Murine cytokine responses following multiple oral immunizations using lipid-formulated mycobacterial antigens

Oral vaccination of mice with live Mycobacterium bovis BCG in lipid-formulation induces a gamma-interferon response that can be measured systemically, and confers protection against aerosolized mycobacterial challenge. Here, we have investigated cytokine responses following the vaccination, drawing comparisons between mice that received single or multiple oral immunizations and between mice receiving formulations containing live BCG or non-replicating mycobacterial antigens. Single oral immunization with lipid-formulated live BCG invoked secreted and cellular IFN-gamma responses in mice 8 weeks post-vaccination, the magnitudes of which were significantly elevated in mice receiving multiple immunizations over the 8-week period. Single oral immunization with live BCG also invoked an interleukin-2 response (but not TNF-alpha or IL-4), although the magnitude was not elevated by multiple immunizations. Multiple immunizations with lipid-formulated soluble or particulate non-replicating mycobacterial antigens failed to invoke cytokine responses, except for a low-level IFN-gamma response in mice multiple immunized with lipid-formulated heat-killed BCG. These results are discussed in contrast to the known patterns of cytokine induction following parenteral-route immunization with live or non-replicating mycobacterial antigens and with practical reference to the development of oral-delivery vaccines against tuberculosis.     

Depletion of CD4+ T cells during immunization with nonviable Listeria monocytogenes causes enhanced CD8+ T cell-mediated protection against listeriosis

Immunization of mice with nonviable Listeria monocytogenes generates an insufficient CD8(+) T cell response and consequently only limited protection against subsequent L. monocytogenes infection. We have recently demonstrated that depletion of regulatory CD4(+) T cells during immunization significantly enhances CD8(+) T cell responses. In the present study, we determined the impact of CD4(+) T cell depletion on the CD8(+) T cell response against heat-killed LISTERIA: Treatment of mice with anti-CD4 mAb during boost immunization with heat-killed Listeria significantly increased numbers of Listeria-specific CD8(+) T cells and improved protection against subsequent infection with L. monocytogenes. During challenge infection, numbers of Listeria-specific CD8(+) T cells were enhanced, and these cells expressed effector functions in terms of IFN-gamma production. In summary, we demonstrate that combining nonviable L. monocytogenes vaccination and CD4(+) T cell depletion improves generation of long-lasting and functional Listeria-specific CD8(+) memory T cells.     

Induced sensitization to nickel in guinea pigs immunized with mycobacteria by injection of purified protein derivative with nickel

Nickel has been reported to be one of the most common causes of allergic contact dermatitis. Despite the fact that nickel is a frequent sensitizer in humans, establishing animal models for nickel allergy has met with considerable difficulties. In clinical cases, allergic contact hypersensitivity to nickel develops much more readily in inflamed skin than normal skin. In this study, we tried to induce nickel sensitization when inflammation has been evoked in guinea pigs immunized with mycobacteria followed by co-administration of a mycobacterial component with nickel. We first examined the delayed-type hypersensitivity (DTH) reaction of mycobacterial components such as the cell wall, cell membrane, 70S ribosomal fraction, cytoplasm, tuberculin purified protein derivative (PPD), RNA and DNA from Mycobacterium bovis BCG in guinea pigs immunized with live M. bovis BCG or heat killed M. tuberculosis. When PPD was used, the hypersensitivity reaction was strongest. Next, we tested whether PPD with nickel could induce nickel sensitivity in guinea pigs immunized with mycobacteria. Strong sensitization to nickel was achieved by injecting PPD with nickel. However, if too large an amount of PPD or nickel salts was used, sensitization to nickel decreased. In this way, sensitization of nickel developed much more easily in guinea pigs immunized with mycobacteria by injection of an appropriate amount of nickel at the inflammation site induced by a suitable amount of PPD.     

Immune Response to Listeria monocytogenes in Rabbits and Humans

Rabbits were immunized with listeria antigens, staphylococcus antigen, or with both, and the course of their immune response was monitored. Antibodies to Listeria and Staphylococcus were produced in both immunoglobulin M (IgM) and immunoglobulin G (IgG) classes in response to inoculation with the specific antigen. Cross-responses occurred in rabbits injected only with Listeria or only with Staphylococcus, as well as in rabbits injected with both antigens. L. monocytogenes serotype 4d appeared to be immunologically distinct from L. monocytogenes serotype 2 and its cross-reaction with S. aureus. Human sera from bacteriologically confirmed cases of listeriosis were examined to determine the nature of the immunological response of man to Listeria. In the sera studied, IgM was the predominant antibody produced to Listeria, whereas cross-reactions with Staphylococcus were observed in both the IgM and the IgG antibody classes.     

The dynamics of the humoral immune response to mycobacterial antigens in mice]

The main regularities of humoral immune response to mycobacterial antigens have been studied in experiments on BALB/c mice immunized with live and thermoinactivated Mycobacterium tuberculosis, var. bovis. As shown in this study, the maximum level of serum antibodies to mycobacterial antigen is achieved in two weeks after immunization irrespective of the dose and viability or mycobacteria, then follows a decrease in the antibody level. The absence of uniformity in the dependence of primary immune response and the formation of immunological memory on the dose and viability of mycobacteria has been demonstrated.     

Protective effect of N-acetyl chitohexaose on Listeria monocytogenes infection in mice

A water-soluble oligosaccharide, N-acetyl chitohexaose (NACOS-6) was able to enhance the protecting effect of BALB/c male mice against Listeria monocytogenes infection, when administered intraperitoneally 24 hr before the challenge with this microbe. Significant decrease in number of microbes within the peritoneal cavity, spleen, and liver from the mice of NACOS-6-administered group was not observed 1 day after the infection but 4 days after the infection. Administration of NACOS-6 enhanced the delayed-type hypersensitivity response against sheep red blood cells (SRBC) or heat-killed L. monocytogenes. Splenic T lymphocytes from mice administered NACOS-6 released macrophage activating factor (MAF). These results suggested that NACOS-6 was also able to elevate the function of cellular immunity. Macrophages treated with a combination of NACOS-6 and the culture supernatant of splenic T lymphocytes from mice administered NACOS-6, "NACOS-6 sup," were found to exert a fairly strong growth-inhibitory effect on L. monocytogenes. Interferon-gamma (IFN-gamma) and interleukin 2 (IL-2) were able to enhance the growth-inhibitory effect on L. monocytogenes by the NACOS-6-treated macrophages.     

Enhanced accumulation of inflammatory neutrophils and macrophages mediated by transfer of T cells from mice immunized with Listeria monocytogenes

We have previously shown that listeria-immunized mice recruit more inflammatory neutrophils and macrophages to the peritoneal cavity after i.p. injection of a sterile irritant than do nonimmune mice. Because the inflammatory phagocytes that were obtained from listeria-immune and nonimmune mice did not differ in their ability to kill Listeria monocytogenes in vitro, this suggested that the rapid recruitment of listericidal inflammatory neutrophils and macrophages may be critically important for resistance to listeriosis. In this study we demonstrate that the transfer of listeria-immune T cells, which enhances recipient resistance to listeriosis, also increases the ability of recipients to mobilize inflammatory neutrophils and macrophages to the peritoneal cavity after the i.p. injection of dead listeria. The transfer of enhanced inflammatory responsiveness was blocked by pretreatment of the transferred cells with anti-Thy-1.2 plus complement, and the magnitude of the inflammatory cell accumulation was dependent on the number of listeria-immune T cells that were injected. Inflammatory neutrophils and macrophages that were obtained from the mice after the transfer of listeria-immune or nonimmune T cells (plus dead listeria) did not differ in their ability to kill L. monocytogenes in vitro. These data suggest that the elicitation of an inflammatory response may be an important event in T cell-mediated resistance to listeriosis.     

Aggravated infection in mice co-administered with Mycobacterium tuberculosis and the 27-kDa lipoprotein

We have previously reported that mice immunized with the mycobacterial 27-kDa lipoprotein were more susceptible to Mycobacterium tuberculosis (Mtb) challenge. We also showed that 27-kDa lipoprotein abrogated the protection afforded by the BCG vaccine when administrated together, suggesting that the 27-kDa lipoprotein may modulate the course of experimental mycobacterial infection. In this study, we address the role of the 27-kDa lipoprotein in modulating the immune response to mycobacteria. Our results show that co-administration of BALB/c mice with Mtb and the 27-kDa lipoprotein (Mtb+27kDa), but not its non-acylated form, increases the susceptibility of mice to Mtb infection. Significantly lower DTH reaction and splenocyte proliferation to PPD stimulation were also observed in Mtb+27kDa-infected mice compared to Mtb-infected mice. Furthermore, during infection, splenocytes and purified T cells lost their ability to proliferate in response to concanavalin A stimulation more rapidly in the Mtb+27kDa-infected mice, which was accompanied by high IFN-gamma and NO production, but low TNF-alpha secretion levels. Addition of L-NMMA, anti-IFN-gamma and anti-TNF-alpha antibodies restored in vitro proliferative responses of T cells from Mtb+27kDa-infected mice. Short-term L-NMMA treatment of Mtb+27kDa-infected mice prevented the 27-kDa-mediated immunosuppression and increase in susceptibility to Mtb. Altogether, these data suggest that the 27-kDa lipoprotein plays a role in Mtb infection by inducing increased suppression of the immune response due to elevated NO production.     

Induction of protective immunity to Listeria monocytogenes in neonates

Neonates suffer unduly from infections and also respond suboptimally to most commonly used vaccines. However, a CD8 T cell response can be elicited in neonates if the Ag is introduced into the cytoplasm of APCs. Listeria monocytogenes (Lm) targets the cytoplasm of APC and is a strong CD8 and CD4 Th1-promoting vaccine vehicle in adult mice. We hypothesized that an attenuated strain of Lm would be safe and induce long-lasting protective immunity, even in neonates. We found that neonatal mice immunized only once with the attenuated strain DeltaactA-Lm developed robust primary and secondary CD8 and CD4 Th1 responses and were fully protected from lethal challenge with virulent wild-type Lm without the need for a booster immunization. Furthermore, DeltaactA-Lm expressing a heterologous recombinant Ag induced a strong CD8 and Th1 memory response to that Ag. Based on these data, we propose that DeltaactA-Lm or derivatives thereof might serve as a vaccine vehicle for neonatal immunization.