Summary
The immune response against hapten is T‐cell‐dependent, and so requires the uptake, processing and presentation of peptides on MHC class II molecules by antigen‐presenting cells to the ...specific T cell. Some haptens, following conjugation to the available free amines on the surface of the carrier protein, can reduce its immunogenicity. The purpose of this study was to explore the mechanism by which this occurs. Four proteins were tested as carriers and six molecules were used as haptens. The immune response to the carrier proteins was reduced > 100‐fold by some of the haptens (termed carrier immunogenicity reducing haptens – CIRH), whereas other haptens did not influence the protein immunogenicity (carrier immunogenicity non‐reducing haptens – nCIRH). Conjugation of the protein to a CIRH affected protein degradation by lysosomal cathepsins, leading to the generation of peptides that differ in length and sequence from those derived from the same native protein or that protein modified with nCIRH. Injection of CIRH‐conjugated protein into mice induced an increase in the population of regulatory T cells. The results of this study provide a putative mechanism of action for the reduction of immune response to haptenated proteins.
Infectious bronchitis virus (IBV) is a highly contagious avian coronavirus. IBV causes substantial worldwide economic losses in the poultry industry. Vaccination with live-attenuated viral vaccines, ...therefore, are of critical importance. Live-attenuated viral vaccines, however, exhibit the potential for reversion to virulence and recombination with virulent field strains. Therefore, alternatives such as subunit vaccines are needed together with the identification of suitable adjuvants, as subunit vaccines are less immunogenic than live-attenuated vaccines. Several glycan-based adjuvants directly targeting mammalian C-type lectin receptors were assessed in vitro using chicken bone marrow-derived dendritic cells (BM-DCs). The β-1-6-glucan, pustulan, induced an up-regulation of MHC class II (MHCII) cell surface expression, potentiated a strong proinflammatory cytokine response, and increased endocytosis in a cation-dependent manner. Ex vivo co-culture of peripheral blood monocytes from IBV-immunised chickens, and BM-DCs pulsed with pustulan-adjuvanted recombinant IBV N protein (rN), induced a strong recall response. Pustulan-adjuvanted rN induced a significantly higher CD4
blast percentage compared to either rN, pustulan or media. However, the CD8
and TCRγδ
blast percentage were significantly lower with pustulan-adjuvanted rN compared to pustulan or media. Thus, pustulan enhanced the efficacy of MHCII antigen presentation, but apparently not the cross-presentation on MHCI. In conclusion, we found an immunopotentiating effect of pustulan in vitro using chicken BM-DCs. Thus, future in vivo studies might show pustulan as a promising glycan-based adjuvant for use in the poultry industry to contain the spread of
as well as of other avian viral pathogens.
Adsorption of proteins to clay minerals is widely studied, mostly in agricultural issues, due to its importance in soil. The poultry industry experiences an increasing need for new generations of ...effective vaccines, requiring minimal handling of bird during administration. Among the problems of vaccination with genetically engineered vaccines is low immunogenicity. Therefore, the development of effective adjuvants for vaccination is a central task. In addition, oral administration of vaccines is more convenient and economic for the poultry industry. The modified heat-labile eteroxin (LT) protein was found to be a safe and immunogenic adjuvant, and showed the ability to enhance immune responses against an antigen co-administered with, for example—the viral protein 2 (VP2). As a model of a possible combination for an oral administrated vaccine, adsorption of VP2 and LT on SWy-2 montmorillonite and on an organo-montmorillonite (berberine adsorbed on SWy-2) were tested. Results of this multidisciplinary study, which combines methods used in clay mineral studies with biological assays, prove feasibility to attach two vaccine-related proteins to organoclay mineral particles, yielding close vicinity between the proteins that may lead to an immune response via the monosialoganglioside (GM1) receptor. Such complexes may be used as vaccine carriers to stimulate desired immune responses.
► Poultry industry experiences need for new generations of effective vaccines. ► Adsorption of VP2 and LT to montmorillonite and organo-montmorillonite were tested. ► Modifying the clay mineral was crucial for efficient co-adsorption of both proteins. ► ELISA assay demonstrates biological activity of the VP2-LT-BER SWy2 complex. ► Results prove feasibility to attach vaccine-related proteins to berberine-SWy2.
Proteins and peptides have been used as drugs for almost a century. Technological advances in the past 30 years have enabled the production of pure, stable proteins in vast amounts. In contrast, ...administration of proteins based on their native active conformation (and thus necessitating the use of subcutaneous injections) has remained solely unchanged. The therapeutic anti-HER2 humanized monoclonal immunoglobulin (IgG) Trastuzumab (Herceptin) is a first line of the treatment for breast cancer. Chicken IgY is a commercially important polyclonal antibody (Ab). These Abs were examined for their ability to self-assemble and form ordered aggregates, by several biophysical methods. Atomic force microscopy analyses revealed the formation of multimeric nanostructures. The biological activity of multimeric IgG or IgY particles was retained and restored, in a dilution/time-dependent manner. IgG activity was confirmed by a binding assay using HER2 + human breast cancer cell line, SKBR3, while IgY activity was confirmed by ELISA assay using the VP2 antigen. Competition assay with native Herceptin antibodies demonstrated that the binding availability of the multimer formulation remained unaffected. Under long incubation periods, IgG multimers retained five times more activity than native IgG. In conclusion, the multimeric antibody formulations can serve as a storage depositories and sustained-release particles. These two important characteristics make this formulation promising for future novel administration protocols and altogether bring to light a different conceptual approach for the future use of therapeutic proteins as self-delivery entities rather than conjugated/encapsulated to other bio-compounds.
Group A rotaviruses (RVA) are one of the most common causes of severe acute gastroenteritis in infants worldwide. Rotaviruses spread from person to person, mainly by faecal⁻oral transmission. Almost ...all unvaccinated children may become infected with RVA in the first two years of life. The establishment of an experimental monkey model with RVA is important to evaluate new therapeutic approaches. In this study, we demonstrated viral shedding and viraemia in juvenile⁻adult
orally inoculated with Wa RVA prototype. Nine monkeys were inoculated orally: seven animals with human RVA and two control animals with saline solution. During the study, the monkeys were clinically monitored, and faeces and blood samples were tested for RVA infection. In general, the inoculated animals developed an oligosymptomatic infection pattern. The main clinical symptoms observed were diarrhoea in two monkeys for three days, associated with a reduction in plasmatic potassium content. Viral RNA was detected in seven faecal and five sera samples from inoculated animals, suggesting virus replication. Cynomolgus monkeys are susceptible hosts for human Wa RVA infection. When inoculated orally, they presented self-limited diarrhoea associated with presence of RVA infectious particles in faeces. Thus, cynomolgus monkeys may be useful as animal models to evaluate the efficacy of new antiviral approaches.
Abstract Melanoma is a highly lethal cancer deriving from transformed dermal melanocytes. Early diagnosed primary melanoma may be curable, but the cure-rate of more advanced stages is limited, with ...high mortality rate. With the progression of the tumor, the melanocytes overexpress intracellular or cell-surface molecules, including ectopic normal and tumor-specific proteins. Some of these induce a specific immune response by T and B lymphocytes. Antibodies raised against melanoma antigens were proposed for differential disease diagnosis, staging, prognosis and evaluation of treatment efficiency. Nevertheless, treatments based on stimulation of specific anti-melanoma immune responses have had only limited success. It seems that efficient immunotherapy should become more feasible pending on finding new adequate antigens to target. New insights into immune regulation of the tumor microenvironment and its progression may help the development of more successful treatments. We present here up-to-date information on known major melanoma-associated antigens, which could serve as tools for diagnosis as well as for clinical immunotherapy. This approach with promising results for treating some other selected malignancies is still experimental with a very limited success in melanoma. The development of new immune modulators of the tumor microenvironment and neo-antigens may be additional promising directions and may open new opportunities for the immunotherapy of melanoma.
The success of adoptive cell transfer in the treatment of metastatic cancer in humans is dependent on the selection of highly active tumor-specific cytotoxic T cells. We report here that CTLs capture ...membrane fragments from their targets while exerting cytotoxic activity and thus gain a detectable functional signature by which they can be identified. Fluorochrome labeling or biotinylation was used to tag tumor cells. CD8(+) T cells were coincubated with the tagged targets, sorted, and functionally evaluated. Our results show that membrane capture by CD8(+) lymphocytes is T-cell receptor dependent, epitope specific, and preferentially associated with highly cytotoxic clonal subsets. CTLs that captured membranes from unmodified melanoma exhibited enhanced cytotoxic activity against tumor cell lines and autologous melanoma. In a human melanoma in vivo model, adoptive transfer of membrane-capturing, peptide-specific T cells, but not noncapturing or bulk CD8(+) T cells, inhibits tumor progression. Membrane capture is therefore a signature of antigen-specific CTLs endowed with high functional avidity and may have direct relevance in the clinical application of adoptive immunotherapy.
Gram-negative bacteria are common and efficient protein expression systems, yet their outer membrane endotoxins can elicit undesirable toxic effects, limiting their applicability for parenteral ...therapeutic applications, e.g., production of vaccine components. In the bacterial genus
Sphingomonas
from the
Alphaproteobacteria
class, lipopolysaccharide (LPS) endotoxins are replaced with non-toxic glycosphingolipids (GSL), rendering it an attractive alternative for therapeutic protein production. To explore the use of
sphingomonas
as a safe expression system for production of proteins for therapeutic applications, in this study,
Sphingobium japonicum
(
SJ
) injected live into embryonated hen eggs proved safe and nontoxic. Multimeric viral polypeptides derived from Newcastle disease virus (NDV) designed for expression in SJ, yielded soluble proteins which were specifically recognized by antibodies raised against the whole virus. In addition, native signal peptide (SP) motifs coupled to secreted proteins in
SJ
identified using whole-genome computerized analysis, induced secretion of α Amylase (αAmy) and mCherry gene products. Relative to the same genes expressed without an SP, SP 104 increased the secretion of αAmy (3.7-fold) and mCherry (16.3-fold) proteins and yielded accumulation of up to 80 µg/L of the later in the culture medium. Taken together, the presented findings demonstrate the potential of this unique LPS-free gram-negative bacterial family to serve as an important tool for protein expression for both research and biotechnological purposes, including for the development of novel vaccines and as a live bacteria delivery system for protein vaccines.
Key points
•
Novel molecular tools for protein expression in non-model bacteria.
•
Bacteria with GSL instead of LPS as a potential vector for protein delivery.
The most common source of foodborne Salmonella infection in humans is poultry eggs and meat, such that prevention of human infection is mostly achieved by vaccination of farm animals. While ...inactivated and attenuated vaccines are available, both present drawbacks. This study aimed to develop a novel vaccination strategy, which combines the effectiveness of live-attenuated and safety of inactivated vaccines by construction of inducible self-destructing bacteria utilizing toxin-antitoxin (TA) systems. Hok-Sok and CeaB-CeiB toxin-antitoxin systems were coupled with three induction systems aimed for activating cell killing upon lack of arabinose, anaerobic conditions or low concentration of metallic di-cations. The constructs were transformed into a pathogenic Salmonella enterica serovar Enteritidis strain and bacteria elimination was evaluated in vitro under specific activating conditions and in vivo following administration to chickens. Four constructs induced bacterial killing under the specified conditions, both in growth media and within macrophages. Cloacal swabs of all chicks orally administered transformed bacteria had no detectable levels of bacteria within 9 days of inoculation. By day ten, no bacteria were identified in the spleen and liver of most birds. Antibody immune response was raised toward TA carrying Salmonella which resembled response toward the wildtype bacteria. The constructs described in this study led to self-destruction of virulent Salmonella enteritidis both in vitro and in inoculated animals within a period which is sufficient for the induction of a protective immune response. This system may serve as a safe and effective live vaccine platform against Salmonella as well as other pathogenic bacteria.
Maternally derived antibodies (MDAs) are critical for offspring protection during the first days of life. However, due to their short half-life (3-5 days), MDA levels decline rapidly and by 8-15 days ...post-hatch drop to below protective levels. In addition, MDAs against a specific pathogen often impede the efficient protective immune response to that pathogen by the new-born following vaccination. The combination of these two phenomena generates a gap in protection in the period between loss of MDA protection and development of vaccine-induced protective antibodies. Herein, a concept is presented that might enable effective vaccination of 1-day-old progeny in the presence of MDAs. The idea is to vaccinate mothers and their progeny with different neutralizing epitopes of the same pathogen. This will allow an effective immune response of the progeny towards neutralizing epitopes while retaining MDA protection until high levels of self-antibodies are produced. This concept is valid for various avian viruses that express two neutralizing proteins or have numerous neutralizing epitopes on the same protein, for example, Newcastle disease virus and infectious bursal disease virus, respectively. These may be used as protein subunit vaccines or live vaccines carried by a vector. This vaccination concept may overcome the gap in protection that occurs when MDAs decrease while self-immunity is still partial, to provide continuous protection at a young age.
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