Stability of cholera and typhoid vaccines

Fluid plain and adsorbed and freeze-dried cholera and tyhpoid vaccines of different composition were examined for thermostability by potency testing (by active mouse protection tests) after exposure to 37 degrees C for 1, 2, 3, 4, 8 and 12 weeks. Loss of potency was evaluated by comparison with samples stored at 4 degrees C. The fluid plain cholera vaccine remained fully potent for 1--3 weeks, the adsorbed vaccine for at least 4 weeks. The fluid typhoid vaccines showed greater thermosensitivity than the fluid plain cholera vaccine. The freeze-dried cholera and typhoid vaccines were both very stable, retaining fully potency after at least 12 weeks' exposure to 37 degrees C. It should be emphasized that the above results apply exclusively to vaccines prepared by the methods used by the authors and formulated to identical compositions.     

The quality standardization of a national vaccine against yellow fever

One of the commercial lots of yellow fever vaccine has been attested as the National Branch Standard (NBS) of yellow fever vaccine. This NBS has been studied in all tests required by the regulations for standard vaccines. The NBS has been found to meet the necessary requirements in all its characteristics. The study of the thermostability of the NBS at temperatures of 4-10 degrees C, 20-22 degrees C, 37 degrees C during storage for 24 hours to 1 year has revealed the rapid loss of the infectious capacity of the virus at the above temperatures and its high stability during storage at -20 degrees C. Thus, the NBS has been found to retain the required level of immunizing potency for 3 months at a temperature of 4-10 degrees C, for 1 month at 20-22 degrees C and for 2 weeks (the term of observation) at 37 degrees C. The heat resistance of the NBS of yellow fever vaccine corresponds to the WHO requirements. The newly developed NBS has been used as the standard preparation for controlling 27 lots of commercial yellow fever vaccine.     

The effects of different inactivating agents on the potency, toxicity and stability of pertussis vaccine

The effect of heat (56 degrees C for 10 min), formaldehyde (0.1% at 37 degrees for 24h), glutaraldehyde (0.05% at room temperature for 10 min), thimerosal (0.02% at 37 degrees C for 24h), acetone-I (three treatments at room temperature) and acetone-II (three treatments at room temperature and fourth treatment at 37 degrees C), when used as inactivating agents in the preparation of pertussis suspension, was studied with regard to potency, toxicity and stability. Five batches each of Bordetella pertussis strains 134 and 509 were used for the study. The thimerosal inactivated pertussis (TIP) preparation was 1.5-2 times more potent than the heat inactivated pertussis (HIP) preparation. The potency values of the formaldehyde inactivated pertussis (TIP) and glutaraldehyde inactivated pertussis (GIP) preparations were similar to those of the HIP preparation, while the potencies of the acetone-I treated pertussis (A(I)TP) and acetone-II treated pertussis (A(II)TP) preparations were about half those of the HIP preparation. The FIP preparation was the least toxic showing maximum weight gain in the mouse weight-gain test (MGWT), while the TIP preparation did not pass the MWGT. The weight gains shown the GIP, A(I)TP and A(II)TP preparations were greater than those shown by the HIP preparation. The potency of pertussis component in the adsorbed diphtheria-pertussis-tetanus (DPT) vaccine was stable at 4-8 degrees C and 25 degrees C for three months for all types of pertussis vaccine. There was about 54-65% loss in the potency of the samples after three months at 35 degrees C. The inactivating agents used in the manufacture of pertussis preparations had no effect on the stability of the vaccine.     

冻干人用狂犬病纯化疫苗稳定剂的筛选

取同一批人用狂犬病纯化疫苗纯化液,分别加入筛选出的A、B、C和D稳定剂,按已确定的适宜冻干曲线进行冻干后,分别检测其安全、效力、外观、水分等,效力结果显示,使用A、B、D稳定剂的制品在37℃放置3个月,效价降低了48%～60%;使用C稳定剂的制品在37℃放置3个月效价仅下降了16.1%。表明C稳定剂为冻干人用狂犬病纯化疫苗适宜的稳定剂配方。     

Relation between storage temperature and fertilizing ability of freeze-dried mouse spermatozoa

The advantage of freeze-dried mouse spermatozoa is that samples can be stored in the refrigerator (+4 degrees C). Moreover, the storage of freeze-dried spermatozoa at ambient temperature would permit spermatozoa to be shipped easily and at low cost around the world. To examine the influence of the storage temperature on freeze-dried spermatozoa, we assessed the fertilizing ability of spermatozoa stored at different temperatures. Cauda epididymal spermatozoa were freeze-dried in buffer consisting of 50 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, 50 mM NaCl, and 10 mM Tris-HCl (pH 8.0). Samples of freeze-dried spermatozoa were stored at -70, -20, +4, or +24 degrees C for periods of 1 week and 1, 3, and 5 months. Sperm chromosomes were maintained well at -70, -20, and + 4 degrees C for 5 months, and oocytes fertilized with these spermatozoa developed to normal offspring. Moreover, the chromosomal integrity of spermatozoa stored at -20 or + 4 degrees C did not decrease even after 17 months. In contrast, the chromosomes of spermatozoa stored at +24 degrees C were maintained well for 1 month but became considerably degraded after 3 months. In addition, to investigate the cause of deterioration of sperm chromosomes during storage at +24 degrees C, spermatozoa were freeze-dried in buffer containing DNase I. The chromosomes of spermatozoa freeze-dried with 1 or 0.2 units/ml of DNase I, 100% or 72%, respectively, exhibited chromosomal abnormalities. Our findings suggest that freeze-dried spermatozoa can be stored long-term with stability at +4 degrees C, and the suppression of nucleases present in the buffer or spermatozoa during storage led to the achievement of long-term storage of freeze-dried spermatozoa.     

Long-term storage of DNA-free RNA for use in vaccine studies

RNA replicons represent potential vaccine delivery vehicles, but are considered too unstable for such use. This study examined the recovery, integrity and function of in vitro transcribed replicon RNA encoding hepatitis C virus (HCV) proteins. To remove residual template DNA, the RNA was digested with TURBO DNase followed by RNeasy DNase set and purified through an RNeasy column. The RNA was freeze-dried in distilled water or trehalose, stored under nitrogen gas for up to 10 months and analyzed at different time points. The recovery of RNA stored at < or = 4 degrees C that was freeze-dried in distilled water varied between 66% to zero of that recovered from RNA freeze-dried in 10% trehalose, a figure that depended on the duration of storage. In contrast, the recovery of the RNA stored in trehalose was consistently high for all time points. After recovery, both RNAs were translationally competent and expressed high levels of proteins after transfection, although the level of expression from the trehalose-stored RNA was consistently higher. Thus the addition of trehalose permitted stable storage of functional RNA at 4 degrees C for up to 10 months and this permits the development of RNA vaccines, even in developing countries where only minimum storage conditions (e.g., 4 degrees C) can be achieved.     

The standardization of the reverse indirect haemagglutination test for the assay of the viral antigen of Japanese encephalitis vaccine

The reverse indirect haemagglutination (RIHA) test has been standardized for the assay of the viral antigen of purified Japanese encephalitis (JE) vaccine. Glutaraldehyde fixed sheep erythrocytes were sensitized with ammonium sulphate purified antibodies to JE vaccine raised in mice. The sensitivity of the erythrocytes fell to about one hundredth of the initial sensitivity in the first two days after preparation. After initial loss in sensitivity the stability of the cells became stabilized and the cells retained their titre for one year at 4-8 degrees C. The initial loss in sensitivity was not reduced by storing the cells at -70 degrees C, but after freeze drying the sensitized cells with a stabilizer one day after their preparation the cells retained their sensitivity. The RIHA test has been found to be a highly reproducible and sensitive method for detecting viral antigen in 5-10 ng of protein nitrogen. The sensitivity of the test was affected by the origins of the erythrocytes, i.e. from the different sheep from which they were drawn. To obtain results more rapidly, goose erythrocytes were used in place of sheep erythrocytes and the sensitized goose erythrocytes gave RIHA results in only 40 min.     

Standardization of an enzyme immunoassay for the in vitro potency assay of inactivated tissue culture rabies vaccines: determination of the rabies virus glycoprotein with polyclonal antisera

A non-competitive enzyme-linked immunoassay (ELISA) has been standardized to supplement the in vivo potency test used for the quality control of inactivated tissue culture vaccines against rabies. The essentials of the ELISA were: fixation of the virus in different dilutions of vaccine on the surface of microtitre plates; testing of the reference and up to six test vaccines on one plate; incubation with polyclonal antisera to rabies virus glycoprotein containing an excess of antibody; further incubation with a species-specific anti-IgG coupled to peroxidase; a final incubation with a substrate. The incubation periods were 1 h, 1 h and 30 min both at +37 degrees C. The relative potency determinations were made graphically or by a computer using a parallel line bioassay in which the potencies of the vaccines of unknown potency were tested against the reference preparation on a single microtitre plate. Under these conditions inactivated rabies vaccines of different types (virus strains, cell substrates, inactivation and concentration procedures) were tested for potency. Furthermore, it was possible with this in vitro method to assay adjuvanted vaccines, in process samples such as tissue culture supernatants with live or inactivated rabies virus, concentrates, and vaccines undergoing thermal stability tests. The rabies glycoprotein antigen-antibody reaction was highly specific according to the results and the glycoprotein content was measured quantitatively. The potency determined by the in vitro ELISA correlated with the in vivo NIH protection potency test. The lower limit of detection of the ELISA was 0.015 IU/ml. Quantitative antigen determination was possible with both homologous and heterologous antisera to rabies virus glycoprotein when vaccines of the same virus strain were tested. When the potencies of vaccines of different virus strain specificity were calculated, it was necessary to take into account the strain-specific antigenicity. Even so vaccines of high potency were found to give a stronger reaction with a heterologous serum than did weak vaccines with a homologous antiserum. Stability tests made on inactivated tissue culture vaccines such as vaccine from the human diploid cell strain (HDCS), from purified chicken embryo cell (PCEC) or from purified Vero cell rabies vaccine (PVRV), showed high stability of the glycoprotein antigen even after four months of storage at +37 degrees C or 24 h at +56 degrees C, provided that the vaccines were stored in a lyophilized state. The antigenicity of liquid vaccines was inactivated after a few hours at +56 degrees C. For tropical areas, therefore, only lyophilized vaccines should be considered.     

Pyrolysis gas chromatography of Streptococcus faecalis: effect of cultural conditions on pyrochromatograms

Streptococcus faecalis 251 was cultured under a variety of different growth conditions, i.e. incubation for 24 or 70 h; at 22 degrees, 37 degrees or 45 degrees C; on blood agar or on MacConkey agar plates; aerobically or anaerobically. Replicate cultures were analysed by pyrolysis-gas liquid chromatography on columns of 7% Carbowax 2 M, TPA on Chromosorb G (AW-DMCS, 80-100 mesh) programmed from 40 degrees C up to 170 degrees C. Culture grown under identical conditions resulted in reproducible pyrochromatograms which were only slightly modified by change in temperature of growth from 37 degrees to 45 degrees C, or length of growth from 24 to 70 h, or growth on MacConkey agar instead of blood agar. Growth under anaerobic conditions resulted in a modified pyrochromatogram; while growth at only 22 degrees C resulted in a major change in pyrochromatogram.     

Improvement in the long-term stability of freeze-dried mouse spermatozoa by adding of a chelating agent

This study demonstrates that a small amount of chelating agent in the freeze-drying solution is necessary to prevent the deterioration of spermatozoa during freeze-drying and subsequent preservation at 4 degrees C. We freeze-dried mouse epididymal spermatozoa in the solutions containing Tris-HCl and ethylenediaminetetraacetic acid (EDTA) as a chelating agent. Spermatozoa stored for various times up to 1 year at 4 degrees C were injected intracytoplasmically into individual oocytes, and the normality of chromosomes in fertilized oocytes was analyzed. In addition, embryos derived from freeze-dried spermatozoa were transferred into recipients to determine their developmental ability. Chromosomes were maintained well when spermatozoa were freeze-dried in a solution containing 10 mM Tris-HCl and 1mM EDTA (73%), and 57% of embryos developed to term. Of embryos derived from spermatozoa stored for 1 year, 65% developed into live offspring. On the other hand, when spermatozoa were freeze-dried in a solution containing 10 mM Tris-HCl and 0 or 50 mM EDTA, spermatozoa that maintained karyotypically normal chromosomes were 64% or 22%, and only 16% or 3% of embryos were developed to term, respectively. This finding suggested that mouse spermatozoa can be freeze-dried in a simple solution containing the same composition as that used to preserve extracted DNA.     

Inactivated Eastern Equine Encephalomyelitis Vaccine Propagated in Rolling-Bottle Cultures of Chick Embryo Cells

A method was developed for the production of Eastern equine encephalomyelitis vaccine from virus grown in rolling-bottle cultures (840 cm(2) growth area) of chick embryo cells. The PE-6 strain of virus was propagated in chick embryo cell roller cultures maintained on serum-free medium 199 containing 0.25% human serum albumin and antibiotics (MM). A multiplicity of inoculum of 0.005 yielded acceptable titers of virus at a convenient harvest time of 18 to 24 hr and reduced the carry-over of extraneous material from the virus seed. Growth studies in which 100, 200, or 300 ml of MM was used showed that use of 300 ml of MM offered two advantages: (i) cytopathic effects were less at the 18- to 24-hr harvest time, thereby decreasing cellular material in the final product, and (ii) total virus yield was not substantially reduced, thus permitting large-scale production of virus for further processing. Studies on formalin inactivation at 37 C indicated that the virus was inactivated by 0.05% formalin within 12 to 16 hr and with 0.1% formalin within 6 to 8 hr. Antigen extinction tests in hamsters revealed excellent potency (e.g., median-effective-dose values of 0.069 to 0.012 ml) for both fluid and freeze-dried products. The advantages of the roller-bottle technique in vaccine production are discussed.     

Effects of preparative procedures on the volume and content of resealed red cell ghosts

The effects of variations in preparative procedures on the volume and content of resealed red cell ghosts have been investigated. Following hypotonic lysis at 0 degrees C, and after a variable delay time (td), concentrated buffer was added to restore isotonicity; resealing was then induced by incubation at 37 degrees C for one hour. Using this procedure, both the resealed ghost volume and the residual hemoglobin (Hb) content decreased for increasing td. If ghosts were maintained at 0 degree C (i.e., no 37 degrees C incubation), they remained nearly spherical until isotonicity was restored. Their volume then fell abruptly, but subsequently increased toward an intermediate level. The fall in volume was greater and the final level achieved was smaller for longer delay times. At 0 degree C, return to isotonicity also halted the otherwise gradual loss of residual Hb from unsealed ghosts. In addition, ghosts with internal osmolality of 40 to 300 mosmol/kg were prepared by adding different amounts of concentrated buffer before resealing for one hour at 37 degrees C. Under these conditions, the final ghost volume was inversely related to the resealing osmolality (i.e., lower osmolality yielded a larger volume). Ghost volume also increased, along with Hb content, if the quantity or concentration of the red cell suspension added to the lysing medium was increased. We conclude that resealed ghost volume is influenced by the ratio of lysate to resealing medium osmolality and by the colloid osmotic pressure of the residual ghost Hb. These data indicate methods by which ghosts with desired characteristics can be prepared, and have potential application for studies of ghost mechanical and biophysical behavior.     

Relative Stability of Pertussis Vaccine Preserved with Merthiolate, Benzethonium Chloride, or the Parabens

When stored at 4 C, or heated at 22 or 35 C followed by storage at 4 C, the potency of pertussis vaccines preserved with Merthiolate was more stable than the potency of vaccines preserved with benzethonium chloride or the parabens (methyl- and propyl-p-hydroxybenzoate). Without preservative, potency was more stable than in the presence of benzethonium chloride or the parabens, but less stable than when Merthiolate was present. The histamine-sensitizing factor of the vaccines likewise decreased with the loss of potency. The deleterious effect of benzethonium chloride and the absence of the stabilizing effect of Merthiolate were contributing factors, if not the sole cause, for the instability of pertussis vaccine in quadruple antigen vaccine (diphtheria and tetanus toxoids and pertussis and poliomyelitis vaccines).     

Stability of T-2 mycotoxin in aqueous media.

3H-labeled T-2 mycotoxin was dissolved in various aqueous media and stored for up to 3 weeks at 4, 22, and 37 degrees C. At periods ranging from 1 to 21 days, aliquots were assayed by thin-layer chromatography. Thin-layer chromatography plates were scanned for breakdown products by use of a radioisotope scanner, and breakdown products were identified based on their comigration with known standards. Results indicated that T-2 toxin was more stable in tissue culture medium with or without serum, than in Hanks balanced salt solution (HBSS), at all temperatures. The metabolites HT-2, T-2 triol, and T-2 tetraol were detected as early as 1 day (HBSS; 37 degrees C) and as late as 3 weeks (HBSS; 4 degrees C) after storage. Stability of the toxin in aqueous media decreased with increasing temperature.     

Stability of T-2 mycotoxin in aqueous media

3H-labeled T-2 mycotoxin was dissolved in various aqueous media and stored for up to 3 weeks at 4, 22, and 37 degrees C. At periods ranging from 1 to 21 days, aliquots were assayed by thin-layer chromatography. Thin-layer chromatography plates were scanned for breakdown products by use of a radioisotope scanner, and breakdown products were identified based on their comigration with known standards. Results indicated that T-2 toxin was more stable in tissue culture medium with or without serum, than in Hanks balanced salt solution (HBSS), at all temperatures. The metabolites HT-2, T-2 triol, and T-2 tetraol were detected as early as 1 day (HBSS; 37 degrees C) and as late as 3 weeks (HBSS; 4 degrees C) after storage. Stability of the toxin in aqueous media decreased with increasing temperature.     

Stability of 17D Yellow Fever Virus Vaccine Using Different Stabilizers

To optimize the thermostability of lyophilized 17D vaccine, the authors investigated parameters important for the freeze-drying process. Six different stabilizers with different sugars and amino acids were analysed in a freeze-thaw cycle for their crystallization characteristics and their stabilizing effect under thermal treatment conditions of 37 degrees C for 28 days. This test indicated that three out of six stabilizers (B, C, F) kept the vaccine significantly more stable than the three others (A, D, E). Under storing conditions of 4 degrees C over 96 days stabilizers A, B and C produced the lowest decrease in titre of about 10% in contrast to stabilizers D, E and F with a higher decrease in infectivity titre. Analysing the stability of the 17D vaccine using five different reconstitution solutions, we found that 90% D2O shows the best stabilizing effect under thermal treatment of 37 degrees C up to 24 h.     

The effect of culture conditions on hydrophobic properties of Pseudomonas aeruginosa

The cell surface hydrophobicity (CSH) plays an important role in a adhesion of bacteria on solid surfaces. CSH of 62 Pseudomonas aeruginosa strains isolated from humans and different animals was assessed using the ammonium sulfate salt aggregation test. Bacteria were grown for 24 h and 48 h at a room temperature (22 degrees C) and 37 degrees C on enrichment broth and agar (Biomed) and tryptic soy agar (Difco). The hydrophobic properties of the Pseudomonas aeruginosa strains were depended on the temperature, time of the culture of bacteria and the kind of media. CSH properties were most frequently expressed when the analyzed strains were cultured in enrichment broth. In a such conditions Pseudomonas aeruginosa strains were more hydrophobic when grown at 22 degrees C (94% after 24 h and 87% after 48%) than those at 37 degrees C (72% after 24 h and 71% after 48 h). Among strains cultured in tryptic soy agar at 37 degrees C, 48% after 24 h and 75% after 48 h were autoaggregating, representing very strong hydrophobic properties.     

Effect of HEPES buffer systems upon the pH, growth and survival of Mycoplasma mycoides subsp. mycoides small colony (MmmSC) vaccine cultures

The use of a buffer system based on N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] (HEPES), in conjunction with standard Gourlay's culture medium was investigated for the growth and maintenance of Mycoplasma mycoides subsp. mycoides SC vaccine strain T(1)44. When the initial pH of the culture medium was adjusted to 8.0, 0.075 M HEPES-NaOH was found to be sufficient to prevent the pH falling below 7.1 at any stage during the growth cycle, even in the presence of 0.5% glucose. Compared to growth in standard unbuffered Gourlay's medium, the final culture titre was found to be one log(10) higher, at 10(11) colour changing units (CCU) per ml, and considerably extended culture survival was observed at 37 degrees C. The titre remained above 10(10) CCU ml(-1) for 4 days, and above 10(8) CCU ml(-1) in excess of 1 month. After 4 month's storage at 37 degrees C the titre had fallen to 5x10(4) CCU ml(-1). In contrast, no viable bacteria could be detected in standard unbuffered medium 3 days after the onset of stationary phase, at which point the pH had dropped to 5.4. No significant difference in growth rate between the two media was observed. Adoption of a HEPES-NaOH buffer system by African vaccine manufacturers should require minimal changes to current formulations and procedures, and should enhance both the final titre and thermostability of freeze-dried and liquid broth vaccines against contagious bovine pleuropneumonia (CBPP).     

Consequences of herpes simplex virus type 2 and human cell interaction at supraoptimal temperatures.

The consequences of herpes simplex virus type 2 (HSV-2) and human embryonic fibroblast cell interaction at different temperatures (37, 40, and 42 degrees C) were investigated. Incubation at 37 or 40 degrees C was permissive for HSV-2 inhibition of host DNA synthesis, induction of virus-specific DNA replication, and infectious virus production. The amount of [methyl-3H]thymidine incorporated into viral DNA and the final yield of new infectious virus were significantly reduced at 40 degrees C compared to 37 degrees C. At 42 degrees C, detectable virus-specific DNA synthesis was totally blocked. Maximum stimulation of host cell DNA synthesis at 42 degrees C was measured after a multiplicity of infection of 0.5 to 1.0 PFU/cell. By autoradiography, data indicated that HSV-2 stimulates host cell chromosomal DNA synthesis. Stimulation of thymidine kinase activity with thermostability properties in common with a virus enzyme was detected during the first 24 h of infection at 42 degrees C, after 24 h the enhanced thymidine kinase activity had properties in common with host cell isozymes. The data obtained during this investigation indicated that stimulation of host cell DNA synthesis does not require viral DNA synthesis.     

Optimising the viability during storage of freeze-dried cell preparations of Campylobacter jejuni

Freeze-dried cultures of Campylobacter jejuni are used in the food and microbiological industry for reference materials and culture collections. However, C. jejuni is very susceptible to damage during freeze-drying and subsequent storage and it would be useful to have longer-lasting cultures. The survival of C. jejuni during freeze-drying and subsequent storage was investigated with the aim of optimising survival. C. jejuni was freeze-dried using cultures of different age (24-120 h), various lyoprotectants (10% phytone peptone, proteose peptone, peptonized milk, trehalose, soytone and sorbitol), various storage (air, nitrogen and vacuum) and re-hydration (media, temperature and time) conditions. One-day-old cultures had significantly greater survival after freeze-drying than older cultures. The addition of trehalose to inositol broth as a lyoprotectant resulted in almost 2 log(10) increase in survival after 2 months storage at 4 degrees C. Storage in a vacuum atmosphere and re-hydration in inositol broth at 37 degrees C increased recovery by 1-2 log(10) survival compared to re-hydration in maximal recovery diluent (MRD) after storage at 4 degrees C. Survival during storage was optimal when a one-day-old culture was freeze-dried in inositol broth plus 10% (w/v) trehalose, stored under vacuum at 4 degrees C and re-hydrated at the same incubation temperature (37 degrees C) in inositol broth for 30 min. The results demonstrate that the survival of freeze-dried cells of C. jejuni during storage can be significantly increased by optimising the culture age, the lyoprotectant, and the storage and re-hydration conditions. The logarithmic rate of loss of viability (K) followed very well an inverse dependence on the absolute temperature, i.e., the Arrhenius rate law. Extrapolation of the results to a more typical storage temperature (4 degrees C) predicted a very low K value of 1.5 x 10(-3). These results will be useful to the development of improved reference materials and samples held in culture collections.