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.     

In vivo modulation of the murine immune response to Francisella tularensis LVS by administration of anticytokine antibodies.

The role(s) of gamma interferon (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), and interleukin-4 (IL-4) in establishment and maintenance of protective immunity to Francisella tularensis LVS in mice (C3H/HeN) was examined by selective removal of these cytokines in vivo with neutralizing antibodies. The 50% lethal dose (LD50) for mice infected intradermally with F. tularensis alone was 136,000 CFU; treatment of mice with anti-IFN-gamma or anti-TNF-alpha at the time of infection significantly reduced (P much less than 0.05) the LD50 to 2 and 5 CFU, respectively. Abrogation of protective immunity, however, was effective only when anti-IFN-gamma or anti-TNF-alpha was administered prior to day 3 postinfection. In contrast, the LD50 for mice treated with anti-IL-4 was repeatedly higher (555,000 CFU) than for controls; this difference, however, was not significant (P greater than 0.05). Thus, IL-4 may be detrimental, while IFN-gamma and TNF-alpha were clearly crucial to the establishment of protective immunity to F. tularensis during a primary infection. The importance of IFN-gamma and TNF-alpha during a secondary immune response to F. tularensis was also investigated. Spleen cells from immune mice passively transfer protective immunity to recipient mice in the absence of confounding antibody-mediated immunity. This passive transfer of immunity, however, was abrogated by treatment of recipient mice with anti-IFN-gamma or anti-TNF-alpha at the time of challenge infection. That anticytokines effectively abrogate protective immunity very early in the course of infection with F. tularensis suggests that T-cell-dependent activation of macrophages for microbicidal activity is unlikely. These T-cell-independent events early in the course of infection may suppress bacterial replication until a T-cell-dependent response ultimately clears the bacteria.     

Inactivated Francisella tularensis live vaccine strain protects against respiratory tularemia by intranasal vaccination in an immunoglobulin A-dependent fashion

Francisella tularensis is a gram-negative intracellular bacterium that is considered to be a potential category A biological weapon due to its extreme virulence. Although vaccination with the attenuated live vaccine strain (LVS) of F. tularensis can protect against lethal challenge, use of inactivated or subunit forms as vaccine candidates for induction of protective antibody responses has not been fully evaluated. In the present study, we examined whether immune protection in the lung could be stimulated by intranasal administration of inactivated LVS together with interleukin-12 (IL-12) as an adjuvant. LVS was inactivated by heat, paraformaldehyde treatment, or exposure to UV, and inactivation of the preparations was confirmed by assessing bacterial growth and the survival of mice after direct inoculation. We found that mucosal vaccination with inactivated LVS provided 90 to 100% protection in mice after lethal intranasal challenge with 10(4) CFU of LVS, and this protection was dependent on inclusion of exogenous IL-12 during vaccine administration. Survival of vaccinated mice after live bacterial challenge was correlated with reduced bacterial burden, decreased pulmonary inflammation, increased serum antibody titers, and lower levels of gamma interferon (IFN-gamma), tumor necrosis factor alpha, and IL-6 in the lungs, livers, and spleens. Whereas NK cells were primarily responsible for the production of IFN-gamma in unvaccinated, challenged animals, vaccinated mice had increased levels of lung IFN-gamma+ CD4+ T cells after challenge. Significantly, mice genetically deficient in immunoglobulin A (IgA) expression were unable to survive lethal challenge after vaccination. These results are the first results to demonstrate that IgA-mediated protection against lethal respiratory tularemia occurs after mucosal vaccination with inactivated F. tularensis LVS.     

In vivo clearance of an intracellular bacterium, Francisella tularensis LVS, is dependent on the p40 subunit of interleukin-12 (IL-12) but not on IL-12 p70

To determine the role of interleukin-12 (IL-12) in primary and secondary immunity to a model intracellular bacterium, we have comprehensively evaluated infection with Francisella tularensis LVS in three murine models of IL-12 deficiency. Mice lacking the p40 protein of IL-12 (p40 knockout [KO] mice) and mice treated in vivo with neutralizing anti-IL-12 antibodies survived large doses of primary and secondary LVS infection but never cleared bacteria and exhibited a chronic infection. In dramatic contrast, mice lacking the p35 protein (p35 KO mice) of heterodimeric IL-12 readily survived large doses of primary sublethal LVS infection as well as maximal secondary lethal challenge, with only a slight delay in clearance of bacteria. LVS-immune wild-type (WT) lymphocytes produced large amounts of gamma interferon (IFN-gamma), but p35 KO and p40 KO lymphocytes produced much less; nonetheless, similar amounts of NO were found in all cultures containing immune lymphocytes, and all immune lymphocytes were equally capable of controlling intracellular growth of LVS in vitro. Purified CD4(+) and CD8(+) T cells from both WT and p40 KO mice controlled intracellular growth, even though T cells from WT mice produced much more IFN-gamma than those from p40 KO mice, and p40 KO T cells did not adopt a Th2 phenotype. Thus, while IL-12 p70 stimulation of IFN-gamma production may be important for bacteriostasis, IL-12 p70 is not necessary for appropriate development of LVS-immune T cells that are capable of controlling intracellular bacterial growth and for clearance of primary or secondary LVS infection. Instead, an additional mechanism dependent on the IL-12 p40 protein, either alone or in another complex such as the newly discovered heterodimer IL-23, appears to be responsible for actual clearance of this intracellular bacterium.     

Protective properties of a human IgG preparation rich in antibodies to a wide spectrum of lipopolysaccharides

Naturally occurring human IgG, rich in antibodies to different lipopolysaccharides was investigated for possible protective effects against lethal endotoxin shock and lethal gram-negative infection in mice. The IgG preparation was obtained from pooled serum of selected blood donors with high concentrations of antibodies to 11 different LPS as measured by ELISA. The human IgG (5 mg/mouse) protected C3H/TifF mice against an otherwise lethal infection with Salmonella typhimurium. The human IgG also inhibited the lethality induced by purified LPS in D-galactosamine sensitized C57B1/6 mice. The protection was dependent on the IgG dose given. However, protection was not obtained against all the LPS preparations tested. Absorption of the IgG with different LPS, showed the protection to be caused by serotype-specific anti-LPS antibodies. Protection against a given LPS was not related directly to the corresponding anti-LPS titer as measured by ELISA and passive hemolysis. The interpretation of these results is discussed.     

Mechanism of protective immunity induced by porin-lipopolysaccharide against murine salmonellosis.

Investigations were undertaken to characterize the protective immunity induced by porin-lipopolysaccharide (LPS) against Salmonella typhimurium infection in mice. Mice immunized with porin-LPS showed higher levels of antiporin immunoglobulin G than mice which received porin alone. Further, T cells from porin-LPS-immunized mice showed an augmented proliferative response to porin in vitro compared with the response of T cells from porin-injected animals. The passive transfer of anti-LPS antibodies conferred significant protection (17%), while antiporin serum failed to protect mice against lethal challenge, indicating the protective ability of anti-LPS antibodies. However, the transfer of serum obtained from porin-LPS-immunized mice resulted in better protection (30%) than did anti-LPS or antiporin antibodies alone. In contrast to LPS, monophosphoryl lipid A completely failed to induce protection against lethal infection. However, comparable to the effect of LPS, injection of porin with monophosphoryl lipid A enhanced antibody response and the protective ability of porin (81.25%). The transfer of T cells from porin-LPS-immunized mice provided higher levels of protection (47%) against lethal challenge than did T cells from porin-immunized mice (23%). The combination of T cells and serum from porin-immunized mice transferred 36% protection. However, a combination of T cells and serum from porin-LPS-immunized mice conferred the highest level of protection (92%), which was reflected by the number of survivors (100%) in the porin-LPS-immunized group. These results demonstrate that besides the protective effect of anti-LPS antibodies, the ability of LPS to augment humoral and cell-mediated immune responses to porin confers effective protection against Salmonella infection.     

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.     

Transfer of immunity against lethal murine Francisella infection by specific antibody depends on host gamma interferon and T cells.

Both serum and spleen cells from mice immune to Francisella tularensis transfer protection to naive recipients. Here we characterize the mechanism of protection induced by transfer of immune mouse serum (IMS). IMS obtained 4 weeks after intradermal infection with 10(3) bacteria of the live vaccine strain (LVS) contained high levels of immunoglobulin G2 (IgG2a) and IgM (end point titers, 1:16,600 and 1:7,200, respectively) and little IgG1, IgG2b, or IgG3. LVS-specific antibodies were detected 5 days after intradermal infection, and reached peak levels by 2 weeks postinfection. Only sera obtained 10 days or more after sublethal infection, when IgG titers peaked, transferred protection against a challenge of 100 50% lethal doses (LD50s). Purified high-titer IgG anti-LVS antibody but not IgM anti-LVS antibody was responsible for transfer of protection against an intraperitoneal challenge of up to 3,000 LD50s. IMS had no direct toxic effects on LVS and did not affect uptake or growth of bacteria in association with peritoneal cells. One day after LVS infection, liver, spleen, and lung tissue from mice treated with IMS contained 1 to 2 log units fewer bacteria than did tissue from mice treated with normal mouse serum or phosphate-buffered saline. Between 2 and 4 days after infection, however, bacterial growth rates in tissues were similar in both serum-protected mice and unprotected mice. Bacterial burdens in IMS-treated, LVS-infected mice declined in infected tissues after day 5, whereas control animals died. This lag phase suggested that development of a host response was involved in complete bacterial clearance. In fact, transfer of IMS into normal recipients that were simultaneously treated with anti-gamma interferon and challenged with LVS did not protect mice from death. Further, transfer of IMS into athymic nu/nu mice did not protect against LVS challenge; protection was, however, reconstituted by transfer of normal T cells into nu/nu mice. Thus, "passive" transfer of protection against LVS with specific antibody is not passive but depends on a host T-cell response to promote clearance of systemic infection and protection against lethal disease.     

Minimal requirements for murine resistance to infection with Francisella tularensis LVS.

Intraperitoneal or intravenous infection of mice with Francisella tularensis LVS is lethal, with an intraperitoneal 50% lethal dose (LD50) approaching a single bacterium. Intradermal (i.d.) LVS infection has a much higher LD50, about 10(6) bacteria in BALB/cByJ mice, and survival of i.d. infection leads to solid generation of immunity against lethal challenge. To define the minimal requirements for both initial and long-term survival of i.d. infection, we characterized the nature of i.d. LVS infection in lymphocyte-deficient BALB/cByJ.scid (scid) mice. scid mice infected i.d. with strain LVS survived for about 20 days and then died from overwhelming disseminated infection. However, scid mice treated with monoclonal antibodies to gamma interferon, tumor necrosis factor alpha, or neutrophils-granulocytes all died within 1 week of infection, indicating that these were essential for early control of infection. Studies using GKO (gamma interferon knockout) mice emphasized that gamma interferon is absolutely required for initial survival of i.d. LVS infection. scid mice could be reconstituted for long-term survival of i.d. LVS infection and clearance of bacteria by intravenous transfer of splenic lymphocytes or purified B220-/T+ lymphocytes but not nu/nu lymphocytes. T cells are therefore required for long-term clearance and survival of i.d. LVS infection; efforts to determine whether CD4+ T cells, CD8+ T cells, or both are involved are ongoing.     

Study of the protective effects of hyperimmune immunoglobulins G and M against endotoxin in mice and rats

We prepared solutions of human IgM and IgG to various lipopolysaccharide (LPS) species. These were then tested, along with solutions of non-LPS specific human IgG or IgM, for their ability to confer passive immunity against experimental endotoxemia in two animal models. The immunoglobulins were first tested for an effect on the lethality induced by seven different LPSs in actinomycin-D sensitized mice, or by three different bacteria in normal mice. When the immunoglobulins were administered 1 h before challenge, a small protective effect was observed. This protection was dependent upon both the anti-LPS agent, the chemical composition of the LPS, or the strain of Gram-negative bacteria used for injection. The anti-LPS IgM and IgG preparations reduced the mortality induced by Escherichia coli but not by Serratia marcescens or Klebsiella pneumoniae, indicating protection by strain-specific antibodies. When the antibodies were preincubated with LPS or bacteria for 30 min before administration, almost complete protection was seen. The influence of these immunoglobulin preparations or of human albumin (as a control) on the hypotensive and vascular-permeabilizing effects of LPS in rats was then studied. A dose-dependent inhibitory effect was observed with IgG preparations and albumin. At 200 mg/kg, anti-LPS IgG reduced the effects of LPS, while at 400 mg/kg, both anti-LPS and normal IgG preparations showed protection, as did human albumin used at the same dose. The IgM-enriched preparation worsened the initial hypotensive phase after LPS, whereas the anti-LPS IgM significantly reduced the second phase of the hypotension, but only at the largest dose of 400 mg/kg. In this second model using the rat, a clear difference between the activity of IgG and IgM was thus observed. We conclude that pretreatment with human immunoglobulins from large plasma pools modestly, but significantly, attenuated the effects of murine and rat Gram-negative sepsis, but that protection was incomplete. Our results suggest that single regimen intervention strategies may not be sufficient to influence the course of the disease. Received: 12 December 1998     

Human IgG but not IgM antibodies can protect mice from the challenge with live O6 Escherichia coli

We evaluated the ability of human anti-lipopolysaccharide (LPS) O6 immunoglobulin G (IgG) and IgM antibodies to protect mice challenged with Escherichia coli serotype O6:K2ac. Purified whole IgG, commercial gammaglobulin, whole IgM-effluent, pool of normal human serum (NHS), agammaglobulinaemic serum (test groups) or phosphate-buffered saline (control group) was injected into adult male 18 h before a challenge with viable O6 E. coli. The mortality rate was assessed over a period of 72 h. To determine the opsonic and phagocytic activity of the antibody isotypes, we incubated peritoneal macrophages from the control and test groups collected at different times after challenge with the live bacteria with acridine orange for fluorescent analysis. Tumour necrosis factor (TNF)-alpha and interleukin (IL)-6 were quantified in serum of both the test and control groups. All mice that received commercial gammaglobulin or NHS survived. Purified whole IgG (containing 1.1 mg/l of anti-LPS O6 IgG antibodies) protected 87.5% of the animals tested in this experiment, while whole IgM-enriched effluent with 1.5 mg/l of anti-LPS O6 IgM antibodies protected only 12.5%. The agamma serum showed no protective capacity compared with PBS (serving as control). The minimal concentration of anti-LPS O6 IgG antibodies able to protect 50% of animals was 0.137 mg/l of purified whole IgG. Whole IgM-enriched effluent showed no protective capacity independently of the concentration tested (0.048-17.0 mg/l of anti-LPS O6 IgM antibodies). Fluorescent analysis of peritoneal macrophages from animals pretreated with purified whole IgG showed no bacteria at 8 h after the challenge. By contrast, whole IgM effluent showed an increasing number of live bacteria at the same time. Mice that had received whole IgM effluent (1.5 mg/l of anti-LPS O6 IgM antibodies) before the challenge with LPS O6 presented 20.5 microg/l of IL-6 and 1.5 microg/l of TNF-alpha. Serum from animals pretreated with purified IgG did not present any detectable pro-inflammatory cytokine. Our findings suggest that IgG but not IgM antibodies protect animals from a challenge with E. coli O6 serotype.     

Rapid generation of specific protective immunity to Francisella tularensis.

Mice inoculated either subcutaneously (s.c.) or intradermally (i.d.) with a sublethal dose of Francisella tularensis LVS are immune to a lethal intraperitoneal (i.p.) or intravenous (i.v.) challenge of LVS. Here, we show that this immunity developed quite rapidly: mice given a sublethal dose of live LVS s.c. or i.d. (but not i.v.) withstood lethal i.p., i.v., or i.d. challenge as early as 2 days after the initial inoculation, despite the presence of bacterial burdens already in tissues. The magnitude of this early protection was quite impressive. The i.p. 50% lethal dose (LD50) in naive C3H/HeN mice was only 2 bacteria, while the i.p. LD50 in mice given 10(4) LVS i.d. 3 days previously was 3 x 10(6) bacteria. Similarly, the i.v. LD50 in C3H/HeN mice shifted from 3 x 10(2) in naive mice to 5 x 10(6) in primed mice within 3 days after i.d. LVS infection. Comparable changes in the i.p. and i.v. LD50 were observed in C57BL/6J mice. This rapid generation of protective immunity was specific for LVS, in that mice given a sublethal i.d. inoculation of LVS did not survive a lethal challenge with either Salmonella typhimurium W118 or Escherichia coli O118 BORT at any time, nor could mice given sublethal doses of S. typhimurium, E. coli, or Mycobacterium bovis BCG survive lethal doses of LVS. Although an increase in the mean time to death from S. typhimurium infection was noted when mice were given a sublethal i.d. dose of LVS 4 to 14 days earlier, no overall increase in protection or change in the S. typhimurium LD50 was observed. Thus, sublethal infection with LVS at skin sites induced rapid and specific protective immunity.     

Francisella tularensis LVS initially activates but subsequently down-regulates intracellular signaling and cytokine secretion in mouse monocytic and human peripheral blood mononuclear cells

Monocytic cells constitute an important defense mechanism against invading pathogens by recognizing conserved pathogens components. The recognition leads to activation of intracellular pathways involving nuclear factor kappa B (NF-kappaB) and mitogen-activated protein kinases (MAPK), such as the c-Jun NH2-terminal kinase (JNK), and p38. We show that in vitro infection with Francisella tularensis results in activation of NF-kappaB, phosphorylation of p38 and c-Jun, and secretion of TNF-alpha in adherent mouse peritoneal cells, in the mouse macrophage-like cell line J774A.1, in the human macrophage cell line THP-1, and in human peripheral blood monocytic cells. This occurred after infection with the human live vaccine strain, F. tularensis LVS or a mutant strain denoted deltaiglC, which lacks expression of a 23-kDa protein, or after addition of killed F. tularensis LVS. Addition of purified F. tularensis LPS resulted in no discernible effects on the cells. When the effects were followed up to 5 h, activation persisted in cultures with killed bacteria or infected with the deltaiglC strain. In contrast, the signal transduction activation and secretion of TNF-alpha were down-regulated within the 5h period in mouse peritoneal cells, J774 cells or human peripheral blood mononuclear cells infected with F. tularensis LVS. Together, the results suggest that infection with live F. tularensis LVS bacteria leads to a rapid induction of a proinflammatory response in mouse and human cells but after internalization of bacteria, this response is completely or partly down-regulated in most cell types. This down-regulation does not occur when cells are infected with the mutant deltaiglC.     

Cytokine expression in the liver during the early phase of murine tularemia.

Cytokine expression was determined in the livers of mice inoculated subcutaneously with Francisella tularensis LVS. During the first 48 h of infection, there was a logarithmic increase of bacteria in the liver, with a doubling time of 2.5 h. Within 48 h, tumor necrosis factor alpha (TNF-alpha), interleukin 10 (IL-10), IL-12, and gamma interferon (IFN-gamma) mRNAs were expressed, and production of TNF-alpha and IFN-gamma was demonstrated. There was no expression within 96 h of mRNA from IL-2, IL-3, or IL-4. After subcutaneous inoculation of heat-killed LVS, no expression of any of the cytokine mRNAs and no increase in the levels of TNF-alpha or IFN-gamma occurred. The expression of TNF-alpha, IL-12, and IFN-gamma is held to be important to evoke an early T-cell-independent host defense against F. tularensis as well as to drive the expansion of a protective Th1 cell response.     

Francisella tularensis LVS grown in macrophages has reduced ability to stimulate the secretion of inflammatory cytokines by macrophages in vitro

The virulence of Francisella tularensis LVS is determined in part by its ability to invade and replicate within macrophages and stimulate the production of inflammatory cytokines. The present study determined the effects of growing F. tularensis in macrophages on its ability to stimulate cytokine secretion by macrophages. F. tularensis grown in Mueller-Hinton broth (FtB) stimulated the secretion of large amounts of TNF-alpha, IL-12p40, IL-6 and MCP-1/CCL2 when incubated with macrophages overnight. In contrast, F. tularensis released from infected macrophages (FtMac) stimulated very little secretion of these cytokines by primary cultures of murine peritoneal macrophages, human monocytes or macrophage cell lines. Stimulation of nitric oxide production by FtMac was also less than that elicited by FtB. FtMac killed with gentamicin or paraformaldehyde also stimulated low levels of cytokine secretion. FtMac recovered the ability to stimulate cytokine secretion after overnight culture in broth. Infection of macrophages with FtMac inhibited the cytokine response to subsequent stimulation with LPS from Escherichia coli but did not affect Fcgamma receptor-mediated phagocytosis. FtMac were ingested by macrophages at about half the rate of FtB, however, this did not account for the lower cytokine secretion. FtMac and FtB replicated at similar rates within macrophages. Finally, Mice infected with FtMac had a higher mortality rate than those infected with FtB. These results reveal that growth in macrophages causes a reversible phenotypic change in F. tularensis that is associated with decreased stimulation of cytokine secretion, inhibition of LPS-stimulated secretion of inflammatory cytokines by macrophages and increased lethality in mice.     

Generation and characterization of hybridoma antibodies for immunotherapy of tularemia

Tularemia is caused by the Gram-negative facultative intracellular bacterium Francisella tularensis, which has been classified as a category A select agent-a likely bioweapon. The high virulence of F. tularensis and the threat of engineered antibiotic resistant variants warrant the development of new therapies to combat this disease. We have characterized 14 anti-Francisella hybridoma antibodies derived from mice infected with F. tularensis live vaccine strain (LVS) for potential use as immunotherapy of tularemia. All 14 antibodies cross-reacted with virulent F. tularensis type A clinical isolates, 8 bound to a purified preparation of LVS LPS, and 6 bound to five protein antigens, identified by proteome microarray analysis. An IgG2a antibody, reactive with the LPS preparation, conferred full protection when administered either systemically or intranasally to BALB/c mice post challenge with a lethal dose of intranasal LVS; three other antibodies prolonged survival. These anti-Francisella hybridoma antibodies could be converted to chimeric versions with mouse V regions and human C regions to serve as components of a recombinant polyclonal antibody for clinical testing as immunotherapy of tularemia. The current study is the first to employ proteome microarrays to identify the target antigens of anti-Francisella monoclonal antibodies and the first to demonstrate the systemic and intranasal efficacy of monoclonal antibodies for post-exposure treatment of respiratory tularemia.     

IL-4 and transforming growth factor-beta suppress human immunoglobulin secretion in vitro by surface IgD- B cells.

The effect of IL-4 and transforming growth factor-beta (TGF-beta) on immunoglobulin secretion in vitro by peripheral blood mononuclear cells (PBMC) or purified B cells activated with murine EL4 thymoma cells and phorbol myristate acetate (PMA) was investigated. As previously reported, IL-4 induced IgE and IgG4 secretion by B cells in PBMC preparations and B cells activated with EL4 cells and PMA. However, when B cells, either in PBMC preparations or purified and activated with EL4 cells and PMA, spontaneously secreted large quantities of immunoglobulin, IL-4 suppressed the immunoglobulin secretion of all isotypes. IL-4 also suppressed the IgE secretion by B cells from an atopic dermatitis patient. This suppressive effect was not reversed by adding IL-2 or interferon-gamma (IFN-gamma) to the cultures. We also showed that TGF-beta suppressed the immunoglobulin secretion by purified B cells activated by EL4 cells and PMA. To investigate whether IL-4 or TGF-beta suppressed immunoglobulin secretion by in vivo 'switched' and isotype-committed B cells, sIgD- B cells were isolated, activated with EL4 cells and PMA and cultured with IL-4 or TGF-beta. Such activated B cells secreted large quantities of IgG1, IgG2, IgG3, IgA1, IgA2 and IgM, and IL-4 and TGF-beta suppressed all these isotypes by greater than 80%. The data demonstrated that IL-4 and TGF-beta suppress immunoglobulin secretion in vitro by in vivo isotype-committed sIgD- B cells, suggesting that these lymphokines may play a down-regulatory role on differentiated isotype-committed B cells in an isotype-unrestricted manner. The data also showed that IL-4 and TGF-beta acted directly on isolated B cells.     

Longitudinal study of antibody response to lipopolysaccharides during chronic Pseudomonas aeruginosa lung infection in cystic fibrosis.

Antibodies to Pseudomonas aeruginosa from 10 cystic fibrosis patients with chronic P. aeruginosa lung infections were quantitatively and qualitatively analyzed. The development of specific antibodies in patient serum was evaluated in a longitudinal study (1972 to 1987). The concentrations and specificities of immunoglobulin G (IgG) and IgM antibodies to purified lipopolysaccharides (LPS) from clinical isolates of P. aeruginosa and to a variety of other gram-negative bacteria were studied by immunoblotting and enzyme-linked immunosorbent assay techniques. Results were compared with the number of immunoprecipitates to P. aeruginosa whole-cell extracts detected by crossed immunoelectrophoresis. IgG, but not IgM, anti-Pseudomonas LPS concentrations increased significantly at the onset of chronic infection and continued to increase during the course of the infection. There was a good positive correlation between the concentration of IgG anti-Pseudomonas LPS antibodies and the number of crossed-immunoelectrophoresis precipitins. The increases in IgG anti-LPS antibody concentrations were much higher to Pseudomonas LPS than to other LPSs. Binding studies demonstrated an increase in binding of IgG anti-Pseudomonas LPS during infection, whereas the binding of other anti-LPS antibodies decreased. Immunoblotting studies confirmed that antibodies reacted strongly with Pseudomonas LPS and weakly with Escherichia coli core-lipid A. The specificity of the reaction with Pseudomonas LPS increased with the duration of infection. It is concluded that anti-LPS response in cystic fibrosis patients during chronic P. aeruginosa infection demonstrates a marked increase in IgG anti-Pseudomonas LPS antibody concentration, specificity, and affinity. The anti-LPS enzyme-linked immunosorbent assay is proposed as a routine test to diagnose and to follow the course of chronic P. aeruginosa lung infection in patients with cystic fibrosis.     

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.