Functional and structural characterization of Francisella tularensis O-antigen antibodies at the low end of antigen reactivity.

The O-antigen (OAg) of the Gram-negative bacterium Francisella tularensis (Ft), which is both a capsular polysaccharide and a component of lipopolysaccharide, is comprised of tetrasaccharide repeats and induces antibodies mainly against repeating internal epitopes. We previously reported on several BALB/c mouse monoclonal antibodies (MAbs) that bind to internal Ft OAg epitopes and are protective in mouse models of respiratory tularemia. We now characterize three new internal Ft OAg IgG2a MAbs, N203, N77, and N24, with 10- to 100-fold lower binding potency than previously characterized internal-OAg IgG2a MAbs, despite sharing one or more variable region germline genes with some of them.

B-cell epitopes in GroEL of Francisella tularensis.

The chaperonin protein GroEL, also known as heat shock protein 60 (Hsp60), is a prominent antigen in the human and mouse antibody response to the facultative intracellular bacterium Francisella tularensis (Ft), the causative agent of tularemia. In addition to its presumed cytoplasmic location, FtGroEL has been reported to be a potential component of the bacterial surface and to be released from the bacteria. In the current study, 13 IgG2a and one IgG3 mouse monoclonal antibodies (mAbs) specific for FtGroEL were classified into eleven unique groups based on shared VH-VL germline genes, and seven crossblocking profiles revealing at least three non-overlapping epitope areas in competition ELISA.

Antibodies to both terminal and internal B-cell epitopes of Francisella tularensis O-polysaccharide produced by patients with tularemia.

Francisella tularensis, the Gram-negative bacterium that causes tularemia, is considered a potential bioterrorism threat due to its low infectivity dose and the high morbidity and mortality from respiratory disease. We previously characterized two mouse monoclonal antibodies (MAbs) specific for the O-polysaccharide (O antigen [OAg]) of F. tularensis lipopolysaccharide (LPS): Ab63, which targets a terminal epitope at the nonreducing end of OAg, and Ab52, which targets a repeating internal OAg epitope.

Discovery of protective B-cell epitopes for development of antimicrobial vaccines and antibody therapeutics.

Protective antibodies play an essential role in immunity to infection by neutralizing microbes or their toxins and recruiting microbicidal effector functions. Identification of the protective B-cell epitopes, those parts of microbial antigens that contact the variable regions of the protective antibodies, can lead to development of antibody therapeutics, guide vaccine design, enable assessment of protective antibody responses in infected or vaccinated individuals, and uncover or localize pathogenic microbial functions that could be targeted by novel antimicrobials. Monoclonal antibodies are required to link in vivo or in vitro protective effects to specific epitopes and may be obtained from experimental animals or from humans, and their binding can be localized to specific regions of antigens by immunochemical assays.

The binding sites of monoclonal antibodies to the non-reducing end of Francisella tularensis O-antigen accommodate mainly the terminal saccharide.

We have previously described two types of protective B-cell epitopes in the O-antigen (OAg) of the Gram-negative bacterium Francisella tularensis: repeating internal epitopes targeted by the vast majority of anti-OAg monoclonal antibodies (mAbs), and a non-overlapping epitope at the non-reducing end targeted by the previously unique IgG2a mAb FB11. We have now generated and characterized three mAbs specific for the non-reducing end of F. tularensis OAg, partially encoded by the same variable region germline genes, indicating that they target the same epitope.

Structural analysis of a protective epitope of the Francisella tularensis O-polysaccharide.

Francisella tularensis (Ft), the Gram-negative facultative intracellular bacterium that causes tularemia, is considered a biothreat because of its high infectivity and the high mortality rate of respiratory disease. The Ft lipopolysaccharide (Ft LPS) is thought to be a main protective antigen in mice and humans, and we have previously demonstrated the protective effect of the Ft LPS-specific monoclonal antibody Ab52 in a mouse model of respiratory tularemia. Immunochemical characterization has shown that the epitope recognized by Ab52 is contained within two internal repeat units of the O-polysaccharide [O-antigen (OAg)] of Ft LPS.

Protective B-cell epitopes of Francisella tularensis O-polysaccharide in a mouse model of respiratory tularaemia.

Antibodies to the lipopolysaccharide (LPS) of Francisella tularensis have been shown to be protective against respiratory tularaemia in mouse models, and we have previously described mouse monoclonal antibodies (mAbs) to non-overlapping terminal and internal epitopes of the F. tularensis LPS O-polysaccharide (OAg). In the current study, we used F.

A typical preparation of Francisella tularensis O-antigen yields a mixture of three types of saccharides.

Tularemia is a severe infectious disease in humans caused by the Gram-negative bacterium Francisella tularensis (Ft). Because of its low infectious dose, high mortality rate, and the threat of its large-scale dissemination in weaponized form, development of vaccines and immunotherapeutics against Ft is essential. Ft lipopolysaccharide (LPS), which contains the linear graded-length saccharide component O-antigen (OAg) attached to a core oligosaccharide, has been reported as a protective antigen.

Characterization of monoclonal antibodies to terminal and internal O-antigen epitopes of Francisella tularensis lipopolysaccharide.

The lipopolysaccharide (LPS) of Francisella tularensis (Ft), the Gram negative bacterium that causes tularemia, has been shown to be a main protective antigen in mice and humans; we have previously demonstrated that murine anti-Ft LPS IgG2a monoclonal antibodies (MAbs) can protect mice against otherwise lethal intranasal infection with the Ft live vaccine strain (LVS). Here we show that four IgG2a anti-LPS MAbs are specific for the O-polysaccharide (O-antigen [OAg]) of Ft LPS. But whereas three of the MAbs bind to immunodominant repeating internal epitopes, one binds to a unique terminal epitope of Ft OAg.

Antibody treatment of human tumor xenografts elicits active anti-tumor immunity in nude mice.

Athymic nude mice bearing subcutaneous tumor xenografts of the human anti-colorectal cancer cell line SW480 were used as a preclinical model to explore anti-tumor immunotherapies. Intratumor or systemic treatment of the mice with murine anti-SW480 serum, recombinant anti-SW480 polyclonal antibodies, or the anti-colorectal cancer monoclonal antibody CO17-1A, caused retardation or regression of SW480 tumor xenografts. Interestingly, when mice that had regressed their tumors were re-challenged with SW480 cells, these mice regressed the new tumors without further antibody treatment.

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.

Production of target-specific recombinant human polyclonal antibodies in mammalian cells.

We describe the expression and consistent production of a first target-specific recombinant human polyclonal antibody. An anti-Rhesus D recombinant polyclonal antibody, Sym001, comprised of 25 unique human IgG1 antibodies, was produced by the novel Sympress expression technology. This strategy is based on site-specific integration of antibody genes in CHO cells, using the FRT/Flp-In recombinase system.

Recombinant polyclonal antibodies for cancer therapy.

Although monoclonal antibodies are increasingly used for cancer therapy, remissions are only temporary due to emergence of tumor cell escape variants that are no longer affected by the antibody. The emergence of escape variants could be minimized by multi-targeting of tumor cells with polyclonal antibodies, which would also be more efficient than monoclonal antibodies at mediating effector functions for target destruction. A technology for generating recombinant polyclonal antibodies for cancer therapy has been developed based on the construction and selection of tumor-reactive Fab phage display libraries.

Generation and preliminary characterization of an antibody library with preferential reactivity to human colorectal cancer cells as compared to normal human blood cells.

We are developing recombinant polyclonal antibody libraries (PCALs) reactive to human colorectal cancer cells as an anti-cancer therapeutic approach. To test the hypothesis that PCALs with preferential recognition of tumor cells as compared to normal cells could be generated, a Fab phage display library reactive to human colorectal cancer cell lines was absorbed with normal human blood cells. ELISA analysis of the absorbed Fab phage display library showed that 70% of tested clones reacted to colorectal cancer cells.

Expression of a prototypic anti-colorectal cancer polyclonal antibody library in mammalian cells.

We describe the production of a prototypic polyclonal antibody library (PCAL), a standardized mixture of full-length IgG polyclonal antibodies for which the genes are available. The PCAL was generated by mass transfer of heavy and light chain variable region gene pairs, selected for binding to human colorectal cancer cells, from a Fab phage display vector to a mammalian IgG expression vector. Following transfection of the IgG vector library into Sp2/0 myeloma cells, clones were characterized for IgG expression and binding to the colorectal cancer cells by ELISA, and for diversity by DNA fingerprinting, nucleotide sequencing, and immunoblot analysis.

Polyclonal Fab phage display libraries with a high percentage of diverse clones to Cryptosporidium parvum glycoproteins.

The protozoan parasite Cryptosporidium parvum is regarded as a major public health problem world-wide, especially for immunocompromised individuals. Although no effective therapy is presently available, specific immune responses prevent or terminate cryptosporidiosis and passively administered antibodies have been found to reduce the severity of infection. Therefore, as an immunotherapeutic approach against cryptosporidiosis, we set out to develop C.

Generation of anti-colorectal cancer fab phage display libraries with a high percentage of diverse antigen-reactive clones.

A combinatorial Fab phage display library was generated from the antibody variable region genes of each of 2 BALB/c mice immunized with the human colorectal cancer cell lines SW480, SW948, and SW837. These libraries were shown to be diverse by nucleotide sequencing and diagnostic restriction enzyme digestion (fingerprinting) of individual members. The two libraries were combined and selected for binding to a suspension of formaldehyde-fixed human colorectal cancer cells in two successive rounds of selection and phage amplification by infection of bacteria.

Polyclonal anti-colorectal cancer Fab phage display library selected in one round using density gradient centrifugation to separate antigen-bound and free phage.

A combinatorial Fab phage display library generated from antibody variable (V) region genes of BALB/c mice immunized with the human colorectal cancer cell lines SW480, SW948, and SW837, was used to isolate an anti-colorectal cancer library. In an attempt to preserve as many anti-colorectal cancer specificities as possible, the original Fab phage display library was selected for binding to a suspension of the human colorectal cancer cells using density gradient centrifugation, instead of washes, to separate cell-bound and free phage. The method consists of placing the cell-phage mixture on a layer of fetal bovine serum (FBS) which had been overlaid on a "cushion" of percoll density medium in a soft, polyallomer tube.