Monophosphoryl Lipid A-Rhamnose Conjugates as a New Class of Vaccine Adjuvants.

Adjuvant is an integral part of all vaccine formulations but only a few adjuvants with limited efficacies or application scopes are available. Thus, developing more robust and diverse adjuvants is necessary. To this end, a new class of adjuvants having α- and ß-rhamnose (Rha) attached to the 1- and 6'-positions of monophosphoryl lipid A (MPLA) was designed, synthesized, and immunologically evaluated in mice. The results indicated a synergistic effect of MPLA and Rha, two immunostimulators that function via interacting with toll-like receptor 4 and recruiting endogenous anti-Rha antibodies, respectively. All the tested MPLA-Rha conjugates exhibited potent adjuvant activities to promote antibody production against both protein and carbohydrate antigens. Overall, MPLA-α-Rha exhibited better activities than MPLA-ß-Rha, and 6'-linked conjugates were slightly better than 1-linked ones. Particularly, MPLA-1-α-Rha and MPLA-6'-α-Rha were the most effective adjuvants in promoting IgG antibody responses against protein antigen keyhole limpet hemocyanin and carbohydrate antigen sTn, respectively.

Rhamnose modified bovine serum albumin as a carrier protein promotes the immune response against sTn antigen.

Rhamnose and sTn antigen were co-conjugated to bovine serum albumin (BSA), a weakly immunogenic carrier protein, for cancer vaccine development. The immune responses against sTn have been significantly augmented with the involvement of Rha-specific antibodies to enhance antigen uptake.

Synthesis and Immunological Evaluation of a Single Molecular Construct MUC1 Vaccine Containing l-Rhamnose Repeating Units.

A rhamnose targeting strategy for generating effective anticancer vaccines was successful in our previous studies. We showed that by utilizing natural anti-rhamnose antibodies, a rhamnose-containing vaccine can be targeted to antigen-presenting cells, such as dendritic cells. In this case, rhamnose (Rha) was linked directly to the liposomes bearing the antigen. However, in the current approach, we conjugated a multivalent Tri-Rha ligand with the antigen itself, making it a single component vaccine construct, unlike the previous two-component vaccine construct where Rha cholesterol and Mucin1 (MUC1) antigen were both linked separately to the liposomes. Synthesis required the development of a linker for coupling of the Rha-Ser residues. We compared those two systems in a mouse model and found increased production of anti-MUC1 antibodies and more primed antigen-specific CD4+ T cells in both of the targeted approaches when compared to the control group, suggesting that this one-component vaccine construct could be a potential design used in our MUC1 targeting mechanisms.

Augmenting Vaccine Immunogenicity through the Use of Natural Human Anti-rhamnose Antibodies.

Utilizing natural antibodies to augment vaccine immunogenicity is a promising approach toward cancer immunotherapy. Anti-rhamnose (anti-Rha) antibodies are some of the most common natural anti-carbohydrate antibodies present in human serum. Therefore, rhamnose can be utilized as a targeting moiety for a rhamnose-containing vaccine to prepare an effective vaccine formulation. It was shown previously that anti-Rha antibody generated in mice binds effectively with Rha-conjugated vaccine and is picked up by antigen presenting cells (APCs) through stimulatory Fc receptors. This leads to the effective uptake and processing of antigen and eventually presentation by major histocompatibility complex (MHC) molecules. In this article, we show that natural human anti-Rha antibodies can also be used in a similar mechanism and immunogenicity can be enhanced by targeting Rha-conjugated antigens. In doing so, we have purified human anti-Rha antibodies from human serum using a rhamnose affinity column. In vitro, human anti-Rha antibodies are shown to enhance the uptake of a model antigen, Rha-ovalbumin (Rha-Ova), by APCs. In vivo, they improved the priming of CD4+ T cells to Rha-Ova in comparison to non-anti-Rha human antibodies. Additionally, increased priming of both CD4+ and CD8+ T cells toward the cancer antigen MUC1-Tn was observed in mice that received human anti-Rha antibodies prior to vaccination with a rhamnose-modified MUC1-Tn cancer vaccine. The vaccine conjugate contained Pam3CysSK4, a Toll-like receptor (TLR) agonist linked via copper-free cycloaddition chemistry to a 20-amino-acid glycopeptide derived from the tumor marker MUC-1 containing the tumor-associated carbohydrate antigen α- N-acetyl galactosamine (GalNAc). The primed CD8+ T cells released IFN-γ and killed tumor cells. Therefore, we have confirmed that human anti-Rha antibodies can be effectively utilized as a targeting moiety for making an effective vaccine.

Biomarker-Based Metabolic Labeling for Redirected and Enhanced Immune Response.

Installation of an antibody-recruiting moiety on the surface of disease-relevant cells can lead to the selective destruction of targets by the immune system. Such an approach can be an alternative strategy to traditional chemotherapeutics in cancer therapy and possibly other diseases. Herein we describe the development of a new strategy to selectively label targets with an antibody-recruiting moiety through its covalent and stable installation, complementing existing methods of employing reversible binding. This is achieved through selective delivery of 1,3,4- O-acetyl- N-azidoacetylmannosamine (Ac3ManNAz) to folate receptor-overexpressing cells using an Ac3ManNAz-folate conjugate via a cleavable linker. As such, Ac3ManNAz is converted to cell surface glycan bearing an azido group, which serves as an anchor to introduce l-rhamnose (Rha), a hapten, via a click reaction with aza-dibenzocyclooctyne (DBCO)-Rha. We tested this method in several cell lines including KB, HEK-293, and MCF7 and were able to demonstrate the following: 1) Rha can be selectively installed to the folate receptor overexpressing cell surface and 2) the Rha installed on the target surface can recruit anti-rhamnose (anti-Rha) antibodies, leading to the destruction of target cells via complement-dependent cytotoxicity (CDC) and antibody-dependent cellular phagocytosis (ADCP).

Chemical remodeling cell surface glycans for immunotargeting of tumor cells.

Recruitment of human endogenous antibodies to target and eliminate tumor cells is a promising therapeutic strategy in the biomedical field. Current antibody-recruiting molecules are typically bi-functional agents that utilize cell-surface receptor binding property for targeting. This approach has intrinsic limitations due to the heterogeneity of tumor cells and the limited number of receptors on the cell surface. Here we report a targeting strategy based on remodeling of cell surface glycans through metabolic engineering and bioorthogonal chemical ligation. In vitro cultured tumor cells and in vivo xenograft tumors were actively remodeled with rhamnose carbohydrate epitopes, which were capable of recruiting endogenous anti-rhamnose antibodies and activating complement-mediated cell cytotoxicity. This study highlights the therapeutic potential for modulating endogenous immune response through cell-surface glycan engineering.

Targeting Tumor Cells by Natural Anti-Carbohydrate Antibodies Using Rhamnose-Functionalized Liposomes.

Recruitment of antibodies in human immune systems for targeted destruction of tumor cells has emerged as an exciting area of research due to its low occurrence of side effects, high efficacy, and high specificity. The presence of large amounts of anticarbohydrate natural antibodies in human sera has prompted research efforts to utilize carbohydrate epitopes for immune recruitment. Here, we have developed a general strategy for specific targeted destruction of tumor cells based on rhamnose-functionalized liposomes. Tumor cells artificially decorated with rhamnose epitopes were subjected to complement-mediated cytotoxicity in vitro and showed delayed tumor growth in vivo. This study highlights the therapeutic potential for activation of endogenous immune response through cell-surface glycan engineering.

Synthesis of a Liposomal MUC1 Glycopeptide-Based Immunotherapeutic and Evaluation of the Effect of l-Rhamnose Targeting on Cellular Immune Responses.

Generation of a CD8(+) response to extracellular antigen requires processing of the antigen by antigen presenting cells (APC) and cross-presentation to CD8(+) T cell receptors via MHC class I molecules. Cross-presentation is facilitated by efficient antigen uptake followed by immune-complex-mediated maturation of the APCs. We hypothesize that improved antigen uptake of a glycopeptide sequence containing a CD8(+) T cell epitope could be achieved by delivering it on a liposome surface decorated with an immune complex-targeting ligand, an l-Rhamnose (Rha) epitope. We synthesized a 20-amino-acid glycopeptide TSAPDT(GalNAc)RPAPGSTAPPAHGV from the variable number tandem repeat region of the tumor marker MUC1 containing an N-terminal azido moiety and a tumor-associated α-N-acetyl galactosamine (GalNAc) at the immunogenic DTR motif. The MUC1 antigen was attached to Pam3Cys, a Toll-like receptor-2 ligand via copper(I)-catalyzed azido-alkyne cycloaddition (CuAAc) chemistry. The Rha-decorated liposomal Pam3Cys-MUC1-Tn 4 vaccine was evaluated in groups of C57BL/6 mice. Some groups were previously immunized to generate anti-Rha antibodies. Anti-Rha antibody expressing mice that received the Rha liposomal vaccine showed higher cellular immunogenicity compared to the control group while maintaining a strong humoral response.

Synthesis of α-L-rhamnosyl ceramide and evaluation of its binding with anti-rhamnose antibodies.

An α-L-rhamnosyl ceramide (1, α-L-RhaCer) has been prepared that was recognized by anti-L-rhamnose (anti-Rha) antibodies. During these studies we explored the use of an α-L-rhamnosyl thioglycoside and a trichloroacetimidate as a glycosyl donors. Subsequently, the acceptors desired for glycosylation, 3-O-benzoylazidosphingosine or 3-O-alloxycarbonylsphingosine, were prepared from D-xylose. The thioglycoside donor, 2,3,4-tri-O-acetyl-1-(4-tolyl)thio-α-L-rhamnopyranoside, and the trichloroacetimidate donor, 2,3,4-tri-O-acetyl-1-(2,2,2-trichloroethanimidate)-α-L-rhamnopyranoside, were synthesized in 50% and 78% yield overall, respectively. The synthesis of the glycosylation acceptor employed an addition-fragmentation olefination that was successfully carried out in 53% yield. With the successful synthesis of key intermediates, α-L-RhaCer (1) was prepared without any insurmountable obstacles. Anti-Rha antibodies were prepared in BALB/c mice by immunizing them with rhamnose-ovalbumin (Rha-Ova) with Sigma Adjuvant System (SAS) and the anti-L-Rha antibodies were isolated from the blood sera. Liposomes and EL4 tumor cells were used as model systems to demonstrate the ability of 1 to insert into a lipid bilayer. The interaction of the liposomes or the EL4 cells with α-L-RhaCer (1) and anti-Rha antibodies were investigated by fluorescence microscopy and flow cytometry, respectively, to confirm the ability of glycolipid 1 to be displayed on the tumor cell surface as well as the ability to be recognized by anti-Rha antibodies.

Rhamnose glycoconjugates for the recruitment of endogenous anti-carbohydrate antibodies to tumor cells.

Immunotherapy is a promising strategy for targeting tumors. One emerging approach is to harness the immune effector functions of natural antibodies to destroy tumor cells. Dinitrophenyl (DNP) and the galactose-α-1,3-galactose (αGal) epitope are two haptens that bind endogenous antibodies. One potential alternative is the deoxysugar L-rhamnose. We compared these candidates by using a biosensor assay to evaluate human sera for endogenous antibody concentration, antibody isotype distribution, and longevity of antibody-hapten interactions. Antibodies recognizing α-rhamnose are of equal or greater abundance and affinity as those recognizing αGal. Moreover, both rhamnose and αGal epitopes are more effective than DNP at recruiting the IgG antibody subtype. Exposure of tumor cells to rhamnose-bearing glycolipids and human serum promotes complement-mediated cytotoxicity. These data highlight the utility of α-rhamnose-containing glycoconjugates to direct the immune system to target cells.

Boosted rat natural xenoantibodies cross-react with Enterococcus faecalis by targeting melibiose and L-rhamnose.

Natural antibodies include a subset described as xenoantibodies considered to be directed at microorganisms and also cross-react with antigens of unrelated species. In this study, we generated T-cell-independent (TI) and T-cell-dependent (TD) xenoantibodies in Lewis rats with hamster and pig blood injections. TI anti-hamster and anti-pig IgM and IgG xenoantibodies cross-reacted with Enterococcus faecalis but not with Escherichia coli isolated from the blood of Lewis rats after cecal ligation and puncture (CLP). TI anti-pig IgM xenoantibodies also showed some reactivity with two human blood isolates of E. faecalis. In contrast, TD xenoantibodies did not show any reactivity with rat or human bacteria. TI and TD anti-hamster and anti-pig IgM and IgG xenoantibodies showed cross-reactivity with lymphocytes and endothelial cells from species distinct to that used for immunization. Glycan array analysis and inhibition assays identified antibodies against melibiose and L-rhamnose as mediators of anti-hamster and anti-porcine xenoantibody cross-reactivity with E. faecalis. A rise in TI anti-hamster and anti-pig xenoantibodies was accompanied by decreased survival of Lewis rats in a low-severity sepsis model of CLP. Therefore, TI xenoantibodies in the rat include anti-carbohydrate antibodies reactive to bacteria of endogenous flora. Enhancement of these antibodies may result in more severe infectious diseases caused by these microorganisms.

Synthesis and immunological evaluation of a MUC1 glycopeptide incorporated into l-rhamnose displaying liposomes.

MUC1 variable number tandem repeats (VNTRs) conjugated to tumor-associated carbohydrate antigens (TACAs) have been shown to break self-tolerance in humanized MUC1 transgenic mice. Therefore, we hypothesize that a MUC1 VNTR TACA-conjugate can be successfully formulated into a liposome-based anticancer vaccine. The immunogenicity of the vaccine should be further augmented by incorporating surface-displayed l-rhamnose (Rha) epitopes onto the liposomes to take advantage of a natural antibody-dependent antigen uptake mechanism. To validate our hypothesis, we synthesized a 20-amino-acid MUC1 glycopeptide containing a GalNAc-O-Thr (Tn) TACA by SPPS and conjugated it to a functionalized Toll-like receptor ligand (TLRL). An l-Rha-cholesterol conjugate was prepared using tetra(ethylene glycol) (TEG) as a linker. The liposome-based anticancer vaccine was formulated by the extrusion method using TLRL-MUC1-Tn conjugate, Rha-TEG-cholesterol, and 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) in a total lipid concentration of 30 mM. The stability, homogeneity, and size characterization of the liposomes was evaluated by SEM and DLS measurements. The formulated liposomes demonstrated positive binding with both anti-Rha and mouse anti-human MUC1 antibodies. Groups of female BALB/c mice were immunized and boosted with a rhamnose-Ficoll (Rha-Ficoll) conjugate formulated with alum as adjuvant to generate the appropriate concentration of anti-Rha antibodies in the mice. Anti-Rha antibody titers were >25-fold higher in the groups of mice immunized with the Rha-Ficoll conjugate than the nonimmunized control groups. The mice were then immunized with the TLRL-MUC1-Tn liposomal vaccine formulated either with or without the surface displaying Rha epitopes. Sera collected from the groups of mice initially immunized with Rha-Ficoll and later vaccinated with the Rha-displaying TLRL-MUC1-Tn liposomes showed a >8-fold increase in both anti-MUC1-Tn and anti-Tn antibody titers in comparison to the groups of mice that did not receive Rha-Ficoll. T-cells from BALB/c mice primed with a MUC1-Tn peptide demonstrated increased proliferation to the Rha-liposomal vaccine in the presence of antibodies isolated from Rha-Ficoll immunized mice compared to nonimmune mice, supporting the proposed effect on antigen presentation. The anti-MUC1-Tn antibodies in the vaccinated mice serum recognized MUC1 on human leukemia U266 cells. Because this vaccine uses separate rhamnose and antigenic epitope components, the vaccine can easily be targeted to different antigens or epitopes by changing the peptide without having to change the other components.

L-rhamnose is often an important part of immunodominant epitope for pneumococcal serotype 23F polysaccharide antibodies in human sera immunized with PPV23.

Streptococcus pneumoniae is a major human pathogen which expresses more than 90 serologically distinct capsular polysaccharides (PS) on the surface. Since pneumococcal PSs elicit protective antibodies against pneumococcal diseases, it is important to identify the immunological epitope eliciting anti-pneumococcal PS antibodies. L-rhamnose is a part of the 23 F PS repeating unit and is known to be a critical part of immunodominant epitope which elicits antibodies against pneumococcal serotype 23 F PS. In order to determine if L-rhamnose is a part of epitope recognized by functional antibodies specific for serotype 23 F PS in human serum samples, we evaluated the opsonophagocytic killing of serotype 23 F pneumococci by serum antibodies specific for L-rhamnose. Using 10 mM L-rhamnose, opsonic capacities (opsonic indices) of serum antibodies were inhibited by 60% in 19 sera (36%) and 30-60% in 16 sera (30%) out of 53 sera from young and old adults immunized with 23-valent pneumococcal polysaccharide vaccine (PPV23). Interestingly, when IgM antibodies were depleted from immune sera in order to preferentially study IgG antibodies, the proportion of young adult sera showing more than 60% inhibition in opsonic capacity by 10 mM of L-rhamnose increased from 33% (11/31) to 68% (21/31). On the other hand, IgM depletion did not alter the proportion for old adult sera. Therefore, young and old adults may produce different antigen binding profiles of IgG antibodies against serotype 23 F PS.

Secondary cell wall polymers of Enterococcus faecalis are critical for resistance to complement activation via mannose-binding lectin.

The complement system is part of our first line of defense against invading pathogens. The strategies used by Enterococcus faecalis to evade recognition by human complement are incompletely understood. In this study, we identified an insertional mutant of the wall teichoic acid (WTA) synthesis gene tagB in E. faecalis V583 that exhibited an increased susceptibility to complement-mediated killing by neutrophils. Further analysis revealed that increased killing of the mutant was due to a higher rate of phagocytosis by neutrophils, which correlated with higher C3b deposition on the bacterial surface. Our studies indicated that complement activation via the lectin pathway was much stronger on the tagB mutant compared with wild type. In concordance, we found an increased binding of the key lectin pathway components mannose-binding lectin and mannose-binding lectin-associated serine protease-2 (MASP-2) on the mutant. To understand the mechanism of lectin pathway inhibition by E. faecalis, we purified and characterized cell wall carbohydrates of E. faecalis wild type and V583ΔtagB. NMR analysis revealed that the mutant strain lacked two WTAs with a repeating unit of →6)[α-l-Rhap-(1→3)]ß-D-GalpNAc-(1→5)-Rbo-1-P and →6) ß-D-Glcp-(1→3) [α-D-Glcp-(1→4)]-ß-D-GalpNAc-(1→5)-Rbo-1-P→, respectively (Rbo, ribitol). In addition, compositional changes in the enterococcal rhamnopolysaccharide were noticed. Our study indicates that in E. faecalis, modification of peptidoglycan by secondary cell wall polymers is critical to evade recognition by the complement system.

Immune roles of a rhamnose-binding lectin in the colonial ascidian Botryllus schlosseri.

The present paper describes the immune role played by a recently identified (Gasparini et al. 2008) member of the rhamnose-binding lectin (RBL) family from the colonial ascidian Botryllus schlosseri. B. schlosseri RBL (BsRBL) can activate phagocytes through: (i) induction of their directional movement towards the source of the molecule; (ii) modification of cytoskeleton, required for shape changes; (iii) stimulation of the respiratory burst, and consequent production of reactive oxygen species (ROS) with microbicidal activity, including superoxide anions and peroxides; and (iv) increase in the ability to phagocytose foreign particles. RBL also induces the synthesis and release, by cytotoxic morula cells (MCs), of cytokines recognised by anti-IL1α and anti-TNFα antibodies. At high concentrations, BsRBL induces degranulation of MCs and the consequent release of the cytotoxic enzyme phenoloxidase into the medium. Results are consistent with the existence of cross-talk between B. schlosseri immunocytes (phagocytes and MCs). In addition, a three-dimensional model for BsRBL is presented.

L-rhamnose antigen: a promising alternative to α-gal for cancer immunotherapies.

The targeting of autologous vaccines toward antigen presenting cells (APCs) via the in vivo complexation between anti α-Gal (anti-Gal) antibodies and α-Gal antigens presents a promising cancer immunotherapy with enhanced immunogenicity. This strategy takes advantage of the ubiquitous anti-Gal antibody in human serum. In contrast to the α-Gal epitope, the recent identification of high titers of anti-l-rhamnose (anti-Rha) antibodies in humans reveals a new approach toward immunotherapy employing l-rhamnose (Rha) monosaccharides. In order to evaluate this simple antigen in preclinical applications, we have synthesized Rha-conjugated immunogens and successfully induced high titers of anti-Rha antibodies in wildtype mice. Moreover, our studies demonstrate for the first time that wildtype mice could replace α1,3galactosyltransferase knockout (α1,3GT KO) mice in such antigen/antibody-mediated vaccine design when developing cancer immunotherapies.

Synthesis of a single-molecule L-rhamnose-containing three-component vaccine and evaluation of antigenicity in the presence of anti-L-rhamnose antibodies.

Carbohydrates are generally considered to be poorly immunogenic. Therefore, new approaches for enhancing their immunogenicity are important for the development of carbohydrates as vaccine components. We hypothesized that conjugation of an l-rhamnose (Rha) moiety to a carbohydrate antigen would enhance the antigenicity of the antigen in mice possessing anti-Rha antibodies via an antibody-dependent antigen uptake mechanism. To explore this hypothesis, we synthesized a single-molecule three-component vaccine containing the GalNAc-O-Thr (Tn) tumor-specific antigen, a 20 amino acid helper T-cell epitope (YAF) derived from an outer-membrane protein of Neisseria meningitides, and a Rha moiety. The vaccine was synthesized by automated Fmoc-based solid-phase peptide synthesis and deacetylated by brief treatment with NaOMe. Groups of female BALB/c mice were immunized and boosted with Rha-ovalbumin (Rha-OVA) formulated with either TiterMax Gold or Sigma Adjuvant System for a period of 35 days in order to determine optimal conditions for generating anti-Rha titers in mice. Anti-Rha antibody titers were >100 fold higher in groups of mice immunized with Rha-OVA than in the control groups. Mice producing anti-Rha were challenged with Rha-YAF-Tn or YAF-Tn. Sera collected from the groups initially immunized with Rha-OVA and later challenged with Rha-YAF-Tn showed a 2-fold increase in anti-Tn titer at 1/100 serum dilution relative to mice not immunized with Rha-OVA. An in vitro T-cell proliferation study using cells primed with either Rha-YAF-Tn or YAF-Tn was done to examine possible differences in antigen uptake and presentation due to anti-Rha antibody and chemical modification. Proliferation of T cells was stimulated by a 10-fold lower antigen concentration in the presence of Rha antibodies. The results strongly suggest that T cells present in the spleen were presented with higher concentrations of Rha-YAF-Tn as a result of the presence of the anti-Rha antibodies.

Manno(rhamno)biose-containing capsular polysaccharides of Klebsiella pneumoniae enhance opsono-stimulation of human polymorphonuclear leukocytes.

We tested the relationship between the capsular and the O-antigen structures and the ability of bacteria to trigger respiratory burst in human polymorphonuclear leukocytes (PMNL). Capsulated and non-capsulated variants as well as capsule-switched derivatives of Klebsiella serotypes bearing or lacking manno(rhamno)biose repeats in their capsular polysaccharides and expressing either mannose-rich or mannose-poor O antigens were tested for their ability to induce respiratory burst and survive in human PMNL. Luminol-enhanced chemiluminescence (CL) was measured to quantify respiratory burst. Intracellular survival was quantified by determining the viable counts of intracellular bacteria. K serotypes and the capsule-switched derivative lacking manno(rhamno)biose induced significantly lower CL than those expressing manno(rhamno)biose. Manno(rhamno)biose-lacking serotypes survived in the cells significantly better than serotypes expressing these repeats. C1q depletion did not affect CL induced by the manno(rhamno)biose-containing serotype, whereas factor B depletion revealed a significantly reduced CL. Likewise, EGTA in the presence of Mg(2+) significantly decreased CL, but the values were higher than those induced by the bacterium opsonized with factor B-depleted serum. In the presence of EGTA, Mg(2+)-treated factor B-depleted serum revealed a significant reduction in the CL response compared with the responses induced by opsonization with factor B-depleted serum alone. These results indicate, in addition to the alternative pathway, a manno(rhamno)biose pattern recognition of Klebsiella by PMNL probably by the complement lectin pathway.

Mycobacterium avium glycopeptidolipids require specific acetylation and methylation patterns for signaling through toll-like receptor 2.

Toll-like receptors (TLRs) recognize pathogen-associated molecules and play a vital role in promoting an immune response against invading microbes. TLR2, one of the key members of the TLR family, recognizes a wide variety of microbial products, including lipoproteins and lipopeptides, from a number of pathogens. Recent studies from our laboratory indicate that glycopeptidolipids (GPLs), a major surface component of Mycobacterium avium and other non-tuberculosis mycobacteria, are ligands for TLR2. However, the molecular requirements necessary for the GPL-TLR2 interaction were not defined in this report. In the present study we isolated different GPL species from M. avium, and using mass spectrometry and NMR analyses, characterized the molecular requirements of the GPL-TLR2 interaction. Interestingly, the extent of the respective acetylation and methylation of the 6-deoxytalose and rhamnose contained within the core GPL structure dictated whether the GPL signaled through TLR2. These experiments illustrate how subtle changes in a complex TLR2 ligand can alter its affinity for this important receptor, and suggest that M. avium could potentially modify its GPL structure to limit its interaction with TLR2.

[Chemical and serological studies of liposaccharides of bacteria of the genus Azospirillum].

The analysis of the lipopolysaccharides (LPS) of nine strains of azospirilla revealed the presence of the characteristic components of these glycopolymers: carbohydrates, hydroxylated fatty acids, and 2-keto-3-deoxyoctonic acid (KDO). SDS electrophoresis revealed the heterogeneous nature and the strains differences in the ratio of the molecular S and R forms present in the LPS. Polyclonal rabbit antibodies (Ab) were obtained against the isolated LPS(Cd), LPS(Sp59b), LPS(Sp7), LPS(S17), and LPS(KBC1) preparations. Based on the results of the serological studies of the LPS, the bacterial strains investigated in the work were divided into two main serogroups. Based on the immunoblotting data, Ab(Sp59b) and Ab(Cd) were found to be formed in response to both the S and R forms of the LPS molecules, whereas all the rest formed in response to the S forms only. It was shown that the heterogeneity of the antigenic determinants is typical of the second LPS group. It was suggested that rhamnose plays one of the key roles in the specific interactions between the azospirillum membrane LPS and Ab.