Collaborative study for the establishment of a European Phamacopoeia Biological reference preparation for Bordetella pertussis mouse antiserum for serological potency testing of acellular pertussis vaccines.

A collaborative study was organised by the European Directorate For the Quality of Medicines (EDQM) to assess the suitability of a candidate mouse antiserum as a European Pharmacopoeia Biological reference preparation (BRP) for acellular pertussis vaccine potency testing. The candidate antiserum was obtained by immunising mice with a five-component acellular pertussis vaccine: pertussis toxin (PT), filamentous haemagglutinin (FHA), pertactin (PRN) and Fimbrial 2/Fimbrial 3 (Fim 2&3).The study has been divided into two separate phases. Phase I was a pre-qualification study including three laboratories. This phase was aimed at pre-qualifying the candidate BRP (cBRP) and at documenting the impact of differences in the antibody detection methodology enzyme linked immunosorbent assay (ELISA) procedures on results of pertussis antisera calibration versus the currently used standard US standard pertussis antiserum (mouse) Lot 1 (SPAM-1) (United States Food and Drug Administration (USFDA) reference serum) and the cBRP. As no significant difference between the antibody titres determined by using the different ELISA methodologies was found, a large-scale study enrolling 13 laboratories (Phase II) was carried out, each participant performing its in-house methodology. Its aim was to calibrate the cBRP (in terms of the SPAM-1 reference) and to demonstrate its equivalence or superiority to internal references. The study showed that there was no difference in positive sera titres expressed relative to their corresponding internal reference (homologous situation) or the proposed standard (heterologous situation) reference. The cBRP can, therefore, reliably act as replacement for the in-house reference preparations. Further analysis of the outcome of this study enabled to assign to the cBRP a potency of 39, 138, 34 and 56 ELISA unit per millilitre, respectively, to its anti-PT, anti-FHA, anti-PRN and anti-Fim 2&3 antibody contents. The cBRP has been adopted by the European Pharmacopoeia Commission at its June 2000 session as Bordetella pertussis mouse anti-serum Ph Eur. BRP batch 1.     

Determination of antibodies to pertussis toxin in working reference preparations of anti-pertussis sera from various national control laboratories.

Working reference preparations of anti-pertussis sera from various National Control Laboratories were assayed for anti-PT antibodies by standardized ELISA and toxin neutralization (Nt) test. Both the ELISA and Nt tests gave highly reproducible results for various preparations when these preparations were assayed repeatedly on different days. Various working reference preparations were assigned units against the proposed International standard for anti-pertussis serum (JNIH-10) assuming its unitage of 250. Assigning unitage to various preparations would help in comparing results of ELISA and Nt tests for anti-PT antibodies reported in various studies.     

Antibody response to pertussis toxin and filamentous haemagglutinin in NIH mice immunized with the International Standard for Pertussis Vaccine

The antibody response to filamentous haemagglutinin and pertussis toxin was studied in N:NIH mice vaccinated according to the WHO recommendations for potency test with the International Standard for Pertussis Vaccine (ISPV). Some of the vaccinated animals were challenged intracerebrally on day 14. All animals, whether challenged or not, were bled on days 7, 14, 21, 28 and 35 after immunization. The relationship between anti-PT and anti-FHA antibodies measured by ELISA and protection from intracerebral challenge was examined. All those mice with anti-PT titres on day 14 higher than 43 EU/ml survived challenge. No relationship was found between anti-FHA antibodies and survival. Anti-PT titres on day 14 below 43 EU/ml were related to the days of survival after challenge; a linear regression curve of y = 13 + 2.4x, with a correlation coefficient r = 0.61 was found. Anti-PT antibodies seem to play an important role in protection when animals are challenged intracerebrally, as is the case in the standard potency test for pertussis vaccine.     

Preparation and Standardization of U.S. Standard Pertussis Vaccine, Lot No. 11

The Center for Biologics Evaluation and Research within the U.S. Food and Drug Administration has prepared a new U.S. Standard Pertussis Vaccine. Whole cell pertussis vaccine concentrate was diluted in 5% (w/v) lactose and lyophilized. The preparation was tested for toxicity, sterility heterogeneity and residual moisture. Based on data from an international sollaborative study involving 11 laboratories, the potency was estimated in relation to the U.S. Master Standard Pertussis Vaccine, Lot 4 and the International Standard for Pertussis Vaccine, Lot 2. The potency of the preparation was defined to be 90 units per ampoule. When reconstituted and stored according to instructions, no significant change in potency was observed in the 14 days following reconstitution. This material was shown to be suitable for a pertussis vaccine standard and accordingly it was designated as U.S. Standard Pertussis Vaccine, Lot 11 on March 22, 1994.     

Standardization of U.S. Reference Rh0 (D) Immune Globulin by quantitative automated hemagglutination

An automated hemagglutination procedure was used to assess the relative potency of US Reference Rh0 (D) Immune Globulin, Lot 3, with respect to the International Reference Preparation, WHO Anti-D immunoglobulin, Lot 68/419. A value of 300 international units (IU) of anti-D per ampoule has been assigned to Lot 68/419. In 25 assays, the mean value for Lot 3 was 820 IU anti-D per milliliter when tested in parallel with Lot 68/419.     

Genetic variability among archival cultures of Salmonella typhimurium

The existence in our laboratory of over 10000 Salmonella typhimurium LT2 cultures sealed in agar stab vials for 33-46 years offers an opportunity for evolutionary and mutational studies. In each of 77 vials examined, 10(3)-10(5) colony forming units per vial were recovered (less than 0.01% of the original population) even after decades of undisturbed storage. Considerable genetic variability was observed in these populations. Three genetic variables, chromosome fragment size as determined by pulsed-field gel electrophoresis, extensive mutational reversions from nutritional auxotrophy to prototrophy, and differences in protein content as assayed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, were measured.     

In vitro induction of antigen specific antibody synthesis and proliferation of T lymphocytes with acellular pertussis vaccines, pertussis toxin and filamentous haemagglutinin in humans

The in vitro response of human B- and T-lymphocytes to the acellular vaccines JNIH-6 (containing pertussis toxoid and filamentous hemagglutinin), and JNIH-7 (containing pertussis toxoid), and to the purified components JNIH-4 (filamentous hemagglutinin) and JNIH-5 (pertussis toxin) was investigated. Pertussis toxoid and filamentous hemagglutinin induced specific Ig synthesis in vitro in lymphocytes obtained from convalescent pertussis patients as target cells. The antigen-dependent Ig production was demonstrated in lymphocyte culture supernatants by ELISA techniques and by a chinese hamster ovary cell toxin neutralization assay. Particularly with JNIH-4, -6 and -7, high antibody titers were obtained. At optimal antigen concentrations a marked lymphocyte blast transformation was found in lymphocyte cultures from whooping cough patients, but not in cultures of lymphocytes obtained from healthy volunteers. At high concentrations native pertussis toxin as well as the B oligomer (S2-5) of the toxin induced a strong proliferation of patient as well as control lymphocytes, indicating non-specific mitogenic activity. At lower concentrations lymphocyte blast transformation was seen in patient cultures only, which indicates an antigen-specific T-cell response. The A protomer (S1), dimer 1 (S2 + 4) and dimer 2 (S3 + 4) induced proliferation of patient lymphocytes, which demonstrates the presence of T-cell epitopes on these peptides. The in vitro B-cell response and the lymphocyte blast transformation assay are both useful tools for estimating the potency of acellular pertussis vaccines in man. Spontaneously acquired and vaccine induced immunity to Bordetella pertussis can be investigated at the level of B- and T-lymphocytes.     

Loss of vacuum in rubber stoppered vials stored in a liquid nitrogen vapor phase freezer

A total of 1885 rubber stoppered 3-ml vials (NS-33 and NS-51 glass (expansion sizes), sealed under vacuum, were subjected to the ultracold temperatures of a liquid nitrogen vapor phase (LNVP) freezer for 16.5 hr and tested for vacuum loss after equilibration back to 25 °C. The presence of a vacuum in each vial was checked before and after freezing. Results with two lots of vials per glass expansion size showed positive or no negative pressure rather than a vacuum in 46.25% (NS-33), 98.25% (NS-33), 64.72% (NS-51), and 99.38% (NS-51) of the vials. Apparently, the fit or slight misfit of the stopper into the vial opening and the hardness of the butyl rubber stopper at ultracold temperatures enhance the chances of leakage around the stopper area under extremely cold temperatures. Storage of rubber stoppered vials containing lyophilized products sealed under vacuum in LNVP freezers is not advisable.     

Protection of histones isolated from elastase-treated mouse epidermis

Small glass shell vials (12 × 35 mm minivials), containing 2.0 ml of a dioxane-based scintillation solution plus a ¹⁴C-labeled sample, were placed in a conventional glass, 20-ml count vial and assayed in a scintillation spectrometer. Statistical comparison of counts recorded from ¹⁴C samples prepared both in the minivial system and conventional 20-ml count vials indicated that the two systems were equivalent with sample volumes of 10 and 100 μliters (1600-cpm solution) and 10 μliters (60-cpm solution). Conventional 20-ml glass or plastic count vials were both acceptable as containers for the minivials.There were no significant differences in the counts from samples in minivials placed on-center and off-center in the container vial. Cost per sample was reduced from 24.8 cents (conventional glass vials) to 4.7 cents (minivial system).     

An economical system for liquid scintillation counting

Small glass shell vials (12 × 35 mm minivials), containing 2.0 ml of a dioxane-based scintillation solution plus a ¹⁴C-labeled sample, were placed in a conventional glass, 20-ml count vial and assayed in a scintillation spectrometer. Statistical comparison of counts recorded from ¹⁴C samples prepared both in the minivial system and conventional 20-ml count vials indicated that the two systems were equivalent with sample volumes of 10 and 100 μliters (1600-cpm solution) and 10 μliters (60-cpm solution). Conventional 20-ml glass or plastic count vials were both acceptable as containers for the minivials.There were no significant differences in the counts from samples in minivials placed on-center and off-center in the container vial. Cost per sample was reduced from 24.8 cents (conventional glass vials) to 4.7 cents (minivial system).     

Third International Standard for pertussis vaccine: international confirmation study of activity of British Standard for pertussis vaccine, coded 66/303.

The first British Standard (BS) (Code 66/303) for pertussis whole cell vaccine was prepared from the same suspension of Bordetella pertussis as the now exhausted Second International Standard for Pertussis Vaccine (2ndIS). The BS and the 2ndIS were compared and calibrated in a previous international study. This report describes a small international study, which included the BS, the 1stIS and the 2ndIS so that the present relationship of these preparations to one another could be assessed. The results of this study show that the relationship of the BS to the 1stIS is consistent with its established unitage of 46 IU per ampoule. The results further show that the potency of the BS is broadly consistent with that of the 2ndIS and that the BS has not lost activity relative to the 2ndIS (from which it was previously found to be indistinguishable). Based on its original calibration and supported by the results of this present study, the BS has been established as the Third International Standard for Pertussis Vaccine, with an assigned unitage of 46 IU per ampoule.     

Increased Parasitization of Aphids on Trap Plants Alongside Vials Releasing Synthetic Aphid Sex Pheromone and Effective Range of the Pheromone

Aphid-infested cereal trap plants were used to detect the effects of aphid sex pheromone components on aphid parasitoid activity in arable field margins. The presence of aphid sex pheromones significantly increased parasitization levels by aphid parasitoids on the plants. By placing plants alongside and at varying distances away from a pheromone-releasing vial, the technique was used to measure the distance over which a point source of aphid sex pheromone could increase parasitization levels. Pheromone-releasing vials significantly increased parasitization by the generalist parasitoid Praon volucre on plants adjacent to vials and on plants placed 20 cm away. When the distance between pheromone-releasing vials and aphid-infested plants was increased to 1 m, parasitization by P. volucre was increased only on the plant adjacent to the vial, whereas parasitization by the specialist parasitoid Aphidius rhopalosiphi was also increased on plants placed 1 m away. This indicates a possible difference between the parasitoids in their foraging behaviour in response to semiochemical cues during host selection. When the experiment was repeated with some trap plants placed 3 m away from the pheromone-releasing vial, parasitization was again concentrated on plants directly alongside the vial, but only P. volucre appeared to be active in the field at the time of this experiment, so the effect on A. rhopalosiphi could not be assessed. The results are encouraging for the prospects of using aphid sex pheromones to manipulate parasitoids in order to improve aphid population control.     

Testing the sterility of injection preparations of penicillin series antibiotics]

The efficiency of 3 variants of the method for determination of microbial flora was compared on the injection preparation of potassium benzylpenicillin artificially infected with Staph. aureus 209P and the spores of Bac. subtilis ATCC 6633 in different doses and with different amounts of the preparation in the vials. The procedure of the preparation dissolution in the vial with the thioglycol medium containing penicillinase proved to be most effective. The microbe detection amounted to 100 per cent. The procedure was less labour- and time-consuming since addition of penicillinase to each vial with the thioglycol medium was excluded. The risk of the medium occasional infection with microbial flora during the assay was decreased.     

Virtual nitrogen tank to monitor frozen cell stocks

We developed a program to facilitate the monitoring of biological samples (cell lines, sera, etc.) that are stored in liquid nitrogen containers. The program consists of a "virtual" container in which scientists can store their samples and a program that records the location of each sample, cell characteristics, storage dates, names of the manipulators and much more. Additional comments and a photograph can be associated with each vial, allowing for reliable tracking of samples. Vials can then be identified according to any parameter of interest to the scientist, including associated comments. Once identified, the program visually presents the location of these vials, which simplifies retrieving them from the real container. The program records the thawing of vials, along with the date and the name of the operator. Any academic laboratory requesting this standalone program will be granted a free license for its use.     

Development and comparisons of efficient gas-cultivation systems for anaerobic carbon monoxide-utilizing microorganisms

We describe a system for the cultivation of gaseous substrate utilizing microorganisms that overcomes some of the limitations of fixed volume culture vessels and the costs associated with sparging. Cali-5-Bond gas-sampling bag was used as the culture vessel. The bags contain approximately six times more mass of CO than the 40 mL vials at 1 atm of pressure and performed equally to the 40 mL vials in terms of their ability to maintain the composition of the gas over extended incubation times. Experiments using Clostridium ljungdahlii and CO as the sole carbon and energy source in both the gas sampling bag cultivation system and the traditional vial system demonstrated that this culture had a 15x increase in optical density in 24 h of incubation. The gas-sampling bags offer a viable alternative to gas sparging while overcoming the limitations of fixed volume culture vessels.     

Impact of Natural Variations in Freeze-Drying Parameters on Product Temperature History: Application of Quasi Steady-State Heat and Mass Transfer and Simple Statistics

Inter- and intra-batch variability in heat and mass transfer during the drying phase of lyophilization is well recognized. Heat transfer variability between individual vials in the same batch arise from both different positions in the vial array and from variations in the bottom contour of the vials, both effects contributing roughly equally to variations in the effective heat transfer coefficient of the vials, K_v. Both effects can be measured in the laboratory, and variations in average K_v values as a function of vial position in the array for lab and production can be calculated by use of the simple steady-state heat and mass transfer theory. Typically, in the laboratory dryer, vials on the edge of the array, “edge vials,” run 2–4°C warmer than “center vials,” but differences between laboratory and manufacturing temperatures are modest. The variability in mass transfer can be assigned to major variations in ice nucleation temperature (both intra-batch and inter-batch), including major differences between laboratory and manufacturing. The net effect of all random variations, for each class of vial, can be evaluated by a simple statistical model-propagation of error, which then allows prediction of the distribution in product temperatures and drying times, and therefore prediction of percent of vials dry and percent of vials collapsed and proximity to the edge of failure for a given process. Good agreement between theoretical and experimentally determined maximum temperatures in primary drying and percent collapsed product demonstrates the calculations have useful accuracy.     

Behavioral and reproductive responses of female opossums to volatile and nonvolatile components of male suprasternal gland secretion

Female gray short-tailed opossums (Monodelphis domestica) lack an estrous cycle and are induced into estrus by exposure to a pheromone in male scent marks. Behavioral and physiological responses of females to the volatile and nonvolatile components of scent marks were examined in two experiments. Young females (n = 9) were tested prior to and during their first estrus for behavioral responses to scent marks, collected on a 7-ml glass vial rubbed over the suprasternal gland of a mature male. The response to volatile components of the scent mark, recorded when marked and unmarked vials were covered with a perforated shield, was compared to the response to these vials when unshielded. Estrous females nuzzled the shields over marked vials (55.8 ± 8.5 nuzzles/10 min) more than the shielded clean vial (10.9 ± 2.4) (P < 0.05); a similar response was observed in anestrous females. Nuzzling of unshielded, scent-marked vials was higher (P < 0.05) during anestrus than in the same females when in estrus. The role of nonvolatile pheromones in reproductive activation was tested in adult females (n = 11) exposed for up to 14 days to a shielded, marked vial or to an unshielded, marked vial in a crossover design. All females exposed to unshielded vials expressed estrus, and 10 copulated. Only 2 females expressed estrus (significantly fewer, P < 0.05), when exposed to shielded marked vials, and neither copulated. These results demonstrate that females detect and respond behaviorally to both volatile and nonvolatile components of male suprasternal gland secretion, but the estrus-inducing pheromone in these secretions is nonvolatile.     

Prediction of growth of Bacillus megaterium spores as affected by gamma radiation dose and spore load

AIMS: To provide data on the interaction of radiation dose (x1) and microbial contamination load (x2), as predictor variables, on the percentage of vials exhibiting growth of Bacillus megaterium spores (y). METHODS AND RESULTS: The influence of a wide range of spore loads (1-50 000 spores of B. megaterium vial-1) and gamma radiation doses (0.2-10 kGy) on the contaminated samples was determined. Each contamination load was studied by adding the specified number of spores to 100 vials containing nutrient broth and exposing them to various doses of gamma radiation. Curves representing the number of contaminated vials against the dose of radiation were sigmoidal in shape and the data showed an indirect relationship. Data were analysed by regression analysis which revealed a significant correlation (R2=0.85). The relationship among the tested variables is exponential and can be described by the following equation: y = 1 - (1 - e(0.0173x(1)))(x(2)) It was also estimated that, for each increase of 1 in the number of spores per vial, there is an increase of 1 in the number of contaminated vials. CONCLUSION: The two variables (x1 and x2) have great influence on the radiation sterilization efficiency and the proposed mathematical model is valid for the prediction of this efficiency. SIGNIFICANCE AND IMPACT OF THE STUDY: The results of the present investigation can be of useful industrial application and can help to set acceptance and rejection limits for the production of materials vulnerable to microbial contamination.     

Interactions among a Fimbrin, a Capping Protein, and an Actin-depolymerizing Factor in Organization of the Fission Yeast Actin Cytoskeleton

We report studies of the fission yeast fimbrin-like protein Fim1, which contains two EF-hand domains and two actin-binding domains (ABD1 and ABD2). Fim1 is a component of both F-actin patches and the F-actin ring, but not of F-actin cables. Fim1 cross-links F-actin in vitro, but a Fim1 protein lacking either EF-hand domains (Fim1A12) or both the EF-hand domains and ABD1 (Fim1A2) has no actin cross-linking activity. Overexpression of Fim1 induced the formation of F-actin patches throughout the cell cortex, whereas the F-actin patches disappear in cells overexpressing Fim1A12 or Fim1A2. Thus, the actin cross-linking activity of Fim1 is probably important for the formation of F-actin patches. The overexpression of Fim1 also excluded the actin-depolymerizing factor Adf1 from the F-actin patches and inhibited the turnover of actin in these structures. Thus, Fim1 may function in stabilizing the F-actin patches. We also isolated the gene encoding Acp1, a subunit of the heterodimeric F-actin capping protein. fim1 acp1 double null cells showed more severe defects in the organization of the actin cytoskeleton than those seen in each single mutant. Thus, Fim1 and Acp1 may function in a similar manner in the organization of the actin cytoskeleton. Finally, genetic studies suggested that Fim1 may function in cytokinesis in cooperation with Cdc15 (PSTPIP) and Rng2 (IQGAP), respectively.     

Purification and immunogenicity of a recombinant Bordetella pertussis S1S3FHA fusion protein expressed by Streptococcus gordonii

Acellular pertussis vaccines typically consist of antigens isolated from Bordetella pertussis, and pertussis toxin (PT) and filamentous hemagglutinin (FHA) are two prominent components. One of the disadvantages of a multiple-component vaccine is the cost associated with the production of the individual components. In this study, we constructed an in-frame fusion protein consisting of PT fragments (179 amino acids of PT subunit S1 and 180 amino acids of PT subunit S3) and a 456-amino-acid type I domain of FHA. The fusion protein was expressed by the commensal oral bacterium Streptococcus gordonii. The fusion protein was secreted into the culture medium as an expected 155-kDa protein, which was recognized by a polyclonal anti-PT antibody, a monoclonal anti-S1 antibody, and a monoclonal anti-FHA antibody. The fusion protein was purified from the culture supernatant by affinity and gel permeation chromatography. The immunogenicity of the purified fusion protein was assessed in BALB/c mice by performing parenteral and mucosal immunization experiments. When given parenterally, the fusion protein elicited a very strong antibody titer against the FHA type I domain, a moderate titer against native FHA, and a weak titer against PT. When given mucosally, it elicited a systemic response and a mucosal response to FHA and PT. In Western blots, the immune sera recognized the S1, S3, and S2 subunits of PT. These data collectively indicate that fragments of the pertussis vaccine components can be expressed in a single fusion protein by S. gordonii and that the fusion protein is immunogenic. This multivalent fusion protein approach may be used in designing a new generation of acellular pertussis vaccines.