Phase variations of Francisella tularensis lipopolysaccharide in human infection and immunization

The comparative study of the specificity of antibodies in human sera after tularemia infection and immunization with live tularemia infection was carried out with the use of passive hemagglutination and immunoblotting techniques. The sera of tularemia patients contained two different types of immunoglobulins: strictly specific to the antigenic epitopes of F. tularensis Iipopolysaccharide (LPS) and strictly specific to F. tularensis subsp. novicida LPS. Such phenomenon may be due to phase variations of the antigenic structure of F. tularensis LPS in the body of a slightly susceptible host. The immune sera of vaccinated were found to contain antibodies, strictly specific only to F. tularensis LPS. At the same time in one vaccinee by the presence of pronounced postvaccinal reactions was found sharply defined interaction between serum imunoglobulins and F. tularensis subsp. novicida LPS. As the result, the data on the possibility of the antigenic modification of F. tularensis in tularemia infection in humans were obtained. At the same time antigenic epitopes, characteristic of faintly pathogenic and closely related F. tularensis novicida LPS, appeared in the structure of F. tularensis LPS.     

Species- and genus-specific antigenic epitopes of Francisella tularensis lipopolysaccharides

Lipopolysaccharide (LPS) antigenic epitopes of natural virulent and isogenic avirulent Francisella tularensis strains and other species of the Francisella genus (F. novicida, F. novicida-like, and F. philomiragia) were studied by dot and immunoblotting. Polyclonal rabbit and human sera to virulent F. tularensis strains and monoclonal antibodies to F. tularensis LPS O-side chain were used for detecting species- and genus-specific LPS epitopes. Typical virulent F. tularensis strains produce two types of S-LPS with different antigenic specificity simultaneously. Antigenic determinants of two LPS types were located in LPS O-polysaccharide but not in the core oligosaccharide. The epitopes of the first LPS type were characterized by species specificity for F. tularensis in contrast to determinants of the second LPS type, which had epitopes common with F. novicida. Cross exhaustion of human and rabbit antitularemic sera by F. tularensis and F. novicida LPS showed that F. novicida LPS molecules contained at least two epitopes--highly specific for F. novicida and common with the second type of F. tularensis LPS. The immune response of rabbits and humans to F. tularensis LPS epitopes was different in principle. Sera from rabbits immunized with vaccine and virulent F. tularensis strains contained antibodies "recognizing" antigenic epitopes of two S-LPS forms of the bacterium: type 1 species-specific (in high titers) and type 2 epitopes common with F. novicida LPS (in low titers). In addition to these, sera from patients with tularemia contain immunoglobulins to species-specific epitopes of F. novicida LPS in high titers. Experiments on avirulent mutants showed that in some cases attenuation of F. tularensis can involve loss of species-specific LPS form, while S-LPS with epitopes common with F. novicida LPS will be retained. The difference in specificity of human and rabbit antitularemic antibodies is due to individual features in the host immune system.     

Interaction of S- and R-lipopolysaccharides of Francisella tularensis with lypopolysaccharide-binding protein of human serum

Investigation of ability of Francisella tularensis S- and R-lypopolysaccharide (LPS) preparations as well as the live bacteria with different chemotypes to interact with human lypopolysaccharide-binding protein (LBP) was carried out. It was found that LPS preparations derived from virulent(S-LPS) or isogenic avirulent mutant (R-LPS) strains of F. tularensis had markedly lower affinity to LBP as compared with typical S-LPS of Salmonella abortus and R-LPS of Yersinia pestis. It was shown that R-LPS preparation from avirulent mutant binds LPB more effectively than S-LPS from F. tularensis virulent strain. Differences in S- and R-LPS affinity were also confirmed for LPS represented by the live cells. Thus, bacteria with S-chemotype of LPS (F. tularensis 15/10) bound only 20.3% of LBP, whereas cells with R-LPS (F. tularensis 543 cap(-)) bound 39.9%. Such pattern was observed in experiments with both normal non-immune human serum and sera from people immunized with live tularemia vaccine. The latter indicates that opsonization of LPS by specific antibodies does not change its affinity to LBP. The observed more efficient binding of avirulent strain R-LPS to LBP is likely determines the more intensive host response directed to destruction and rapid elimination of the causative agent. At the same time, weak affinity of the vaccine and virulent strains S-LPS to LBP probably allows the bacterium to avoid activation of host defense mechanisms thus contributing to its long-term persistence in microorganism and development of specific immunity against tularemia.     

Comparative analysis of the immune response of a rabbit to antigens to live and killed Francisella species bacteria]

Serum antibodies were analyzed in rabbits immunized with live and formalin-killed Francisella (F. tularensis, F. novicida, F. novicida-like, and F. philomiragia). Passive hemagglutination test with erythrocytes sensitized by these bacteria' LPS showed much higher titers of species-specific antibodies in all sera to live microorganisms than sera to killed bacteria. The results of immunoblotting with purified LPS and bacterial lysates indicate that sera to live bacteria contained mainly immunoglobulins to species-specific antigenic epitopes of LPS O-polysaccharide chain and few antibodies to the protein component of the cell. By contrast, killed bacterial cells induced weak production of antibodies to S-LPS and a pronounced antibody response to protein antigens. Besides the quantitative differences, live and killed bacteria differed by the qualitative spectrum of immunodominant proteins. Serum to live F. tularensis 15/10 contained antibodies to at least 3 immunodominant antigens of the cell, while serum to killed bacteria contained antibodies to only two of these. Immunoglobulins to protein antigens, absent in homologous sera to live bacteria, were detected in the sera to killed F. novicida and F. novicida-like bacteria. Both sera to F. philomiragia had antibodies reacting with LPS epitopes and immunodominant complex containing protein. In contrast to other Francisella, F. philomiragia was found to synthesize an uncommon LPS representing two major lipooligosaccharides with different molecular weights and antigenic specificity. Therefore, immune response of the host to live and killed Francisella is different: live cells more effectively induce the production of antibodies to S-LPS epitopes, while killed ones to protein antigens.     

The opsonizing activity of the sera of hamadryas baboons immunized with a preparation of the outer membranes of Francisella tularensis (based on data from the luminol-dependent luminescence method)]

The opsonizing properties of sera obtained from hamadryas baboons immunized with the preparation of F. tularensis outer membranes (OM) were studied with the use of luminol-dependent chemiluminescence (CL) of whole blood. The immunization of monkeys with the OM preparation was shown to lead to the formation of functionally active antibodies possessing opsonizing properties with respect to virulent F. tularensis. Immune sera obtained from the animals immunized with live vaccine and from those immunized with OM preparation had no essential differences in their opsonizing properties. The level of IgG antibodies in immune sera correlated with the CL parameters of whole blood in the presence of F. tularensis opsonized with these sera. Increased CL of phagocytes observed after addition of bacteria and immune sera under test to whole blood taken from a nonimmune donor made it possible to evaluate the functional activity of antibodies, thus permitting its use as a test for the evaluation of the effectiveness of new vaccine preparations.     

Francisella tularensis selectively induces proinflammatory changes in endothelial cells

Naturally acquired infections with Francisella tularensis, the bacterial agent of tularemia, occur infrequently in humans. However, the high infectivity and lethality of the organism in humans raise concerns that it might be exploited as a weapon of bioterrorism. Despite this potential for illicit use, the pathogenesis of tularemia is not well understood. To examine how F. tularensis interacts with cells of its mammalian hosts, we tested the ability of a live vaccine strain (LVS) to induce proinflammatory changes in cultured HUVEC. Living F. tularensis LVS induced HUVEC to express the adhesion molecules VCAM-1 and ICAM-1, but not E-selectin, and to secrete the chemokine CXCL8, but not CCL2. Stimulation of HUVEC by the living bacteria was partially suppressed by polymyxin B, an inhibitor of LPS, but did not require serum, suggesting that F. tularensis LVS does not stimulate endothelium through the serum-dependent pathway that is typically used by LPS from enteric bacteria. In contrast to the living organisms, suspensions of killed F. tularensis LVS acquired the ability to increase endothelial expression of both E-selectin and CCL2. Up-regulation of E-selectin and CCL2 by the killed bacteria was not inhibited by polymyxin B. Exposure of HUVEC to either live or killed F. tularensis LVS for 24 h promoted the transendothelial migration of subsequently added neutrophils. These data indicate that multiple components of F. tularensis LVS induce proinflammatory changes in endothelial cells in an atypical manner that may contribute to the exceptional infectivity and virulence of this pathogen.     

Immunogenicity and toxicity of lipopolysaccharide from Francisella tularensis LVS

Abstract Lipopolysaccharide (LPS) from the live vaccine strain of Francisella tularensis ( F . tularensis LVS) was isolated and purified. The LPS did not stimulate lymphocytes from previously tularaemia-vaccinated individuals or lymphocytes from nonprimed individuals. However, serum antibodies from tularaemia vaccines reacted with the LPS whereas virtually no reactivity was found with antibodies from individuals not exposed to F. tularensis LVS. Antibodies of immunoglobulin class M displayed the antibody reactivity predominantly. The LPS failed to induce the mononuclear cell-derived cytokine interleukin-1 and only low levels of tumour necrosis factor were detected. Furthermore, no LPS endotoxin properties were found in galactosamine-treated mice or in the Limulus amoebocyte lysate assay. From these results it can be concluded that F. tularensis LVS possesses a lipopolysaccharide-like molecule, which does not exhibit properties of a classical endotoxin.     

The use of solid-phase liposomal immunoassay for determining a bacterial antigen of lipopolysaccharide nature]

The solid-phase liposomal immunoassay procedure for the determination of Francisella tularensis lipopolysaccharide (LPS) has been developed. This assay has been made with the use of monolayer liposomes, on the average, 360 nm in diameter with their phospholipid bilayer modified with F. tularensis LPS and their internal space filled with calcein used as fluorescent marker. The assay is based on the principle of the competitive immunosorption of liposomes and antigenic LPS on the surface of polystyrene plates sensitized with specific monoclonal antibodies. The dynamic range of the method is 50-2,500 mg/ml, the variation index being 2.3-11.5%. Optimization of this method and its comparison with the enzyme immunoassay system for the given antigen-antibody pair has been made.     

Natural IgM Mediates Complement-Dependent Uptake of Francisella tularensis by Human Neutrophils via Complement Receptors 1 and 3 in Nonimmune Serum

A fundamental step in the life cycle of Francisella tularensis is bacterial entry into host cells. F. tularensis activates complement, and recent data suggest that the classical pathway is required for complement factor C3 deposition on the bacterial surface. Nevertheless, C3 deposition is inefficient and neither the specific serum components necessary for classical pathway activation by F. tularensis in nonimmune human serum nor the receptors that mediate infection of neutrophils have been defined. In this study, human neutrophil uptake of GFP-expressing F. tularensis strains live vaccine strain and Schu S4 was quantified with high efficiency by flow cytometry. Using depleted sera and purified complement components, we demonstrated first that C1q and C3 were essential for F. tularensis phagocytosis, whereas C5 was not. Second, we used purification and immunodepletion approaches to identify a critical role for natural IgM in this process, and then used a wbtA2 mutant to identify LPS O-Ag and capsule as prominent targets of these Abs on the bacterial surface. Finally, we demonstrate using receptor-blocking Abs that CR1 (CD35) and CR3 (CD11b/CD18) acted in concert for phagocytosis of opsonized F. tularensis by human neutrophils, whereas CR3 and CR4 (CD11c/CD18) mediated infection of human monocyte-derived macrophages. Altogether, our data provide fundamental insight into mechanisms of F. tularensis phagocytosis and support a model whereby natural IgM binds to surface capsular and O-Ag polysaccharides of F. tularensis and initiates the classical complement cascade via C1q to promote C3 opsonization of the bacterium and phagocytosis via CR3 and either CR1 or CR4 in a phagocyte-specific manner.     

Prevalence of antibodies to Francisella tularensis in forest workers from different regions of Poland

In the present study we evaluate the prevalence of antibodies to F. tularensis in 480 serum samples obtained from healthy forest workers from different regions of Poland. The investigations were performed using the tube agglutination test and ELISA. The cut-off limit of serum antibodies was set at mean antibody titre determined in the sera of 115 blood donors exceeded by three standard deviations. In none serum samples we detected antibodies to F. tularensis by tube agglutination test. Of the 480 tested sera IgA antibodies were detected by ELISA in 4.6%, antibodies IgG in 3.8% and antibodies IgM in 2.70% serum samples. The results of our study showed that antibodies to F. tularensis were slightly, but not statistically significant, more often diagnosed in healthy forest workers than healthy blood donors.     

Survival and growth of Francisella tularensis in Acanthamoeba castellanii

Francisella tularensis is a highly infectious, facultative intracellular bacterium which causes epidemics of tularemia in both humans and mammals at regular intervals. The natural reservoir of the bacterium is largely unknown, although it has been speculated that protozoa may harbor it. To test this hypothesis, Acanthamoeba castellanii was cocultured with a strain of F. tularensis engineered to produce green fluorescent protein (GFP) in a nutrient-rich medium. GFP fluorescence within A. castellanii was then monitored by flow cytometry and fluorescence microscopy. In addition, extracellular bacteria were distinguished from intracellular bacteria by targeting with monoclonal antibodies. Electron microscopy was used to determine the intracellular location of F. tularensis in A. castellanii, and viable counts were obtained for both extracellular and intracellular bacteria. The results showed that many F. tularensis cells were located intracellularly in A. castellanii cells. The bacteria multiplied within intracellular vacuoles and eventually killed many of the host cells. F. tularensis was found in intact trophozoites, excreted vesicles, and cysts. Furthermore, F. tularensis grew faster in cocultures with A. castellanii than it did when grown alone in the same medium. This increase in growth was accompanied by a decrease in the number of A. castellanii cells. The interaction between F. tularensis and amoebae demonstrated in this study indicates that ubiquitous protozoa might be an important environmental reservoir for F. tularensis.     

Immunoproteomic analysis of the murine antibody response to successful and failed immunization with live anti-Francisella vaccines

Francisella tularensis subspecies tularensis is one of the most virulent of bacterial pathogens for humans. Protective immunity against the pathogen can be induced in humans and some, but not all, mouse strains by vaccination with live, but not killed, vaccines. In mice, this protection is mediated predominantly by CD4+ and CD8+ T cells. This is thought to be the case too for humans. Nevertheless, it is possible that successful vaccination elicits antigen-specific antibodies that can serve as correlates of protection. To test this hypothesis we examined the repertoire of antibodies induced following successful immunization of BALB/c and CH3/HeN mice versus unsuccessful vaccination of C57BL/6 and DBA\2 mice with F. tularensis Live Vaccine Strain or following unsuccessful vaccination of BALB/c mice with highly related subspecies, F. novicida. The results showed that successful vaccination elicited antibodies to at least six proteins that were not recognized by antisera from vaccinated but unprotected mice.     

Prevalence of selected pathogenic microbial agents in the red fox (Vulpes fulva) and gray fox (Urocyon cinereoargenteus) of southwestern Wisconsin

Free-ranging red foxes (Vulpes fulva) and gray foxes (Urocyon cinereoargenteus) were trapped in southwestern Wisconsin. Fox sera were tested to determine the prevalence of antibody for five different Leptospira interrogans serovars, canine distemper virus (CDV), infectious canine hepatitis virus (ICHV), and Franciscella tularensis infections. Grippotyphosa was the most prevalent leptospiral serovar antibody observed. Twenty-five of 53 (47%) red foxes and 11 of 36 (31%) gray foxes had specific antibodies to grippotyphosa. Juvenile foxes had geometric mean antibody titers to grippotyphosa significantly higher (P less than 0.05) than those of the adults of both species. CDV antibody was detected in sera of red foxes only. Six of 57 (11%) red foxes had CDV antibody. ICHV antibody was detected in 2 of 57 (3%) red foxes and 3 of 32 (9%) gray foxes. Antibody to F. tularensis was not detected in any fox sera.     

Francisella novicida LPS has greater immunobiological activity in mice than F. tularensis LPS,and contributes to F. novicida murine pathogenesis

To further understand the role of LPS in the pathogenesis of Francisella infection, we characterized murine infection with F. novicida, and compared immunobiological activities of F. novicida LPS and the LPS from F. tularensis live vaccine strain (LVS). F. novicida had a lower intradermal LD(50) in BALB/cByJ mice than F. tularensis LVS, and mice given a lethal F. novicida dose intraperitoneally died faster than those given the same lethal F. tularensis LVS dose. However, the pattern of in vivo dissemination was similar, and in vitro growth of both bacteria in bone marrow-derived macrophages was comparable. F. novicida LPS stimulated very modest in vitro proliferation of mouse splenocytes at high doses, but F. tularensis LVS LPS did not. Murine bone marrow macrophages treated in vitro with F. novicida LPS produced IL12 and TNF-alpha, but did not produce detectable interferon-gamma, IL10, or nitric oxide; in contrast, murine macrophages treated with F. tularensis LVS LPS produced none of these mediators. In contrast to clear differences in stimulation of proliferation and especially cytokines, both types of purified LPS stimulated early protection against lethal challenge of mice with F. tularensis LVS, but not against lethal challenge with F. novicida. Thus, although LPS recognition may not be a major factor in engendering protection, the ability of F. novicida LPS to stimulate the production of proinflammatory cytokines including TNF-alpha likely contributes to the increased virulence for mice of F. novicida compared to F. tularensis LVS.     

Lipopolysaccharide Specific Immunochromatography Based Lateral Flow Assay for Serogroup Specific Diagnosis of Leptospirosis in India

Background

Leptospirosis is a re-emerging infectious disease that is under-recognized due to low-sensitivity and cumbersome serological tests. MAT is the gold standard test and it is the only serogroup specific test used till date. Rapid reliable alternative serogroup specific tests are needed for surveillance studies to identify locally circulating serogroups in the study area.

Methods/Principal Findings

In the present investigation the serological specificity of leptospiral lipopolysaccharides (LPS) was evaluated by enzyme linked immunosorbent assay (ELISA), dot blot assay and rapid immunochromatography based lateral flow assay (ICG-LFA). Sera samples from 120 MAT positive cases, 174 cases with febrile illness other than leptospirosis, and 121 seronegative healthy controls were evaluated for the diagnostic sensitivity and specificity of the developed assays. LPS was extracted from five locally predominant circulating serogroups including: Australis (27.5%), Autumnalis (11.7%), Ballum (25.8%), Grippotyphosa (12.5%), Pomona (10%) and were used as antigens in the diagnostics to detect IgM antibodies in patients’ sera. The sensitivity observed by IgM ELISA and dot blot assay using various leptospiral LPS was >90% for homologous sera. Except for Ballum LPS, no other LPS showed cross-reactivity to heterologous sera. An attempt was made to develop LPS based ICG-LFA for rapid and sensitive serogroup specific diagnostics of leptospirosis. The developed ICG-LFA showed sensitivity in the range between 93 and 100% for homologous sera. The Wilcoxon analysis showed LPS based ICG-LFA did not differ significantly from the gold standard MAT (P>0.05).

Conclusion

The application of single array of LPS for serogroup specific diagnosis is first of its kind. The developed assay could potentially be evaluated and employed for as MAT alternative.     

金标银染免疫渗滤法检测土拉弗朗西斯菌

目的:建立金标银染免疫渗滤法检测土拉弗朗西斯菌(土拉菌)的方法,评价其灵敏度、特异性、重复性及其应用。方法:以小鼠抗土拉菌脂多糖单克隆抗体作为捕获抗体包被硝酸纤维素膜、兔抗土拉菌多克隆抗体作为检测抗体标记胶体金,通过金标银染技术放大检测信号,建立金标银染免疫渗滤法检测土拉弗朗西斯菌体系;评价该方法的灵敏度、特异性和重复性;以经荧光定量PCR定量的土拉弗朗西斯菌为检测对象,比较金标银染免疫渗滤法和免疫层析法。结果:金标银染免疫渗滤法检测土拉弗朗西斯菌的最小检出量为1.0×103 CFU/mL,灵敏度高于免疫层析法;检测大肠杆菌、炭疽芽孢杆菌、布鲁菌和鼠疫耶尔森菌的结果均为阴性;密封保存的检测卡80 d内重复性良好,100 d后反应强度略有降低。结论:金标银染免疫渗滤法检测土拉弗朗西斯菌敏感性高、特异性强、重复性好,且方便快捷,不需要仪器设备,可作为快速检测土拉弗朗西斯菌的首选方法。     

Serologic evidence of human infection by Francisella tularensis in the population of Castilla y León (Spain) prior to 1997

Prior to an outbreak in Castilla y León in December 1997, tularaemia was practically non-existent in Spain. In this paper we studied the prevalence of antibodies against Francisella tularensis in a representative sample of the population (4825 people) from Castilla y León (Spain) in samples collected before this outbreak. Antibodies against F. tularensis were detected in nine (0.19%) of the 4825 sera, with antibody titres ranging from 1/20 to 1/160. Of these nine sera, one was positive in seroagglutination against Brucella. Seroagglutination against other bacteria (Yersinia enterocolitica O:9 and O:3 and Proteus OX19) was negative in all sera. Seroprevalence of antibodies in females was 0.20% and 0.17% in males; no statistically significant differences were found in prevalence in terms of sex, age or province.     

Evaluation of the enzyme-linked immunosorbent assay for the serodiagnosis of tularemia

The usefulness of the ELISA using as antigen prepared in our laboratory supernatant obtained after centrifugation of sonicated F. tularensis cell suspension was compared with the tube agglutination test with commercial available antigen. Paired serum specimens obtained from 6 patients with ulceroglandular syndrome of tularemia were tested in both tests. The cut-off limit of serum antibodies was set at mean antibody titre determined in the sera of 115 blood donors exceeded by three standard deviations. Antibodies to F. tularensis in diagnostically significant titre were detected in all 12 serum samples by both tests. However the titres obtained in ELISA were several times higher than in tube agglutination test. In the second serum sample the level of IgA and IgM was lower but the level of IgG higher than in the first sample. We could not observe any difference in the level of antibodies between paired serum specimens in tube agglutination test.     

Characterisation of the core part of the lipopolysaccharide O-antigen of Francisella novicida (U112)

Francisella novicida (U112), a close relative of the highly virulent bacterium F. tularensis, is known to produce a lipopolysaccharide that is significantly different in biological properties from the LPS of F. tularensis. Here we present the results of the structural analysis of the F. novicida LPS core part, which is found to be similar to that of F. tularensis, differing only by one additional alpha-Glc residue:where R is an O-chain, linked via a beta-bacillosamine (2,4-diamino-2,4,6-trideoxyglucose) residue. The lipid part of F. novicida LPS contains no phosphate substituent and apparently has a free reducing end, a feature also noted in F. tularensis LPS.