A novel staphylococcal enterotoxin B subunit vaccine candidate elicits protective immune response in a mouse model.

Staphylococcal enterotoxin B (SEB), produced by the gram-positive bacterium Staphylococcus aureus, is responsible for food poisoning and toxic shock syndrome, and is considered a potential bioterrorism agent. Unfortunately, still now no approved vaccines are available against SEB. In this study, we constructed a series of nontoxic SEB mutants (mSEBs) and examined whether these mSEBs provide protective immunity against SEB challenge. These mSEB vaccine candidates did not demonstrate superantigen activity in mouse splenocyte cultures. Immunization with the vaccine candidates triggered the production of IgG-antibodies with neutralizing activity. In addition, increased production of IgG1 and IgG3 was observed after immunization, which signifies both Th1- and Th2-induced immune responses. Among the vaccine candidates tested, S9, a double mutant (N23A and Y90A) and S19, a quadruple mutant (N23A, Y90A, R110A, and F177A), demonstrated complete protection against a lethal SEB challenge. Altogether, our results strongly suggest that these mSEBs could be an effective recombinant SEB vaccine candidates for further/future preclinical and clinical studies.

Construction and characterization of VL-VH tail-parallel genetically engineered antibodies against staphylococcal enterotoxins.

Staphylococcal enterotoxins (SEs) produced by Staphylococcus aureus have increasingly given rise to human health and food safety. Genetically engineered small molecular antibody is a useful tool in immuno-detection and treatment for clinical illness caused by SEs. In this study, we constructed the V(L)-V(H) tail-parallel genetically engineered antibody against SEs by using the repertoire of rearranged germ-line immunoglobulin variable region genes. Total RNA were extracted from six hybridoma cell lines that stably express anti-SEs antibodies. The variable region genes of light chain (V(L)) and heavy chain (V(H)) were cloned by reverse transcription PCR, and their classical murine antibody structure and functional V(D)J gene rearrangement were analyzed. To construct the eukaryotic V(H)-V(L) tail-parallel co-expression vectors based on the "5'-V(H)-ivs-IRES-V(L)-3'" mode, the ivs-IRES fragment and V(L) genes were spliced by two-step overlap extension PCR, and then, the recombined gene fragment and V(H) genes were inserted into the pcDNA3.1(+) expression vector sequentially. And then the constructed eukaryotic expression clones termed as p2C2HILO and p5C12HILO were transfected into baby hamster kidney 21 cell line, respectively. Two clonal cell lines stably expressing V(L)-V(H) tail-parallel antibodies against SEs were obtained, and the antibodies that expressed intracytoplasma were evaluated by enzyme-linked immunosorbent assay, immunofluorescence assay, and flow cytometry. SEs can stimulate the expression of some chemokines and chemokine receptors in porcine IPEC-J2 cells; mRNA transcription level of four chemokines and chemokine receptors can be blocked by the recombinant SE antibody prepared in this study. Our results showed that it is possible to get functional V(L)-V(H) tail-parallel genetically engineered antibodies in same vector using eukaryotic expression system.

Development and evaluation of IgY ImmunoCapture PCR ELISA for detection of Staphylococcus aureus enterotoxin A devoid of protein A interference.

In the present study, a sensitive and specific IgY mediated ImmunoCapture-PCR-ELISA (IC-PCR-ELISA) was developed for the detection of staphylococcal enterotoxin A (SEA) from culture supernatants and suspected contaminated samples. Due to the virtue of avian immunoglobulins (IgY) to have the least affinity towards staphylococcal protein A (SpA) responsible for false positives, we employed anti-SEA IgY for capture of SEA toxin and revealed with SEA specific rabbit antibodies conjugated to a 524bp DNA marker. Biotin-11-dUTP was incorporated during PCR amplification and post PCR analysis was performed by PCR-ELISA. Unlike IgG immunocapture, IgY mediated immunocapture of SEA was free from false positives due to protein A. The developed assay was specific to SEA except for minor cross reactivity with staphylococcal enterotoxin E (SEE). Several raw milk samples were evaluated for the presence of SEA with and without enrichment. Three samples were found to be positive for SEA after enrichment for 8h. Though IC-PCR-ELISA for SEA showed 100% correlation with PCR analysis for sea gene, the assay was unique in terms of sensitivity of detecting ~10pg/ml of SEA toxin from spiked milk samples. Result of IC-PCR-ELISA was further confirmed by conventional methods of isolation and characterization. The presented method can be very useful for rapid analysis of milk samples for SEA contamination and can be further extended for detection of multiple SE's in different wells of same PCR plate using common DNA substrate.

Improvement of strain discrimination by combination of superantigen profiles, PFGE, and RAPD for Staphylococcus aureus isolates from clinical samples and food-poisoning cases.

Staphylococcus aureus is one of the major bacterial species that may cause clinical infection and food-poisoning cases. Strains of this bacterial species may produce a series of superantigens (SAgs) (i.e., staphylococcal enterotoxins [SEs], staphylococcal enterotoxin-like toxins, and toxic shock syndrome toxin). In this study, S. aureus strains from clinical samples and food-poisoning cases in Taiwan were collected; their SAg profiles, and SmaI digestion patterns determined by pulsed-field gel electrophoresis (PFGE), were then analyzed. Results showed that their SAg gene profiles and SmaI digestion patterns of chromosomal DNA were highly diverse. Although PFGE has been used as a criterion standard for typing of S. aureus strains, and the SAg profiles have been used in combination with PFGE for typing of S. aureus strains, we found that strains grouped in these combined patterns could be further discriminated by the random amplified polymorphic DNA (RAPD) method. Thus, the combined use of SAg profiles, PFGE, and RAPD patterns permits high discrimination for typing of S. aureus strains from not only the clinical samples but also the food-poisoning cases. Such a combined method may be used as a highly accurate approach for epidemiological study and tracing of the contamination origin of staphylococcal infections either in hospitals or food-poisoning cases.

Identification and characterization of two novel staphylococcal enterotoxins, types S and T.

In addition to two known staphylococcal enterotoxin-like genes (selj and selr), two novel genes coding for two superantigens, staphylococcal enterotoxins S and T (SES and SET), were identified in plasmid pF5, which is harbored by food poisoning-related Staphylococcus aureus strain Fukuoka 5. This strain was implicated in a food poisoning incident in Fukuoka City, Japan, in 1997. Recombinant SES (rSES) specifically stimulated human T cells in a T-cell receptor Vbeta9- and Vbeta16-specific manner in the presence of major histocompatibility complex (MHC) class II(+) antigen-presenting cells (APC). rSET also stimulated T cells in the presence of MHC class II(+) APC, although its Vbeta skewing was not found in reactive T cells. Subsequently, we examined the emetic activity of SES and SET. We also studied SElR to determine emetic activity in primates. This toxin was identified in previous studies but was not examined in terms of possession of emetic activity for primates. rSES induced emetic reactions in two of four monkeys at a dose of 100 microg/kg within 5 h of intragastric administration. In one monkey, rSET induced a delayed reaction (24 h postadministration) at a dose of 100 microg/kg, and in the other one, the reaction occurred 5 days postadministration. rSElR induced a reaction in two of six animals within 5 h at 100 microg/kg. On this basis, we speculate that the causative toxins of vomiting in the Fukuoka case are SES and SER. Additionally, SES, SER, and SET also induced emesis in house musk shrews as in the monkeys.

Superantigens: structure and relevance to human disease.

Superantigens are a class of immunostimulatory molecules produced by bacteria and viruses. Their potent immune effects are due to their unique ability to bind to the major histocompatibility complex (MHC) outside the antigen-binding cleft and to stimulate T cells in a T-cell receptor (TCR) Vbeta-specific manner. Structural studies have revealed the binding sites involved in the MHC/superantigen/TCR complex. The bacterial superantigens are responsible for a number of syndromes, including food poisoning and toxic shock syndrome, but their effects may be not only acute but also chronic and complex. Recent evidence suggests that superantigens may be relevant to the pathogenesis of autoimmune and immunodeficiency disorders. To illustrate the detrimental effects of superantigens on disease outcome, evidence demonstrating the modulation of experimental allergic encephalomyelitis, an animal model for multiple sclerosis, by superantigen, as well as the potential role of superantigens in HIV pathogenesis of AIDS, will be presented. The information presented may provide valuable insight into the role of superantigens in autoimmunity and HIV infection.

Immunoregulatory effects of superantigens: interactions of staphylococcal enterotoxins with host MHC and non-MHC products.

Staphylococcus aureus carries a highly conserved set of genes which encode a set of secreted enterotoxins. Although it is likely that these enterotoxins affect the host/parasite in favor of the bacterium, we do not understand the molecular basis of this interaction. We summarize recent evidence that defines two types of interaction between the bacterial toxin and host cellular receptors that may subvert the host immune response to S. aureus. An interaction between the toxin and class II products on APC can result in inhibition of costimulatory activity and thus impair clonal expansion of T cells specific for bacterial antigens. Studies using anti-class II antibodies suggest that this may reflect transmission of a negative signal to APC after ligation of class II products. A second interaction between a subset of toxins, including SEC, with non-MHC products stimulates both T-cell proliferation as well as toxin-specific cytotoxic T cells (CTL). We put forward the hypothesis that this interaction reflects binding of a VCAM-1-like subsequence of SEC to VLA-4 expressed by activated target cells. We suggest that this interaction may serve to inhibit the host response by subversion of lymphocyte homing to sites of infection by SEC-producing staphylococci and by local elimination of (VLA-4+) memory T cells.

Purificación de anticuerpos policlonales a la enterotoxina estafilocóccica C por cromatografía de afinidad / Purification of policlonals antibodies a the staphylococcal C enterotoxins by affinity chromatography

Se describe un método para la purificación de anticuerpos policlonales, con el objetivo de utilizarlos en la detección de enterotoxinas estafilocóccicas en los alimentos. Se seleccionó la técnica de Cromatografía de Afinidad empleando como inmunoadsorbente la toxina C ligada a Sepharosa 4B activada con Bromuro de Cianógeno. El procedimiento permite la purificación en un paso, donde se obtuvo un alto rendimiento (97 por ciento de la IgG fijada), rapidez y eficiencia

Interaction of staphylococcal protein A in enzyme-linked immunosorbent assays for detecting staphylococcal antigens.

ELISA procedures for detecting staphylococcal antigens may be subject to interference by reactions between staphylococcal protein A (SPA) and IgG molecules. It was found that rabbit IgG reacted with SPA, both in the native state and after conjugation with peroxidase. Sheep IgG, however, did not react with SPA if conjugated with peroxidase. Peroxidase conjugated SPA reacted with rabbit IgG but not with sheep IgG. These results demonstrate that the source of IgG used in an ELISA system is of major importance to correct quantitation of staphylococcal antigens.

[Serologic shifts in food poisoning by 1 and 2 infectious agents]. / Serologicheskie sdvigi pri pishchevykh toksikoinfektsiiakh, vyzvannykh odnim i dvumia vozbuditeliami.

Features of serological shifts in patients with alimentary toxicoinfections (ATI) caused by one infective agent (E. coli, staphilococci, Proteus, enterococci) and by two infective agents in various combinations have been studied. The suppression of immunogenesis has been found to be most pronounced in patients having ATI mixed according to agglutinin titers to the autostrains of infective agents. The features of serological shifts in mixed ATI should be taken into consideration in evaluating the diagnostic significance of this reaction in the time course of the disease.