Native Antigen Research Articles

Use of recombinant calflagin protein as a potential candidate for diagnosis of Trypanosoma evansi infection

Serodiagnosis of surra, caused by Trypanosoma evansi, is still based on native antigens purified from bloodstream form of T. evansi grown in rodents. In order to investigate prospective diagnostic possibilities as an alternative for native antigens, we cloned, expressed 26 kDa calflagin protein containing 218 amino acids from T. evansi (Indian Strain) in Escherichia coli. The potential of recombinant calflagin (rCLF) protein as diagnostic antigen was evaluated in immunoblot and indirect ELISA using experimentally infected equine serum samples from 0 to 84 days post infection. The antibodies against T. evansi were detected with rCLF antigen in serum samples of experimentally infected equines as early as 10 days and 14 days post infection, using immunoblot and ELISA respectively. No cross-reactivity was observed with rCLF antigen in ELISA with different serum samples of equines positive for Equine herpesvirus 1, Burkholderia mallei, and Theileria equi infections. Several immunoreactive regions ranging from 10 to 28 kDa were detected using distinct T. evansi isolates (pony, cattle, donkey and camel origin) indicating presence of multiple calflagin family members in a single trypanosome. Indirect immunofluorescence antibody test with anti-CLF rabbit hyperimmune serum showed localisation of native immunogenic protein near attachment of flagellum. The rCLF protein was found to be a potential diagnostic candidate for distinguishing T. evansi positive and negative equine serum sample, suggesting that it could be used for serological surveys in animals for surra. In addition, it could be used with other potential diagnostic candidates to improve the diagnostic efficiency.

Trypanosoma evansi RoTat 1.2 variant surface antigen mimotopes selected by panning of the random peptide phage-display library against monoclonal antibodies.

Mimotope peptides of native antigens are valuable for diverse applications such as diagnostics, therapeutics and modern vaccine design. Here, we report for the first time the selection and identification of peptide mimotopes of Trypanosoma evansi RoTat 1.2 variant surface glycoprotein (VSG) for their potential uses in surra diagnostics and multi-epitope vaccine research. First, we produced the mouse monoclonal antibodies (mAbs), designated as 2E11 (IgG1) and 1C2 (IgG1), against the antigens in T. evansi RoTat 1.2 lysates. We then used 2E11 mAb to immunoprecipitate the target antigen. The immunoprecipitated antigen was then identified to be the VSG by mass spectrometry. Both 2E11 and 1C2 mAbs reacted with the VSG in immunoblots. The surface plasmon resonance immunosensors developed with both the mAbs detected VSG in the parasite lysates as well as in the rodent sera. Further, the mAbs were biotinylated and used in three rounds of panning to select peptide mimotopes from the random peptide phage display library (PhD-12; New England Biolabs, USA). The phage clones selected against each mAb were amplified and tested by phage capture ELISA for specificity. The peptide coding regions of the selected phages were sequenced and the protein blast search of the deduced amino acid sequences was performed by accessing the non-redundant protein database at https://blast.ncbi.nlm.nih.gov/. The conformational B epitope prediction of the selected mimotope sequences was done by using 3D Pepitope algorithms accessed at: http://pepitope.tau.ac.il/. The potential applications of the selected mimotopes in surra diagnostics and research are being explored.

An alphavirus-derived self-amplifying mRNA encoding PpSP15-LmSTI1 fusion protein for the design of a vaccine against leishmaniasis.

The main aims of the present study were to design a fusion protein of Leishmania major stress-inducible protein 1 (LmSTI1) and Phlebotomus papatasi SP15 (PpSP15), and to express it in the form of alphavirus packaged Self-amplifying mRNA (SAM). Two combinations, PpSP15-LmSTI1 and LmSTI1-PpSP15 fusion forms, were analyzed for folding and minimum free energies of the mRNA. Conformational studies on 3D modeled fusion and native forms were performed, and the Root-Mean-Square-distance (RMSD) of the Cα atomic coordinates were calculated. Antigenicity and stability were predicted using bioinformatics tools. The coding sequences of PpSP15-LmSTI1 fusion, PpSP15, and LmSTI1 were cloned into an alphavirus-based vector and used to produce the SAM constructs. All the subcloned constructs were then subjected to packaging in the form of viral replicon particles (VRPs),and were evaluated for their ability to infect BHK-21 cells and express the recombinant fusion proteins. The in-silico analysis indicated that the PpSP15-LmSTI1 combination could be a promising candidate based on lower folding ΔG of mRNA, higher protein antigenicity and lower instability indexes, and less conformational changes compared to the native proteins and the LmSTI1-PpSP15 fusion form. Packaged SAM encoding fusion and native antigens are used for infection of mammalian cells and for recombinant protein expression. This is the first study on in silico designing and successful packaging of an alphavirus-derived SAM in the form of the VRPs to target leishmaniasis.

Enhancing adoptive T cell therapy for solid tumor with cell-surface anchored immune checkpoint inhibitor nanogels

The efficacy of Adoptive Cell Therapy (ACT) for solid tumor is still mediocre. This is mainly because tumor cells can hijack ACT T cells' immune checkpoint pathways to exert immunosuppression in the tumor microenvironment. Immune Checkpoint Inhibitors such as anti-PD-1 (aPD1) can counter the immunosuppression, but the synergizing effects of aPD1 to ACT was still not satisfactory. Here we demonstrate an approach to safely anchor aPD1-formed nanogels onto T cell surface via bio-orthogonal click chemistry before adoptive transfer. The spatial-temporal co-existence of aPD1 with ACT T cells and the responsive drug release significantly improved the treatment outcome of ACT in murine solid tumor model. The average tumor weight of the group treated by cell-surface anchored aPD1 was only 18 % of the group treated by equivalent dose of free aPD1 and T cells. The technology can be broadly applicable in ACTs employing natural or Chimeric Antigen Receptor (CAR) T cells.

TCR-like antibodies targeting autoantigen-mhc complexes: a mini-review.

T cell receptors (TCRs) recognize peptide antigens bound to major histocompatibility complex (MHC) molecules (p/MHC) that are expressed on cell surfaces; while B cell-derived antibodies (Abs) recognize soluble or cell surface native antigens of various types (proteins, carbohydrates, etc.). Immune surveillance by T and B cells thus inspects almost all formats of antigens to mount adaptive immune responses against cancer cells, infectious organisms and other foreign insults, while maintaining tolerance to self-tissues. With contributions from environmental triggers, the development of autoimmune disease is thought to be due to the expression of MHC risk alleles by antigen-presenting cells (APCs) presenting self-antigen (autoantigen), breaking through self-tolerance and activating autoreactive T cells, which orchestrate downstream pathologic events. Investigating and treating autoimmune diseases have been challenging, both because of the intrinsic complexity of these diseases and the need for tools targeting T cell epitopes (autoantigen-MHC). Naturally occurring TCRs with relatively low (micromolar) affinities to p/MHC are suboptimal for autoantigen-MHC targeting, whereas the use of engineered TCRs and their derivatives (e.g., TCR multimers and TCR-engineered T cells) are limited by unpredictable cross-reactivity. As Abs generally have nanomolar affinity, recent advances in engineering TCR-like (TCRL) Abs promise advantages over their TCR counterparts for autoantigen-MHC targeting. Here, we compare the p/MHC binding by TCRs and TCRL Abs, review the strategies for generation of TCRL Abs, highlight their application for identification of autoantigen-presenting APCs, and discuss future directions and limitations of TCRL Abs as immunotherapy for autoimmune diseases.

Immunogenic properties of SARS-CoV-2 inactivated by ultraviolet light.

Vaccination against COVID-19 is the most effective method of controlling the spread of SARS-CoV-2 and reducing mortality from this disease. The development of vaccines with high protective activity against a wide range of SARS-CoV-2 antigenic variants remains relevant. In this regard, evaluation of the effectiveness of physical methods of virus inactivation, such as ultraviolet irradiation (UV) of the virus stock, remains relevant. This study demonstrates that the UV treatment of SARS-CoV-2 completely inactivates its infectivity while preserving its morphology, antigenic properties, and ability to induce the production of virus-neutralizing antibodies in mice through immunization. Thus, the UV inactivation of SARS-CoV-2 makes it possible to obtain viral material similar in its antigenic and immunogenic properties to the native antigen, which can be used both for the development of diagnostic test systems and for the development of an inactivated vaccine against COVID-19.

Cover Feature: Structurally Constrained MUC1‐Tn Mimetic Antigen as Template for Molecularly Imprinted Polymers (MIPs): A Promising Tool for Cancer Diagnostics (ChemPlusChem 9/2022)

The cover feature image shows the possibility to replace the use of monoclonal antibodies with an innovative class of catecholamine-based Molecularly Imprinted Polymers (MIPs). MIPs are synthetic receptors characterized by high sensitivity and specificity towards the imprinted target. Here, the imprinted targets are the native TnThr mucin antigen, over-expressed in several adenocarcinomas including the very aggressive Triple Negative Breast Cancer (TNBC), and the structurally locked TnThr mimetic. Cover design by Francesco Papi. More information can be found in the Research Article by C. Nativi, S. Scarano, and co-workers.

Sero-Epidemiological Study of Human Hydatidosis in Semnan and Sorkheh, Semnan Province, Iran.

To determine the seroprevalence of human cystic echinococcosis/hydatidosis which is one of the most important zoonotic diseases by ELISA using native antigen B in Semnan and Sorkheh, Semnan province, Iran, where no significant information about human infection exists. Overall, 957 human serum samples were randomly prepared from Semnan, Sorkheh, and its 13 surrounding villages in different seasons from 2017 to 2018. Antigen B was prepared from native hydatid cyst fluid of domestic sheep. All serum samples were evaluated by ELISA while the suspected cases were rechecked. The cut-off was calculated as the X ¯ ±2SD. Overall, 48(5%) out of 957 (422 males and 535 females) were positive for hydatidosis. The seropositivity based on sex showed 20(2.1%) out of 422 in males and 28(2.9%) out of 535 in females. The distribution of seropositive samples based on residence area showed 41 (4.3) out of 882 in urban and 7 (0.7) out of 75 in rural areas. The highest seroprevalence cases was among housewives (2.1%) followed by employers (1.5%). Based on education, source of drinking water, and age groups the highest seropositivity was observed in high school and less, in the plumping water consumers, and 50 to 59 yr old age group, respectively. There was a significant difference between seropositivity with occupation, literacy, and age group (P<0.05). Semnan with 4% seropositivity had the highest prevalence followed by Sorkheh, county. High prevalence of the disease in this area emphasizes the importance of increasing people's awareness about hydatidosis.

Characterization of Proteasome-Generated Spliced Peptides Detected by Mass Spectrometry.

CD8+ T cells recognize peptides displayed by HLA class I molecules and monitor intracellular peptide pools. It is known that the proteasome splices two short peptide fragments. Recent studies using mass spectrometry (MS) and bioinformatics analysis have suggested that proteasome-generated spliced peptides (PSPs) may account for a substantial proportion of HLA class I ligands. However, the authenticity of the PSPs identified using bioinformatics approaches remain ambiguous. In this study, we employed MS-based de novo sequencing to directly capture cryptic HLA ligands that were not templated in the genome. We identified two PSPs originating from the same protein in a human colorectal cancer line with microsatellite instability. Healthy donor-derived CD8+ T cells readily responded to the two PSPs, showing their natural HLA presentation and antigenicity. Experiments using minigene constructs demonstrated proteasome-dependent processing of two PSPs generated by standard and reverse cis splicing, respectively. Our results suggest a broader diversity of HLA class I Ag repertoires generated by proteasomal splicing, supporting the advantage of MS-based approaches for the comprehensive identification of PSPs.

Obtaining of native microbial antigens with anti-adhesive properties

Annotation. Traditional methods of sanitation of bacterial carriers of nosocomial infections of staphylococcal genesis using antibacterial drugs are ineffective and lead to the formation of antibiotic resistance in strains of microorganisms. Currently, when developing new classes of immunobiological drugs, it is recommended to use pathogen-associated molecular structures (PAMS) of bacteria that block surface epitopes and trigger the mechanisms of innate and adaptive immunity. The aim of the work was to obtain native surface antigens of Staphylococcus aureus using physical factors. Quantification of protein content in the obtained antigen preparations was performed using spectrophotometry according to the method of O. H. Lowry et al. (1951). The content of teichoic acids was determined according to the method of A. R. Archibald (2012). Adhesive properties were studied according to the method of V. I. Brilis et al. (1986). Each sample was tested in three to four replicates. Data were statistically processed using Statistica 8.0 (StatSoft Inc., USA) or Excel 2003, 2010 (Microsoft, USA). S. aureus surface antigens (Ag) were obtained as follows: a test culture of S. aureus ATСС 25923 was pre-irradiated with 61.0 GHz millimeter frequency range waves for eight hours, and then sonicated with a GZ-109 (60 kHz) or UZIN-2T (44 kHz) instrument and the protein and teichoic acid contents were measured. It was found that pretreatment of suspension S. aureus ATСС 25923 by 61.0 GHz millimeter frequency range waves increased the protein content in the samples by 1.5-1.9 times (p&lt;0.01). Lipoteichoic acid content exceeded ribiteichoic acid content, on average, by 16.7 times (p&lt;0.001) in samples obtained with the UZIN device and by 28.5 times (p&lt;0.001) in samples obtained with the G3-10 device. All of the antigenic (Ag) samples obtained had anti-adhesive activity against S. aureus ATСС 25923, with the Ag5 sample (obtained using the GZ-109 instrument) exhibiting a more pronounced anti-adhesive activity. The use of physical factors will contribute to the standardization of the process of obtaining surface native structures and reduce the toxicity and reactogenicity of immunobiological preparations.

Strategies to Screen Anti-AQP4 Antibodies from Yeast Surface Display Libraries.

A rapid and effective method to identify disease-specific antibodies from clinical patients is important for understanding autoimmune diseases and for the development of effective disease therapies. In neuromyelitis optica (NMO), the identification of antibodies targeting the aquaporin-4 (AQP4) membrane protein traditionally involves the labor-intensive and time-consuming process of single B-cell sorting, followed by antibody cloning, expression, purification, and analysis for anti-AQP4 activity. To accelerate patient-specific antibody discovery, we compared two unique approaches for screening anti-AQP4 antibodies from yeast antibody surface display libraries. Our first approach, cell-based biopanning, has strong advantages for its cell-based display of native membrane-bound AQP4 antigens and is inexpensive and simple to perform. Our second approach, FACS screening using solubilized AQP4 antigens, permits real-time population analysis and precision sorting for specific antibody binding parameters. We found that both cell-based biopanning and FACS screening were effective for the enrichment of AQP4-binding clones. These screening techniques will enable library-scale functional interrogation of large natively paired antibody libraries for comprehensive analysis of anti-AQP4 antibodies in clinical samples and for robust therapeutic discovery campaigns.

Mapping of SARS-CoV-2 IgM and IgG in gingival crevicular fluid: Antibody dynamics and linkage to severity of COVID-19 in hospital inpatients

Mapping of SARS-CoV-2 IgM and IgG in gingival crevicular fluid: Antibody dynamics and linkage to severity of COVID-19 in hospital inpatients

Structural Characterization and Evaluation of an Epitope at the Tip of the A-Band Rhamnan Polysaccharide of Pseudomonas aeruginosa.

Pseudomonas aeruginosa produces a variety of cell surface glycans. Previous studies identified a common polysaccharide (PS) antigen often termed A-band PS that was composed of a neutral d-rhamnan trisaccharide repeating unit as a relatively conserved cell surface carbohydrate. However, nuclear magnetic resonance (NMR) spectra and chemical analysis of A-PS preparations showed the presence of several additional components. Here, we report the characterization of the carbohydrate component responsible for these signals. The carbohydrate antigen consists of an immunogenic methylated rhamnan oligosaccharide at the nonreducing end of the A-band PS. Initial studies performed with the isolated antigen permitted the production of conjugates that were used to immunize mice and rabbits and generate monoclonal and polyclonal antibodies. The polyclonal antibodies were able to recognize the majority of P. aeruginosa strains in our collection, and three monoclonal antibodies were generated, one of which was able to recognize and facilitate opsonophagocytic killing of a majority of P. aeruginosa strains. This monoclonal antibody was able to recognize all P. aeruginosa strains in our collection that includes clinical and serotype strains. Synthetic oligosaccharides (mono- to pentasaccharides) representing the terminal 3-O-methyl d-rhamnan were prepared, and the trisaccharide was identified as the antigenic determinant required to effectively mimic the natural antigen recognized by the broadly cross-reactive monoclonal antibody. These data suggest that there is considerable promise in this antigen as a vaccine or therapeutic target.

Development and characterization of a novel mouse anti-canine oncostatin M receptor beta monoclonal antibody

Oncostatin M receptor beta (OSMRβ) mediates signaling of Oncostatin M (OSM) and interleukine-31 (IL-31), two key cytokines involved in many important biological processes including inflammation and cancer progression. More importantly, OSMRβ might be a potential biomarker and therapeutic target for some diseases, such as inflammatory bowel disease, pruritus and ovarian cancer. In this study, soluble recombinant canine OSMRβ (cOSMRβ) was experimentally expressed as a native antigen to develop an effective cOSMRβ-specific monoclonal antibody (mAb), 2O2, using hybridoma technology. It was demonstrated that 2O2 is able to detect OSMRβ expressed on cell surface using immunofluorescence assay (IFA) and flow cytometry (FACS). This mAb exhibits very high binding affinity to cOSMRβ with the KD and half-maximal effective concentration (EC50) values of 2.49 nM and 96.96 ng/ml, respectively. Meanwhile, it didn't show any cross-relativities with feline OSMRβ (fOSMRβ) and human OSMRβ (hOSMRβ). Moreover, we determined the binding epitope of 2O2, which localizes in the domain VI (DVI, amino acids 623–734) of cOSMRβ. In conclusion, this novel mAb, 2O2, can be used in immunoassays, including IFA, FACS and enzyme-linked immunosorbent assay (ELISA) to facilitate studies in dogs.

Structurally Constrained MUC1-Tn Mimetic Antigen as Template for Molecularly Imprinted Polymers (MIPs): A Promising Tool for Cancer Diagnostics.

Abnormal glycoconjugates have distinctly been recognized as potential biomarkers for cancer diagnosis. A great deal of attention has been focused on Tn antigen, an oversimplified mucin-1 O-glycan, over-expressed in different cancers. Herein, we investigate the possibility to replace the use of anti-Tn monoclonal antibodies with an innovative class of catecholamine-based Molecularly Imprinted Polymers (MIPs), emerging in recent years as promising tools for bioanalytical applications. MIPs are synthetic receptors characterized by high sensitivity and specificity towards the imprinted target. Here, original polynorepinephrine-based MIPs coupled to Surface Plasmon Resonance biosensing for Tn antigen recognition are reported. We have verified the imprinting and binding capacity of these MIPs towards very small antigenic entities, represented by the natural Tn antigen and the TnThr mimetic 1 (conjugated to BSA or linked to a MUC1 hexapeptide analogue), and compared the biosensor performances with an anti-Tn monoclonal antibody. The results clearly display the effectiveness of the pursued imprinting strategies.

Discovery of Antigen Specific CD4+ T cells in anti-3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) Immune Mediated Necrotizing Myopathy (IMNM)

Abstract Anti-HMGCR+ IMNM is characterized by IgG antibodies against HMGCR and a strong association with HLA-DRB1* 11:01. Although this implicates HMGCR-specific CD4+ T cells in disease pathogenesis, no such cells have been identified thus far. Monocyte derived dendritic cells from 6 anti-HMGCR+ IMNM patients were incubated with HMGCR protein and presented peptides were identified using a natural antigen processing assay (NAPA). Briefly, HLA-DR/peptide complexes were isolated by immunoprecipitation and bound peptides were sequenced by mass spectrometry. HMGCR peptides corresponding to putative T cell epitopes were synthesized and used to stimulate peripheral blood mononuclear cells from: 10 anti-HMGCR+ IMNM patients, 10 dermatomyositis patients (DM), 5 HLA-DRB1* 11:01+ healthy controls (HC). HMGCR-reactive CD4+ T cells were identified by flow cytometry based on CD154 upregulation. A total of 7 different naturally processed HMGCR peptides were identified. The number of distinct peptides presented per patient ranged from 1 to 5, with 5 being presented by at least 2 patients. All HMGCR peptides elicited robust CD4+ T cell responses in 9/10 anti-HMGCR+ IMNM patients, compared to 4/10 DM patients (p=0.017), 1/5 of HC (p=0.0119). The anti-HMGCR+ IMNM patients responded to more epitopes (p=0.0163) and had higher total number of positive CD4+ T cell cells (p= 0.0001). Our findings represent the first report of antigen-specific CD4+ T cells in anti-HMGCR+ IMNM. Leveraging NAPA, we defined a core set of naturally presented HMGCR peptides defining precise immunologically relevant CD4+ T cell epitopes. Definition of these epitopes is key in understanding disease pathogenesis and will aid in the development of antigen-specific therapeutic tools. Supported by grants from Rheumatology Research Foundation and the Buck Foundation.

Citrullination modulates MHC class II antigen processing and presentation by revealing cryptic epitopes

Abstract Cryptic peptides, hidden from the immune system under physiologic conditions, are revealed by changes to MHC class II processing and can facilitate loss of tolerance to self-antigens. Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by immune responses to citrullinated self-antigens, in which arginine residues are converted to citrullines; however, the initiating events in RA remain poorly understood. We investigated the hypothesis that citrullination exposes cryptic epitopes by modifying protein structure and proteolysis in a manner sufficient to activate a previously ignorant repertoire of self-reactive T cells. We first utilized in vitro proteolytic mapping to define the effect of citrullination on four well-defined RA autoantigens-. We then used a natural antigen processing assay to elucidate the molecular mechanisms of citrullination-modulated processing and evaluated autoantigen-specific T cell responses against the unique peptide repertoire. We show that citrullination alters MHC class II processing and presentation of RA autoantigens, resulting in the destruction of dominant epitopes and the generation and presentation of cryptic epitopes, composed primarily of native peptide sequences. The citrullination-dependent repertoire stimulates T cells from RA patients with anti-citrullinated protein antibodies more robustly than the native repertoire and control T cells. The generation of this unique repertoire is achieved through altered protease affinity and protein destabilization, rather than direct presentation of citrulline-containing epitopes, suggesting a novel paradigm for the role of protein citrullination in the breach of immune tolerance in RA. Supported by a grant from the Rheumatology Research Foundation and by the Luke Evnin and Deann Wright Fellowship

Serological Assays for Alveolar and Cystic Echinococcosis-A Comparative Multi-Test Study in Switzerland and Kyrgyzstan.

Both alveolar (AE) and cystic echinococcosis (CE) are lacking pathognomonic clinical signs; consequently imaging technologies and serology remain the main pillars for diagnosis. The present study included 100 confirmed treatment-naïve AE and 64 CE patients that were diagnosed in Switzerland or Kyrgyzstan. Overall, 10 native Echinococcus spp. antigens, 3 recombinant antigens, and 4 commercial assays were comparatively evaluated. All native E. multilocularis antigens were produced in duplicates with a European and a Kyrgyz isolate and showed identical test values for the diagnosis of AE and CE. Native antigens and three commercial tests showed high diagnostic sensitivities (Se: 86–96%) and specificities (Sp: 96–99%) for the diagnosis of AE and CE in Swiss patients. In Kyrgyz patients, values of sensitivities and specificities were 10–20% lower as compared to the Swiss patients’ findings. For the sero-diagnosis of AE in Kyrgyzstan, a test-combination of an E. multilocularis protoscolex antigen and the recombinant antigen Em95 appears to be the most suitable test strategy (Se: 98%, Sp: 87%). For the diagnosis of CE in both countries, test performances were hampered by major cross-reactions with AE patients and other parasitic diseases as well as by limited diagnostic sensitivities (93% in Switzerland and 76% in Kyrgyzstan, respectively).

Synthesis and Immunological Evaluation of Pentamannose-Based HIV-1 Vaccine Candidates.

Dense glycosylation and the trimeric conformation of the human immunodeficiency virus-1 (HIV-1) envelope protein limit the accessibility of some cellular glycan processing enzymes and end up with high-mannose-type N-linked glycans on the envelope spike, among which the Man5GlcNAc2 structure occupies a certain proportion. The Man5GlcNAc2 glycan composes the binding sites of some potent broadly neutralizing antibodies, and some lectins that can bind Man5GlcNAc2 show HIV-neutralizing activity. Therefore, Man5GlcNAc2 is a potential target for HIV-1 vaccine development. Herein, a highly convergent and effective strategy was developed for the synthesis of Man5 and its monofluoro-modified, trifluoro-modified, and S-linked analogues. We coupled these haptens to carrier protein CRM197 and evaluated the immunogenicity of the glycoconjugates in mice. The serological assays showed that the native Man5 conjugates failed to induce Man5-specific antibodies in vivo, while the modified analogue conjugates induced stronger antibody responses. However, these antibodies could not bind the native gp120 antigen. These results demonstrated that the immune tolerance mechanism suppressed the immune responses to Man5-related structures and the conformation of glycan epitopes on the synthesized glycoconjugates was distinct from that of native glycan epitopes on gp120.

The Tip of Brucella O-Polysaccharide Is a Potent Epitope in Response to Brucellosis Infection and Enables Short Synthetic Antigens to Be Superior Diagnostic Reagents.

Brucellosis is a global disease and the world’s most prevalent zoonosis. All cases in livestock and most cases in humans are caused by members of the genus Brucella that possess a surface O-polysaccharide (OPS) comprised of a rare monosaccharide 4-deoxy-4-formamido-D-mannopyranose assembled with α1,2 and α1,3 linkages. The OPS of the bacterium is the basis for serodiagnostic tests for brucellosis. Bacteria that also contain the same rare monosaccharide can induce antibodies that cross-react in serological tests. In previous work we established that synthetic oligosaccharides, representing elements of the Brucella A and M polysaccharide structures, were excellent antigens to explore the antibody response in the context of infection, immunisation and cross reaction. These studies suggested the existence of antibodies that are specific to the tip of the Brucella OPS. Sera from naturally and experimentally Brucella abortus-infected cattle as well as from cattle experimentally infected with the cross-reactive bacterium Yersinia enterocolitica O:9 and field sera that cross react in conventional serological assays were studied here with an expanded panel of synthetic antigens. The addition of chemical features to synthetic antigens that block antibody binding to the tip of the OPS dramatically reduced their polyclonal antibody binding capability providing conclusive evidence that the OPS tip (non-reducing end) is a potent epitope. Selected short oligosaccharides, including those that were exclusively α1,2 linked, also demonstrated superior specificity when evaluated with cross reactive sera compared to native smooth lipopolysaccharide (sLPS) antigen and capped native OPS. This surprising discovery suggests that the OPS tip epitope, even though common to both Brucella and Y. enterocolitica O:9, has more specific diagnostic properties than the linear portion of the native antigens. This finding opens the way to the development of improved serological tests for brucellosis.