Suzanne Lacelle

Normal and Malignant Human Colonic Tissues Investigated by Pressure-Tuning FT-IR Spectroscopy

Infrared spectra of the epithelial, the connective, the mucosa, and the malignant tissues of the human colon have been measured as a function of pressure. Infrared spectra of collagen proteins have also been measured and compared with the connective tissue. The infrared spectra of different types of colon tissues exhibit significantly different patterns. With these specific infrared patterns, the tissue types of the colon can be differentiated unambiguously. Many structural changes at the molecular level from normal epithelium to malignant tumor have been derived from the spectral features of these two related tissues. These structural changes in carcinogenesis of the colon are comparable with those in other human cancers. The present results suggest that determination of these infrared spectra may be applied to the rapid identification of normal tissue types of the colon and evaluation of colon cancer.

Development, Characterization, and Functional Activity of a Panel of Specific Monoclonal Antibodies to Inner Core Lipopolysaccharide Epitopes in Neisseria meningitidis

ABSTRACT A panel of six murine monoclonal antibodies (MAbs) recognizing inner core lipopolysaccharide (LPS) epitopes of Neisseria meningitidis was prepared and characterized in order to determine the diversity of inner core LPS glycoforms among disease and carrier isolates. Two of these MAbs, L2-16 (immunoglobulin G2b [IgG2b]) and LPT3-1 (IgG2a), together with a third, previously described MAb, L3B5 (IgG3), showed reactivity, either individually or in combination, with all except 3 of 143 disease and carriage isolates (125 of 126 strains from blood, cerebrospinal fluid, or skin biopsy samples and 15 of 17 from nasopharyngeal cultures). MAbs L3B5, L2-16, and LPT3-1 were further characterized in an indirect immunofluorescence assay. All three MAbs bound to the bacterial cell surface, findings that correlated strongly with whole-cell enzyme-linked immunosorbent assay and immunodot blots. However, in contrast to our findings with L3B5, cell surface binding of L2-16 or LPT 3-1 did not correlate with functional activity as determined by bactericidal or infant rat passive protection assays against wild-type N. meningitidis strains. These findings are provocative with respect to the requirements for protective activity of antibodies and the development of inner core LPS vaccines against invasive meningococcal disease.

Naturally-occurring human serum antibodies to inner core lipopolysaccharide epitopes of Neisseria meningitidis protect against invasive meningococcal disease caused by isolates displaying homologous inner core structures.

Sera from healthy infants (under 1 year old), toddlers (3-4 years) and adults (18-65 years) were assayed for their ability to bind to inner core (ic) lipopolysaccharide (LPS) epitopes of Neisseria meningitidis. Antibodies (Abs) reacting to inner core structures, including different substitutions of the first heptose (HepI) and second heptose (HepII) residues of the LPS backbone, truncated and fully extended LPS glycoforms, were detected and for each structure, these inner core antibodies showed an age-related pattern of acquisition. A novel column-based methodology was used to affinity purify IgG antibodies in which purified inner core LPS (derived from a mutant MC58) was covalently linked to Sepharose 4B. Comparison of reactivity before and after affinity purification of the pooled sera showed that the purified Abs bound to the surface of N. meningitidis organisms displaying truncated and extended LPS with a homologous inner core region, promoted the deposition of C3b, were opsonophagocytic in vitro and decreased bacteraemia when used to passively protect infants rats. In addition, the purified Abs were bactericidal in vitro against the mutant strain displaying truncated LPS with a homologous inner core region. These results demonstrate that naturally occurring serum human antibodies to N. meningitidis LPS can access inner core epitopes of encapsulated organisms with a fully extended LPS.

Investigating the potential of conserved inner core oligosaccharide regions of Moraxella catarrhalis lipopolysaccharide as vaccine antigens: accessibility and functional activity of monoclonal antibodies and glycoconjugate derived sera.

We investigated the conservation and antibody accessibility of inner core epitopes of Moraxella catarrhalis lipopolysaccharide (LPS) in order to assess their potential as vaccine candidates. Two LPS mutants, a single mutant designated lgt2 and a double mutant termed lgt2/lgt4, elaborating truncated inner core structures were generated in order to preclude expression of host-like outer core structures and to create an inner core structure that was shared by all three serotypes A, B and C of M. catarrhalis. Murine monoclonal antibodies (mAbs), designated MC2-1 and MC2-10 were obtained by immunising mice with the lgt2 mutant of M. catarrhalis serotype A strain. We showed that mAb MC2-1 can bind to the core LPS of wild-type (wt) serotype A, B and C organisms and concluded that mAb MC2-1 defines an immunogenic inner core epitope of M. catarrhalis LPS. We were unsuccessful in obtaining mAbs to the lgt2/lgt4 mutant. MAb MC2-10 only recognised the lgt2 mutant and the wt serotype A strain, and exhibited a strong requirement for the terminal N-acetyl-glucosamine residue of the lgt2 mutant core oligosaccharide, suggesting that this residue was immunodominant. Subsequently, we showed that both mAbs MC2-1 and MC2-10 could facilitate bactericidal killing of the lgt2 mutant, however neither mAb could facilitate bactericidal killing of the wt serotype A strain. We then confirmed and extended the candidacy of the inner core LPS by demonstrating that it is possible to elicit functional antibodies against M. catarrhalis wt strains following immunisation of rabbits with glycoconjugates elaborating the conserved inner core LPS antigen. The present study describes three conjugation strategies that either uses amidases produced by Dictyostelium discoideum, targeting the amino functionality created by the amidase activity as the attachment point on the LPS molecule, or a strong base treatment to remove all fatty acids from the LPS, thus creating amino functionalities in the lipid A region to conjugate via maleimide-thiol linker strategies targeting the carboxyl residues of the carrier protein and the free amino functionalities of the derived lipid A region of the carbohydrate resulted in a high loading of carbohydrates per carrier protein from these carbohydrate preparations. Immunisation derived antisera from rabbits recognised fully extended M. catarrhalis LPS and whole cells. Moreover, bactericidal activity was demonstrated to both the immunising carbohydrate antigen and importantly to wt cells, thus further supporting the consideration of inner core LPS as a potential vaccine antigen to combat disease caused by M. catarrhalis.

Investigating the candidacy of LPS-based glycoconjugates to prevent invasive meningococcal disease: chemical strategies to prepare glycoconjugates with good carbohydrate loading

In previous studies protective antibodies that could facilitate bactericidal killing of Neisseria meningitidis (Nm) serogroup B strains were derived from immunisation with glycoconjugates prepared from O-deacylated lipopolysaccharide (LPS-OH) via direct reductive amination between the reducing end of the oligosaccharide molecule, created by treatment with alkaline phosphatase, and amino functionalities on the CRM197 carrier protein. These glycoconjugates proved difficult to prepare because the presence of amide linked fatty-acyl groups results in glycolipids that are relatively insoluble and aggregate. Therefore, we have examined several strategies to prepare glycoconjugates in order to identify a robust, consistently reproducible strategy that produces glycoconjugates with a high loading of LPS derived oligosaccharides. Initially we used completely deacylated LPS molecules, but lacking phosphoethanolamine (PEtn) from the core OS as the strong basic conditions required to completely deacylate the LPS would modify the PEtn residue. We utilised a squarate linker and conjugated via the reducing end of the carbohydrate antigen following removal of the glycosidic phosphate to amino groups on CRM197, however carbohydrate loading on the carrier protein was low. Glycoconjugates were then produced utilising amidases produced by Dictyostelium discoideum (Dd), which partially remove N-linked fatty acids from the lipid A region of the Nm LPS molecule, which enabled the retention of the PEtn residue. LPS-OH was treated with Dd amidase, the reducing glycosidic phosphate removed, and using a cystamine linker strategy, conjugated to the carrier protein. Carbohydrate loading was somewhat improved but still not high. Finally, we have developed a novel conjugation strategy that targets the amino functionality created by the amidase activity as the attachment point. The amino functionality on the PEtn residue of the inner core was protected via a novel blocking and unblocking strategy with t-butyl oxycarbonyl. A maleimide-thiol linker strategy, targeting lysine residues on the carrier protein did not result in high loading of the carbohydrate molecules, however when we targeted the carboxyl residues we have consistently obtained a high loading of carbohydrate antigens per CRM197, which can be controlled by variation in the amount of activated carbohydrate utilised in the conjugation reaction.

Investigating the candidacy of LPS-based glycoconjugates to prevent invasive meningococcal disease: immunology of glycoconjugates with high carbohydrate loading

We investigated the immune responses of rabbits that were immunised with lipopolysaccharide (LPS)-based glycoconjugates by measuring the reactivity of the derived sera to a panel of selected wild-type and mutant strains of Neisseria meningitidis. In all cases, high titers of antibodies capable of recognising LPS elaborating the identical structure as presented on the immunising glycoconjugate were obtained, and in most cases the derived sera also recognised heterologous strains including wild-type, but at lower titers. However, although serum bactericidal antibodies were consistently obtained against strains elaborating the same LPS structure as the immunising antigen, this functional response was not observed against wild-type strains. We identified several potentially competing neo-epitopes that had been introduced via our conjugation strategies, which might compete with the conserved inner core oligosaccharide target region, thus reducing the antibody titers to epitopes which could facilitate bactericidal killing. This study has therefore identified key factors that are crucial to control in order to increase the likelihood of obtaining bactericidal antibodies to wild-type meningococcal cells with LPS-derived glycoconjugates. Glycoconjugates utilised in this study, have been found to contain epitopes that do not contribute to the derivation of antibodies that may facilitate bactericidal killing of wild-type strains and must be avoided in future LPS-based glycoconjugate preparations.

Investigating the candidacy of lipopolysaccharide-based glycoconjugates as vaccines to combat Mannheimia haemolytica

Inner core lipopolysaccharide (LPS) has been shown to be conserved in the majority of veterinary strains from the species Mannheimia haemolytica, Actinobacillus pleuropneumoniae and Pasteurella multocida and as such is being considered as a possible vaccine antigen. The proof-in-principle that a LPS-based antigen could be considered as a vaccine candidate has been demonstrated from studies with monoclonal antibodies raised to the inner core LPS of Mannheimia haemolytica, which were shown to be both bactericidal and protective in a mouse model of disease. In this study we confirm and extend the candidacy of the inner core LPS by demonstrating that it is possible to elicit functional antibodies against Mannheimia haemolytica wild-type strains following immunisation of rabbits with glycoconjugates elaborating the conserved inner core LPS antigen. The present study describes a conjugation strategy that uses amidases produced by Dictyostelium discoideum, targeting the amino functionality created by the amidase activity as the attachment point on the LPS molecule. To protect the amino functionality on the phosphoethanolamine (PEtn) residue of the inner core, we developed a novel blocking and unblocking strategy with t-butyl oxycarbonyl. A maleimide-thiol linker strategy with the thiol linker on the carboxyl residues of the carrier protein and the maleimide linker on the carbohydrate resulted in a high loading of carbohydrates per carrier protein. Immunisation derived antisera from rabbits recognised fully extended Mannheimia haemolytica LPS and whole cells from serotypes 1 and 2, despite a somewhat immunodominant response to the linkers also being observed. Moreover, bactericidal activity was demonstrated to a strain elaborating the immunising carbohydrate antigen and crucially to wild-type cells of serotypes 1 and 2, thus further supporting the consideration of inner core LPS as a potential vaccine antigen to combat disease caused by Mannheimia haemolytica.

Effect of daptomycin on the barotropic behavior of dioleoylphosphatidylglycerol: an infrared spectroscopic investigation

Abstract According to our infrared spectra, the pressure-induced liquid-crystal to gel transition of dioleoylphosphatidylglycerol (DOPG) involves a conformational change making the glycerol Csn−1 − Csn−2 bond more parallel to the bilayer surface and resulting in increased carbonyl groups hydrogen bonding. This conformational change allows a more compact packing of the cis unsaturated acyl chains. The lipopeptide daptomycin, at lipid/daptomycin molar ratios of 4:1, impedes the DOPG conformational change and induces an increase of transition pressure from 5.5 (pure lipid) to 6.8 kbar and a higher disorder in the gel state. The analogue (LY298862) of daptomycin containing a myristoyl instead of a decanoyl chain shifts the phase transition to 7.8 kbar at the same molar ratio and reduces more efficiently the lipid conformational change. The lipopeptide hydrocarbon chain length is thus a critical parameter in the modulation of the physical effects of this type of compounds, often used as antibiotics.