Allogeneic CD3/CD28 cross-linked Th1 memory cells provide potent adjuvant effects for active immunotherapy of leukemia/lymphoma.

The breaking of peripheral T-cell tolerance toward self-antigens expressed by tumor cells and the subsequent establishment of an effective tumor protective immune response remains a major challenge for cancer immunotherapy. We report that both protective and therapeutic anti-tumor immune responses can be achieved in a mouse leukemia/lymphoma tumor model through the strong adjuvant effects provided by allogeneic CD3/CD28 cross-linked Th1 memory cells. The adjuvant effect of these cells is mediated by their ability to produce a variety of 'danger signals' which serve to deviate native non-protective Th2 anti-leukemia immune responses to effective Th1 immune responses.

Recombinant egg drop syndrome subunit vaccine offers an alternative to virus propagation in duck eggs.

Egg drop syndrome (EDS) virus vaccines are routinely produced in embryonated duck eggs (Solyom et al., 1982). This procedure poses the risk of dissemination of pathogens, such as avian influenza virus, as the eggs used are not from specific pathogen free birds. To address this problem, the knob and part of the shaft domain of the fibre protein of the EDS virus (termed knob-s) were expressed in Escherichia coli and assessed as a subunit vaccine. A single vaccination with the recombinant protein induced the production of anti-EDS virus antibodies, as detected by haemagglutination inhibition, enzyme-linked immunosorbent assay and virus neutralization tests, for at least 20 weeks. A positive correlation was demonstrated between these three assays. A dose-response assessment showed that the vaccine was effective over the range of 2 to 64 microg protein per dose. Two vaccinations with the recombinant protein, administered before the onset of lay, induced high haemagglutination inhibition antibody titres, comparable with those induced by an inactivated whole-virus vaccine. The vaccine did not have any adverse effects on egg production, quality or weight. The present study has shown that two vaccinations with the recombinant knob-s protein elicited high neutralizing antibody titres that persisted for more than 50 weeks of lay.

B subunit of E. coli enterotoxin as adjuvant and carrier in oral and skin vaccination.

Mucosal sites are one of the main natural ports of entry into the body. Stimulation of a local response by antibodies as the systemic protection may enhance the efficacy of non-living vaccines, and allow for vaccination by subunit vaccines without the need for injection. Mucosal or skin vaccination necessitates a suitable adjuvant and carrier. Escherichia coli heat-labile enterotoxin (LT) and its B subunit (LTB) have been found to be effective adjuvants. The aim of this study was to efficiently produce and purify recombinant LTB (brLTB), and examine its adjuvant and carrier properties. The gene encoding LTB was cloned and expressed in E. coli, and the product was found to have a pentameric form with the ability to bind the cell receptor, GM1 ganglioside. A one-step method for efficient purification and concentration of brLTB was developed. Both oral and intramuscular vaccination with purified brLTB yielded high antibody titers, which detected the whole toxin. In an attempt to test its adjuvant characteristics, brLTB was mixed with either BSA or a recombinant protein (rKnob of egg drop syndrome adenovirus) and delivered intramuscularly, orally or transcutaneously. The addition of brLTB significantly elevated the antibody response in groups vaccinated orally and transcutaneously, but had no influence in injected groups. Vaccination with another recombinant protein, (viral protein 2 of infectious bursal disease virus) supplemented with brLTB did not elevate the antibody response, as compared to vaccination with the antigen alone. These results demonstrate that the addition of brLTB makes oral and transcutaneous vaccination with protein antigens possible.

A subunit vaccine against hemorrhagic enteritis adenovirus.

Hemorrhagic enteritis virus (HEV) is an adenovirus that infects turkeys and causes immunosuppression and mortality. The virus used for the inactivated vaccine is extracted from spleens of infected turkeys, since its propagation in tissue cultures or embryonated eggs is unsuitable for mass production. The aim of this study was to develop a subunit vaccine based on a capsid protein of the virus. The knob protein, together with an adjacent part of the shaft domain pertaining to the fiber protein of HEV, was expressed in Escherichia coli and tested as a vaccine. Vaccination with this recombinant protein conferred protection against challenge in controlled and in floor-pen experiments. This finding suggests that the knob protein may be used as safe and efficient vaccine against hemorrhagic enteritis of turkeys. The possibility that the knob proteins of other adenoviruses may be protective and serve as vaccine is also discussed.

Vaccine and adjuvant activity of recombinant subunit B of E. coli enterotoxin produced in yeast.

Escherichia coli heat-labile enterotoxin (LT) and cholera toxin (CT) have been studied intensively as vaccines against diseases caused by those bacteria and as adjuvants for mucosal vaccination. Two major problems interfere with the use of these promising adjuvants: their toxicity and the residual bacterial endotoxins mixed with the desired LT. In this study, subunit B of LT was expressed in Pichia pastoris yeast cells (yrLTB) and the recombinant protein was purified and concentrated by ion-exchange chromatography. The final yield of the recombinant protein was 5-8 mg/l induction medium. The molecule is in pentameric form and binds to GM1 gangliosides. When given orally to chickens, anti-LTB antibodies were produced, exhibiting its ability to cross the digestive system and induce an immune response. The adjuvant activity of yrLTB was proven by fusing it to viral protein 2 (VP2) of infectious bursal disease virus. Birds intramuscularly vaccinated with this molecule exhibit 70-100% protection, in a dose-response-dependent manner. This method eliminated the bacterial endotoxins and enabled the production of large quantities of LTB. Expression in a eukaryotic system allows the production of fusion proteins that require post-translational modifications. This may allow oral vaccination with a protein fused to yrLTB. The approach described in this study will enable the efficient production of a non-toxic, eukaryotically expressed enterotoxin as a vaccine against the toxin itself or as a carrier or adjuvant for foreign vaccine molecules.

A subunit vaccine against the adenovirus egg-drop syndrome using part of its fiber protein.

In this study, the effectiveness of antibodies against the hexon, fiber or a fiber fragment of an avian adenovirus egg-drop syndrome (EDS), in neutralizing the virus was tested. The fiber protein is responsible for binding the virus to the target cell. The fiber fragment knob-s comprises the carboxy-terminal knob domain and 34 amino acids of the immediately adjacent shaft domain of the adenovirus fiber protein. The hexon, fiber capsid protein and knob-s were produced in E. coli and injected into chickens. Antibodies that were produced against the whole fiber protein showed some hemagglutination inhibition (HI) activity. Antibodies produced against the knob-s protein showed HI activity and serum neutralization (SN) activity similar to the positive control-whole virus vaccine. We assume that production of only part of the fiber enables the protein produced in E. coli to fold correctly. Antibodies produced against the hexon protein showed no SN activity. In summary, knob-s induced SN and HI antibodies against EDS virus at a rate similar to the whole virus and were significantly more efficient than the full-length fiber. The recombinant knob-s protein may be used as a vaccine against pathogenic adenovirus infections.