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The 2018 12 th Workshop on Recent Issues in Bioanalysis took place in Philadelphia, PA, USA on April 9-13, 2018 with an attendance of over 900 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5-day, week-long event - a full immersion week of bioanalysis, biomarkers and immunogenicity. As usual, it was specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small- and large-molecule bioanalysis involving LCMS, hybrid LBA/LCMS and LBA/cell-based assays approaches. This 2018 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2018 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 2) covers the recommendations for PK, PD and ADA assays by hybrid LBA/LCMS and regulatory agencies' input. Part 1 (LCMS for small molecules, peptides, oligonucleotides and small molecule biomarkers) and Part 3 (LBA/cell-based assays: immunogenicity, biomarkers and PK assays) are published in volume 10 of Bioanalysis , issues 22 and 24 (2018), respectively.

The 2018 12 th Workshop on Recent Issues in Bioanalysis took place in Philadelphia, PA, USA on April 9-13, 2018 with an attendance of over 900 representatives from pharmaceutical/biopharmaceutical companies, biotechnology companies, contract research organizations and regulatory agencies worldwide. WRIB was once again a 5-day full immersion in bioanalysis, biomarkers and immunogenicity. As usual, it was specifically designed to facilitate sharing, reviewing, discussing and agreeing on approaches to address the most current issues of interest including both small- and large-molecule bioanalysis involving LCMS, hybrid LBA/LCMS and LBA/cell-based assays approaches. This 2018 White Paper encompasses recommendations emerging from the extensive discussions held during the workshop and is aimed to provide the bioanalytical community with key information and practical solutions on topics and issues addressed, in an effort to enable advances in scientific excellence, improved quality and better regulatory compliance. Due to its length, the 2018 edition of this comprehensive White Paper has been divided into three parts for editorial reasons. This publication (Part 3) covers the recommendations for large molecule bioanalysis, biomarkers and immunogenicity using LBA and cell-based assays. Part 1 (LCMS for small molecules, peptides, oligonucleotides and small molecule biomarkers) and Part 2 (hybrid LBA/LCMS for biotherapeutics and regulatory agencies' inputs) are published in volume 10 of Bioanalysis , issues 22 and 23 (2018), respectively.

In this work, we report the development of a general strategy for enhancing the efficiency of target capture in immunoassays, using a bifunctional fusion protein construct which incorporates a substrate-anchoring moiety for the high-abundance immobilization of an antigen-binding domain. This approach was informed by the development of a pseudo first-order rate constant model, and tested in a paper-based assay format using a fusion construct consisting of an rcSso7d binding module and a cellulose-binding domain. These rcSso7d-CBD fusion proteins were solubly expressed and purified from bacteria in high molar yields, and enable oriented, high-density adsorption of the rcSso7d binding species to unmodified cellulose within a 30-second incubation period. These findings were validated using two distinct, antigen-specific rcSso7d variants, which were isolated from a yeast surface display library via flow cytometry. Up to 1.6 micromoles of rcSso7d-CBD was found to adsorb per gram of cellulose, yielding a volume-averaged binder concentration of up to 760μM within the resulting active material. At this molar abundance, the target antigen is captured from solution with nearly 100% efficiency, maximizing the attainable sensitivity for any given diagnostic system., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Roach (Rutilus rutilus), from a unpolluted lake, were caged in a lake receiving treated waste waters from a pulp and paper mill 16 km upstream, and simultaneously a group of fish were held in cages in the unpolluted reference lake. The level of serum immunoglobulin of the fish kept in cages in these lakes was quantified. Serum immunoglobulin levels decreased when the fish were transferred to the contaminated lake and remained significantly reduced for the 8-week period fish were monitored. In another experiment fish were immunized with bovine gamma-globulin (BGG) after 5 weeks caging in the lakes. The kinetics of specific antibody synthesis were determined by assaying the number of antibody secreting cells in the spleen using enzyme-linked immunospot assay and by measuring the concentration of specific antibody in the serum. The peak response of the effluent exposed fish against BGG was lower than that for fish kept in the unpolluted reference lake, and the antibody titers, due to immunization, increased more slowly in the exposed fish. These results demonstrate that subchronic exposure of fish in a lake receiving treated waste waters from a bleached kraft mill affects the antibody-mediated immunity resulting in, besides lower levels of Ig, weak responsiveness against antigen.

Vaccines are an effective intervention for preventing infectious diseases. Currently many vaccine strategies are designed to improve vaccine efficacy by controlling antigen release, typically involving various approaches at the injection site. Yet, strategies for intracellular slow-release of antigens in vaccines are still unexplored. Our study showed that controlling the degradation of antigens in dendritic cells and slowing their transport from early endosomes to lysosomes markedly enhances both antigen-specific T-cell immune responses and germinal center B cell responses. This leads to the establishment of sustained humoral and cellular immunity in vivo imaging and flow cytometry indicated this method not only prolongs antigen retention at the injection site but also enhances antigen concentration in lymph nodes, surpassing traditional Aluminium (Alum) adjuvants. Additionally, we demonstrated that the slow antigen degradation induces stronger follicular helper T cell responses and increases proportions of long-lived plasma cells and memory B cells. Overall, these findings propose that controlling the speed of antigens transport in dendritic cells can significantly boost vaccine efficacy, offering an innovative avenue for developing highly immunogenic next-generation vaccines., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Masson SAS.)

The incorporation of molecular adjuvants has revolutionized vaccine by boosting overall immune efficacy. While traditional efforts have been concentrated on the quality and quantity of vaccine components, the impact of adjuvant and antigen delivery kinetics on immunity remains to be fully understood. Here, we employed poly (lactic-co-glycolic acid) nanoparticle (PLGA NP) -stabilized Pickering emulsion (PPE) to refine the delivery kinetics of molecular adjuvant CpG and antigen, aiming to optimize immune responses. The hierarchical structure of PPE enabled spatially differential loading of CpG and antigen. The component inserted on the oil-water interphase exhibited a rapid release profile, while the one encapsulated in the PLGA NPs demonstrated a sustained release. This led to distinct intracellular spatial-temporal release kinetics. Compared to the PPE with sustained CpG release and burst release of antigen, we found that the PPE with rapid CpG release and sustained antigen release triggered an early and robust activation of Toll-like receptor 9 (TLR9) in direct way. This fostered a more immunogenic microenvironment, significantly outperforming the inverted delivery profile in dendritic cells (DCs) activation, resulting in higher CD40 expression, elevated proinflammatory cytokine levels, sustained antigen cross-presentation, an enhanced Th1 response, and increased CD8 + T cells. Moreover, prior exposure of CpG led to suppressed tumor growth and enhanced efficacy in Varicella-zoster virus (VZV) vaccine. Our findings underscore the importance of tuning adjuvant and antigen delivery kinetics in vaccine design, proposing a novel path for enhancing vaccination outcomes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Neutrophils are the most abundant leukocytes in circulation and are key "first responders" in the immune response to infectious and non-infectious stimuli. Unlike other immune cells, neutrophils can mount a robust response (including a change in surface markers and the production of extracellular traps and reactive oxygen species) just minutes after sensing a disturbance. It has been speculated that, in some individuals, the activation of neutrophils inadvertently leads to the generation of anti-neutrophil cytoplasmic autoantibodies (ANCA) against particular neutrophil proteins (antigens) such as myeloperoxidase (MPO) and proteinase 3 (PR3). In these individuals, continuous ANCA-antigen interactions are thought to drive persistent activation of neutrophils, chronic immune activation, and disease, most notably, small vessel vasculitis. There are significant gaps however in our understanding of the underlying mechanisms and even the pathogenicity of ANCA given that vasculitis can develop in the absence of ANCA, and that ANCA have been found in circulation in other conditions with no apparent contribution to disease. These gaps are particularly evident in the context of human studies. Herein, we review knowledge on neutrophil-derived ANCA antigens PR3 and MPO, ANCA generation, and ANCA-antigen interaction(s) that may promote immune activation and disease., (©2020 Society for Leukocyte Biology.)

The structural determinants for antigen 34 in the E group salmonella are glucosyl substituents on the galactosyl units of the O antigen which has a mannosylrhamnosylgalactose repeating sequence. The temperate bacteriophage epsilon(34) brings about the production of antigen 34. It has been shown here that glucose is transferred from uridine diphosphate glucose to the O antigen via a glucosyl-lipid intermediate in a two-step reaction. Glucose is linked through carbon 1 to the lipid by a phosphodiester bridge, the glucosyl bond having the beta-anomeric configuration. The lipid is a C(55)-polyisoprenoid alcohol, each isoprene unit having one double bond. It is the same lipid which is involved in the synthesis of the O antigen repeating sequence.

More than 300 sera from patients with a connective tissue disease were analyzed with the immunoblotting technique. The presence of autoantibodies against an 86,000-mol wt marker antigen for diffuse scleroderma (Scl-86) was found in 14 out of 33 patients with scleroderma. The presence of anti-Scl-86 antibodies seemed to correlate with the diagnosis of diffuse scleroderma since they were found in 13 out of 22 diffuse scleroderma patients and in only one out of 11 patients with limited scleroderma. All scleroderma sera (33 patients' sera and 13 reference sera) were also tested for the presence of anti-Scl-70 antibodies. It was found that all anti-Scl-70 positive sera (n = 25) contained anti-Scl-86 antibody as well, suggesting a relationship between these two antigens. However, the Scl-86 antigen was shown to be an extremely insoluble nonchromosomal protein, resistant to boiling in sodium dodecyl sulfate. This contrasts with the Scl-70 antigen, which has been described as a thermolabile, soluble antigen present in the chromatin fraction. Together, our results are consistent with the idea that Scl-70 is a degradation product of Scl-86. The Scl-86 antigen is present in freshly prepared rabbit thymus, spleen, and liver nuclei as well as in nuclei from various cultured cell lines, but is not detectable in extractable nuclear antigen from rabbit thymus. In a limited retrospective study, the anti-Scl-86 antibodies were found in two sera from patients with Raynaud's phenomenon before the development of diffuse scleroderma. Therefore, it is possible that screening of patients' serum for this antibody might predict the development of diffuse scleroderma.

The epidermolysis bullosa acquisita antigen is a major constituent of the basement membrane zone beneath stratified squamous epithelium. The antigen which is recognized in extracts of skin basement membrane by Western blot analysis with polyclonal or monoclonal antiepidermolysis bullosa acquisita antigen antibodies as 2 chains (a major chain of 290,000 and a minor chain of 145,000) has a native molecular weight over 800,000. Both epidermolysis bullosa acquisita antigen chains contain carbohydrate and the 290K chain is sensitive to collagenase.

Monoclonal antibodies were prepared to identify molecular components specific to the mitotic apparatus of sea urchin eggs. The mitotic apparatus or asters induced within unfertilized eggs by taxol treatment were isolated from Strongylocentrotus purpuratus and used for immunization of mice. After fusion with spleen cells, the supernatant of hybridomas were screened in two stages by indirect immunofluorescence staining, first on isolated sea urchin mitotic spindles in 96-well microtiter plates to identify rapidly potential positive hybridomas, and second, on whole mitotic eggs on coverslips to distinguish between spindle-specific staining and adventitious contamination. Two hybridomas, SU4 and SU5, secreted antibodies reactive to microtubule-containing structures in eggs during the course of development. They preferentially stained the centrosphere both in isolated mitotic apparatus and in whole metaphase eggs, which was further confirmed by staining the isolated centrospheres with these antibodies. SU4 recognized a major 190-kD polypeptide on immunoblots as well as a species at 180 and 20 kD, whereas hybridoma SU5 stained a species at 50 kD. Thus, these polypeptides may be components of the centrosphere.

Antigenic extracts of Streptococcus group E (SGE) were subjected to fractional ethanol precipitation, block (preparative) electrophoresis, and gel filtration for the purpose of separating the type antigen from the group antigen. Ethanol precipitation was ineffective in separating the substances. Block electrophoresis yielded serologically pure group antigen and a mixture of type and group antigen. Serologically pure type antigen was obtained by gel filtration. In some cases, pure group antigen was also recovered; in others, it was contaminated with type antigen. Gel filtration column effluents of antigenic extracts of SGE serotypes, I, II, III, IV, V and "untypable" isolates, collected from the region in which type antigen was eluted, were studied by paper chromatography and infrared spectrophotometry in an effort to develop a nonserological means of detecting type antigen. Hydrolysates of type antigens or suspect type antigens of serotypes I through V contained l-rhamnose, d-glucose, and several unidentified substances. d-Galactose also was present in hydrolysates of serotypes III and V. Untypable isolates and negative controls contained traces of d-glucose only. The data suggested that serotypes I through V contained a type antigen and that the untypable isolates were devoid of type antigen. Infrared absorbance spectra seemed to support the paper chromatography data.

Bahn, Arthur N. (Northwestern University, Chicago, Ill.), Patrick C. Y. Kung, and James A. Hayashi. Chemical composition and serological analysis of the cell wall of Peptostreptococcus. J. Bacteriol. 91:1672-1676. 1966.-Chemical and serological analyses were made of the cell wall of Peptostreptococcus to characterize taxonomically this genus of anaerobic streptococci. Cell wall hydrolysates of P. putridus strains 06 and 85, P. intermedius strains 11 and 87, and P. elsdenii strain B-159 were prepared, and the cell wall sugars were measured quantitatively by paper chromatography. Strain 85 contained only glucose, whereas strain 06 contained 93% glucose and 7% mannose. Strain 87 contained only rhamnose, and strain 11 contained approximately equal amounts of glucose and rhamnose. Strain B-159 differed from all the other strains in having a low (3.1%) content of total carbohydrate, consisting of rhamnose, galactose, and glucose. Quantitative amino acid analyses showed that the major amino compounds present in the cell wall were glutamic and aspartic acids, alanine, lysine, muramic acid, glucosamine, and galactosamine. Strains 06 and 85 possessed this complement of amino compounds, but strains 11 and 87 had relatively little aspartic acid. Strain B-159 was markedly different in having a high content of glycine and diaminopimelic acid, with only traces of lysine; it was the only strain in which teichoic acid was found. Serological analyses were made with the use of cell wall extracts as antigenic material and with homologous antisera, as well as streptococcal group antisera for groups A through S. The only strong agglutination was obtained between strain 87 antigen and group C antisera; weak agglutination was obtained with 87 against N, O, and K, and between strain 11 and groups E and F. All other antisera gave negative reactions. It is concluded that strain B-159 does not belong to the genus Peptostreptococcus, that strains 06 and 85 are members of P. putridus, and that strains 11 and 87 may be members of two different genera.

A soluble polysaccharide antigen from the cell wall of Rothia dentocariosa ATCC 17931 has been isolated, purified and characterized by serological and chemical procedures. The polysaccharide (RPS) was found to be located at the surface of cells grown under diverse environmental conditions, and could be easily detected on cells in pure culture or in clinical samples from humans and experimentally infected hamsters by fluorescent-antibody techniques. Fructose, glucose, galactose, and ribose were the major constituents of RPS. Although purified RPS was not immunogenic in rabbits, it was presumed to be a major antigen of the cell because it could specifically absorb approximately one-third of the antibody nitrogen in antisera prepared against whole cells of R. dentocariosa. Haptene inhibition studies indicated that fructose was the principal determinant of serological specificity in RPS. This polysaccharide was found to be serologically unique and did not cross-react with the polysaccharides and surface polymers of other oral actinomycetes and filamentous organisms.

A procedure has been described for the purification of the carcinoembryonic antigens (CEA) of the human digestive system. Tumor tissue extraction in 0.6 M perchloric acid followed by paper block electrophoresis and column chromatography on Sephadex G-200 resulted in highly purified CEA preparations as determined by both immunological and physicochemical criteria. The properties and composition of five different purified CEA preparations derived from digestive system cancer metastases were examined. The findings demonstrated a high degree of uniformity amongst these samples. Sedimentation coefficients ranged from 6.9 to 8S. Each sample showed the presence of 14 different amino acid residues and six different carbohydrate constituents (four of which could be quantitated with the amount of material available for analyis). Studies of a purified CEA preparation from a primary hepatoma yielded results which, in some respects, differed from those obtained with the CEA samples of metastatic tumor origin. The implications of these variations were discussed with regard to the probable presence of non-CEA components in the hepatoma preparation. Of primary importance was the observation that the few normal adult digestive system tissues tested failed to show the presence of constituents similar to the CEA. This finding would seem to indicate that, in the adult, the carcinoembryonic antigens of the human digestive system are qualitatively tumor-specific and are not dectectable in comparable normal tissues.

Bolaños, Roger (Tulane University, New Orleans, La.), and Charles W. DeWitt. Isolation and characterization of the K1 (L) antigen of Escherichia coli. J. Bacteriol. 91:987-996. 1966.-An acidic polysaccharide with the serological properties of the K1 (L) antigen has been isolated from Escherichia coli O2:K1:H4 by means of phenol-water extraction, fractionation with hexadecyltrimethylammonium bromide (Cetavlon), filtration through Sephadex G-200, and chromatography on anion-exchange cellulose columns. Nucleic acid and protein content were reduced to a nondetectable level. There is no contamination with O antigen. The active material appears in two different positions in the Cetavlon fractionation, each with a slightly different serological specificity, as followed by the inhibition of passive hemagglutination. By paper chromatography, the polysaccharide moiety of the O antigenic fraction is composed of glucose, galactose, hexosamine and rhamnose. The absence of colanic acid in either K1 fraction was not proven, although its participation in our assay system, as well as the participation of the common or cross-reacting antigen, was ruled out by serological means.

A particulate fraction from a T1 form of Salmonella typhimurium incorporated radioactivity from uridine diphosphate (UDP)-(14)C-glucose into products associated with the particulate enzyme. A major fraction of the incorporated radioactivity was found in the cell wall lipopolysaccharide fraction. Acid hydrolysis of incorporation products produced labeled galactose, ribose, and also glucose. The incorporation of glucose could be dissociated from the incorporation of galactose and ribose under certain conditions, and was assumed to represent incorporation into a polymer not related to T1 antigen. The incorporation of galactose and ribose probably represented the synthesis of T1 side chains of lipopolysaccharide, because (i) particulate fractions from non-T1 strains incorporated much less of these sugars and (ii) periodate oxidation and borohydride reduction converted a large portion of incorporated galactose residues into arabinose. The latter finding indicates that the galactose residues are galactofuranosides substituted either at C2 or C3; about 70% of the galactose residues in T1 side chains are known to be galactofuranosides substituted at C3. UDP-(14)C-galactose preparation used was not contaminated by UDP-(14)C-galactofuranose; therefore pyranose-to-furanose conversion must have taken place at some step during the reactions described above. The mechanism of conversion of galactose to ribose is not clear, but it was not found to involve a selective elimination of C1 or C6 of galactose or glucose.

The "T1 side chain" portion of cell wall lipopolysaccharide from T1 strains of Salmonella contains d-galactofuranose and d-ribofuranose residues. Isotope labeling studies, using intact cells of mutants each blocked at either of the two different steps of d-galactose metabolism (uridine diphosphate-glucose 4-epimerase and galactose-1-P uridylyl transferase) or at phosphoglucoisomerase, led to the following conclusions. (i) d-Galactofuranose residues are synthesized from d-galactopyranose or its derivatives, rather than by a direct conversion from other hexopyranoses or their derivatives. (ii) The pyranose-to-furanose conversion does not appear to take place at the level of the free d-galactose or d-galactose 1-phosphate. This result suggests that the conversion may occur at the stage of uridine diphosphate-d-galactose. (iii) In a mutant lacking phosphoglucoisomerase, d-ribofuranose residues in T1 side chains contained (14)C derived from exogenous d-fructose-U-(14)C, but little (3)H from exogenous d-glucose-1-(3)H. Thus, no evidence was found for a direct pathway of aldohexose-to-ribose conversion involving a loss of one of the carbons in the C2-C6 moiety of aldohexoses. This suggests, but does not prove, that the T1 ribofuranose residues are synthesized by conventional mechanisms involving hexose monophosphate shunt and transketolase-transaldolase reactions.

Enzyme-linked Immunosorbent Assays (ELISAs) techniques are widely known for the robust signal amplification with high sensitivity and specificity for the quantitation of antibodies and antigens in complex biological fluids. However, strict caution in the pre-analytical, analytical, and post-analytical phases is crucial to warrant an immunoassay quality. Since analytical errors are commonly seen in papers, this study aimed to discuss the main principles and guidelines to guarantee the specificity and sensitivity in the post-analytical phase of the ELISA method. Briefly, we highlighted the relevance of the analytical noise, as well as the limit of detection and quantitation, in the sensitivity of an immunoassay. Moreover, strategies involving the use of reverse pipetting and the spike-and-recovery test can decrease the inter-assay variance, while the linearity-of-dilution assay may protect against the prozone (or hook) effect. Addressed to researchers from different fields, we offer recommendations to limit variability and increase the usefulness of the ELISA assay in a practical way that will assist scientists to prevent future analytical errors., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Currently approved replication-competent and inactivated vaccines are limited by excessive reactogenicity and poor safety profiles, while subunit vaccines are often insufficiently immunogenic without co-administering exogenous adjuvants. Self-assembling peptide-, peptidomimetic-, and protein-based biomaterials offer a means to overcome these challenges through their inherent modularity, multivalency, and biocompatibility. As these scaffolds are biologically derived and present antigenic arrays reminiscent of natural viruses, they are prone to immune recognition and are uniquely capable of functioning as self-adjuvanting vaccine delivery vehicles that improve humoral and cellular responses. Beyond this intrinsic immunological advantage, the wide range of available amino acids allows for facile de novo design or straightforward modifications to existing sequences. This has permitted the development of vaccines and immunotherapies tailored to specific disease models, as well as generalizable platforms that have been successfully applied to prevent or treat numerous infectious and non-infectious diseases. In this review, we briefly introduce the immune system, discuss the structural determinants of coiled coils, β-sheets, peptide amphiphiles, and protein subunit nanoparticles, and highlight the utility of these materials using notable examples of their innate and adaptive immunomodulatory capacity. STATEMENT OF SIGNIFICANCE: Subunit vaccines have recently gained considerable attention due to their favorable safety profiles relative to traditional whole-cell vaccines; however, their reduced efficacy requires co-administration of reactogenic adjuvants to boost immune responses. This has led to collaborative efforts between engineers and immunologists to develop nanomaterial-based vaccination platforms that can elicit protection without deleterious side effects. Self-assembling peptidic biomaterials are a particularly attractive approach to this problem, as their structure and function can be controlled through primary sequence design and their capacity for multivalent presentation of antigens grants them intrinsic self-adjuvanticity. This review introduces the various architectures adopted by self-assembling peptides and discusses their application as modulators of innate and adaptive immunity., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021. Published by Elsevier Ltd.)

VHH stands for the variable regions of heavy chain only of camelid IgGs. The VHH family forms a set of interesting proteins derived from antibodies that maintain their capacity to recognize the antigen, despite their relatively small molecular weight (in the 12,000 Da range). Continuing our exploration of the possibilities of those molecules, we chose to design alternative molecules with maintained antigen recognition, but enhanced capacity, by fusing four VHH with one Fc, the fragment crystallizable region of antibodies. In doing so, we aimed at having a molecule with superior quantitative antigen recognition (×4) while maintaining its size below the 110 kDa. In the present paper, we described the building of those molecules that we coined VHH 2 -Fc-VHH 2 . The structure of VHH 2 -Fc-VHH 2 in complex with HER2 antigen was determined using electronic microscopy and modeling. The molecule is shown to bind four HER2 proteins at the end of its flexible arms. VHH 2 -Fc-VHH 2 also shows an internalization capacity via HER2 receptor superior to the reference anti-HER2 monoclonal antibody, Herceptin®, and to a simple fusion of two VHH with one Fc (VHH 2 -Fc). This new type of molecules, VHH 2 -Fc-VHH 2 , could be an interesting addition to the therapeutic arsenal with multiple applications, from diagnostic to therapy., (© 2021 The Protein Society.)

The presence of anti-citrullinated protein antibodies (ACPAs) in the serum is one of the immunological features of rheumatoid arthritis (RA). Anti-cyclic citrullinated peptide (CCP) assay has been widely used in clinic for the diagnosis of RA. However, up to 40% of RA patients are anti-CCP negative and the diagnostic sensitivity in this population needs to be improved for better clinical management. In this study, peptides with Multiple Citrulline Similar Motif (MCSM) were synthesized and a new ELISA system, which we called RA_CP, was developed to detect citrullinated antigens with MCSM present in the serum. 106 RA,48 other arthritis patients and 41 sex- and age-matched healthy controls (HCs) were included in this study. Patients with RA have a significantly higher amount of citrullinated antigens with MCSM than other arthritis patients and HCs. RA patients with positive anti-CCP are also MCSM positive, whereas 75% anti-CCP negative patients are positive for MCSM. The diagnostic sensitivity for anti-CCP and MCSM was 81.1% and 95.3%, while the specificity was 100% and 94.4%, respectively. ROC curve analyses showed that the area under the curve (AUC) values were 0.906 (95% CI: 0.860-0.951) for anti-CCP and 0.948 (95% CI: 0.912-0.985) for MCSM while the combination of MCSM and anti-CCP test has the highest AUC (0.971, 95% CI: 0.946-0.996). Our results suggest that detection of citrullinated antigens with MCSM has improved sensitivity compared with anti-CCP assay and could serve as a biomarker in diagnosis of RA patients., Competing Interests: The authors declare that there is no conflict of interest regarding the publication of this paper., (Copyright © 2021 Zhu Chen et al.)

Biomaterial delivery systems offer unique potential to improve cancer vaccines by offering targeted delivery and modularity to address disease heterogeneity. Here, we develop a simple platform using a conserved human melanoma peptide antigen (Trp2) modified with cationic arginine residues that condenses an anionic toll-like receptor agonist (TLRa), CpG, into polyplex-like nanoparticles. We reasoned that these structures could offer several useful features for immunotherapy - such as tunable loading, co-delivery of immune cues, and cargo protection - while eliminating the need for synthetic polymers or other complicating delivery systems. We demonstrate that Trp2/CpG polyplexes can readily form over a range of Trp2:CpG ratios and improve antigen uptake by primary antigen presenting cells. We show antigen loading can be tuned by interchanging Trp2 peptides with defined charges and numbers of arginine residues. Notably, these polyplexes with greater antigen loading enhance the functionality of Trp-2 specific T cells and in a mouse melanoma model, decrease tumor burden and improve survival. This work highlights opportunities to control the biophysical properties of nanostructured materials built from immune signals to enhance immunotherapy, without the added complexity or background immune effects often associated with synthetic carriers., Competing Interests: CJ is an employee of the VA Maryland Health Care System. The views reported in this paper do not reflect the views of the Department of Veterans Affairs or the United States Government. CJ has an equity position in Cellth Systems, LLC and Avidea Technologies. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Tsai, Amerman and Jewell.)

ABH antigens are histo-antigens, but were first described on the surface of human erythrocytes. They are found in those cells only in great apes and humans, while in more primitive animals they are found in tissues and body fluids. ABH antigens are mainly distributed in tissues that are in contact with the external environment and may serve as ligands for pathogens in tissues or block their connection. Description of the distribution of these molecules in non-human primate tissues is restricted to a few tissues and species. This paper describes the expression of human A, B and H type antigens in different organs from four species of New World Primates, obtained from the Centro Nacional de Primatas, as well as comparing that expression with what has been described for humans. In this study, although the tissue description of the antigens is similar to the genetic model for humans, some differences in expression between some organs from those species and those of humans were found. The differences occurred mainly in endodermal organs that have secretory functions and are probably under the control of the human-type FUT-2 enzyme. In the mesodermal-origin organs there was a reduction or absence of A and B antigen marking, particularly in the H precursor substance, indicating that those organs are under the control of the human-type FUT-1 enzyme. These findings have demonstrated that there is similar ABH antigen reactivity in tissue distribution between the species, although there are some species-specific cases., Competing Interests: The authors have declared that no competing interests exist.

Enolase, a multifunctional glycolytic enzyme, is known to act as a plasminogen receptor in many species, involved in the pivotal processes such as motility, adhesion, invasion, growth, and differentiation of the parasites. Knowledge on the function of enolase from Dermanyssus gallinae is very limited. Here we report on the molecular cloning, enzymatic activity, tissue distribution and plasminogen binding activity of enolase from D. gallinae (DgENO). The full-length of cDNA was 1305 bp, specifying a peptide of 434 amino acids. Bioinformatics analysis showed that DgENO was highly conserved compared with a range of organisms, indicating the potentially similar functions in D. gallinae. A recombinant DgENO (rDgENO) protein was produced and characterized, it catalyzed the dehydration of 2-phospho-D-glycerate to phosphoenolpyruvate, the optimal pH was 7.5. Polyclonal antibodies were generated in mice and western blotting indicated that antiserum specifically recognized the native enolase in the somatic extracts from D. gallinae. Immunohistochemical staining of mite sections revealed that the distribution of DgENO was ubiquitous with high level in salivary gland, mite digestive tissues and fat bodies in D. gallinae. Expression level of DgENO was observed mostly in engorged adult mites. Moreover, ELISA binding assay showed that rDgENO could bind plasminogen, and lysine analog ε-aminocaproic acid significantly inhibited this binding activity, indicating that D. gallinae enolase is a receptor of plasminogen. The present study provided foundation for understanding of the biological functions of DgENO and its application in development of vaccines against D. gallinae., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)