Immunization Of Naive Mice Research Articles

Immunization with lytic polysaccharide monooxygenase CbpD induces protective immunity against Pseudomonas aeruginosa pneumonia

Pseudomonas aeruginosa (PA) CbpD belongs to the lytic polysaccharide monooxygenases (LPMOs), a family of enzymes that cleave chitin or related polysaccharides. Here, we demonstrate a virulence role of CbpD in PA pneumonia linked to impairment of host complement function and opsonophagocytic clearance. Following intratracheal challenge, a PA ΔCbpD mutant was more easily cleared and produced less mortality than the wild-type parent strain. The x-ray crystal structure of the CbpD LPMO domain was solved to subatomic resolution (0.75Å) and its two additional domains modeled by small-angle X-ray scattering and Alphafold2 machine-learning algorithms, allowing structure-based immune epitope mapping. Immunization of naive mice with recombinant CbpD generated high IgG antibody titers that promoted human neutrophil opsonophagocytic killing, neutralized enzymatic activity, and protected against lethal PA pneumonia and sepsis. IgG antibodies generated against full-length CbpD or its noncatalytic M2+CBM73 domains were opsonic and protective, even in previously PA-exposed mice, while antibodies targeting the AA10 domain were not. Preexisting antibodies in PA-colonized cystic fibrosis patients primarily target the CbpD AA10 catalytic domain. Further exploration of LPMO family proteins, present across many clinically important and antibiotic-resistant human pathogens, may yield novel and effective vaccine antigens.

Spirochete antigens persist near cartilage after murine Lyme borreliosis therapy

An enigmatic feature of Lyme disease is the slow resolution of musculoskeletal symptoms that can continue after treatment, with some patients developing an inflammatory arthritis that becomes refractory to antibiotic therapy. Using intravital microscopy and the mouse model of Lyme borreliosis, we observed that Borrelia burgdorferi antigens, but not infectious spirochetes, can remain adjacent to cartilage for extended periods after antibiotic treatment. B. burgdorferi was not recovered by culture or xenodiagnosis with ticks after antibiotic treatment of WT mice and all but one of the immunodeficient mice with heightened pathogen burden due to impaired TLR responsiveness. Amorphous GFP+ deposits were visualized by intravital microscopy in the entheses of antibiotic-treated mice infected with GFP-expressing spirochetes and on the ear cartilage surface in sites where immunofluorescence staining detected spirochete antigens. Naive mice were not infected by tissue transplants from antibiotic-treated mice even though transplants contained spirochete DNA. Tissue homogenates from antibiotic-treated mice induced IgG reactive with B. burgdorferi antigens after immunization of naive mice and stimulated TNF-α production from macrophages in vitro. This is the first direct demonstration that inflammatory B. burgdorferi components can persist near cartilaginous tissue after treatment for Lyme disease. We propose that these deposits could contribute to the development of antibiotic-refractory Lyme arthritis.

Suppression of experimental systemic lupus erythematosus (SLE) with specific anti-idiotypic antibody-saporin conjugate.

The importance of the idiotypic network is represented in experimental SLE induced by active immunization of naive mice with an anti-DNA idiotype (Ab1) emulsified in adjuvant. The mice after 4 months of incubation generate Ab3 having anti-DNA activity. In addition, the mice develop other serological markers for SLE associated with clinical and histopathological manifestations characteristic of the disease. To confirm further the etiological role of the idiotype in this experimental model, the mice were treated with specific anti-idiotypic antibodies (anti-Id) which were also conjugated to a toxin-saporin (Immunotoxin (IT)). Pretreatment of hybridoma cell line producing the anti-anti-Id (anti-DNA = (Ab3)) for 48 h with the anti-Id MoAb (Ab2) reduced the production of anti-DNA by 58%, while pretreatment with the IT resulted in 86% decrease in anti-DNA secretion (saporin alone had only 12% effect). The anti-Id MoAb had no effect on the production of immunoglobulin by an unrelated cell line. In vivo treatment of mice with experimental SLE led to a significant decrease in titres of serum autoantibodies, with diminished clinical manifestations. The results were more remarkable when the IT was employed. These suppressive effects were specific, since an anti-Id treatment of experimental anti-phospholipid syndrome was of no avail. The anti-Id effect was mediated via a reduction in specific anti-DNA antibody-forming cells, and lasted only while anti-Id injections were given. Discontinuation of the anti-Id injection was followed by a rise in titres of anti-DNA antibodies. No immunological escape of new anti-DNA Ids was noted. Our results point to the importance of pathogenic idiotypes in SLE and to the specific potential of implementing anti-idiotypic therapy, enhanced by the conjugation of the anti-Id to an immunotoxin, in particular one with low spontaneous toxicity.

The therapeutic effect of glatiramer acetate in a murine model of inflammatory bowel disease is mediated by anti-inflammatory T-cells

Inflammatory bowel diseases (IBDs) are complex multifactorial immunological disorders characterized by dysregulated immune reactivity in the gut and imbalance between pro-inflammatory and anti-inflammatory reactivity. The therapeutic effect of the immunomodulatory drug glatiramer acetate (GA, Copaxone, copolymer 1) has been established in several IBD models, including trinitrobenzene sulfonic acid (TNBS) and dextran sulfate sodium (DSS)-induced colitis, as well as in a spontaneous colitis model. In the present study we investigated the mechanism of action of GA and cells specifically induced by it. Immunization of naive mice by GA, generated a lymphocyte population of the Th2/3 subtype, that drastically reduced disease manifestations upon their adoptive transfer to mice with DSS colitis. This was demonstrated by the substantial decrease in weight loss, intestinal bleeding and diarrhea, as well as by the prevention of macroscopic and microscopic colonic damage. In contrast, adoptive transfer of control lysozyme-specific cells did not induce any beneficial effect on the disease. Moreover, GA-specific short-term T-cell lines, either exogenously labeled or genetically marked, adoptively transferred by the intraperitoneal route to colitis-induced mice, localized in the inner layers of the colon and secreted in situ the regulatory cytokine TGF-β. These results demonstrate the accumulation of GA-specific Th2/3 cells secreting regulatory cytokines in the injured colon, and thus draw a direct linkage between the therapeutic effect of GA in IBD and an immunomodulatory effect at the site in which the pathological process occurs.

Surrogate Tumor Antigen Vaccination Induces Tumor-Specific Immunity and the Rejection of Spontaneous Metastases

The nonimmunogenic 4T1 murine mammary carcinoma model and a model surrogate tumor antigen (sTA) were employed to explore the possibility of inducing tumor-specific immunity through active immunization in the absence of defined tumor-associated antigens. Immunization of naive mice with protein-based sTA resulted in protection from s.c. challenge, with 4T1 modified to express the sTA (4T1.sTA), or from a sTA-expressing unrelated tumor cell line (mKSA). Immunization had no effect on parental 4T1 tumor growth or the formation of parental 4T1 spontaneous lung metastases. Mice that were sTA immunized and successfully rejected 4T1.sTA challenge also rejected a subsequent challenge in the contralateral flank with parental 4T1 and strikingly prevented the formation of spontaneous parental 4T1 lung metastases. The rejection of parental 4T1 seemed to be specific for and associated with unknown 4T1 tumor-associated antigens, because rejection of mKSA did not induce cross-protection against a challenge with parental 4T1. To evaluate the effect of this vaccine approach on established disease, mice were simultaneously challenged on day 0 with 4T1.sTA and parental 4T1 in contralateral flanks and then immunized on days 3, 10, 17, and 24 with sTA protein. Tumor growth and metastasis were delayed in four of five animals, and 20% (2 of 5) of the animals were tumor free at the completion of the experiment. Together, these data suggest that prior vaccination with a sTA followed by inoculation with poorly immunogenic tumor cells modified to express the sTA activates determinant spreading and the induction of systemic tumor immunity resulting in indigenous tumor rejection.

Immunization of naive BALB/c mice with human beta2-glycoprotein I breaks tolerance to the murine molecule.

Immunization of naive mice with beta2-glycoprotein I (beta2GPI) leads to the generation of pathogenic anticardiolipin antibodies associated with clinical manifestations of the antiphospholipid syndrome (APS). The aim of this study was to determine whether immunization of naive mice with human beta2GPI, which shares homology with mouse beta2GPI molecules, breaks tolerance to murine beta2GPI and leads to the generation of anti-mouse beta2GPI. Twenty-four female BALB/c mice were immunized in the footpads with 10 microg of human beta2GPI. Twelve age- and sex-matched BALB/c mice were immunized in the same manner with Freund's complete adjuvant and served as controls. The reactivity of whole sera, polyclonal IgG, and affinity-purified anti-beta2GPI IgG antibodies against human, bovine, and mouse beta2GPI was evaluated by enzyme-linked immunosorbent assay. High titers of anti-human beta2GPI IgG antibodies were detected 1 month after immunization. Progressively increasing titers against murine and bovine beta2GPI were recorded 1-4 months after injection. Immunization of mice with human beta2GPI resulted in the generation of antibodies reacting with human, bovine, and murine beta2GPI. The loss of tolerance to mouse beta2GPI is attributable to the high interspecies homology of beta2GPI. These results may point to molecular mimicry as a possible cause of APS.

Cutting Edge: Physical Interaction Between Dendritic Cells and Tumor Cells Results in an Immunogen That Induces Protective and Therapeutic Tumor Rejection

AbstractDendritic cells (DCs) are potent professional APCs capable of presenting Ag in the context of costimulatory signals necessary for T cell activation. Although tumor cells express target Ags, they are generally incapable of stimulating an immune response. We show that the short term physical interaction of DCs and tumor cells, with or without cell fusion, results in rapid, efficient, and stable DC-tumor cell association. Immunization of naive mice with unselected, irradiated DC-tumor cell conjugates induces tumor-specific CD8+ cytotoxic T cells and protection from lethal tumor challenge. Furthermore, the immunogenicity of this cellular vaccine is dependent on the physical interaction of DCs and tumor cells before injection. Immunization with DCs and tumor cells after physical interaction can result in the regression of established tumors and persistent antitumor immunity. These results suggest that immunization with DC-tumor cell vaccines may be a simple, rapid, and potent strategy for tumor immunotherapy.

Antigen-specific regression of established tumors induced by active immunization with irradiated IL-12- but not B7-1-transfected tumor cells.

Transfection of modestly immunogenic tumors to express B7 family co-stimulator molecules results in their rejection by syngeneic mice, suggesting a possible clinical application in cancer patients. Immunization of naive mice with irradiated B7-1-transfected P1.HTR cells is sufficient to induce specific cytolytic T lymphocytes (CTL) and to protect against tumor challenge. However, patients to be treated will have an existing tumor burden; thus, preclinical models should examine therapeutic efficacy in an established tumor setting. Contrary to expectations, immunization of mice with irradiated B7-1-transfected P1.HTR cells had no impact on the growth of pre-established control-transfected tumors. Mice bearing control-transfected P1.HTR tumors successfully rejected living B7-1 transfectants on the contralateral flank, demonstrating the ability of tumor-bearing mice to respond to B7 co-stimulation. Inasmuch as IL-12 is another important factor for CTL maturation, P1.HTR transfectants expressing B7-1 and/or IL-12 were then constructed. Remarkably, regression of pre-established tumors was achieved following immunization with irradiated IL-12 transfectants, even without co-expression of B7-1. Rejection required a shared antigen with the tumor used for immunization, could not be reproduced with rIL-12 alone, depended on host T lymphocytes and correlated with a high IFN-gamma-producing T cell phenotype. In addition, IL-12-facilitated tumor rejection required co-operation with a CTLA-4 ligand provided by the host, and correlated with up-regulation of B7-1 and B7-2 on host antigen-presenting cells. Thus, active immunization in the established tumor setting is benefitted greatly by the provision of IL-12, which may recruit participation of sufficient B7 co-stimulation from the host that it need not be provided exogenously.

Natural autoantibodies in sera of patients with Gaucher's disease.

Gaucher's disease (GD) is associated with hyperactivity of the immune system, which manifests by polyclonal hypergamma-globulinemia and an increased incidence of monoclonal gammopathies in GD patients. We analyzed sera of 43 patients with GD for the presence of autoantibodies against 14 autoantigens. The results demonstrated a significant increase in the incidence of all autoantibodies tested, ranging from 11% for anti-RNP, pyruvate dehydrogenase (PDH), and DNA antibodies to 57% for rheumatoid factor. The autoantibodies were of all three isotypes, namely, IgG, IgM, and IgA. There was no correlation between the levels of immunoglobulins in the serum and the titer of autoantibodies found. Immunization of naive mice with a pool of purified anti-DNA antibodies form GD patients did not result in induction of experimental systemic lupus erythematosus (SLE), suggesting that they may represent natural autoantibodies that are not pathogenic. In conclusion, we found high titers of natural, polyspecific, nonpathogenic autoantibodies in the sera of GD patients.

Immunization with Anticardiolipin Cofactor (Beta-2-Glycoprotein I) Induces Experimental Antiphospholipid Syndrome in Naive Mice

Beta-2-GPI is a 50 kDa glycoprotein which is known to be a serum co-factor, with a role in determining the binding of pathogenic anticardiolipin antibodies to phospholipids. Immunization of naive mice with beta-2-GPI resulted in elevated levels of antibodies directed against negatively charged phospholipids (cardiolipin, phosphotidylserine, phosphatidylinositol). The presence of increased titres of antiphospholipid antibodies in the sera of the mice was later followed by prolonged activated partial thromboplastin time (APTT), thrombocytopenia, and when the mice were mated, by a high percentage of fetal resorptions in the uterus. These data point to the ability of beta-2-GPI to induce pathogenic anti-cardiolipin antibodies following active immunization.

Mimicry of a neutralizing epitope of the major outer membrane protein of Chlamydia trachomatis by anti-idiotypic antibodies.

The major outer membrane protein (MOMP) is a primary target antigen for the development of chlamydial vaccine. This protein is composed of four variable domains (I to IV) flanked by constant regions. Some of the variable domains contain antigenic determinants that elicit a neutralizing antibody response. Murine monoclonal antibodies (MAbs) against three nonoverlapping epitopes of MOMP were developed. One of these, called DP10, bound to all serovars, as shown by immunoblot analysis, and neutralized chlamydial infectivity for hamster kidney (HaK) cells in a complement-independent in vitro assay. Furthermore, analysis of the fine specificity of this MAb showed that it recognized a synthetic peptide contained within variable domain IV of the MOMP. Anti-idiotypic antibodies (aId) directed against this anti-MOMP MAb were produced in rabbits. These aId specifically bound to the relevant idiotype (DP10) and inhibited the binding of anti-MOMP MAb (DP10) to MOMP preparations in a dose-dependent fashion. The specificity of our aId for the binding site of anti-MOMP MAb is further suggested by the binding inhibition of affinity-purified aId to DP10 by the synthetic peptide defined by the idiotype. In addition, these aId also reacted with anti-MOMP antisera from rats and mice, suggesting an idiotypic cross-reactivity between these species. Finally, immunization of naive mice with aId induced an antibody response directed against the peptide defined by our anti-MOMP MAb and with neutralizing activity. Taken together, these data suggest that aId mimic a neutralization site on MOMP and could serve as a surrogate antigen to induce protective immunity against Chlamydia trachomatis.

Idiotype and anti-anti-idiotype antibodies to Neisseria gonorrhoeae lipooligosaccharides with bactericidal activity but no cross-reactivity with red blood cell antigens.

A panel of murine mAb against three different epitopes of Neisseria gonorrhoeae lipooligosaccharides (LOS) was developed. Only one of these, 3G5, displays bactericidal activity against all in vitro serum-resistant strains of N. gonorrhoeae. Evidence suggests that sialylation, which could occur in vivo, modifies some LOS epitopes in such a way that the strains become resistant to bactericidal activity and are no longer recognized by specific antibodies. The epitope recognized by our bactericidal mAb is not affected by sialylation as shown by immunoblot analysis. We also provided evidence that the 3G5 epitope is different from RBC precursor Ag, since our mAb did not induce RBC agglutination. Since LOS induce an immune response in a T cell-independent fashion and are highly toxic, they cannot be used for immunization. Use of anti-idiotypic antibodies (aId) could be a way to bypass these difficulties. Therefore, in the present study, aId were produced in rabbits and rendered idiotype-specific by appropriate adsorption. These aId specifically bind to the relevant Id but not to LOS, and inhibit only the binding of anti-LOS mAb (3G5) to LOS preparations from N. gonorrhoeae in a dose-response fashion. The specificity of our aId for the binding site of anti-LOS mAb is suggested by the binding inhibition of affinity-purified aId to Id by LOS. In addition, the capacity of aId to inhibit bactericidal activity of this anti-LOS mAb and the idiotypic cross-reactivity between rat and mouse anti-LOS antibodies support this point. Finally, the elicitation of anti-LOS activity with bactericidal activity upon immunization of naive mice with aId confirms the internal image properties of the aId. These data suggest that a bactericidal mAb suitable for immunoprotection was obtained, and the production of aId opens the door for development of a vaccine.

A "network antigen" for human CD4. A murine monoclonal anti-idiotype to Leu-3a induces an anti-CD4 response in naive mice.

Previous studies have evaluated anti-CD4 mAb as idiotypic models of the HIV gp120-binding site for CD4. The success of this strategy depends upon the concept of internal image, whereby the binding paratope of the anti-CD4 structurally mimics the equivalent binding surface on HIV gp120. To test this concept of internal image, anti-idiotypic antibodies were raised against the anti-CD4, Leu-3a. If any of these anti-Id detect the paratopic idiotope on the anti-CD4 antibody, their own respective paratopes should structurally model the corresponding binding epitope on CD4 bound by Leu-3a. Consequently, the immunization of naive mice with the selected anti-Id should induce an anti-CD4 response which reflects the binding specificities of Leu-3a. Four anti-Id to Leu-3a were characterized and tested for their ability to induce anti-CD4 responses in naive animals. Although one anti-Id induced an anti-CD4 response in mice, no such response could be detected in other species. Thus the failure to raise anti-Id with internal image characteristics may provide an explanation for the lack of anti-gp120 activity reported in anti-Id antisera raised to multiple anti-CD4 antibodies.

The Importance of the Pathogenic 16/6 Idiotype in the Induction of SLE in Naive Mice

We have previously demonstrated the pathogenicity of the common anti-DNA idiotype designated 16/6 Id. Immunization of naive mice with the 16/6 Id induced SLE-like disease characterized by serological (e.g. anti-dsDNA and anti-Sm auto-antibodies), clinical (increased ESR, leucopenia and proteinuria), and pathological (16/6 Id deposition in kidneys) parameters. To elucidate further the role of the 16/6 Id in SLE induction the following studies were carried out: BALB/c mice were immunized with SA-1, a human anti-DNA monoclonal antibody carrying the 16/6 Id; TB-68, a mouse monoclonal anti-tuberculosis (TB) glycolipid, which binds dsDNA and carries the 16/6 Id; TB-72, a mouse monoclonal anti-TB glycolipid that binds DNA and does not harbour the 16/6 Id; and 4B4, a human anti-Sm antibody that carries the 16/6 Id. SLE was induced in BALB/c mice only when immunized with SA-1, TB-68, and 4B4, namely antibodies with diverse binding capacities albeit having the 16/6 Id. Our studies further support previous evidence on the pathogenic role attributed to the 16/6 Id in SLE, and suggest that SLE is most probably an idiotype-induced disease.