Monoclonal antibodies to Legionella pneumophila cytolysin

The fusion of spleen cells, taken from BALB/c mice immunized with the purified preparation of L. pneumophila cytolysin, with cells Sp2/0 and NP has been carried out. As a result, hybridoma cells producing IgG1, IgG3 and IgM antibodies to this protein have been obtained. All monoclonal antibodies (McAb) thus obtained react with L. pneumophila strain lysates in the precipitation test, while IgG3 and IgM antibodies react with erythrocyte diagnostic agents prepared from the lysate of L. pneumophila cells in the hemagglutination test. In the Western blot assay, McAb react with the 37 KD protein (cytolysin) and a number of other proteins from L. pneumophila cultures and L. pneumophila cell lysate, but do not react with the species-specific protein with a molecular weight of 29 KD, contained in the outer membrane of L. pneumophila, as well as with other species: L. bozemanii, L. dumoffii, L. longbeachae, L. micdadei. The possibility of using these McAb conjugated with FITC and peroxidase for the rapid diagnosis of Legionella infection is shown.     

The biological properties of monoclonal antibodies to the cytolysin of Legionella pneumophila

The study of the biological properties of monoclonal antibodies (McAb) to L. pneumophila cytolysin has been carried out. These McAb have been shown to possess no capacity for the in vitro neutralization of cytolysin and the protection of guinea pigs from aerosol infection with L. pneumophila. Still the protective effect of the McAb under study has been observed in experiments with the intraperitoneal infection of guinea pigs, which is indicative of the possibility, in principle, of involving humoral immunity into the protection of the body from Legionella infection.     

The typing of Legionella pneumophila strains by using monoclonal antibodies to the cytolysin]

Studies on the typing of L. pneumophila strains of serogroup 1, isolated from patients and environmental objects, have been made with the use of monoclonal antibodies (McAb) to cytolysin. The results of the comparison of the specificity of our McAb with that of a commercial set McAb obtained from the USA make it possible to recommend preparations based on McAb to cytolysin for the detection of L. pneumophila pathogenic strains of serovar 1. The use of FITC- and peroxidase-labeled McAb to cytolysin permits the reduction of the time necessary for the diagnosis of Legionella infections and the detection of the antigen in a dose of 10 ng/ml.     

Induction of immune response to influenza virus with anti-idiotypic antibodies.

Anti-idiotypic (anti-Id) antibodies were raised in rabbits against five monoclonal antibodies (MAbs) specific for different antigenic sites on the hemagglutinin (HA) of influenza virus Mem71H-BelN (H3N1) [A/Memphis/1/71 (H3N2) x A/Bel/42 (H1N1)]. Each of the anti-Id sera was directed predominantly towards a unique (private) idiotype of the immunizing MAb, none of the five idiotypes being detectable in pooled BALB/c antisera against Mem71H-BelN virus or on most other anti-HA MAbs tested. Partial idiotypic sharing was observed, however, between certain MAbs, from different mice, having the same or similar epitope specificity for HA. When used as immunogens in BALB/c mice, two of the five anti-Id preparations induced antibodies that reacted with Mem71H-BelN virus and displayed neutralizing activity. Mice of other inbred strains responded similarly, indicating that the response was not genetically restricted by the Igh locus. From their pattern of reactivity with mutants of Mem71H-BelN virus with known single amino acid substitutions in the HA molecule, the antiviral antibodies elicited by anti-Id antibodies were shown to be directed to the same antigenic site on A/Memphis/1/71 HA as the original immunizing MAb (site A or site E, respectively). However, several of these antisera were shown to contain additional distinct subpopulations of antibodies specific for heterologous influenza A virus strains, either of the H3 subtype or of a different HA subtype (H1 or H2). Since the induction of antibodies to HA of different subtypes is not a feature of the antibody response to influenza virus itself, their induction by anti-Id antibodies merits further investigation.     

Characteristics of the cytolysin of Legionella pneumophila

A thermolabile cytolysin was purified from liquid culture of L. pneumophila. Its homogenicity was determined by the Ouchterlony double immunodiffusion and SDS-electrophoresis in polyacrylamide gel. The molecular weight of cytolysin was ca. 37 kDa. Analysis of amino acid composition revealed a high proportion of aromatic, dicarbonic amino acids, and methionine. The minimal cytolytic concentration for CHO cells and erythrocytes was ca. 1 microgram/ml: Purified cytolysin in doses of 10-60 micrograms caused haemorrhage and necrosis when injected i. c. into guinea pigs.     

Production of monoclonal antibodies against Legionella pneumophila serogroup 1

Four monoclonal antibodies to Legionella pneumophila Philadelphia 1 were produced by the fusion of immunized BALB/c lymphocytes to a murine myeloma cell line. Two (Lp1-1 and Lp1-3) of the four monoclonal antibodies reacted with 14 L. pneumophila serogroup 1 strains, and the other (Lp1-2 and Lp1-4) reacted with only three out of 20 strains tested. These four monoclonal antibodies did not bind to any strains of L. pneumophila serogroup 2-7 and other Legionella species. In addition, it has been shown that these monoclonal antibodies may be useful not only for subserotyping of L. pneumophila but also for retrospective diagnosis using immunopathological methods.     

Induction of a neutralizing immune response to human respiratory syncytial virus with anti-idiotypic antibodies.

Anti-idiotypic (anti-Id) antibodies were raised in rabbits against monoclonal antibodies that recognized either F glycoprotein 47F or G glycoprotein 63G, 62G, or 74G of the human respiratory syncytial virus Long strain. Anti-Id sera inhibited the virus binding of the immunizing monoclonal antibodies and in some cases the binding of other antibodies reacting with overlapping epitopes. The anti-Id sera also inhibited virus neutralization mediated by the original monoclonal antibodies. Affinity purified anti-Id antibodies were subsequently used to raise a homologous anti-anti-Id response in rabbits. One of the rabbits, inoculated with anti-Id 63G, generated antibodies that reacted with the G protein of respiratory syncytial virus and neutralized the virus to high titers. The antiviral antibodies induced by anti-Id 63G were broadly cross-reactive with strains of the A and B subtypes. However, the specificities of monoclonal antibody 63G and anti-anti-Id 63G were not exactly the same, as indicated by their reaction with escape mutants to antibody 63G. These results demonstrate for the first time the induction of an anti-respiratory syncytial virus response by anti-Id antibodies.     

Induction of an anti-human type II collagen response by monoclonal anti-idiotypic antibody

Several distinct epitopes on human type II collagen were defined by using mAb. The presence of species-specific and species-nonspecific (common) epitopes was thus clarified. Anti-idiotypic mAb (Ab2) was developed against one of the antibodies (Ab1) reactive with species-specific epitopes. Thus Ab2 was demonstrated to recognize an idiotope expressed on the Ag-binding site (paratope) of Ab1, since the binding of Ab1 to human type II collagen was blocked by Ab2, and the binding of Ab2 to Ab1 was inhibited by soluble human type II collagen, but not by murine and bovine type II collagens. DBA/1 mice immunized with Ab2 coupled to keyhole limpet hemocyanin produced an antibody (Ab3) specifically reactive with human type II collagen. It was also demonstrated that Ab3 expressed an idiotype similar to that of Ab1. These findings indicate that anti-idiotypic antibody directed against mAb to human type II collagen mimics a species-specific epitope on human type II collagen. The anti-idiotypic antibody bearing internal image of type II collagen will open the way to isolation of the arthritogenic epitope on type II collagen.     

Suppression of the immune response to ovalbumin in vivo using anti-idiotypic antibodies

The effect of anti-idiotype antibodies (a-IdpI) on primary immune response to ovalbumin (OVA) was studied. A-IdpI against OVA antibodies were obtained with pI 6.2-6.7 from BALB/c mice. About 30-40% of IgG plaque cell formation (PCF) was inhibited if a-IdpI antiserum was added in situ, which served as a test for the evaluation of idiotype-positive (Id+) PCF. Up to 70% of common PCF and 70-80% of Id+ PCF were suppressed in mice that were injected with a-IdpI antibodies prior to immunization. This suppression was antigen- and allo-specific and depended upon the time of a-IdpI injection. If a-IdpI antibodies were disaggregated (DA) the Id+ suppression increased. A-IdpI antibodies also decreased IgE response to OVA, the suppression being most pronounced with the use of DA samples.     

Studies on the suppression of the immune response to a defined protein epitope by anti-idiotypic antibodies

We have previously reported that C3H.SW (CSW) and A/J mice immunized with the tobacco mosaic virus protein (TMVP) produce antibodies to a decapeptide epitope corresponding to amino acid residues 103-112 of the protein. In the C3H.SW (CSW) strain, the antibodies to the decapeptide contain major crossreactive idiotope, C10-IdX, which is found on a CSW-derived monoclonal antidecapeptide antibody, designated as C10. The in vivo administration of anti-C10 antibodies suppresses the response to the decapeptide epitope in CSW mice. The present communication describes experiments designed to elucidate several parameters responsible for the suppression produced by the in vivo administration of anti-C10. It was found that 50 micrograms of anti-C10 was required to suppress the response to the decapeptide in CSW mice when 2 weeks elapsed between administration of the anti-C10 and immunization with TMVP; however, only 10 ng was required when one injection of antigen instead of two was administered and when the interval between treatment with anti-C10 and immunization was extended to 6 weeks. This suggests that the anti-C10 induces alterations in the idiotypic network which are not yet fully developed after 2 weeks. Furthermore, experiments presented herein demonstrate that decapeptide-binding antibodies from A/J mice, which lack the C10-IdX, were also suppressed by pretreatment with anti-C10. Interestingly, unlike the case with the CSW strain, the titer to TMVP was decreased by the administration of anti-C10 to A/J mice which were subsequently immunized with TMVP. These findings suggest that the polyclonal anti-C10 contains antibodies to an idiotype which is a major component of the overall anti-TMVP response of A/J mice and may be important in the overall regulation of the anti-TMVP response.     

Differential regulation of the immune response to SRBC by monoclonal antibodies to interferon-gamma

Three monoclonal antibodies against different epitopes of interferon-gamma (IFN-gamma) were used to assess its role as a normal immunomodulatory molecule. Two of these antibodies were able to reduce significantly the primary antibody response to sheep erythrocytes in an in vitro culture system. One of these two antibodies has been reported to suppress both the antiviral and macrophage activation factor activities of IFN-gamma by binding to its carboxyl terminus. These findings indicate that IFN-gamma is an important lymphokine for the maximum expression of the immune response and that it acts via the carboxyl terminus of the molecule. This antibody suppressed the immune response only when added at the initiation of culture, suggesting that the action of IFN-gamma is on an early component of the response. The third monoclonal antibody, which binds to the amino end of IFN-gamma, did not suppress the in vitro response. However, it was able to block the effects exerted by an immunosuppressive dosage of exogenous IFN-gamma on in vitro antibody production. These results indicate that the immunosuppression vitro antibody production. These results indicate that the immunosuppression induced by the addition of IFN-gamma to a primary antibody response and the role that it plays in that response are mediated through different sites on the molecule and, therefore, probably by different mechanisms.     

Heat-shock response in Legionella pneumophila

The heat-shock response of Legionella pneumophila was examined by radiolabelling bacterial cell proteins with [35S]methionine following a temperature shift from 30 to 42 degrees C. Five heat-shock proteins were identified as having molecular masses of 17, 60, 70, 78, and 85 kilodaltons (kDa). The 85- and 60-kDa proteins were equally distributed between supernatant and pellet fractions following ultracentrifugation at 100,000 x g, the 70- and 78-kDa proteins were found primarily in the supernatant, and the 17-kDa protein was found primarily in the pellet. Synthesis of subsets of the heat-shock proteins could be stimulated by novobiocin, patulin, or puromycin. Ethanol, an effector of the heat-shock response in other microorganisms, had little effect on L. pneumophila, even at the highest concentration tolerated by the bacterial cells (1.9%). Finally, the 60-kDa heat-shock protein of L. pneumophila was immunologically cross-reactive with a polyclonal antibody prepared to the Escherichia coli groEL protein. However, a mouse monoclonal antibody reactive with the 60-kDa protein of all legionellae tested did not cross-react with the E. coli groEL protein, suggesting that the Legionella 60-kDa protein contains common and unique epitopes.     

Random mutagenesis of Legionella pneumophila reveals genes associated with lipopolysaccharide synthesis and recognition by typing monoclonal antibodies

AIMS: To use random mutagenesis for the characterization of Legionella pneumophila lipopolysaccharide (LPS) components and serotypes. METHODS AND RESULTS: Five strains belonging to different serogroups and/or monoclonal subgroups were mutagenized using a mini-Tn10 transposon. Exactly 11 819 mutants were checked for alterations in LPS using at least 11 monoclonal antibodies (mAbs) that define L. pneumophila serotypes. Among the mutants, five different mini-Tn10 insertions were identified. Four mutants originating from serogroup-1 did not lose their serogroup-specific epitope, but did sustain subtler changes that resulted in switches to different mAb subgroups. In contrast, a mutant from serogroup-6 lost its serogroup-specific epitope, while retaining a serogroup-cross-reacting epitope. CONCLUSIONS: Random mutagenesis is a valuable tool for LPS epitope mapping. While some characteristics of L. pneumophila LPS can be altered, others appear resistant to mutagenesis. This underscores both the flexibility and rigidity of LPS architecture in L. pneumophila. SIGNIFICANCE AND IMPACT OF THE STUDY: Losses of L. pneumophila LPS epitopes can result in new serotypes, changes that might escape detection by current DNA-based typing schemes. But, as the frequency of these changes is rare, based upon our observations, serotyping should remain an important tool for identifying L. pneumophila in water systems that are implicated in human infection.     

Recognition of Legionella pneumophila nucleic acids by innate immune receptors

Innate immune receptors evolved to sense conserved molecules that are present in microbes or are released during non-physiological conditions. Activation of these receptors is essential for early restriction of microbial infections and generation of adaptive immunity. Among the conserved molecules sensed by innate immune receptors are the nucleic acids, which are abundantly contained in all infectious organisms including virus, bacteria, fungi and parasites. In this review we focus in the innate immune proteins that function to sense nucleic acids from the intracellular bacterial pathogen Legionella pneumophila and the importance of these processes to the outcome of the infection.     

Induction of delayed-type hypersensitivity responses by monoclonal anti-idiotypic antibodies to tumor cells expressing carcinoembryonic antigen and tumor-associated glycoprotein-72

The use of anti-idiotypic antibodies as immunogens represents one potential approach to active specific immunotherapy of cancer. Two panels of syngeneic monoclonal anti-idiotypic antibodies were generated. One panel was directed against mAb CC49 and the other to mAb COL-1. mAb CC49 recognizes the pancarcinoma antigen (Ag), tumor-associated glycoprotein-72 (TAG-72), and mAb COL-1 recognizes carcinoembryonic antigen (CEA). Seven anti-idiotypic (AI) antibodies (Ab2) designated AI49-1–7 were generated that recognize the variable region of mAb CC49. These mAb were shown to inhibit the interaction of mAb CC49 (Ab1) with TAG-72 (Ag). Five anti-idiotypic antibodies designated CAI-1–5 were also generated to the anti-CEA mAb, COL-1 (Ab1). These Ab2 were shown to inhibit the interaction between COL-1 (Ab1) and CEA (Ag). Immunization of mice, rats, and rabbits with Ab2 directed against CC49 or COL-1 could not elicit specific Ab3 humoral immune responses, i.e., antibody selectively reactive with their respective target antigens. However, immunization of mice with the CC49 anti-idiotypic antibody (Ab2), designated AI49-3, could induce a delayed-type hypersensitivity response (DTH) specific for tumor cells that express TAG-72. Similarly, immunization of mice with an anti-idiotypic antibody directed against COL-1, designated CAI-1, could induce specific DTH cell-mediated immune responses to murine tumor cells that express human CEA on their surface. These results thus demonstrate that while some anti-idiotype mAb may not be potent immunogens in eliciting Ab3 humoral responses, they are capable of eliciting specific cellular immune responses against human carcinoma-associated antigens. This type of mAb may ultimately be useful in active immunotherapy protocols for human carcinoma.Some of the studies described in this paper were in partial fulfillment of requirements for the completion of Dr. Irvine's dissertation at the George Washington University     

Autophagy is an immediate macrophage response to Legionella pneumophila

After ingestion by macrophages, Legionella pneumophila enter spacious vacuoles that are quickly enveloped by endoplasmic reticulum (ER), then slowly transferred to lysosomes. Here we demonstrate that the macrophage autophagy machinery recognizes the pathogen phagosome as cargo for lysosome delivery. The autophagy conjugation enzyme Atg7 immediately translocated to phagosomes harbouring virulent Legionella. Subsequently, Atg8, a second autophagy enzyme, and monodansyl-cadaverine (MDC), a dye that accumulates in acidic autophagosomes, decorated the pathogen vacuoles. The autophagy machinery responded to 10-30 kDa species released into culture supernatants by Type IV secretion-competent Legionella, as judged by the macrophages' processing of Atg8 and formation of vacuoles that sequentially acquired Atg7, Atg8 and MDC. When compared with autophagosomes stimulated by rapamycin, Legionella vacuoles acquired Atg7, Atg8 and MDC more slowly, and Atg8 processing was also delayed. Moreover, compared with autophagosomes of Legionella-permissive naip5 mutant A/J macrophages, those of resistant C57BL/6 J macrophages matured quickly, preventing efficient Legionella replication. Accordingly, we discuss a model in which macrophages elevate autophagy as a barrier to infection, a decision influenced by regulatory interactions between Naip proteins and caspases.     

Induction of allergen-specific IgE and IgG responses by anti-idiotypic antibodies

In order to explore idiotypic, anti-idiotypic, and anti-anti-idiotypic responses to allergens, BALB/c mice were immunized with affinity-purified human idiotypic antibodies directed against a highly purified shrimp allergen. This resulted in the production of anti-idiotypic antibodies which were quantitated by using rabbit idiotypic antibodies raised against the same purified allergen. The mouse anti-idiotypic antibodies recognized shrimp-specific human idiotypic antibodies of the IgE isotype from 18 of 20 individuals, and IgG antibodies from 14 of 20 shrimp-sensitive patients. Immunization of BALB/c mice with affinity-purified, allergen-specific anti-idiotypic antibodies induced anti-allergen IgE and IgG responses in the absence of the allergen. This paper thus presents evidence that anti-idiotypic antibodies raised against allergen-specific idiotypic antibodies may substitute for the original allergen in the induction of allergen-specific idiotypic antibodies. The demonstration of shared idiotopes on IgG and IgE antibodies in the sera of shrimp-sensitive patients supports the use of allergen-specific anti-idiotypic antibodies as surrogate allergens.     

Induction of interleukin 1 by Legionella pneumophila in murine peritoneal macrophage cultures

Legionella pneumophila-induced production of both membrane-associated and secreted interleukin 1 (mIL-1 and sIL-1, respectively) was examined utilizing peritoneal macrophages from BALB/c mice. The Legionella preparations for these studies included viable bacteria and formalin-killed whole cell preparations. Both of the preparations induced mIL-1 and sIL-1 in a dose-dependent fashion. However, the viable bacteria required about 1 log lower concentrations than the formalin-killed bacteria to induce the same level of IL-1 activity measured in the thymocyte proliferation assay. Kinetic studies showed that mIL-1 and sIL-1 were detectable within 4 hr after addition of either of the L. pneumophila preparations to the peritoneal macrophage cultures, with peak levels achieved within 24 hr. These results indicate that L. pneumophila is a potent inducer of both mIL-1 and sIL-1 in normal mouse peritoneal macrophage cultures.     

Syngeneic anti-idiotypic antisera to murine anti-HLA class II monoclonal antibodies

BALB/c mice have been immunized with six anti-HLA Class II monoclonal antibodies (MoAb); the latter included MoAb CR11-462, Q5/6, and Q5/13 to monomorphic determinants, the anti-HLA-DR1,4,w6,w8,w9 MoAb AC1.59, the anti-HLA-DRw9 MoAb AB7ae9, and the anti-HLA-DQw3 MoAb AC6G. The six monoclonal antibodies markedly differ in their ability to induce anti-idiotypic antibodies, because the latter were not detected in the sera from the mice immunized with the MoAb AB7ae9 and with the MoAb AC6G. The MoAb AC1.59 and CR11-462 elicited antibodies to private idiotypes, and the MoAb Q5/6 and Q5/13 elicited antibodies to private and public idiotypes. The titer of the latter in the anti-MoAb Q5/6 antiserum appears to be markedly lower than that of the former ones; no marked difference was detected in the titer of the two types of antibodies in the anti-MoAb Q5/13 antiserum. Blocking experiments with a panel of monoclonal antibodies showed that the MoAb Q5/13 shares idiotypes with the anti-HLA Class I MoAb Q5/6, 127, and 441 and with the anti-HLA Class I MoAb CR11-351, Q1/28, Q6/64, and 6/31, but does not share idiotypes with any of the nine anti-human melanoma-associated antigen MoAb tested. The spectrotypes of the anti-MoAb CR11-462 and anti-MoAb Q5/13 antisera comprise two major components in the pH 6.2 to 6.7 range, that of the anti-MoAb Q5/6 antiserum comprises two major components in the pH 6.5 to 6.8 range, and that of the anti-MoAb AC1.59 antiserum comprises a number of components in the pH 5.6 to 7.2 range.     

Iron Acquisition by Legionella pneumophila

For nearly 20 years, it was believed that Legionella pneumophila does not produce siderophores. Yet, we have now determined that L. pneumophila secretes a siderophore (legiobactin) that is detectable by the CAS assay. We have optimized conditions for legiobactin expression, shown its biological activity, and found genes (lbtAB) involved in its production and secretion. LbtA is homologous with siderophore synthetases from E. coli (aerobactin), Sinorhizobium (rhizobactin), and Bordetella (alcaligin), while LbtB is a member of the major facilitator superfamily of multidrug efflux pumps. Mutants lacking lbtAB produce 40–70% less CAS reactivity. The lbtA mutant is also defective for growth in deferrated media containing citrate, indicating that legiobactin is required in conditions of severe iron limitation. lbtAB mutants grow normally in macrophages and amoebae host cells as well as within the lungs of mice. L. pneumophila does express lbtA in macrophages, suggesting that legiobactin has a dispensable role in infection. Legiobactin is iron repressed and does not react in the Csáky and Arnow assays. Anion-exchange HPLC has been used to purify legiobactin, and thus far, structural analysis suggests that the molecule is similar but not identical to rhizobactin, rhizoferrin, and alcaligin. The residual CAS reactivity present in supernatants of the lbtAB mutants suggests that L. pneumophila might produce a second siderophore. Besides siderophores, we have determined that ferrous iron transport, encoded by feoB, is critical for L. pneumophila growth in low-iron conditions, in host cells, and in the mammalian lung. Some of our other studies have discovered a critical, yet undefined, role for the L. pneumophila cytochrome c maturation locus in low-iron growth, intracellular infection, and virulence.