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Klebsiella pneumoniae (Kp) poses an escalating threat to public health, particularly given its association with nosocomial infections and its emergence as a leading cause of neonatal sepsis, particularly in low- and middle-income countries (LMICs). Host cell adherence and biofilm formation of Kp is mediated by type 1 and type 3 fimbriae whose major fimbrial subunits are encoded by the fimA and mrkA genes, respectively. In this study, we focus on MrkA subunit, which is a 20 KDa protein whose 3D molecular structure remains elusive. We applied solution NMR to characterize a recombinant version of MrkA in which the donor strand segment situated at the protein's N-terminus is relocated to the C-terminus, preceded by a hexaglycine linker. This construct yields a self-complemented variant of MrkA. Remarkably, the self-complemented MrkA monomer loses its capacity to interact with other monomers and to extend into fimbriae structures. Here, we report the nearly complete assignment of the 13C,15N labelled self-complemented MrkA monomer. Furthermore, an examination of its internal mobility unveiled that relaxation parameters are predominantly uniform across the polypeptide sequence, except for the glycine-rich region within loop 176–181. These data pave the way to a comprehensive structural elucidation of the MrkA monomer and to structurally map the molecular interaction regions between MrkA and antigen-induced antibodies. [ABSTRACT FROM AUTHOR]

The effect of the composition and size of polymeric particles on the immunogenicity of the fibrillogenic β2-microglobulin protein immobilized on their surface was studied. For this purpose, nanoparticles (NP) based on a copolymer of L-glutamic acid and L-phenylalanine (P(Glu-co-Phe)) and a block copolymer of poly(ethylene glycol) with poly(lactic acid) (PEG-b-PLA) as well as microparticles (MP) based on poly(lactic acid) (PLA) were selected. Materials and Methods. α-L-amino acid copolymer-based nanoparticles were prepared by gradient phase inversion, and PEG-b-PLA-based nanoparticles by nanoprecipitation. Double emulsion method was used to form polymeric microparticles based on PLA. Recombinant chimeric model protein beta-2-microglobulin-green fluorescent protein (β2M-sfGFP) was used to covalently modify all types of polymeric particles followed by immunization of four groups of laboratory animals equal in number. An enzyme immunoassay method was used to evaluate the humoral immune response. Results and discussion. In three experimental groups, mice were immunized using poly(amino acid)-based (NP-PAA) and PEG-b-PLA (NP-PLA) nanoparticles as well as PLA microparticles containing immobilized β2M-sfGFP on the surface. The control group was immunized using a physical mixture of PEG-b-PLA nanoparticles and free β2M-sfGFP. The highest level of antibodies to sfGFP in blood serum was found when mice were immunized with a mixture of protein and nanoparticles. When mice were immunized with β2M-sfGFP-modified nanoparticles, the amount of antibodies to sfGFP was statistically significantly lower (p < 0.001) compared to the control group. However, the groups immunized with nanoparticles of similar size but different composition conjugated to the model proteins did not differ significantly among themselves. It was also found that the size of the particles affects the immunogenicity of the associated protein. A similar pattern of relative antibody content in the sera of mice was maintained at all steps of immunization. [ABSTRACT FROM AUTHOR]

• A fluorescence-based DSF provides rapid assessment of adjuvant-adsorbed protein in a microplate format. • The effect of multi-factors in formulation development can be well-explored using smaller sample amount than DSC. • Good correlation of the results from well-established DSC and from high-throughput, automation amenable DSF is observed. Nano/microparticles are widely used as vaccine adjuvants to improve antigen stability and enhance immune response. Conformational stability of a given protein was normally assessed using differential scanning calorimetry (DSC) for the optimization of formulation and for ensuring antigen stability in vaccine products. Here, a higher throughput version, namely the microtiter plate-based differential scanning fluorimetry (DSF) method was developed and optimized for assessing the protein thermal stability in the particulate adjuvant-adsorbed form. Using recombinant human papillomavirus (HPV) vaccine antigens, along with several model proteins, enhanced sensitivity and correlation to the well-established differential scanning calorimetry were demonstrated. Higher throughput and much smaller sample consumption (1/10 ∼ 1/20 of the amount needed as compared to DSC) make the plate-based DSF a method of choice for formulation development, particularly during the early developmental phase of a project where the sample amount is usually quite limited. [ABSTRACT FROM AUTHOR]

In a companion paper, a two-step developability assessment is presented to rapidly evaluate low-cost formulations (multi-dose, aluminum-adjuvanted) for new subunit vaccine candidates. As a case study, a non-replicating rotavirus (NRRV) recombinant protein antigen P[4] was found to be destabilized by the vaccine preservative thimerosal, and this effect was mitigated by modification of the free cysteine (C173S). In this work, the mechanism(s) of thimerosal-P[4] protein interactions, along with subsequent effects on the P[4] protein's structural integrity, are determined. Reversible complexation of ethylmercury, a thimerosal degradation byproduct, with the single cysteine residue of P[4] protein is demonstrated by intact protein mass analysis and biophysical studies. A working mechanism involving a reversible S-Hg coordinate bond is presented based on the literature. This reaction increased the local backbone flexibility of P[4] within the helical region surrounding the cysteine residue and then caused more global destabilization, both as detected by HX-MS. These effects correlate with changes in antibody-P[4] binding parameters and alterations in P[4] conformational stability due to C173S modification. Epitope mapping by HX-MS demonstrated involvement of the same cysteine-containing helical region of P[4] in antibody-antigen binding. Future formulation challenges to develop low-cost, multi-dose formulations for new recombinant protein vaccine candidates are discussed. [ABSTRACT FROM AUTHOR]

Several vaccines targeting bacterial pathogens show reduced efficacy upon concurrent viral infection, indicating that a new vaccinology approach is required. To identify antigens for the human pathogen Streptococcus pneumoniae that are effective following influenza infection, we performed CRISPRi-seq in a murine model of superinfection and identified the conserved lafB gene as crucial for virulence. We show that LafB is a membrane-associated, intracellular protein that catalyzes the formation of galactosyl-glucosyl-diacylglycerol, a glycolipid important for cell wall homeostasis. Respiratory vaccination with recombinant LafB, in contrast to subcutaneous vaccination, was highly protective against S. pneumoniae serotypes 2, 15A, and 24F in a murine model. In contrast to standard capsule-based vaccines, protection did not require LafB-specific antibodies but was dependent on airway CD4+ T helper 17 cells. Healthy human individuals can elicit LafB-specific immune responses, indicating LafB antigenicity in humans. Collectively, these findings present a universal pneumococcal vaccine antigen that remains effective following influenza infection. [Display omitted] • CRISPRi-seq in pneumococcal superinfection identified lafB as crucial for virulence • LafB catalyzes the formation of a glycolipid important for cell wall homeostasis • Intranasal vaccination with LafB protects against pneumococcal non-vaccine serotypes • Nasal vaccine-induced protection depends on lung Th17 lymphocytes with TRM features Liu et al. identify lafB as crucial for Streptococcus pneumoniae replication in vivo using CRISPRi-seq. Intranasal vaccination with flagellin-adjuvanted LafB induces lung Th7 lymphocytes that protect against superinfections with various pneumococcal serotypes in mice. Healthy individuals can elicit LafB-specific immune responses, suggesting that LafB is a universal, capsule-independent pneumococcal antigen. [ABSTRACT FROM AUTHOR]

Pneumococcal whole cell vaccines (WCVs) could cost-effectively protect against a greater strain diversity than current capsule-based vaccines. Immunoglobulin G (IgG) responses to a WCV were characterised by applying longitudinally-sampled sera, available from 35 adult placebo-controlled phase I trial participants, to a panproteome microarray. Despite individuals maintaining distinctive antibody ‘fingerprints’, responses were consistent across vaccinated cohorts. Seventy-two functionally distinct proteins were associated with WCV-induced increases in IgG binding. These shared characteristics with naturally immunogenic proteins, being enriched for transporters and cell wall metabolism enzymes, likely unusually exposed on the unencapsulated WCV’s surface. Vaccine-induced responses were specific to variants of the diverse PclA, PspC and ZmpB proteins, whereas PspA- and ZmpA-induced antibodies recognised a broader set of alleles. Temporal variation in IgG levels suggested a mixture of anamnestic and novel responses. These reproducible increases in IgG binding to a limited, but functionally diverse, set of conserved proteins indicate WCV could provide species-wide immunity. Clinical trial registration: The trial was registered with ClinicalTrials.gov with Identifier NCT01537185; the results are available from https://clinicaltrials.gov/ct2/show/results/NCT01537185.

Oil-based emulsions are commonly used adjuvants for veterinary vaccines. After formulation, it is required to extract protein antigens from emulsified vaccines for testing their stability and antigenicity. To establish a simple method to extract the protein antigens, two emulsified vaccines, designated as Cap-206 and Cap-35, were prepared by formulating a 28-kDa capsid protein (Cap) of PCV2b with ISA 206, a water-in-oil-in-water emulsion, or ISA 35, an oil-in-water emulsion. We found that the freeze-thaw-centrifugation method with steps of freezing at −20 °C for 12 h, thawing at room temperature and centrifuging at 9000× g for 10 min could separate the aqueous phase from Cap-206, and a centrifugation method by centrifuging at 9000× g for 10 min could isolate the aqueous portion from Cap-35. The Cap proteins were recovered from the aqueous phase and could be evaluated for their stability and antigenicity by SDS-PAGE, Western blot and ELISA. The freeze-thaw-centrifugation or the centrifugation method could also be used to recover recombinant mycobacterial heat-shock protein 65, a larger protein with molecular weight of 57-kDa, from ISA 206 or ISA 35 emulsions. The methods could be used to recover protein antigens from oil-based emulsion formulated vaccines for monitoring their stability and antigenicity during vaccine manufacture and storage. [ABSTRACT FROM AUTHOR]

In the past few decades there has been a progressive understanding that epicutaneous sensitization with protein antigen is an important sensitization route in patients with atopic dermatitis. A murine protein-patch model has been established, and an abundance of data has been obtained from experiments using this model. This review discusses the characteristics of epicutaneous sensitization with protein antigen, the induced immune responses, the underlying mechanisms, and the therapeutic potential.

Most pathogens initiate their infections at the human mucosal surface. Therefore, mucosal vaccination, especially through oral or intranasal administration routes, is highly desired for infectious diseases. Meanwhile, protein-based antigens provide a safer alternative to the whole pathogen or DNA based ones in vaccine development. However, the unique biopharmaceutical hurdles that intranasally or orally delivered protein vaccines need to overcome before they reach the sites of targeting, the relatively low immunogenicity, as well as the low stability of the protein antigens, require thoughtful and fine-tuned mucosal vaccine formulations, including the selection of immunostimulants, the identification of the suitable vaccine delivery system, and the determination of the exact composition andmanufacturing conditions. This reviewaims to provide an up-to-date survey of the protein antigen-based vaccine formulation development, including the usage of immunostimulants and the optimization of vaccine delivery systems for intranasal and oral administrations. [ABSTRACT FROM AUTHOR]

Poly(lactic-co-glycolic acid) (PLGA) particles have been extensively studied as biodegradable delivery system to improve the potency and safety of protein-based vaccines. In this study we analyzed how the size of PLGA particles, and hence their ability to be engulfed by dendritic cells (DC), affects the type and magnitude of the immune response in comparison to sustained release from a local depot. PLGA microparticles (MP, volume mean diameter ≈ 112 μm) and nanoparticles (NP, Z -average diameter ≈ 350 nm) co-encapsulating ovalbumin (OVA) and poly(I:C), with comparable antigen (Ag) release characteristics, were prepared and characterized. The immunogenicity of these two distinct particulate vaccines was evaluated in vitro and in vivo . NP were efficiently taken up by DC and greatly facilitated MHC I Ag presentation in vitro , whereas DC cultured in the presence of MP failed to internalize significant amounts of Ag and hardly showed MHC I Ag presentation. Vaccination of mice with NP resulted in significantly better priming of Ag-specific CD8 + T cells compared to MP and OVA emulsified with incomplete Freund's adjuvant (IFA). Moreover, NP induced a balanced T H 1/T H 2-type antibody response, compared to vaccinations with IFA which stimulated a predominant T H 2-type response, whereas MP failed to increase antibody titers. In conclusion, we postulate that particle internalization is of crucial importance and therefore particulate vaccines should be formulated in the nano- but not micro-size range to achieve efficient uptake, significant MHC class I cross-presentation and effective T and B cell responses. [ABSTRACT FROM AUTHOR]

Linking physicochemical characterization to functional properties is crucial for defining critical quality attributes during development of subunit vaccines toward optimal safety and efficacy profiles. We investigated how the trehalose 6,6′-diester (TDX) chain length influenced the physicochemical and immunopotentiating properties of the clinically tested liposomal adjuvant composed of dimethyldioctadecylammonium (DDA) bromide and analogues of trehalose-6,6′-dibehenate (TDB). TDB analogues with symmetrically shortened acyl chains [denoted X: arachidate (A), stearate (S), palmitate (P), myristate (Myr) and laurate (L)] were incorporated into DDA liposomes and characterized with respect to size, polydispersity index, charge, thermotropic phase behavior and lipid–lipid interactions. Incorporation of 11 mol% TDX into DDA liposomes significantly decreased the polydispersity index when TDA, TDS, TDP and TDMyr were incorporated, whereas both the initial size and the charge of the liposomes were unaffected. The long-term colloidal stability was only decreased when including TDL in DDA liposomes. The fatty acid length of TDX affected the phase transition of the liposomes, and for the DDA/TDP and DDA/TDS liposomes a homogeneous distribution of the lipids in the bilayer was indicated. The membrane packing was studied further by using the Langmuir monolayer technique. Incorporation of TDS improved the packing of the lipid monolayer, as compared to the other analogues, suggesting the most favorable stability. Finally, immunization of mice with the recombinant tuberculosis fusion antigen Ag85B-ESAT-6-Rv2660c (H56) and the physicochemically most optimal formulations (DDA/TDB, DDA/TDS and DDA/TDP) induced comparable T-cell responses. In conclusion, of the investigated TDB analogues, incorporation of 11 mol% TDS or TDP into DDA liposomes resulted in an adjuvant system with the most favorable physicochemical properties and an immunological profile comparable to that of DDA/TDB. [ABSTRACT FROM AUTHOR]

Highlights: [•] Cellular immunity is a necessary component for vaccines directed to some pathogens. [•] Full-protein antigens rather than peptide epitopes ensure broad population coverage. [•] High-throughput T cell antigen identification technologies are rapidly evolving. [•] Advantages and disadvantages of T cell antigen identification methods are discussed. [ABSTRACT FROM AUTHOR]

Abstract: Oral tolerance is defined as the specific suppression of humoral and/or cellular immune responses to an antigen by administration of the same antigen through the oral route. Due to its absence of toxicity, easy administration, and antigen specificity, oral tolerance is a very attractive approach to prevent unwanted immune responses that cause a variety of diseases or that complicate treatment of a disease. Many researchers have induced oral tolerance to efficiently treat autoimmune and inflammatory diseases in different animal models. However, clinical trials yielded limited success. Thus, understanding the mechanisms of oral tolerance induction to therapeutic proteins is critical for paving the way for clinical development of oral tolerance protocols. This review will summarize progress on understanding the major underlying tolerance mechanisms and contributors, including antigen presenting cells, regulatory T cells, cytokines, and signaling pathways. Potential applications, examples for therapeutic proteins and disease targets, and recent developments in delivery methods are discussed. [Copyright &y& Elsevier]

Abstract: By screening a whole-genome λ-display library of Streptococcus pneumoniae, we have previously identified a novel surface protein, named Spr1875, that exhibited immunogenic properties and was closely related to pneumococcal virulence. In the present study, we investigated the role of the Spr1875 antigen in the interaction of S. pneumoniae with microglia, the resident brain macrophages. By using an in vitro infection model, the BV2 microglial cell line was challenged with the S. pneumoniae strain DP1004 and its isogenic spr1875-deleted mutant (Δspr1875). Both strains were phagocytosed by microglia efficiently and to a similar extent; however, the DP1004 strain was more resistant than the Δspr1875 mutant to the intracellular killing, as assessed by antibiotic protection and phagosome maturation assays. Moreover, significant differences between the two strains were also observed in terms of susceptibility to microglia-mediated killing. Taken together, these results indicate that S. pneumoniae-microglial cell interplay is influenced by the presence of Spr1875, suggesting that this protein may play a role in the pathogenesis of pneumococcal meningitis. [Copyright &y& Elsevier]

Abstract: In the past few decades there has been a progressive understanding that epicutaneous sensitization with protein antigen is an important sensitization route in patients with atopic dermatitis. A murine protein-patch model has been established, and an abundance of data has been obtained from experiments using this model. This review discusses the characteristics of epicutaneous sensitization with protein antigen, the induced immune responses, the underlying mechanisms, and the therapeutic potential. [Copyright &y& Elsevier]

Antibody molecules interact with antigen proteins through the epitope area, where the epitope residues are found to be discontinuous or spatial or conformational rather than linear on the protein surface. There are various computational algorithms to predict the spatial epitopes, and each of them have an outstanding performance based on their individual testing dataset. In this work, an independent dataset was created through collection of the epitope residual sites which have been confirmed by experiments. Based on this dataset, 6 popular web-servers developed for B-cell structural epitope prediction, including SEPPA, CEP, DiscoTope, ElliPro, PEPOP and BEpro, were evaluated and compared according to sensitivity, the positive predictive value, the successful pick-up rate and the area under the curve of the receiver operator characteristic (AUC). The results showed that the general performance of spatial epitope prediction tools did obtain substantial advancement, and SEPPA gave the best performance among the 6 tools. However, the current prediction accuracy was still far from satisfaction. Moreover, our comparison elucidated that the performance of the web-servers was significantly affected by their training datasets and the algorithms adopted. In this sense, the results of our research may improve the design of B-cell epitope prediction tools and provide additional clues when the users utilize these tools in their related research. [ABSTRACT FROM AUTHOR]

Abstract: With respect to liposomes as delivery vehicles and adjuvants for vaccine antigens, the role of vesicle surface charge remains disputed. In the present study we investigate the influence of liposome surface charge and antigen–liposome interaction on the antigen depot effect at the site of injection (SOI). The presence of liposome and antigen in tissue at the SOI as well as the draining lymphatic tissue was quantified to analyse the lymphatic draining of the vaccine components. Furthermore investigations detailing cytokine production and T-cell antigen specificity were undertaken to investigate the relationship between depot effect and the ability of the vaccine to induce an immune response. Our results suggest that cationic charge is an important factor for the retention of the liposomal component at the SOI, and a moderate to high (>50%) level of antigen adsorption to the cationic vesicle surface was required for efficient antigen retention in the same tissue. Furthermore, neutral liposomes expressing poor levels of antigen retention were limited in their ability to mediate long term (14days) antigen presentation to circulating antigen specific T-cells and to induce the Th1 and Th17 arms of the immune system, as compared to antigen adsorbing cationic liposomes. The neutral liposomes did however induce the production of IL-5 at levels comparable to those induced by cationic liposomes, indicating that neutral liposomes can induce a weak Th2 response. [Copyright &y& Elsevier]

Abstract: Liposomes have been widely exploited as antigen delivery systems for a variety of diseases including leishmaniasis. These vesicles can be prepared in various ways which may affect the immunogenicity of the encapsulated antigens. In this study we compared the vaccine potentiality of three cationic formulations with Leishmania donovani promastigote membrane antigens (LAg) and the best vesicle was evaluated for long-term protection against experimental visceral leishmaniasis. We immunized mice with LAg encapsulated in multilamellar vesicles (MLV), dehydration–rehydration vesicles (DRV) and reverse-phase evaporation vesicles (REV) and challenged them with parasites ten days after vaccination. LAg in MLV or DRV induced almost complete protection, while LAg alone or entrapped in REV exhibited partial resistance. Protection observed with antigen incorporated MLV or DRV was predominantly Th1 as evidenced by elicitation of significantly high DTH, IgG2a antibodies and IFN-γ. MLV encapsulated LAg demonstrated durable cell-mediated immunity and mice challenged ten weeks after vaccination could also resist experimental challenge strongly. Field trials of L. donovani vaccine were unsatisfactory mainly due to lack of an appropriate adjuvant. Cationic MLV when used as adjuvant with protein antigens induced sustained Th1 immunity. Adjuvant potential of cationic MLV can be utilized to design subunit vaccines. [Copyright &y& Elsevier]

Pneumonia is a deadly respiratory disease inflicting millions of people in third world countries. Current vaccination strategies which include polysaccharide-based vaccines are not effective against all strains of pneumonia. Furthermore, these vaccines are given through parenteral route instead of the more preferred mucosal route which has the obvious advantage of eliciting both systemic as well as mucosal immune responses. This review intends to understand the etiology, treatment options, and potential delivery systems for the vaccination against pneumonia via mucosal route. Various vaccination options including current polysaccharide-based vaccinations along with potential protein-based vaccines have been discussed. [ABSTRACT FROM AUTHOR]

Abstract: Background: Th17 is a newly identified effector T cell lineage which plays a central role in many human inflammatory diseases and experimental animal models. Epicutaneous sensitization with a protein antigen has been proven to induce a Th2-predominant immune response and lead to development of atopic diseases in a murine protein-patch model. Objective: We sought to assess the generation of Th17 cells in epicutaneous sensitization with a protein antigen and its regulation by environmental elements and genetic background. Methods: BALB/c, C57BL/6, and DO11.10 mice were epicutaneously immunized by patch application of the following: ovalbumin alone, or co-administration of one of TLR ligands, irritant, hapten or superantigens. IL-17 and IL-22 contents in supernatants of in vitro reactivation culture of lymph nodes cells were determined by ELISA. Frequency of IL-17-secreting CD4 T cells was measured by ELISPOT. Results: Small but significant amounts of IL-17 and IL-22 could be detected in supernatants of in vitro reactivation culture of lymph nodes cells of mice receiving patch application of ovalbumin. ELISPOT assay for IL-17 also revealed low frequency of IL-17-secreting CD4 T cells in lymph nodes cells in ovalbumin group. All TLR ligands tested including agonists for TLR2, TLR3, TLR4, TLR5, TLR7 and TLR9 as well as many environmental elements including irritant, hapten and superantigen could further promote the generation of Th17 cells. In addition, C57BL/6 mice generate less Th17 cells than BALB/c mice in epicutaneous sensitization. Conclusion: This study demonstrates Th17 generation and its regulation by environmental elements and genetic background to a protein antigen by epicutaneous route. [Copyright &y& Elsevier]

Abstract: Pseudomonas aeruginosa is a major cause of nosocomal and community acquired chronic infections in subjects with compromised respiratory function. The microbe is environmentally ubiquitious and has a high level of innate antimicrobial resistance. This has led researchers to investigate vaccine and immunotherapeutic approaches to prevent and treat P. aeruginosa infections. Seven cytosolic non-integral proteins were studied as vaccine candidates in an acute lung infection model in the rat. Five of these (amidase, amidopeptidase, KatE, KatE and Pa13 a novel 13kDa protein) enhanced bacterial clearance from the lung compared to control animals following challenge and are worthy of further study. Immune mechanisms stimulated by these proteins in response to both immunization and infection varied. The most pronounced degree of bacterial clearance from the lung was associated with antigens, which demonstrated greater surface exposure and induced an increase in phagocyte recruitment, in particular, an increased proportion of polymorphonuclear leukocytes. Lymphocytic proliferation and specific antibody responses in the absence of enhanced clearance were less informative as immune correlates. [Copyright &y& Elsevier]

Abstract: Recently, we have developed a simple and potent therapeutic cancer vaccine consisting of a cationic lipid and a peptide antigen. In this report, we expanded the utility of this formulation to protein based vaccines. First, we formulated the human papillomavirus (HPV) 16 E7 protein (E7) in different doses of DOTAP liposome. The results showed that these formulations failed to regress an established tumor. However, when sodium chloride (30mM) was added to the DOTAP (100nmol)/E7 (20μg) formulation, anti-tumor activity was generated in the immunized mice. Correlatively, 30mM NaCl in the DOTAP/E7 protein formulation increased the particle size from ∼350 to 550nm, decreased the protein loading capacity (from 95 to 90%), and finally increased the zeta potential (from 29 to 38mV). Next, a model protein antigen ovalbumin (OVA) was formulated in different doses of DOTAP liposomes. Similarly, the results showed that 20μg OVA formulated in 200nmol DOTAP with 30mM NaCl had the best OVA-specific antibody response, including both IgG1 and IgG2a, suggesting both Th1 and Th2 immune responses were generated by this formulation. In conclusion, we have expanded the application of cationic DOTAP liposome formulation to protein based vaccines and also identified that small amounts of salt could change the physicochemical properties of the vaccine formulation and enhance the activity of the DOTAP/protein based vaccine. The enhancement of immune responses by salt is possibly due to its interference of the electrostatic interaction between the cationic lipid and the protein antigen to facilitate the antigen release from the carrier and at the same time activate the antigen presenting cells. [Copyright &y& Elsevier]

Streptococcus mutans, a major pathogen of dental caries and infective endocarditis, is classified into serotypes c, e, f, and k, with serotype k strains recently reported to be frequently detected in persons with infective endocarditis. Thus, we hypothesized that common properties associated with infective endocarditis are present in those strains. Fifty-six oral S. mutans strains, including 11 serotype k strains, were analyzed. Western blotting analysis revealed expression of the 3 types of glucosyltransferases in all strains, while expression of the approximately 190-kDa cell-surface protein (PA) was absent in 12 strains, among which the prevalence of serotype k (7/12) was significantly high. Furthermore, cellular hydrophobicity and phagocytosis susceptibility were lower in the group of serotype k strains. These results indicate that the absence of PA expression, low cellular hydrophobicity, and phagocytosis susceptibility are common bacterial properties associated with serotype k strains, which may be associated with virulence for infective endocarditis. [ABSTRACT FROM AUTHOR]

Background/aims: The cell surface protein antigen (PAg) and glucosyltransferases (GTFs) produced by Streptococcus sobrinus are considered to be major colonization factors of the organism. Methods: We constructed a fusion gene encoding a protein composed of the alanine-rich region of PAg (PAgA) and the glucan-binding domain (GB) of GTF-I, which catalyzes the synthesis of water-insoluble glucan in S. sobrinus. The fusion protein PAgA-GB was purified from cell extracts of Escherichia coli harboring the fusion gene, and antibodies against the fusion protein were prepared in rabbits. Results: In the presence of sucrose, the antibody against PAgA-GB significantly inhibited the adhesion of both S. sobrinus MT8145 and Streptococcus mutans Xc to saliva-coated hydroxyapatite beads, and the inhibitory effect on S. sobrinus was stronger than that on S. mutans. In the absence of sucrose, the antibody against PAgA-GB significantly inhibited the adhesion of both S. sobrinus and S. mutans, however the inhibitory effect on S. sobrinus was unexpectedly weaker than that on S. mutans. A similar result was observed with the antibody against the intact recombinant PAg protein (rPAg), while the same antibody reacted more strongly against S. sobrinus than against S. mutans cells. Conclusion: Taken together, these results show that the antibody against S. sobrinus GTF-I may be useful for effective inhibition of the sucrose-dependent adhesion of S. sobrinus. However, PAg of S. sobrinus may not function primarily as a receptor for acquired pellicles, and other cell surface proteins may be involved in the sucrose-independent adhesion of S. sobrinus. [ABSTRACT FROM AUTHOR]

Molecular pathogenesis of Tannerella forsythia, a putative periodontal pathogen, has not yet been adequately elucidated due to limited information on its virulence factors. Here, identification of in vivo expressed antigens of T. forsythia is reported using in vivo-induced antigen technology (IVIAT). Among 13 000 recombinant clones screened, 16 positive clones were identified that reacted reproducibly with sera obtained from patients with periodontal disease. DNA sequences from 12 of these in vivo-induced genes were determined. IVIAT-identified protein antigens of T. forsythia include: BspA, a well-defined virulence factor of T. forsythia; enzymes involved in housekeeping functions (tRNA synthetases, glycine hydroxymethyltransferase, and glucoside glucohydrolase); enzymes implicated in tissue destruction (dipeptidyl peptidase IV); a DNA mismatch repair protein; and putative outer membrane proteins of unknown function. The in vivo gene expression of these IVIAT-identified antigens was confirmed by a quantitative real-time PCR analysis. This is, to the best of the authors' knowledge, the first report using IVIAT in T. forsythia. It is anticipated that detailed analysis of the in vivo-induced genes identified by IVIAT in this study will lead to a better understanding of the molecular mechanisms mediating periodontal infection by T. forsythia. [ABSTRACT FROM AUTHOR]

It is well known that Leptospira vaccine prevents the disease. However specificity for serovars limits the efficacy of killed whole cell vaccines. Leptospiral antigens that induce cross-protective immunity to the various serovars are sought as new vaccine candidates. In this paper, we have summarized both past and current findings about leptospiral antigens that are conserved among pathogenic leptospires and that induce protective immunity in animal models. The full-length genome sequences of two Leptospira strains have been published and reverse vaccinology has been used to identify leptospiral vaccine candidates. Although humoral immunity is thought to be dominant in protection from leptospiral infection, a role for cell-mediated immunity is now being explored. [ABSTRACT FROM AUTHOR]

The vaccine directed against human chorionic gonadotropin (hCG) has previously undergone clinical test demonstrating the feasibility of the approach in preventing pregnancy in women. Some individuals, however, did not response adequately despite employing highly immunogenic bacterial toxoids as carriers. In this study, we investigated the potential of three copies of C3d as a new molecular adjuvant to enhance the immunogenicity of hCGβ protein antigen. The antibody response to the hCGβ-C3d3 fusion protein immunization was compared with those resulting from immunization with the hCGβ alone and the hCGβ plus CFA/IFA either in BALB/c mice or in C57BL/6J mice. Our results showed that the fusion of C3d3 to hCGβ protein antigen resulted in a significant elevation of the serum anti-hCGβ antibody level in the two mouse strains and the antibodies were capable of effectively neutralizing the bioactivity of hCG. The immunization with C3d3 as a molecular adjuvant favored Th2 bias of immune response. The immunity-enhancing effect of the C3d3 was 10-fold (initial) and 20–32-fold (booster) greater than CFA/IFA. These findings indicated that fusion of C3d3 to hCGβ, as a means of harnessing the adjuvant potential of the innate immune system, may improve immunogenicity of the hCGβ contraceptive vaccine, which is useful to produce a cost-effective vaccine and for the less-responsive population. [Copyright &y& Elsevier]

Drainage and retention of brain-derived antigens are important factors in initiating and regulating immune responses in the central nervous system (CNS). We investigated distribution, immunological processing and retention of intracerebrally infused protein antigen, ovalbumin (OVA), and the subsequent recruitment of CD8+ T cells into the CNS. We found that protein antigens infused into the CNS can drain rapidly into the cervical lymph node and initiate antigen-specific immune response in the periphery. A portion of the antigens are also retained by CD11b/MAC-1+ cells in the brain parenchyma where they are recognized by antigen-specific CD8+ T cells. [Copyright &y& Elsevier]

BackgroundIn recent years considerable interest in the pathogenetic role of aeroallergens exacerbating atopic dermatitis (AD) has emerged. The ‘atopy patch test’ with aeroallergens was introduced by Platts-Mills et al. as an experimental model and as a diagnostic tool. However, its relevance for the clinical manifestation of AD is still not clear. ObjectiveWe asked whether there is a relationship between the individual antigen exposure to the major allergen of Dermatophagoides pteronyssinus (Der p 1) and the immunological markers of sensitization to Der p 1 or the clinical severity of AD. MethodsWe investigated 92 patients with moderate to severe AD. For clinical evaluation the SCORAD severity score was used. Patch tests were performed with purified Der p 1. Specific IgE was measured by a commercial assay. Der p 1 exposure was quantified in a sample of the patient's mattress dust by using a commercial ELISA. ResultsNo correlation between SCORAD, Der p 1 exposure and RAST could be established. However, there was an unexpected significant inverse correlation between the quantity of mite antigen in the mattress dust and patch test reactivity. Patients with a high antigen load (> 25 μg/g) mostly had a negative patch test. Also, when Der p 1 was correlated to the mattress area (m2) in this group all patch tests were negative. A possible explanation could be that continuous exposure of the skin to house dust mite allergen Der p 1 may induce a down-regulation of the skin immune system of patients with AD. ConclusionAlthough the mechanism of this phenomenon is presently unknown, our study shows that a positive allergen patch test alone should not be an indication to undertake allergen exclusion measures in AD patients. [ABSTRACT FROM AUTHOR]

There is a need for non-living adjuvant vectors that will allow a full range of local and systemic immune responses to orally administered purified antigens. Here we describe our experience with lipophilic immune-stimulating complexes (ISCOMs) containing the saponin adjuvant Quil A. When given orally, ISCOMs containing the model protein antigen ovalbumin (OVA) induce a wide range of systemic immune responses, including Th1 and Th2 CD4-dependent activity, serum IgG antibodies and class I MHC-restricted cytotoxic T cell responses. In addition, there is local production of secretory IgA antibodies in the intestine itself, as well as priming of CD4 and CD8 T cell responses in the draining lymphoid tissues. Preliminary results indicate that the mucosal adjuvant properties of ISCOMs may reflect their ability to deliver antigen combined with the pro-inflammatory properties of Quil A in a particulate form. Of the many inflammatory mediators induced, interleukin-12, derived from dendritic cells and/or macrophages, appears to be of central importance. These results indicate that ISCOMs may prove to be useful mucosal vaccine vectors with functions which are distinct from existing vectors of this type. [ABSTRACT FROM AUTHOR]

Culture filtrate proteins were obtained from Mycobacterium tuberculosis cultures after 7 days growth in Proskauer and Beck medium. The protein yield increased substantially to peak about the time the number of viable organisms reached its maximum level (day 8). Examination of the protein concentrate by SDS PAGE revealed the presence of at least 12 separate protein bands varying from 10 to 90 kD. Mice were injected subcutaneously with 20μg of M. tuberculosis culture filtrate (MTCF) protein suspended in saline or Freund's complete or incomplete adjuvant. The vaccinated mice were subjected to an acrogenic challenge with 103 colony-forming unit (CFU) M. tuberculosis Erdman and a significant reduction in the number of viable organisms was observed in the spleens and lungs determined over a 21-day period compared with age-matched normal controls. Mice immunized with the same culture filtrate proteins bound to nitrocellulose particles also showed some resistance to the virulent challenge, suggesting that individual antigens present in the culture filtrate were able lo induce a protective T cell-mediated immune response in appropriately immunized mice. [ABSTRACT FROM AUTHOR]

Current vaccination against Streptococcus pneumoniae uses vaccines based on capsular polysaccharides from selected serotypes and has led to nonvaccine serotype replacement disease. We have investigated an alternative serotype-independent approach, using multiple-antigen vaccines (MAV) prepared from S. pneumoniae TIGR4 lysates enriched for surface proteins by a chromatography step after culture under conditions that induce expression of heat shock proteins (Hsp; thought to be immune adjuvants). Proteomics and immunoblot analyses demonstrated that, compared to standard bacterial lysates, MAV was enriched with Hsps and contained several recognized protective protein antigens, including pneumococcal surface protein A (PspA) and pneumolysin (Ply). Vaccination of rodents with MAV induced robust antibody responses to multiple serotypes, including nonpneumococcal conjugate vaccine serotypes. Homologous and heterologous strains of S. pneumoniae were opsonized after incubation in sera from vaccinated rodents. In mouse models, active vaccination with MAV significantly protected against pneumonia, while passive transfer of rabbit serum from MAV-vaccinated rabbits significantly protected against sepsis caused by both homologous and heterologous S. pneumoniae strains. Direct comparison of MAV preparations made with or without the heat shock step showed no clear differences in protein antigen content and antigenicity, suggesting that the chromatography step rather than Hsp induction improved MAV antigenicity. Overall, these data suggest that the MAV approach may provide serotype-independent protection against S. pneumoniae.

Plasmodium falciparum-infected erythrocytes (IEs) bind various host receptors via members of the P. falciparum erythrocyte membrane protein 1 (PfEMP1) family expressed on the surface of the IEs. Antibody reagents are needed to investigate interactions between specific PfEMP1 proteins and receptors expressed by human endothelial cells. This protocol describes the production of rat and mouse polyclonal anti-PfEMP1 antibodies. Polyclonal antibodies are relatively easy to produce and have advantages compared to monoclonal antibodies (see Chapters 28 - 30 ) for some applications. An ELISA-based method to test the polyclonal antibodies before their use in more advanced procedures is also presented., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Vasculitis has been linked to several processes, like infections, drugs and allergic, rheumatologic and neoplastic diseases. Neoplasm-associated vasculitis described in the medical literature has mostly been reported in association with haemotological neoplasms. Adenocarcinoma of rectum presenting as leucocytoclastic vasculitis is rare. We present a case of a 43-year-old male with paraneoplastic leucocytoclastic vasculitis preceding the manifestation of adenocarcinoma rectum. The vasculitis subsided on resection of the rectal malignancy and the patient did not require steroid therapy thereafter. [ABSTRACT FROM AUTHOR]

Current vaccination against Streptococcus pneumoniae uses vaccines based on capsular polysaccharides from selected serotypes and has led to nonvaccine serotype replacement disease. We have investigated an alternative serotype-independent approach, using multiple-antigen vaccines (MAV) prepared from S. pneumoniae TIGR4 lysates enriched for surface proteins by a chromatography step after culture under conditions that induce expression of heat shock proteins (Hsp; thought to be immune adjuvants). Proteomics and immunoblot analyses demonstrated that, compared to standard bacterial lysates, MAV was enriched with Hsps and contained several recognized protective protein antigens, including pneumococcal surface protein A (PspA) and pneumolysin (Ply). Vaccination of rodents with MAV induced robust antibody responses to multiple serotypes, including nonpneumococcal conjugate vaccine serotypes. Homologous and heterologous strains of S. pneumoniae were opsonized after incubation in sera from vaccinated rodents. In mouse models, active vaccination with MAV significantly protected against pneumonia, while passive transfer of rabbit serum from MAV-vaccinated rabbits significantly protected against sepsis caused by both homologous and heterologous S. pneumoniae strains. Direct comparison of MAV preparations made with or without the heat shock step showed no clear differences in protein antigen content and antigenicity, suggesting that the chromatography step rather than Hsp induction improved MAV antigenicity. Overall, these data suggest that the MAV approach may provide serotype-independent protection against S. pneumoniae ., (Copyright © 2019 Chan et al.)

The success of polysaccharide conjugate vaccines represents a major advance in the prevention of pneumococcal disease, but the power of these vaccines is limited by partial spectrum of coverage and high cost. Vaccines using immunoprotective proteins are a promising alternative type of pneumococcal vaccines. In this study, we constructed a library of antisera against conserved pneumococcal proteins predicted to be associated with cell surface or virulence using a combination of bioinformatic prediction and immunization of rabbits with recombinant proteins. Screening of the library by an opsonophagocytosis killing (OPK) assay identified the OPK-positive antisera, which represented 15 (OPK-positive) proteins. Further tests showed that virtually all of these OPK-positive antisera conferred passive protection against lethal infection of virulent pneumococci. More importantly, immunization with recombinant forms of three OPK-positive proteins (SP148, PBP2b, and ScpB), alone or in combination, conferred significant protection against lethal challenge of pneumococcal strains representing capsular serotypes 3, 4, and 6A in a mouse sepsis model. To our best knowledge, this work represents the first example in which novel vaccine candidates are successfully identified by the OPK screening. Our data have also provided further confirmation that the OPK activity may serve as a reliable in vitro surrogate for evaluating vaccine efficacy of pneumococcal proteins., (Copyright © 2018 American Society for Microbiology.)

Background: The clarification of cutaneous dendritic cell subset and the role of thymic stromal lymphopoietin (TSLP) signaling in epicutaneous sensitization with protein antigens, as in the development of atopic dermatitis, is a crucial issue. Objectives: Because TSLP is highly expressed in the vicinity of Langerhans cells (LCs), we sought to clarify our hypothesis that LCs play an essential role in epicutaneous sensitization with protein antigens through TSLP signaling. Methods: By using Langerin-diphtheria toxin receptor knock-in mice and human Langerin-diphtheria toxin A transgenic mice, we prepared mice deficient in LCs. We also prepared mice deficient in TSLP receptors in LCs by using TSLP receptor–deficient mice with bone marrow chimeric technique. We applied these mice to an ovalbumin (OVA)-induced epicutaneous sensitization model. Results: Upon the epicutaneous application of OVA, conditional LC depletion attenuated the development of clinical manifestations as well as serum OVA-specific IgE increase, OVA-specific T-cell proliferation, and IL-4 mRNA expression in the draining lymph nodes. Consistently, even in the steady state, permanent LC depletion resulted in decreased serum IgE levels, suggesting that LCs mediate the TH2 local environment. In addition, mice deficient in TSLP receptors on LCs abrogated the induction of OVA-specific IgE levels upon epicutaneous OVA sensitization. Conclusion: LCs initiate epicutaneous sensitization with protein antigens and induce TH2-type immune responses via TSLP signaling. [Copyright &y& Elsevier]

Recent studies have suggested that a successful subunit human cytomegalovirus (CMV) vaccine requires improved formulation to generate broad-based anti-viral immunity following immunization. Here we report the development of a non-live protein-based vaccine strategy for CMV based on a polyepitope protein and CMV glycoprotein B (gB) adjuvanted with TLR4 and/or TLR9 agonists. The polyepitope protein includes contiguous multiple MHC class I-restricted epitopes with an aim to induce CD8(+) T cell immunity, while gB is an important target for CD4(+) T cell immunity and neutralizing antibodies. Optimal immunogenicity of this bivalent non-live protein vaccine formulation was dependent upon the co-administration of both the TLR4 and TLR9 agonist, which was associated with the activation of innate immune signatures and the influx of different DC subsets including plasmacytoid DCs and migratory CD8-DEC205+CD103-CD326- langerin-negative dermal DCs into the draining lymph nodes. Furthermore these professional antigen presenting cells also expressed IL-6, IL-12p70, TNFα, and IFNα which play a crucial role in the activation of adaptive immunity. In summary, this study provides a novel platform technology in which broad-based anti-CMV immune responses upon vaccination can be maximised by co-delivery of viral antigens and TLR4 and 9 agonists which induce activation of innate immune signatures and promote potent antigen acquisition and cross-presentation by multiple DC subsets.