Humoral and cell-mediated immunity in mice to a 17-kilodalton lipoprotein of Francisella tularensis expressed by Salmonella typhimurium.

A 17-kDa lipoprotein, TUL4, of the facultative intracellular bacterium Francisella tularensis is one of several membrane proteins that induce an in vitro response in T cells from F. tularensis-primed humans. A DNA fragment of the live vaccine strain F. tularensis LVS encoding TUL4 was cloned into Salmonella typhimurium chi 4072, an attenuated delta cya delta crp mutant. Expression of the protein by the recombinant S. typhimurium chi 4072 (pTUL4-15) was maintained after passage in BALB/cJ mice. When mice were immunized with S. typhimurium chi 4072(pTUL4-15), some animals showed an antibody response and a T-cell response to TUL4. When the immunized mice were challenged with the live vaccine strain F. tularensis LVS, bacterial counts in the liver and spleen were lower than in animals immunized with S. typhimurium chi 4072. Immunization with F. tularensis LVS caused a much stronger protection against the challenge than did immunization with S. typhimurium chi 4072(pTUL4-15). The present study demonstrated that the 17-kDa lipoprotein TUL4 of F. tularensis is involved in a protective immunity to tularemia. Possibly, several T-cell-reactive proteins of the organism have to contribute for optimal protection to be achieved.     

Murine model for study of cell-mediated immunity: protection against death from fully virulent Francisella tularensis infection.

To assess cell-mediated immunity in terms of host protection, an experimental model was developed in which passively transferred spleen cells from immunized AKR/J mice enabled nonimmume syngeneic recipients to survive an otherwise fatal infection with fully virulent Francisella tularensis. Donor immunization was achieved by administering live attenuted tularemia vaccine and, subsequently, the virulent streptomycin-sensitive SCHU S4 strain of F. tularensis. At selected intervals after immunization, donor spleen cells were transferred to streptomycin-treated recipients challenged subcutaneously, intravenously, or intraperitoneally with 25 to 50 minimal lethal doses of virulent streptomycin-resistant F. tularensis SCHU S5. The protection afforded by immune spleen cells was maximal (essentially 100%) 12 days after the SCHU S4 secondary immunization.     

Intranasal vaccination induces protective immunity against intranasal infection with virulent Francisella tularensis biovar A

The inhalation of Francisella tularensis biovar A causes pneumonic tularemia associated with high morbidity and mortality rates in humans. Exposure to F. tularensis usually occurs by accident, but there is increasing awareness that F. tularensis may be deliberately released in an act of bioterrorism or war. The development of a vaccine against pneumonic tularemia has been limited by a lack of information regarding the mechanisms required to protect against this disease. Vaccine models for F. tularensis in inbred mice would facilitate investigations of the protective mechanisms and significantly enhance vaccine development. Intranasal vaccination with the attenuated live vaccine strain (LVS) of F. tularensis reproducibly protected BALB/c mice, but not C57BL/6 mice, against intranasal and subcutaneous challenges with a virulent clinical isolate of F. tularensis biovar A (NMFTA1). The resistance of LVS-vaccinated BALB/c mice to intranasal NMFTA1 challenge was increased 100-fold by boosting with live NMFTA1 but not with LVS. The protective response was specific for F. tularensis and required both CD4 and CD8 T cells. The vaccinated mice appeared outwardly healthy for more than 2 months after NMFTA1 challenge, even though NMFTA1 was recovered from more than half of the vaccinated mice. These results show that intranasal vaccination induces immunity that protects BALB/c mice from intranasal infection by F. tularensis biovar A.     

Immunization against experimental murine salmonellosis with liposome-associated O-antigen.

Partially delipidated Salmonella typhimurium (O-1,4,5,12) lipopolysaccharide was incorporated into small multilamellar liposomes composed of either naturally occurring or synthetic phospholipids. Vaccination of mice with the liposome-lipopolysaccharide complexes induced a cellular response specific for O-1,4,5,12 determinants, as determined by the development of a delayed-type hypersensitivity reaction. The liposome-lipopolysaccharide vaccines were significantly more effective, compared with other nonviable vaccines tested, in protecting mice against a lethal intravenous challenge infection with virulent S. typhimurium. Protection afforded by the liposome-lipopolysaccharide vaccines was comparable to that conferred by a live S. typhimurium vaccine. Results suggest that liposome-induced modulation of the host immune response in favor of cell-mediated immunity may be more efficacious in preventing diseases in which cell-mediated immunity is of prime importance.     

Protection of C3H/HeJ mice from lethal Salmonella typhimurium LT2 infection by immunization with lipopolysaccharide-lipid A-associated protein complexes.

C3H/HeJ mice were immunized intraperitoneally (i.p.) with lipopolysaccharide (LPS)-lipid A-associated protein (LAP) complexes or with purified protein-free LPS prior to lethal i.p. or intravenous Salmonella typhimurium LT2 challenge. Our results demonstrated that these Salmonella-hypersusceptible mice can be effectively protected against 1,000 100% lethal doses of S. typhimurium LT2 (i.e., 1,000 viable bacteria) administered by intravenous challenge when previously immunized with LAP-LPS complexes. In contrast to these results, immunization with LPS afforded markedly less protection regardless of the route of challenge, thus suggesting that the LAP portion of LAP-LPS complexes may be necessary for inducing protection against Salmonella infections. For most experiments, antigens were emulsified in complete Freund adjuvant (CFA); however, the CFA portion of the vaccine was suggested not to be an essential component for the induction of immunity to Salmonella infections, since equivalent levels of protection were obtained when it was omitted from the vaccine. The induction of immunity to murine salmonellosis by prior immunization with CFA-LAP-LPS was demonstrated not to be a transient phenomenon, since C3H/HeJ mice were still protected against lethal S. typhimurium LT2 challenge as late as 225 days postimmunization.     

Attenuated Francisella novicida transposon mutants protect mice against wild-type challenge

Francisella tularensis is the bacterial pathogen that causes tularemia in humans and a number of animals. To date, there is no approved vaccine for this widespread and life-threatening disease. The goal of this study was to identify F. tularensis mutants that can be used in the development of a live attenuated vaccine. We screened F. novicida transposon mutants to identify mutants that exhibited reduced growth in mouse macrophages, as these cells are the preferred host cells of Francisella and an essential component of the innate immune system. This approach yielded 16 F. novicida mutants that were 100-fold more attenuated for virulence in a mouse model than the wild-type parental strain. These mutants were then tested to determine their abilities to protect mice against challenge with high doses of wild-type bacteria. Five of the 16 attenuated mutants (with mutations corresponding to dsbB, FTT0742, pdpB, fumA, and carB in the F. tularensis SCHU S4 strain) provided mice with protection against challenge with high doses (>8 x 10(5) CFU) of wild-type F. novicida. We believe that these findings will be of use in the design of a vaccine against tularemia.     

Mice sublethally infected with Francisella novicida U112 develop only marginal protective immunity against systemic or aerosol challenge with virulent type A or B strains of F. tularensis

The current study determined the ability of Francisella novicida to act as a live vaccine against the much more virulent, but closely related pathogen, Francisella tularensis. Live attenuated strains of the latter are effective vaccines against human tularemia. However, the molecular cause of their attenuation remains unknown, and this is a regulatory barrier for licensing such vaccines. Moreover, F. tularensis is exceptionally difficult to manipulate genetically. This is hampering the development of rationally attenuated vaccine strains. F. novicida shares a lot of genetic homology with F. tularensis and is more amenable to genetic manipulation. If the former naturally expresses the protective antigens of the latter, it could be used to develop a defined tularemia vaccine. However, the results presented herein show that wild-type F. novicida elicits almost no protection in mice against challenge with virulent F. tularensis.     

Mechanisms of protective immunogenicity of microbial vaccines: effects of cyclophosphamide pretreatment in Venezuelan encephalitis, Q fever and tularaemia.

Administration of high-dose (250 mg/kg) cyclophosphamide (CY) to guinea-pigs and mice 3 days prior to immunization with inactivated vaccine derived from Venezuelan encephalitis virus (VE), Coxiella burnetii and Francisella tularensis resulted in accentuated and prolonged delayed-type hypersensitivity (DTH) and in vitro cellular immunity (CMI) to specific antigen. Humoral antibody were either absent or significantly lower in CY-pretreated animals compared to immunized non-pretreated controls. CY pretreatments precluded protection in the VE virus model, suggesting that resistance is related to antibody. In the Q fever model, the protective immunogenicity of vaccine was preserved or increased by CY pretreatment suggesting that cell-mediated immunity is the important factor. In the tularaemia bacterial system, there was a complex effect of CY pretreatment on the low-grade protection afforded by killed vaccine against virulent infection. These findings suggest that the inability of killed vaccines to induce high-grade resistance against tularaemia and Q fever may be due in part to a suppressive B cell response which is eliminated by CY. These studies have given useful information on the relative significance of components of the specific immune response and may lead to an increased understanding of the mechanisms of action of vaccines and adjuvants.     

Ribonucleic Acid-Protein Fractions of Virulent Salmonella typhimurium as Protective Immunogens

Mice were injected with virulent Salmonella typhimurium SR-11 subfractions containing varied amounts of ribonucleic acid (RNA) and protein or with living attenuated S. typhimurium RIA. In these mice, maximal resistance to lethal infection by 1,000 or 5,000 median lethal doses of S. typhimurium SR-11 was seen 2 to 3 weeks after immunization. The S. typhimurium RIA vaccine and a crude ethanol-precipitated RNA fraction (E-RNA) prepared from lysates of S. typhimurium SR-11 were the most efficient immunogens inducing protection against salmonellosis. The contribution of the components present in the E-RNA fractions to host protection against lethal salmonella infection was also examined. RNA-rich fractions (P-RNA) prepared from lysates of the virulent salmonellae contained several bands of protein when examined by disc electrophoresis. P-RNA fractions stimulated protective immunity in mice to infection with S. typhimurium SR-11 but to a much lesser degree than did the E-RNA fractions or strain RIA vaccine. Protein-rich fractions (NP), separated from E-RNA by salt precipitation, exhibited the same number and distribution of protein bands by disc electrophoresis as did the parent E-RNA fractions. Mixtures of either bovine liver soluble RNA or various synthetic polynucleotides and NP were examined, as was NP fraction alone, for the ability to confer protection in mice to challenge infections by the virulent strain of salmonella. Polyadenylic-uridylic acid plus NP conferred significant protective immunity to challenge infections in mice immunized with this mixture, being nearly as effective an immunogen as were the E-RNA fractions of S. typhimurium SR-11 or the attenuated S. typhimurium RIA.     

Intranasal interleukin-12 treatment for protection against respiratory infection with the Francisella tularensis live vaccine strain

Francisella tularensis is a gram-negative intracellular bacterium that can induce lethal respiratory infection in humans and rodents. However, little is known about the role of innate or adaptive immunity in protection from respiratory tularemia. In the present study, the role of interleukin-12 (IL-12) in inducing protective immunity in the lungs against intranasal infection of mice with the live vaccine strain (LVS) of F. tularensis was investigated. It was found that gamma interferon (IFN-gamma) and IL-12 were strictly required for protection, since mice deficient in IFN-gamma, IL-12 p35, or IL-12 p40 all succumbed to LVS doses that were sublethal for wild-type mice. Furthermore, exogenous IL-12 treatment 24 h before intranasal infection with a lethal dose of LVS (10,000 CFU) significantly decreased bacterial loads in the lungs, livers, and spleens of wild-type BALB/c and C57BL/6 mice and allowed the animals to survive infection; such protection was not observed in IFN-gamma-deficient mice. The resistance induced by IL-12 to LVS infection was still observed in NK cell-deficient beige mice but not in CD8-/- mice. These results demonstrate that exogenous IL-12 delivered intranasally can prevent respiratory tularemia through a mechanism that is at least partially dependent upon the expression of IFN-gamma and CD8 T cells.     

H-2 restriction in acquired cell-mediated immunity to infection with Francisella tularensis LVS

The H-2 restriction imposed on the T-lymphocyte-macrophage interaction leading to the expression of acquired cellular immunity was evaluated in an experimental model of infection with the live vaccine strain of Francisella tularensis. Restriction between T cells and macrophages was examined in vitro in cultures containing macrophages from C57BL/10 (B10) mice, T cells from immune B10 H-2 congenic mice, and F. tularensis antigen. The cellular interaction was assayed by the production in the cultures of factors which stimulate thymocyte DNA synthesis. It was observed that homology at the I-A region of the H-2 complex was required for productive T-cell-macrophage cooperation to occur. Restriction was also investigated in an in vivo passive cell transfer system. Spleen cells from immunized B10 mice were injected into naive B10 H-2 congenic mice, which were then challenged with F. tularensis. Enhanced resistance to the challenge infection in recipient mice was used as a marker of a successful T-cell-macrophage interaction. It was found that when the recipient strain shared H-2 I-A region homology with the donor strain, enhanced antitularemic resistance was expressed, whereas homology at the H-2 K or D region was insufficient. Thus, macrophage--T-cell cooperation in immunity to experimental tularemia was restricted at the level of class II determinants.     

Purified lipopolysaccharide from Francisella tularensis live vaccine strain (LVS) induces protective immunity against LVS infection that requires B cells and gamma interferon

Previous results have demonstrated that nonspecific protective immunity against lethal Francisella tularensis live vaccine strain (LVS) or Listeria monocytogenes infection can be stimulated either by sublethal infection with bacteria or by treatment with bacterial DNA given 3 days before lethal challenge. Here we characterize the ability of purified lipopolysaccharide (LPS) from F. tularensis LVS to stimulate similar early protective immunity. Treatment of mice with surprisingly small amounts of LVS LPS resulted in very strong and long-lived protection against lethal LVS challenge within 2 to 3 days. Despite this strong protective response, LPS purified from F. tularensis LVS did not activate murine B cells for proliferation or polyclonal immunoglobulin secretion, nor did it activate murine splenocytes for secretion of interleukin-4 (IL-4), IL-6, IL-12, or gamma interferon (IFN-gamma). Immunization of mice with purified LVS LPS induced a weak specific anti-LPS immunoglobulin M (IgM) response and very little IgG; however, infection of mice with LVS bacteria resulted in vigorous IgM and IgG, particularly IgG2a, anti-LPS antibody responses. Studies using various immunodeficient mouse strains, including LPS-hyporesponsive C3H/HeJ mice, muMT(-) (B-cell-deficient) knockout mice, and IFN-gamma-deficient mice, demonstrated that the mechanism of protection does not involve recognition through the Lps(n) gene product; nonetheless, protection was dependent on B cells as well as IFN-gamma.     

Biochemical and immunological properties of ribonucleic acid-rich extracts from Francisella tularensis.

Ribonucleic acid (RNA)-rich extracts derived from the attenuated strain of Francisella tularensis (strain LVS) protected Swiss mice against lethal challenge with F. tularensis strain 425 but not against strain SCHU S4. No killed preparation, including an RNA-rich extract from SCHU S4 itself, offered protection against strain SCHU S4 in contrast to the high level of protection offered against this strain by vaccination with live strain LVS. The protective activity observed against strain 425 was sensitive to ribonuclease but not to Pronase. Protective activity is not a general property of bacterial RNA, since RNA-rich extracts from Staphylococcus aureus offered no protection against tularemia, although disc gel electrophoresis showed similar kinds and amounts of RNA in preparations form F. tularensis and S. aureus. Furthermore, inability to localize activity to a specific region in sucrose gradients suggests a structural rather than an informational role for the RNA in such extracts. RNA-rich extracts from F. tularensis but not from S. aureus were efficient inducers of F. tularensis opsonins in mouse serum, suggesting one mechanism by which such extracts confer protection.     

Francisella tularensis-induced in vitro gamma interferon, tumor necrosis factor alpha, and interleukin 2 responses appear within 2 weeks of tularemia vaccination in human beings.

Cell-mediated immunity is essential for protection against the intracellular bacterium Francisella tularensis, which causes tularemia. Positive in vitro T-cell responses in the form of lymphocyte proliferation and lymphokine interleukin 2 (IL-2) and gamma interferon (IFN-gamma) secretion are found in memory immunity. Studies on the secretion of lymphokines with regard to the developing immunity to F. tularensis have not been published. Therefore, 14 subjects with no clinical history of tularemia were vaccinated with a live F. tularensis vaccine strain. The in vitro responses of five subjects (antigen-induced mononuclear cell and whole blood culture DNA synthesis and cytokine secretion) were measured twice a week throughout the period from 0 to 35 days after vaccination, and the peripheral blood lymphocyte subpopulations of nine subjects were determined between days 0 and 14. Positive reactions, i.e., responses exceeding those on day 0, were reached on day 10 with regard to the whole blood culture DNA synthesis response and IL-2 and IFN-gamma secretion and on day 14 with regard to the mononuclear cell DNA synthesis response and tumor necrosis factor alpha (TNF-alpha) secretion. No measurable IL-4 was found in either the immune or nonimmune supernatants. Since the secretion of TNF-alpha was related to immunization, this points to the specificity of the phenomenon, even though the type of secreting cell is not yet known. If it is shown later that specific T cells produce it, the TNF-alpha response and the negative IL-4 finding may speak for the importance of the Th1-like pattern in immunity to F. tularensis.     

Experimental murine tularemia caused by Francisella tularensis, live vaccine strain: a model of acquired cellular resistance

We have established a model of experimentally-induced tularemia in mice, using the live vaccine strain of Francisella tularensis. A sublethal, intravenous inoculation of this organism caused in C57BL/6 strain mice an acute infection which lasted approximately 12 days. The clearance of Francisella from the bloodstream was shown to be complete by 5.5 hours postinfection. At this time, approximately twice as many bacteria were isolated from the spleen as from the liver. Mice which had recovered from a primary infection demonstrated a significant resistance to re-infection with autologous Francisella, a memory which persisted for at least 15 weeks. Resistance to experimental tularemia could be passively transferred from infected mice to naive mice by means of non-adherent spleen cells. Cells capable of adoptive transfer of resistance were present at a maximal concentration 7 days following infection, and persisted in significant numbers within the spleen cell population for at least 20 days after infection. Treatment of mice with serum from recovered animals caused a decrease in resistance when measured in the livers, and an increase in resistance when measured in the spleens. Suppression of T cell-mediated immunity during infection by treatment with cyclosporin A resulted in a dramatic increase in the tissue bacterial counts. Cyclosporin A-induced suppression of antitularemic resistance was first noted 2-3 days following infection and remained apparent for at least 8 days. The results of these experiments demonstrate that resistance to experimental murine tularemia is mediated predominantly by a cell-mediated mechanism. This mechanism involves T cells which become activated as early as 2-3 days following infection. Experimental, non-lethal infection with Francisella tularensis is thus an excellent model for investigating the mechanisms of acquired cellular immunity.     

Neisseria meningitidis PorB, a Toll-like receptor 2 ligand, improves the capacity of Francisella tularensis lipopolysaccharide to protect mice against experimental tularemia

Francisella tularensis causes severe pneumonia that can be fatal if it is left untreated. Due to its potential use as a biological weapon, research is being conducted to develop an effective vaccine and to select and study adjuvant molecules able to generate a better and long-lasting protective effect. PorB, a porin from Neisseria meningitidis, is a well-established Toll-like receptor 2 ligand and has been shown to be a promising vaccine adjuvant candidate due to its ability to enhance the T-cell costimulatory activity of antigen-presenting cells both in vitro and in vivo. BALB/c mice were immunized with lipopolysaccharide (LPS) isolated from the F. tularensis subsp. holarctica live vaccine strain (LVS), with or without PorB from N. meningitidis, and the antibody levels induced during the vaccination regimen and the level of protection against intranasal challenge with LVS were determined. Antigen administered alone induced a specific F. tularensis LPS immunoglobulin M (IgM) response that was not maintained over the weeks and that conferred protection to only 25% of the mice. In contrast, F. tularensis LPS given in combination with neisserial PorB induced consistent levels of specific IgM throughout the immunization and increased the proportion of surviving mice to 70%. Postchallenge cytokine analysis showed that interleukin-6 (IL-6), monocyte chemoattractant protein 1, and gamma interferon were markers of mortality and that IL-1beta was a correlate of survival, independent of the presence of PorB as an adjuvant. These data indicate that neisserial PorB might be an optimal candidate adjuvant for improving the protective effect of F. tularensis LPS and other subunit vaccines against tularemia, but there is still a need to test its efficacy against virulent type A and type B F. tularensis strains.     

Immunization with heat-killed Francisella tularensis LVS elicits protective antibody-mediated immunity

Francisella tularensis (FT) has been classified by the CDC as a category A pathogen because of its high virulence and the high mortality rate associated with infection via the aerosol route. Because there is no licensed vaccine available for FT, development of prophylactic and therapeutic regimens for the prevention/treatment of infection is a high priority. In this report, heat-killed FT live vaccine strain (HKLVS) was employed as a vaccine immunogen, either alone or in combination with an adjuvant, and was found to elicit protective immunity against high-dose FT live vaccine strain (FTLVS) challenge. FT-specific antibodies produced in response to immunization with HKLVS alone were subsequently found to completely protect naive mice against high-dose FT challenge in both infection-interference and passive immunization experiments. Additional passive immunization trials employing serum collected from mice immunized with a heat-killed preparation of an O-antigen-deficient transposon mutant of FTLVS (HKLVS-OAg(neg)) yielded similar results. These findings demonstrated that FT-specific antibodies alone can confer immunity against high-dose FTLVS challenge, and they reveal that antibody-mediated protection is not dependent upon production of LPS-specific antibodies.     

Specific antibodies contribute to the host protection against strains of Francisella tularensis subspecies holarctica

T cells are crucial to the control and eradication of the facultative intracellular bacterium Francisella tularensis. A contributory role of humoral antibodies in the host defence remains to be assessed. We used B-cell-deficient mice to study the possible contribution of antibodies to the defence against the live vaccine strain (LVS) or a clinical isolate of F. tularensis, both belonging to the subspecies holarctica (type B). When B-cell-deficient (Igmu(-/-)) mice of the C57BL/10 background were administered immune serum one day before intradermal injection of LVS, they developed lower bacterial numbers in skin, liver, and spleen than did mice receiving normal serum, and survived a challenge inoculum that was lethal for mice given normal serum. Administration of immune serum to C57BL/10 mice afforded protection also against infection with the clinical isolate of F. tularensis subsp. holarctica. Five days after intradermal inoculation of bacteria of the isolate, animals receiving immune serum showed 4log10 lower bacterial counts in liver and spleen than mice administered normal serum. In mice primed by LVS infection, T-cell immunity and host protection were strong and only a marginal contribution of immune serum against a secondary intradermal infection was demonstrated. Together, these findings show that specific antibodies contribute to the host defence of mice against F. tularensis subsp. holarctica.     

Ribosomal Vaccines II. Specificity of the Immune Response to Ribosomal Ribonucleic Acid and Protein Isolated from Salmonella typhimurium

Ribosomal proteins isolated from Salmonella typhimurium were effective in inducing immunity in mice. This immunity was specific since animals immunized with ribosomal proteins from S. typhimurium were not protected against challenge with S. cholerae-suis or S. enteritidis. Immunity was evident as early as 5 days after immunization. Ribosomal ribonucleic acid (RNA) failed to provide any effective immunity in mice. Polyinosinic acid: polycytidylic acid (poly I: C) induced a rapid, short-lived immunity to all three Salmonella species. In contrast, ribosomal RNA failed to elicit any rapid nonspecific response to infection.     

Successful Protection against Tularemia in C57BL/6 Mice Is Correlated with Expansion of Francisella tularensis-Specific Effector T Cells

Francisella tularensis is an intracellular, Gram-negative bacterium that causes the fatal disease tularemia. Currently, there are no licensed vaccines for tularemia and the requirements for protection against infection are poorly defined. To identify correlates of vaccine-induced immunity against tularemia, we compared different strains of the live vaccine strain (LVS) for their relative levels of virulence and ability to protect C57BL/6 mice against challenge with virulent F. tularensis strain SchuS4. Successful vaccination, as defined by survival of C57BL/6 mice, was correlated with significantly greater numbers of effector T cells in the spleen and lung. Further, lung cells and splenocytes from fully protected animals were more effective than lung cells and splenocytes from vaccinated but nonimmune animals in limiting intracellular replication of SchuS4 in vitro. Together, our data provide a unique model to compare efficacious vaccines to nonefficacious vaccines, which will enable comprehensive identification of host and bacterial components required for immunization against tularemia.