Engineered human antibodies for the opsonization and killing of Staphylococcus aureus.

Gram-positive organisms with their thick envelope cannot be lysed by complement alone. Nonetheless, antibody-binding on the surface can recruit complement and mark these invaders for uptake and killing by phagocytes, a process known as opsonophagocytosis. The crystallizable fragment of immunoglobulins (Fcγ) is key for complement recruitment. The cell surface of S. aureus is coated with Staphylococcal protein A (SpA). SpA captures the Fcγ domain of IgG and interferes with opsonization by anti-S. aureus antibodies. In principle, the Fcγ domain of therapeutic antibodies could be engineered to avoid the inhibitory activity of SpA. However, the SpA-binding site on Fcγ overlaps with that of the neonatal Fc receptor (FcRn), an interaction that is critical for prolonging the half-life of serum IgG. This evolutionary adaptation poses a challenge for the exploration of Fcγ mutants that can both weaken SpA-IgG interactions and retain stability. Here, we use both wild-type and transgenic human FcRn mice to identify antibodies with enhanced half-life and increased opsonophagocytic killing in models of S. aureus infection and demonstrate that antibody-based immunotherapy can be improved by modifying Fcγ. Our experiments also show that by competing for FcRn-binding, staphylococci effectively reduce the half-life of antibodies during infection. These observations may have profound impact in treating cancer, autoimmune, and asthma patients colonized or infected with S. aureus and undergoing monoclonal antibody treatment.

The effects of Staphylococcus aureus protein a (SpA) on the expression of inflammatory cytokines in autoimmune patients and their probable immune response modulation mechanisms.

The recombinant Staphylococcal protein A (SpA) is widely used in biotechnology to purify polyclonal and monoclonal IgG antibodies. At very low concentrations, the highly-purified form of the protein A can down-regulate the activation of human B-lymphocytes and macrophages which are the key cells in determining autoimmune diseases. In the present study, the efficiency of three different forms of protein A, including native full-length SpA, the recombinant full-length SpA, and a recombinant truncated form of SpA on the reduction of 4 inflammatory cytokines, including IL-8, IL-1ß, TNF-α, and IL-6 by peripheral blood mononuclear cell (PBMCs) were studied and compared to an anti-rheumatoid arthritis commercial drug, Enbrel. The recombinant proteins were expressed in E. coli and the native form of SpA was commercially provided. PBMCs were obtained from adult patients with active rheumatoid arthritis (RA) and healthy control donors. Then, the effect of different doses of the three pure forms of SpA in comparison with Enbrel was investigated by analyzing the expression of selected cytokines using ELISA. The results showed that the truncated form of recombinant SpA significantly reduced the expression of cytokines more effectively than the other full-length formulations as well as the commercial drug Enbrel. In silico analysis shows that in the truncated protein, as the radius of gyration increases, the structure of IgG-binding domains become more open and more exposed to IgG. To summarize, our findings indicate that the truncated form of protein A is the most efficient form of SpA as it significantly decreases the secretion of evaluated cytokines from PBMCs in vitro.

Glycosylation-dependent opsonophagocytic activity of staphylococcal protein A antibodies.

Antibodies may bind to bacterial pathogens or their toxins to control infections, and their effector activity is mediated through the recruitment of complement component C1q or the engagement with Fcγ receptors (FcγRs). For bacterial pathogens that rely on a single toxin to cause disease, immunity correlates with toxin neutralization. Most other bacterial pathogens, including Staphylococcus aureus, secrete numerous toxins and evolved multiple mechanisms to escape opsonization and complement killing. Several vaccine candidates targeting defined surface antigens of S. aureus have failed to meet clinical endpoints. It is unclear that such failures can be solely attributed to the poor selection of antibody targets. Thus far, studies to delineate antibody-mediated uptake and killing of Gram-positive pathogens remain extremely limited. Here, we exploit 3F6-hIgG1, a human monoclonal antibody that binds and neutralizes the abundant surface-exposed Staphylococcal protein A (SpA). We find that galactosylation of 3F6-hIgG1 that favors C1q recruitment is indispensable for opsonophagocytic killing of staphylococci and for protection against bloodstream infection in animals. However, the simple removal of fucosyl residues, which results in reduced C1q binding and increased engagement with FcγR, maintains the opsonophagocytic killing and protective attributes of the antibody. We confirm these results by engineering 3F6-hIgG1 variants with biased binding toward C1q or FcγRs. While the therapeutic benefit of monoclonal antibodies against infectious disease agents may be debatable, the functional characterization of such antibodies represents a powerful tool for the development of correlates of protection that may guide future vaccine trials.

Antibody orientational labeling via staphylococcus A protein to improve the sensitivity of gold immunochromatography assays.

The Cry1Ab toxin is usually expressed in genetically modified crops in order to control chewing pests. Although the gold immunochromatography assay (GICA) based on the double-antibody sandwich method has been developed to detect this toxin, its detection sensitivity needs improvement. In this study, Cry1Ab-51 antibodies were immobilized orientationally in a simple and effective way on colloidal gold nanoparticles (CGNPs) using the affinity of staphylococcal protein A (SPA) towards the fragment crystallizable (FC) fragment of mouse immunoglobulin G (IgG). Lateral flow detection test strips, assembled with probes labeled with orientational methods under optimal operational conditions (new probe), were 10 times more sensitive than test strips assembled with probes labeled by adsorption (conventional probe). Experiments showed that the affinity of the new probe was much higher than the conventional probe. The immunochromatography gold strip (ICG strip) assembled using the new probe was highly specific to Cry1Ab with no cross-reaction with other transgenic proteins, and it was proved that the specificity of the new probe had no change. Furthermore, the ICG strips assembled with the new probe could be stored for 12 months under dry conditions without a significant loss of sensitivity. The orientational labeling of the antibodies with SPA on colloidal gold proved to be suitable for improving the sensitivity of the ICG strips.

IgG immune complexes with Staphylococcus aureus protein A enhance osteoclast differentiation and bone resorption by stimulating Fc receptors and TLR2.

Staphylococcus aureus is a main pathogen of osteomyelitis and protein A is a virulence factor with high affinity for IgG. In this study, we investigated whether S. aureus affects the differentiation and bone resorption of osteoclasts through the IgG-binding capacity of protein A. Staphylococcus aureus pre-treated with serum or IgG showed marked enhancement in osteoclastogenesis and bone resorption compared to non-treated S. aureus or a protein A-deficient mutant. Blocking of the Fc receptor and deletion of the Fcγ receptor gene in osteoclast precursor cells showed that enhanced osteoclastogenesis stimulated by S. aureus IgG immune complexes (ICs) was mediated by the Fc receptor on osteoclast precursor cells. In addition, osteoclastogenesis stimulated by S. aureus ICs but not the protein A-deficient mutant was markedly reduced in osteoclast precursor cells of Myd88-knockout mice. Moreover, NFATc1, Syk and NF-κB signals were necessary for osteoclastogenesis stimulated by S. aureus ICs. The results suggest the contribution of a of Toll-like receptor 2 (TLR2)-Myd88 signal to the activity of S. aureus ICs. We further examined the expression of pro-inflammatory cytokines that is known to be enhanced by FcγR-TLR cross-talk. Osteoclasts induced by S. aureus ICs showed higher expression of TNF-α and IL-1ß, and marked stimulation of proton secretion of osteoclasts activated by pro-inflammatory cytokines. Finally, injection of S. aureus, but not the protein A-deficient mutant, exacerbated bone loss in implantation and intra-peritoneal administration mouse models. Our results provide a novel mechanistic aspect of bone loss induced by S. aureus in which ICs and both Fc receptors and TLR pathways are involved.

Evaluating a new mixed-mode resin Diamond MMC Mustang using Capto MMC ImpRes as a benchmark.

Diamond MMC Mustang is a relatively new mixed-mode resin, which mediates both cation exchange and hydrophobic interactions. In this work, we evaluated this resin using Cytiva's Capto MMC ImpRes, a well-established mixed-mode resin with similar properties, as a benchmark. The data suggest that in comparison with Capto MMC ImpRes, Diamond MMC Mustang exhibits comparable binding capacity and resolution. In addition, the resin under evaluation shows good lot-to-lot consistency. The information provided in this study allows users to have additional options when selecting mixed-mode resin for intermediate purification or final polishing, which is favourable especially at the present time when the supply chains of many manufacturers are negatively impacted by the coronavirus pandemic.

Simultaneously targeting nitrocellulose and antibody by a dual-headed protein.

Immobilizing antibodies on the nitrocellulose membrane is an important step to increase the sensitivity of the Lateral Flow Test strip for detecting pathogenic antigen. In our research, the fusion protein between nitrocellulose-binding anchor protein 3-Helix - a protein that has a strong affinity to nitrocellulose membrane and protein A - a protein that can bind to the Fc tail of IgG antibody was generated. This fusion protein was expected to help IgG antibodies to be more strongly binding and oriented immobilized onto the nitrocellulose membrane. The recombinant vector pET22b-proA and pET22b-proA-3-Helix coded for protein A and protein A-3-Helix were cloned. These proteins were overexpressed in BL21 and purified by immobilized metal affinity chromatography with purity above 90%. The purified protein was used to evaluate the orientation binding on nitrocellulose membranes by lateral flow challenge. Results showed that protein A-3-Helix binding to nitrocellulose membrane was better than that of protein A. The former protein increased antibody binding and stereochemical immobilizing onto nitrocellulose membrane compared to its protein A counterpart. In summary, we have succeeded in cloning, purifying, and characterizing a dual-head recombinant protein A and protein A-3-Helix. The results show the potential application of protein A-3-Helix in the immobilizing antibody on the test strip.

The first report on the sortase-mediated display of bioactive protein A from Staphylococcus aureus (SpA) on the surface of the vegetative form of Bacillus subtilis.

Protein A (SpA) is one of the most important Staphylococcus aureus cell wall proteins. It includes five immunoglobulin (Ig)-binding domains which can bind to immune complexes through the Fc region of immunoglobulins. The binding of SpA to the polymeric supports can be used to prepare affinity chromatography resins, which are useful for immunoprecipitation (IP) of antibodies. Protein A is also used to purify many anti-cancer antibodies. In this study, SpA was displayed on the surface of Bacillus subtilis cells using a sortase-mediated system to display the target protein to the B. subtilis cell wall. A series of plasmids consisting of cassettes for cell wall-directed protein A as well as negative controls were constructed and transformed into B. subtilis WASD (wprA sigD) cells. SDS-PAGE, western blot, flow cytometry, functional IgG purification assay, and a modified ELISA assay were used to confirm the surface display of SpA and evaluate its function. Semi-quantitative ELISA results showed that the binding capacity of lyophilized Bs-SpA is 100 µg IgG from rabbit serum per 1 mg of cells under optimal experimental conditions. Low production costs, optimal performance, and the use of a harmless strain compared to a similar commercial product predict the possible use of SpA immobilization technology in the future.

Protein A-Mediated Binding of Staphylococcus spp. to Antibodies in Flow Cytometric Assays and Reduction of This Binding by Using Fc Receptor Blocking Reagent.

Staphylococcus aureus and other coagulase-positive Staphylococcus spp. bind the Fc region of IgG antibodies through expression of protein A (SpA). These species have consequently been a source of false-positive signals in antibody-based assays designed to detect other target bacteria. Here, flow cytometry was used to study the influence of a number of factors on the SpA-mediated binding of single cells to an anti-human IgG antibody, including strain, heat killing, overnight storage, growth phase, cell physiology, surface adhesion, and growth in model food systems. Through the costaining of antibody-stained cells with the permeability dye propidium iodide and calcein violet AM, the cell physiological status was related to SpA-mediated antibody binding. Generally, permeabilized cells lacking esterase activity did not strongly bind antibody. The binding of a number of commercially available polyclonal IgG antibodies to non-Staphylococcus spp. was also characterized. Not all SpA-expressing species showed strong binding of mouse IgG, and one species not known to express SpA showed strong binding. Most SpA-expressing strains bound rabbit IgG antibodies to some extent, whereas only one strain bound goat IgG. To reduce or eliminate SpA-mediated IgG binding, the following products were evaluated as blocking reagents and applied prior to staining with primary or secondary antibody: normal rabbit serum, mouse IgG isotype control, goat IgG, and a commercial FcR blocking reagent. Only the FcR blocking reagent consistently reduced SpA-mediated binding of Staphylococcus spp. to antibodies against other species and could be recommended as a blocking reagent in immunoassays designed to detect non-Staphylococcus species.IMPORTANCE This study characterizes a widespread but little-studied problem associated with the antibody-based detection of microbes-the Staphylococcus protein A (SpA)-mediated binding of IgG antibodies-and offers a solution: the use of commercial FcR blocking reagent. A common source of false-positive signals in the detection of microbes in clinical, food, or environmental samples can be eliminated by applying this study's findings. Using flow cytometry, the authors demonstrate the extent of heterogeneity in a culture's SpA-mediated binding of antibodies and that the degree of SpA-mediated antibody binding is strain, growth phase, and food matrix dependent and influenced by simulated food processing treatments and cell adherence. In addition, our studies of SpA-mediated binding of Staphylococcus spp. to antibodies against other bacterial species produced a very nuanced picture, leading us to recommend testing against multiple strains of S. aureus and S. hyicus of all antibodies to be incorporated into any immunoassay designed to detect a non-Staphylococcus spp.

Investigation of the antibacterial activity of Ag-NPs conjugated with a specific antibody against Staphylococcus aureus after photoactivation.

AIM: This work reports a new method for the use of lasers for the selective killing of bacteria targeted using light-absorbing Silver nanoparticles (Ag-NPs) conjugated with a specific antibody against the Gram-positive bacterium Staphylococcus aureus (S. aureus). METHODS AND RESULTS: Ag-NPs were synthesized using a chemical reduction method and characterized with respect to their surface plasmon resonance, surface morphology via transmission electron microscopy (TEM) and dynamic light scattering (DLS). The bacterial surface was targeted using 20 nm Ag-NPs conjugated with an anti-protein A antibody. Labelled bacteria were irradiated with blue visible laser at 2·04 W/cm2 . The antibacterial activity of functionalized Ag-NPs was investigated by fluorescence microscopy after irradiation, and morphological changes in S. aureus after laser treatment were assessed using scanning electron microscopy (SEM). The laser-irradiated, functionalized Ag-NPs exhibited significant bactericidal activity, and laser-induced bacterial damage was observed after 10 min of laser irradiation against S. aureus. The fluorescence microscopic analysis results supported that bacterial cell death occurred in the presence of the functionalized Ag-NPs. CONCLUSIONS: The results of this study suggest that a novel method for the preparation of functionalized nanoparticles has potential as a potent antibacterial agent for the selective killing of resistant disease-causing bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows that Ag-NPs functionalized with a specific antibody, could be used in combination with laser radiation as a novel treatment to target resistant bacterial and fungal pathogens with minimal impact on normal microflora.

Label-Free Impedimetric Immunosensors Modulated by Protein A/Bovine Serum Albumin Layer for Ultrasensitive Detection of Salbutamol.

The sensing properties of immunosensors are determined not only by the amount of immobilized antibodies but also by the number of effective antigen-binding sites of the immobilized antibody. Protein A (PA) exhibits a high degree of affinity with the Fc part of IgG antibody to feasibly produce oriented antibody immobilization. This work proposes a simple method to control the PA surface density on gold nanostructure (AuNS)-deposited screen-printed carbon electrodes (SPCEs) by mixing concentration-varied PA and bovine serum albumin (BSA), and to explore the effect of PA density on the affinity attachment of anti-salbutamol (SAL) antibodies by electrochemical impedance spectroscopy. A concentration of 100 µg/mL PA and 100 µg/mL BSA can obtain a saturated coverage on the 3-mercaptoproponic acid (MPA)/AuNS/SPCEs and exhibit a 50% PA density to adsorb the amount of anti-SAL, more than other concentration-varied PA/BSA-modified electrodes. Compared with the randomly immobilized anti-SAL/MPA/AuNS/SPCEs and the anti-SAL/PA(100 µg/mL):BSA(0 µg/mL)/MPA/AuNS/SPCE, the anti-SAL/PA(100 µg/mL): BSA(100 µg/mL)/MPA/AuNS/SPCE-based immunosensors have better sensing properties for SAL detection, with an extremely low detection limit of 0.2 fg/mL and high reproducibility (<2.5% relative standard deviation). The mixture of PA(100 µg/mL):BSA(100 µg/mL) for the modification of AuNS/SPCEs has great promise for forming an optimal protein layer for the oriented adsorption of IgG antibodies to construct ultrasensitive SAL immunosensors.

Staphylococcus aureus Decolonization of Mice With Monoclonal Antibody Neutralizing Protein A.

Staphylococcus aureus persistently colonizes the nasopharynx of about one-third of the human population, a key risk factor for community- and hospital-acquired invasive infections. Current strategies for S. aureus decolonization include topical and systemic administration of antibiotics, which is associated with selection for antibiotic resistance and posttreatment recolonization. Using a mouse model for S. aureus colonization, we show here that systemic administration of a recombinant monoclonal antibody neutralizing staphylococcal protein A (SpA) can stimulate antibacterial immunoglobulin G and immunoglobulin A responses and promote S. aureus decolonization. These results suggest that antibody neutralizing SpA, a B-cell superantigen, may also be useful for S. aureus decolonization in humans.

A comprehensive review on staphylococcal protein A (SpA): Its production and applications.

The Staphylococcus aureus protein A (SpA) can be obtained through the culture of wild-type S. aureus and also as a recombinant protein in safe bacterial hosts. Several methods have been used to purify SpA among which ion-exchange chromatography, affinity chromatography, gel filtration, and per aqueous liquid chromatography (PALC) are common. SpA has a wide range of biochemical, biotechnological, and medical applications and is most commonly used in test methods such as immunoprecipitation, enzyme-linked immunosorbent assay, and Western blotting. SpA has also been widely utilized in pharmaceutical applications to bind to immune complexes and serum immunoglobulins. SpA also directly binds to the B-cells preventing initiation of infectious diseases as well as having a role in the development of various autoimmune diseases. This review considers different applications of SpA in biotechnology and its novel clinical application for effective treatment of autoimmune diseases. It also discusses various strategies for expression and purification of the SpA including types of column chromatography that are commonly used in protein purification and developing SpA surface display technologies. Finally, this review highlights the potential and novel applications of SpA immobilization, SpA typing, protein engineering for further development of immunological and biochemical research, and also application of SpA as a diagnostic biosensor.

Mechanisms of Immune Evasion and Bone Tissue Colonization That Make Staphylococcus aureus the Primary Pathogen in Osteomyelitis.

PURPOSE OF REVIEW: Staphylococcus aureus is the primary pathogen responsible for osteomyelitis, which remains a major healthcare burden. To understand its dominance, here we review the unique pathogenic mechanisms utilized by S. aureus that enable it to cause incurable osteomyelitis. RECENT FINDINGS: Using an arsenal of toxins and virulence proteins, S. aureus kills and usurps immune cells during infection, to produce non-neutralizing pathogenic antibodies that thwart adaptive immunity. S. aureus also has specific mechanisms for distinct biofilm formation on implants, necrotic bone tissue, bone marrow, and within the osteocyte lacuno-canicular networks (OLCN) of live bone. In vitro studies have also demonstrated potential for intracellular colonization of osteocytes, osteoblasts, and osteoclasts. S. aureus has evolved a multitude of virulence mechanisms to achieve life-long infection of the bone, most notably colonization of OLCN. Targeting S. aureus proteins involved in these pathways could provide new targets for antibiotics and immunotherapies.

Peptidoglycan-linked protein A promotes T cell-dependent antibody expansion during Staphylococcus aureus infection.

A hallmark of Staphylococcus aureus disease in humans is persistent infections without development of protective immune responses. Infected patients generate VH3 plasmablast expansions and increased VH3 idiotype Ig; however, the mechanisms for staphylococcal modification of immune responses are not known. We report here that S. aureus-infected mice generate VH3 antibody expansions via a mechanism requiring MHC-restricted antigen presentation to CD4(+) T cells and staphylococcal protein A (SpA), a cell wall-anchored surface molecule that binds Fcγ and VH3 variant heavy chains of Ig. VH3 expansion occurred with peptidoglycan-linked SpA from the bacterial envelope but not with recombinant SpA, and optimally required five tandem repeats of its Ig-binding domains. Signaling via receptor-interacting serine/threonine protein kinase 2 (RIPK2) was essential for implementing peptidoglycan-linked SpA superantigen activity. VH3 clan IgG from S. aureus-infected or SpA-treated animals was not pathogen-specific, suggesting that SpA cross-linking of VH3 idiotype B-cell receptors and activation via attached peptidoglycan are the determinants of staphylococcal escape from adaptive immune responses.

A novel bivalent fusion vaccine induces broad immunoprotection against Staphylococcus aureus infection in different murine models.

With more and more drug-resistant Staphylococcus aureus strains emerging in hospitals, there is an urgent need to develop an effective vaccine to combat S. aureus infection. In this study, we constructed a novel bivalent fusion vaccine, SpA-DKKAA-FnBPA37-507 (SF), based on the D domain of staphylococcal protein A (SpA) and the A domain of fibronectin-binding protein A (FnBPA). Immunisation with SF induced a more ideal protective effect compared with the single components alone in a sepsis model. It also showed broad immunoprotection against seven FnBPA isotypes. Vaccination with SF induced strong antibodies responses and Th1/Th17 polarized cellular responses. Further we demonstrated the protective effect of antibodies by the opsonophagocytic assay (OPA) and passive immunisation. Moreover, vaccination with SF showed protective efficacy in a murine pneumonia model and skin abscess model. These results suggest that SF can be regarded as a promising vaccine candidate for the prevention of S. aureus infections.

Carbon dots based immunosorbent assay for the determination of GFAP in human serum.

Glial fibrillary acidic protein (GFAP) is expressed in the central nervous system and the level of GFAP normally rises with brain injury and astroglial tumors. So, serum GFAP is used as a marker for diagnosing various types of brain damage and astroglial tumors. In this study, a new sensor based on carbon dots (CDs) linked with antibodies to specifically detect GFAP in human serum was developed. Anti-GFAP (Ab1) linked with protein A/G agarose resin (PA/G) as a capture antibody (PA/G-Ab1) and anti-GFAP (Ab2) labeled with CDs as a detection antibody (CDs-Ab2) were prepared firstly. Then the CD-linked antibody immunosorbent assay (CLAISA) method was constructed based on the sandwich conjunction reaction among PA/G-Ab1, GFAP, and CDs-Ab2. CLAISA, using the fluorescence of PA/G-Ab1-GFAP-Ab2-CDs as the direct signal, enabled the proposed immunosensor to detect GFAP sensitively with a linear range of 0.10-8.00 ng ml-1 and a detection limit of 25 pg ml-1. This method was applied to the determination of GFAP in human serum by the standard addition method, and the results showed high accuracy and precision. Considering the easy synthetic process and excellent performance of CLAISA, this method has great potential to be used to monitor GFAP in the clinic.

Protein a Immunoadsorption May Hamper the Decision to Transplant Due to Interference With CDC Crossmatch Results.

Transplanting immunized patients requires immunological monitoring in the pretransplant phase to follow reduction of donor specific HLA antibodies (DSA) after Staphylococcus aureus protein A (SPA) immunoadsorption (IA) or therapeutic plasma exchange followed by IVIG and Rituximab administration. Pretreatment aims to significantly reduce DSA strength. The Tissue Typing Lab at Aarhus University Hospital performs immunological monitoring of approximately 150 kidney transplantation patients per year from two transplant centers. From 2012 to 2013, we experienced seven patients desensitized using SPA IA, initially presenting negative cytotoxic complement dependent (CDC) T-cell crossmatches but positive B and T cell flowcytometric crossmatch, who despite significant DSA reduction developed weakly positive CDC T-cell crossmatch shortly prior to transplantation. We hypothesised that leached SPA during IA could be the cause, as the complication was not observed in patients who received plasma exchanges. We found that the positive CDC was not donor specific and SPA column material incubated with control serum reproduced a positive CDC T-cell crossmatch. Finally, we detected leached SPA in one of the patient samples using a highly sensitive time-resolved fluorescent assay. In conclusion, the results emphasize the importance of carefully considering CDC crossmatch results subsequent to IA, before a planned transplantation is either postponed or cancelled. J. Clin. Apheresis 32:163-169, 2017. © 2016 Wiley Periodicals, Inc.

Confounding B-cell defences: lessons from a staphylococcal superantigen.

Studies of microbial superantigens that target large clonal sets of B cells through conserved antigen-receptor-variable-region sites are providing new insights into the mechanisms of B-cell activation-induced cell death. These investigations have shown differences between the clonal regulation of follicular B cells (B2 cells) and the innate-like marginal-zone B cells and B1 cells, and have also shown how B-cell superantigens can affect specialized host defences against infection. Agents designed to emulate the properties of B-cell superantigens might also provide new approaches for the treatment of B-cell-mediated autoimmune and neoplastic diseases.

Production of antigen-specific human IgGs by in vitro immunization.

BACKGROUND: We previously developed in vitro immunization based on a fusion protein containing the transcriptional transactivator (Tat) of human immunodeficiency virus and a double domain, called ZZ, derived from protein A of Staphylococcus aureus. In this approach, naïve human peripheral blood mononuclear cells (PBMCs) trigger a specific IgM antibody (Ab) response in the presence of ZZTat. In the present study, we attempted to raise a specific IgG Ab response. RESULTS: We found that PBMCs incubated with ZZTat and a mixture containing anti-CD40, IL4 and IL21 secrete anti-Tat IgG Abs in their supernatants, indicating that the cytokine cocktail provides an isotypic switch. Then, we deciphered the Tat determinant involved in the phenomenon and found that it is located in the region 22-57 and that, within this region, the cysteine-rich domain and the basic residues play a crucial role. Finally, we prepared a fusion protein containing a fragment derived from the NY-ESO-1 cancer/testis antigen (Ag) and showed that PBMCs incubated with ZZfNY-ESO-1Tat trigger a specific anti-fNY-ESO-1 IgG Ab response, which demonstrates the possibility of transferring immunizing ability to an Ag unrelated to Tat. CONCLUSION: Our ZZTat-based in vitro immunization approach that offers the possibility to raise an IgG Ab response against NY-ESO-1 might represent a valuable first stage for the generation of fully human IgG specific Abs.