Jeanne B. Kaufman

The Efficacy of a Salmonella typhi Vi Conjugate Vaccine in Two-to-Five-Year-Old Children

BACKGROUND Typhoid fever is common in developing countries. The licensed typhoid vaccines confer only about 70 percent immunity, do not protect young children, and are not used for routine vaccination. A newly devised conjugate of the capsular polysaccharide of Salmonella typhi, Vi, bound to nontoxic recombinant Pseudomonas aeruginosa exotoxin A (rEPA), has enhanced immunogenicity in adults and in children 5 to 14 years old and has elicited a booster response in children 2 to 4 years old. METHODS In a double-blind, randomized trial, we evaluated the safety, immunogenicity, and efficacy of the Vi-rEPA vaccine in children two to five years old in 16 communes in Dong Thap Province, Vietnam. Each of the 11,091 children received two injections six weeks apart of either Vi-rEPA or a saline placebo. Cases of typhoid, diagnosed by the isolation of S. typhi from blood cultures after 3 or more days of fever (a temperature of 37.5 degrees C or higher), were identified by active surveillance over a period of 27 months. We estimated efficacy by comparing the attack rate of typhoid in the vaccine group with that in the placebo group. RESULTS S. typhi was isolated from 4 of the 5525 children who were fully vaccinated with Vi-rEPA and from 47 of the 5566 children who received both injections of placebo (efficacy, 91.5 percent; 95 percent confidence interval, 77.1 to 96.6; P<0.001). Among the 771 children who received only one injection, there was 1 case of typhoid in the vaccine group and 8 cases in the placebo group. Cases were distributed evenly among all age groups and throughout the study period. No serious adverse reactions were observed. In all 36 children studied four weeks after the second injection of the vaccine, levels of serum IgG Vi antibodies had increased by a factor of 10 or more. CONCLUSIONS The Vi-rEPA conjugate typhoid vaccine is safe and immunogenic and has more than 90 percent efficacy in children two to five years old. The antibody responses and the efficacy suggest that this vaccine should be at least as protective in persons who are more than five years old.

Adaptive control strategy for maintaining dissolved oxygen concentration in high density growth of recombinant E. coli.

Large amounts of bacterial biomass are needed for the production of various bacterial products, especially for the production of recombinant proteins from E. coli-carrying recombinant DNA. The method of choice for obtaining the proper amount of the biomass is to grow the bacteria to high density, thereby increasing the production of bacterial biomass per unit of fermentor volume. The high density approach is of special importance when the recombinant proteins are produced after an induction step because the bacteria cannot be reused after the induction. There are several methods for obtaining high density growth of E. coli,14 all of which are based on supplying the growing bacterial culture with its nutrient needs. A nutrient of special importance is oxygen, which has limited solubility in aqueous solutions. Therefore, most of the high density methods specifically address the question of how to supply the needed soluble oxygen. The majority of the high density growth methods are based on a fed-batch approach that controls the carbon source (usually glucose) concentration of the media and thus also the bacterial growth rate and the oxygen c o n s ~ m p t i o n . ~ ~ ~ The main reason for using the fed-batch approach is to prevent inefficient utilization of the carbon source. The fed-batch control strategy is

Use of Streamline chelating for capture and purification of poly-His-tagged recombinant proteins

Expression of recombinant proteins with poly-histidine tags enables their convenient capture and purification using immobilized metal affinity chromatography (IMAC). The 6×His-tagged protein binds to a chelating resin charged with metal ions such as Ni2+, Cu2+ or Zn2+, and can therefore be separated from proteins which have lower, or no, affinity for the resin. Two recombinant proteins, a malaria transmission-blocking vaccine candidate secreted extracellularly by S. cerevisiae and a modified diphtheria toxin produced intracellularly by E. coli, were expressed with 6×His tags and could therefore be purified using IMAC. In an effort to further simplify the initial capture of these proteins, an expanded bed adsorption technique using a chelating resin (Streamline Chelating) was introduced. It was possible to capture the intracellular diphtheria protein from E. coli directly after cell lysis, without prior centrifugation or filtration. The extracellular malaria vaccine candidate was also directly captured from a high cell density yeast culture. Detailed information on the experimental work performed, and the capture processes developed, is provided.

Production of HIV-1 gp120 in Packed-Bed Bioreactor Using the Vaccinia Virus/T7 Expression System

The HeLa cell‐vaccinia virus system is an attractive method for producing recombinant mammalian proteins with proper post‐translation modifications. This approach is especially important for the production of HIV‐1 envelope glycoprotein, gp120, since more than half of its total mass is due to carbohydrates. A recombinant vaccinia virus/T7 RNA polymerase expression system was developed to express and produce large amounts of gp120 tagged with six histidine residues. In this system, the expressed T7 RNA polymerase from one virus drives the transcription of the gp120 encoded in the second virus. During the process development phase, the following parameters were studied: infection time, infection duration, multiplicity of infection, ratio of the two viruses, medium composition, and medium replacement strategy during the infection phase. The chosen production method was based on using the packed‐bed bioreactor. The HeLa cells were immobilized on fibrous disks (Fibra‐Cel) packed in an internal basket positioned in a vertically mixed bioreactor (Celligen Plus), and 25 g of carriers were packed in a 1.6‐L (working volume) reactor. The process included a growth stage followed by a production stage. In the growth stage, the bed was perfused with a serum‐containing medium, allowing the cells to grow to saturation, and in the production stage, done using serum‐free medium, the cells were infected with the two recombinant viruses. The expressed protein was secreted, collected from the culture fluid, and purified. The specific production was found to be between 2 and 3 μg of protein/106 cells, and the volumetric production was around 10 mg/50 g carriers.

The Combined Use of Expanded-Bed Adsorption and Gradient Elution for Capture and Partial Purification of Mutant Diphtheria Toxin (CRM 9) from Corynebacterium diphtheriae

Expanded-bed adsorption (EBA) is a new approach for performing the initial recovery or capture of proteins from various crude feedstocks. The essence of the method is direct adsorption of the desired protein from the unclarified suspension by passing it through a stable expanded bed of the adsorbent. This type of operation replaces centrifugation, clarification, dialysis, and concentration with one simple unit operation. The recovery is done by pumping the feedstock upward on the expanded column and eluting it downward in a step mode from the packed bed. One of the unique properties of the expanded bed is its behavior as a true plug-flow column, which makes it possible to use gradient elution and to achieve better purification in addition to the other benefits. In this work the “traditional” recovery and purification process of the extracellular mutant diphtheria toxin (CRM 9) was replaced with an expanded bed adsorption process in which the protein was eluted using a linear salt gradient in an upward mod...

Hollow Fiber Microfiltration Methods for Recovery of Rat Basophilic Leukemia Cells (RBL—2H3) From Tissue Culture Media

Recovery of mammalian cells from tissue culture media presents significant problems, especially when large volumes of media must be rapidly processed. These cells are characteristically fragile and present at low cell densities; the presence of other large biomolecules in tissue culture media further complicates the separation problem. Recovery of mammalian cells must be done expeditiously to avoid deterioration of the intracellular compounds and membraneous material that are often the desired products. In some situations, preservation of cellular integrity is not critical during recovery operations. As such, low-speed centrifugation can be used for recovery of extracellular products, or intracellular products after cell lysis. This approach, however, is impractical for recovery of intact cells, especially when cellular integrity and viability must be maintained. In most cases, microfiltration techniques are the methods of choice for mammalian cell recovery [I-61. Several different approaches are possible based on tangential-flow filtration. The configurations most widely used are either based on plate-and-frame arrangements or involve hollow fibers. In either case, few guidelines have been developed for mammalian cell recovery from tissue culture media especially when large volumes are involved. This can be, at least in part, attributed to the lack of operating data for such applications and the expense associated with any comprehensive testing program. The purpose here is to discuss some operating experience with the use of hollow fiber tangential flow filters for recovery of rat basophilic leukemia cells (RBG2H3) grown in suspension culture. These cells have histamine-containing granules and receptors for IgE and have been used as a model to study the IgEreceptor interaction. The receptors are apparently involved in the antigen-induced IgE-mediated histamine release. In recovery of these cells from tissue culture media, it is important to avoid cell damage as this will lead to the impaired function of the receptors [ 71. Although this work addresses the recovery of cells from a specific mammalian cell line, the results should be useful for addressing separation and recovery by hollow fiber tangential flow filtration of other mammalian cells grown in suspension.

Continuous Production of the Extracellular Domain of Recombinant Human Ca++ Receptor from HEK 293 Cells Using Novel Serum Free Medium

Large amounts of the extracellular domain of human parathyroid Ca++ receptor were needed for structural studies and clinical evaluation1. The domain was cloned into human embryonic kidney cells (HEK 293) which produced the protein constitutively and secreted it to the media. To obtain the quantity of protein required for evaluation an optimal production procedure was developed. Since the cells cannot grow in suspension, solid support was required. Several solid support arrangements are currently available and these arrangements may be divided into two major groups. The first group consists of supports that allow growth in a stirred tank reactor (i.e microcarriers)2. The second group of supports allow growth in arrangements other than a stirred tank reactor, for example hollow fibers3 and flat sheets. When cells grow on solid supports, medium replacement (perfusion)4 is the method of choice to sustain growth and protein production. Medium replacement operation maintains a relatively constant supply of growth factors such as carbon source, amino acids and vitamins, while lowering the concentration of inhibitory metabolites such as lactic acid and ammonia5. The perfusion may be performed in several ways: if small variations in metabolite concentration affects cellular metabolism and protein biosynthesis, continuous perfusion is the optimal method. However, if cellular metabolism and protein biosynthesis are stable during changes in nutrient concentration and metabolite formation, batch or semi batch replacement may be performed. In each case, the important parameter is the amount of medium needed per cell, per unit time.

High-yield production of diphtheria toxin mutants by high-density culture of C7(β)tox+ strains grown in a non-deferrated medium

A high-density growth approach was utilized to produce mutated diphtheria toxin from two strains of Corynebacterium diphtheria: C7 (β)(tox-201, tox-9) and C7 (β)(tox-107). The cross-reacting mutants (CRM) of the diphtheria toxin are CRM9 and CRM107; both of them carry the mutation in their binding site and, as a result, have 1/300 of the systemic toxicity of the wild-type diphtheria toxin. Since iron inhibits diphtheria toxin production, the traditional approach has been to grow the bacteria in a very low iron concentration. The procedure described here involved the use of a modified, non-deferrated, growth medium that provided fast and high-density growth of the bacteria, and which, when associated with simultaneous depletion of glucose and iron, enhanced the toxin production. Oxygen-enriched air was supplied to enable the bacteria to grow to a cell density giving an absorbance of 70 at 600 nm (15–20 g/l dry weight). The maximum toxin concentration in the culture supernatant was 150 mg/l. The CRM products, which remained stable following microfiltration and ultrafiltration, could be easily purified using a two-step chromatography procedure.

Large-Scale Growth of Bordetella pertussis for Production of Extracellular Toxin

The bacterium, Bordetella pertussis, is responsible for the upper respiratory infection known as whooping cough. The organism secretes several proteins that have been proven to play a role in its virulence.’ One of the proteins, pertussis toxin, is an oligomeric protein that conforms to the A-B model of other bacterial toxins.2 It enzymatically transfers the adenosine diphosphate (ADP) ribosyl moiety of NAD to certain acceptor proteins that are involved in the regulation of cyclic nucleotide m e t a b ~ l i s m . ~ Pertussis toxin is an effective immunogen in human volunteers4 and has been suggested as the basis for the development of a new acellular vaccine.’ To facilitate the testing of whooping cough vaccine, we have undertaken to isolate large amounts of purified pertussis toxin from the supernatant of Bordetella pertussis grown in submerged culture. A major obstacle in this endeavor is the difficulty associated with the submerged growth of this microorganism. The problem lies in the tendency of the bacteria to float on the air bubbles and, as a result, to be removed from the media and stop growing. Therefore, complete elimination of foam is needed. Bordetella pertussis is a slow-growing microorganism with a doubling time of 3.5 to 4 hours; consequently, its oxygen consumption rate is relatively low. Thus, the traditional solution for the foam problem was vortex agitation and surface aeration,6 which eliminated the foam production. This type of operation was not a satisfactory one because only one-third of the available fermentor volume was being used and the dissolved oxygen level became limiting. Recently, we reported on the use of bottom aeration with oxygen-enriched air in an antifoam-containing media.’ This approach