Daniel Tusé

Plant-produced idiotype vaccines for the treatment of non-Hodgkin's lymphoma: Safety and immunogenicity in a phase I clinical study

Plant-made vaccines have been the subject of intense interest because they can be produced economically in large scale without the use of animal-derived components. Plant-made therapeutic vaccines against challenging chronic diseases, such as cancer, have received little research attention, and no previous human clinical trials have been conducted in this vaccine category. We document the feasibility of using a plant viral expression system to produce personalized (patient-specific) recombinant idiotype vaccines against follicular B cell lymphoma and the results of administering these vaccines to lymphoma patients in a phase I safety and immunogenicity clinical trial. The system allowed rapid production and recovery of idiotypic single-chain antibodies (scFv) derived from each patients tumor and immunization of patients with their own individual therapeutic antigen. Both low and high doses of vaccines, administered alone or co-administered with the adjuvant GM-CSF, were well tolerated with no serious adverse events. A majority (>70%) of the patients developed cellular or humoral immune responses, and 47% of the patients developed antigen-specific responses. Because 15 of 16 vaccines were glycosylated in plants, this study also shows that variation in patterns of antigen glycosylation do not impair the immunogenicity or affect the safety of the vaccines. Collectively, these findings support the conclusion that plant-produced idiotype vaccines are feasible to produce, safe to administer, and a viable option for idiotype-specific immune therapy in follicular lymphoma patients.

Individualized human scFv vaccines produced in plants: humoral anti-idiotype responses in vaccinated mice confirm relevance to the tumor Ig

We have developed a method for rapidly producing in plants the idiotype regions of the tumor-specific Ig as single-chain Fv (scFv) proteins for use in the treatment of non-Hodgkins lymphoma. Variable region gene sequences were generated from either a tumor hybridoma or human tumor biopsy cells, and idiotype domains were joined by a novel linker and cloned into a modified tobacco mosaic virus (TMV) vector designed to secrete the scFv protein in infected Nicotiana benthamiana plants. Thirty-eight out of 44 human scFv proteins showed Coomassie visible material in crude secretory (interstitial fluid, IF) extracts, 21 of those between 100 and 800 microg/ml. Eight of these proteins were tested for appropriate idiotype responses in vaccinated mice. In all eight cases, anti-idiotype immune responses were induced with minimal cross reactivity to irrelevant Ig or scFv proteins. Four out of four anti-scFv sera were also shown to recognize the Ig on human tumor cells by flow cytometry analysis.

Single‐cell protein: Current status and future prospects

The consumption of microorganisms by man and animals is not a revolutionary new idea. For thousands of years man has consumed, either intentionally or unintentionally, such products as alcoholic beverages, cheeses, yogurt, and soya sauce and, along with these products, the microbial biomass responsible for their production. The rapid growth rate and high protein content of microbes and their ability to utilize inexpensive feedstocks as sources of carbon and energy for growth have made microorganisms prime candidates for use as human food and animal feed protein supplements. Yet, in spite of their promise, only a limited number of commercial-scale, single-cell protein (SCP) processes have been seen. Recently, with the advent of recombinant DNA technology a rebirth of interest in SCP has resulted. This review analyzes the answers to two questions: (1) how far have we come?; and (2) what impact, if any, will the new biotechnologies have in this field?

Glucans as Immunological Adjuvants

β-1,3-Linked glucopyranose (β-glucan), a major structural component of yeast, fungi, and algae [1], has a wide range of biological activities. Systemic administration of β-glucan (1) provides nonspecific resistance in experimental animals against a variety of pathogenic challenges [2–7]; (2) prolongs the survival time of tumor-bearing animals [8,9]; (3) enhances bone marrow recovery and survival of lethally irradiated mice [10]; (4) promotes wound healing when applied topically [11,12]; and (5) displays an adjuvant effect when coadministered with either bacterial, fungal, protozoan, or viral agents[13–18].

Broad and efficient control of major foodborne pathogenic strains of Escherichia coli by mixtures of plant-produced colicins

Significance Enterohemorrhagic Escherichia coli-contaminated food products are among the leading causes of bacterial enteric infections in the United States and worldwide. Currently, other than thermal inactivation, there are no effective methods to control pathogenic bacteria in food. We investigated colicins, nonantibiotic antimicrobial proteins produced by certain E. coli strains and active against other strains of the species, as potential pathogen control agents. We demonstrate that most colicins can be expressed at high yields in plants and are fully functional. We show that mixtures of colicins applied at low concentrations are highly and broadly active against all major pathogenic E. coli strains of concern for foodborne illness. We propose plant-produced colicins as an inexpensive food treatment for the broad control of pathogenic E. coli strains. Enterohemorrhagic Escherichia coli (EHEC) is one of the leading causes of bacterial enteric infections worldwide, causing ∼100,000 illnesses, 3,000 hospitalizations, and 90 deaths annually in the United States alone. These illnesses have been linked to consumption of contaminated animal products and vegetables. Currently, other than thermal inactivation, there are no effective methods to eliminate pathogenic bacteria in food. Colicins are nonantibiotic antimicrobial proteins, produced by E. coli strains that kill or inhibit the growth of other E. coli strains. Several colicins are highly effective against key EHEC strains. Here we demonstrate very high levels of colicin expression (up to 3 g/kg of fresh biomass) in tobacco and edible plants (spinach and leafy beets) at costs that will allow commercialization. Among the colicins examined, plant-expressed colicin M had the broadest antimicrobial activity against EHEC and complemented the potency of other colicins. A mixture of colicin M and colicin E7 showed very high activity against all major EHEC strains, as defined by the US Department of Agriculture/Food and Drug Administration. Treatments with low (less than 10 mg colicins per L) concentrations reduced the pathogenic bacterial load in broth culture by 2 to over 6 logs depending on the strain. In experiments using meats spiked with E. coli O157:H7, colicins efficiently reduced the population of the pathogen by at least 2 logs. Plant-produced colicins could be effectively used for the broad control of pathogenic E. coli in both plant- and animal-based food products and, in the United States, colicins could be approved using the generally recognized as safe (GRAS) regulatory approval pathway.

Clinical Safety and Immunogenicity of Tumor-Targeted, Plant-Made Id-KLH Conjugate Vaccines for Follicular Lymphoma

We report the first evaluation of plant-made conjugate vaccines for targeted treatment of B-cell follicular lymphoma (FL) in a Phase I safety and immunogenicity clinical study. Each recombinant personalized immunogen consisted of a tumor-derived, plant-produced idiotypic antibody (Ab) hybrid comprising the hypervariable regions of the tumor-associated light and heavy Ab chains, genetically grafted onto a common human IgG1 scaffold. Each immunogen was produced in Nicotiana benthamiana plants using twin magnICON vectors expressing the light and heavy chains of the idiotypic Ab. Each purified Ab was chemically linked to the carrier protein keyhole limpet hemocyanin (KLH) to form a conjugate vaccine. The vaccines were administered to FL patients over a series of ≥6 subcutaneous injections in conjunction with the adjuvant Leukine (GM-CSF). The 27 patients enrolled in the study had previously received non-anti-CD20 cytoreductive therapy followed by ≥4 months of immune recovery prior to first vaccination. Of 11 patients who became evaluable at study conclusion, 82% (9/11) displayed a vaccine-induced, idiotype-specific cellular and/or humoral immune response. No patients showed serious adverse events (SAE) related to vaccination. The fully scalable plant-based manufacturing process yields safe and immunogenic personalized FL vaccines that can be produced within weeks of obtaining patient biopsies.

Plant-made Salmonella bacteriocins salmocins for control of Salmonella pathovars

Salmonella enterica causes an estimated 1 million illnesses in the United States each year, resulting in 19,000 hospitalizations and 380 deaths, and is one of the four major global causes of diarrhoeal diseases. No effective treatments are available to the food industry. Much attention has been given to colicins, natural non-antibiotic proteins of the bacteriocin class, to control the related pathogen Escherichia coli. We searched Salmonella genomic databases for colicin analogues and cloned and expressed in plants five such proteins, which we call salmocins. Among those, SalE1a and SalE1b were found to possess broad antimicrobial activity against all 99 major Salmonella pathovars. Each of the two salmocins also showed remarkably high potency (>106 AU/µg recombinant protein, or >103 higher than colicins) against major pathogenic target strains. Treatment of poultry meat matrices contaminated with seven key pathogenic serovars confirmed salmocin efficacy as a food safety intervention against Salmonella.

Conservation of receptor expression and phagocytic activity of murine macrophages exposed to various ultrasonic regimens in vitro

Murine peritoneal macrophages insonated in vitro at 37 degrees C were assayed for impairment of adhesion to and spreading on glass coverslips, expressions of Fc gamma and C3b receptors, and phagocytosis. Insonation conditions were typical for exposures by B‐mode imaging equipment and approximated the most severe exposures anticipated in use of pulsed Doppler equipment. In no case were the assay results for insonated samples significantly different from those for the sham‐exposed controls.

Conservation of bactericidal activity in ultrasound-exposed murine peritoneal phagocytic cells

Murine peritoneal exudate cells (PEC), predominantly macrophages, were insonated in vitro with burst-mode ultrasound and assayed for their ability to phagocytose and kill Staphylococcus aureus. PEC were exposed at 37 degrees C in rotating tubes to 1-MHz, burst-mode (10 ms on, 10 ms off) ultrasound at 3.7 +/- 0.2 W/cm2 ISPTA (7.4 +/- 0.4 W/cm2 ISPBA) for 150 s. Bactericidal activity was assayed at 1, 2, and 3 h after exposure and subsequent 37 degrees C incubation with the bacteria for 20 min. In these experiments, which comprised 17 treated and 7 sham-treated control samples, there was no significant difference in results between treated and control samples (p > 0.29).