Glutaraldehyde inactivated pertussis vaccine: a less histamine sensitizing vaccine

The effects of different inactivating agents on the biological activity of the histamine sensitization factor of Bordetella pertussis toxin were examined. The agents were used for inactivation in the preparation of whole cell pertussis suspension. The histamine sensitizing activity was reduced to 36.9-13.3% by treatment with glutaraldehyde, to about 50% by treatment with formaldehyde and by the acetone-II treatment, relative to the reduction by heat treatment. Treatment with thimerosal and the acetone-I treatment did not reduce the histamine sensitizing activity as the 50% histamine sensitizing doses of the heat inactivated pertussis preparation, the thimerosal inactivated pertussis preparation and the acetone-I treated pertussis preparation were very similar. Glutaraldehyde has thus been found to be a better inactivating agent for the preparation of a safe pertussis suspension as it considerably reduced the histamine sensitizing activity of pertussis toxin.     

Determination of the epinephrine-refractory hypoglycaemic activity of whole-cell pertussis vaccine in mice

A new assay method has been developed for the quantitative estimation of the inhibitory effect of pertussis vaccine on epinephrine-induced hyperglycaemia in mice. The statistical analysis of the assay was based on logarithm-transformed estimates of the blood glucose levels. The method was sufficiently sensitive to detect the activity of 0.004 millilitre of commercial combined diphtheria-tetanus-whole cell pertussis vaccine. The estimated common variance was as small as 0.0034 and the assay was highly reproducible. Among commercial vaccines there was a significant difference in activity. The activity of a stock pertussis vaccine was inactivated by 5 mM glutaraldehyde at 37 degrees C for 30 min, but resisted treatment with 40 mM formaldehyde at 37 degrees C for 5 days. The extent of inactivation with the chemicals was calculated by a parallel line assay as the activity relative to that of untreated control pertussis vaccine.     

The detoxification of Bordetella pertussis with glutaraldehyde

To improve the whole-cell pertussis vaccine we have studied the inactivation of the biological properties characteristic of Bordetella pertussis phase I bacteria, i.e. histamine-sensitizing, lymphocytosis-promoting and mouse protective activities, by treating a concentrated bacterial suspension with various concentrations of glutaraldehyde. Under the experimental conditions, treatment with 10 mM glutaraldehyde at 37 degrees C for 30 min resulted in a marked reduction of the toxic activities without grossly diminishing the protective potency. Further tests were performed on the stability of the protective potency, on the agglutinin production in mice, and on the freedom from abnormal toxicity in guinea-pigs.     

吸附无细胞百白破(DPTa)的稳定性试验

采用半综合液体培养基（含０．０５％ＭｅβＣＤ）,大罐培养百日咳Ⅰ相ＣＳ菌制备的吸附无细胞百日咳菌苗、白喉、破伤风类毒素混合制剂（ＤＰＴａ）置４－８℃分别保存一年、保存两年,测三种抗原成分的效力及毒性,以及将该制剂于３７℃分别放置三周、三个月,测定有无毒性逆转。结果表明,该制剂质量稳定,无毒性逆转     

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.     

Antigens of Bordetella pertussis. IV. Effect of heat, merthiolate, and formaldehyde on histamine-sensitizing factor and protective activity of soluble extracts from Bordetella pertussi.s

Munoz, J. (Rocky Mountain Laboratory, Hamilton, Mont.), and B. M. Hestekin. Antigens of Bordetella pertussis. IV. Effect of heat, Merthiolate, and formaldehyde on histamine-sensitizing factor and protective activity of soluble extracts from Bordetella pertussis. J. Bacteriol. 91:2175-2179. 1966.-Both histamine-sensitizing and protective activities of soluble preparations from Bordetella pertussis cells are destroyed by heating at 80 C for 0.5 hr. The histamine-sensitizing activity appeared to be more susceptible to inactivation by heat than the protective activity. Formaldehyde in a final concentration of 0.5% rapidly diminished the histamine-sensitizing ability of saline extract (SE) held at 37 C. The protective activity was clearly more resistant to inactivation by formaldehyde at similar temperature. The inactivating action of formaldehyde was slower when the concentration of SE was increased or when the mixture was kept at 2 to 5 C. Merthiolate in a final concentration of 1:10,000 had no demonstrable deleterious effects on either protective or histamine-sensitizing activity of SE.     

Thermostability of Japanese encephalitis vaccine produced in India.

Different batches of bulk vaccine, final bulk at in-process level, finished freeze-dried and reconstituted Japanese encephalitis vaccine were assayed for their stability at temperatures of 22, 37 and 40 degrees C. After ultrazonal purification of 50 times concentrated brain suspension, JE Bulk vaccine was found to be stable for up to 2 years at 4 degrees C, however, the percentage loss in potency (log 10 N antibody titre) after 2.5 years was 24%. Three-times concentrated final bulk showed rapid deterioration by the fourth week at 37 and 40 degrees C. Freeze-dried JE vaccine maintained at 22 degrees C for 28 weeks did not show perceptible deterioration. At 37 degrees C, the same vaccine started showing deterioration (14%) after 8 weeks whereas at 40 degrees C the loss of potency was 24% after 8 weeks. The freeze-dried vaccine was found to be stable for up to 2 weeks duration at 40 degrees C.     

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。     

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.     

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.     

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 development of a new generation of pertussis vaccine

The results of the weight gain test on mice have shown that acellular pertussis vaccine is less toxic than the pertussis component of adsorbed diphtheria-pertussis-tetanus (DPT) vaccine due to a lower content of endotoxin in the acellular vaccine; but the leukocytosis-promoting and histamine-sensitizing activities of JNIH-6 and adsorbed DPT vaccines are indicative of incomplete inactivation of Bordetella pertussis toxin. The content of incompletely inactivated B. pertussis toxin is practically the same in both preparations, constituting 1/100-1/200 of the calculated initial activity. For this reason, the use of the new pertussis vaccine also involves a risk of development of serious postvaccinal reactions and/or complications caused by this toxin. Search for the optimum method of inactivation of B. pertussis main toxin should be continued. As shown by the enzyme immunoassay, acellular pertussis vaccine used in the same immunizing dose as adsorbed DPT vaccine induces a more intensive immune response to hemagglutinin and B. pertussis toxin. This is due to higher residual toxicity of the corpuscular component of adsorbed DPT vaccine. Induction of antibodies to B. pertussis toxin has been shown to decrease in response to injection of acellular pertussis vaccine containing a certain residual amount of incompletely inactivated B. pertussis toxin.     

The evaluation of the mutagenic activity of pertussis vaccinal preparations]

Two vaccine preparations obtained from Bordetella pertussis, whole-cell vaccine constituting one of the components of adsorbed DPT vaccine and acellular vaccine, were tested for mutagenicity. The doses of the preparations covered the range 1-100 ED50. Ames' test and the metaphase analysis of marrow cells of C57BL/6J mice were used. The acellular preparation was also tested on thymectomized mice, taking into consideration chromosomal aberrations in marrow metaphases. Whole-cell and acellular pertussis vaccines did not induce mutations in Salmonella typhimurium and chromosomal aberrations in mouse marrow cells.     

Acellular pertussis vaccines: evaluation of reversion in a nude mouse model

An animal model has been developed to assess the safety of acellular pertussis vaccines in terms of reversion to toxicity. Adsorbed pertussis toxoid preparations, alone or combined in a DTP formulation, were administered to nude mice intraperitoneally. In parallel, groups of positive and negative control mice received pertussis toxin and buffer, respectively. The circulating white blood cells of the animals were monitored for 28 days. Mice immunized with glutaraldehyde toxoid preparations did not develop a lymphocytosis during the observation period, whereas mice immunized with an experimental formalin pertussis toxoid vaccine exhibited a high lymphocytosis six days after vaccine administration, demonstrating, in this model, a reversion of the toxoid. The nude mouse model thus appears to reveal the in-vivo reversion of pertussis toxoids and could be included in the quality control panel for the assessment of the safety of acellular pertussis vaccine.     

Stabilization of restriction endonuclease Bam HI by cross-linking reagents

Bacillus amyloliquefaciens H produces a restriction endonuclease enzyme BamHl which is heat labile even at low temperatures. Studies were conducted to enhance thermal stability of BamHl using cross-linking reagents, namely, glutaraldehyde, dimethyl adipimidate (DMA), dimethyl suberimidate (DMS), and dimethyl 3,3'-dithiobispropionimidate (DTBP). Reaction with glutaraldehyde did not result in a preparation with enhanced thermal stability. However, the DMA-, DMS-, and DTBP-cross-linked preparations of BamHI exhibited significant improvement in thermal stability. Studies on thermal denaturation of the cross-linked enzyme preparations revealed that these do not follow a true first-order kinetics A possible deactivation scheme has been proposed in which the enzyme has been envisaged to go through a fully active but more susceptible transient state which, on prolonged heat exposure, exhibits a first-order decay kinetics. At 35 degrees C, which is close to the optimum reaction temperature of 37 degrees C for BamHl activity, the half-line of DMA-, DMS-, and DTBP-cross-linked preparations were 4.0, 5.25, and 5.5 h, respectively, whereas the native enzyme exhibited a half-line of 1.2 h only. The apparent values of deactivation rate constants for native, DMA-, DMS-, and DTBP-cross-linked BamHl were 1.13, 0.39, 0.29, and 0.26 h(-1), respectively, at the same temperature, and the apparent values of activation energies for denaturation of native, DMA-, DMS-, and DTBP-cross-linked BamHl were 2.63, 5.24, 6.55, and 9.2 kcal/mol, respectively. The DTBP-cross-linked Bam HI was, therefore, the best heat-stable preparation among those tested. The unusually low values of activation energies for denaturation of Bam Hl represent their highly thermolabile nature compared to other commonly encountered enzymes such as trypsin, having activation energies of more than 40 kcal/mol for their denaturation.     

Acetone-treated pertussis vaccine--a potent and safer new pertussis vaccine

A vaccine was prepared from the growth of Bordetella pertussis by repeated treatment with acetone. The vaccine has been designated as acetone-treated pertussis vaccine (ATPV). A total of ten batches of ATPV were prepared, five each from B. pertussis strains 134 and 509. These strains are routinely employed at this Institute for the production of conventional whole-cell pertussis vaccine (WCPV) for blending in diphtheria-pertussis-tetanus vaccine. The mouse protective and histamine sensitizing activities of ATPV and WCPV were compared. The ATPV showed 1.5- to 2-fold higher potency than the WCPV. The histamine sensitizing activity of ATPV was much reduced compared with that of the WCPV. No appreciable difference was observed in the results of a mouse weight-gain tests between the ATPV and WCPV. The details of the preparation of ATPV have been described. Because of higher potency and reduced histamine sensitizing activity, the ATPV may prove more acceptable in immunization programmes against pertussis, even in countries where WCPV is unpopular due to its suspected reactogenicity.     

Cold chain monitoring of OPV at transit levels in India: correlation of VVM and potency status

We have conducted a study to analyze monitoring of the cold chain of 674 OPV field samples collected at four different levels of vaccine distribution viz., immunization clinics, district stores, hospitals and Primary Health Centers (PHC) from states of Uttar Pradesh, Madhya Pradesh, and Delhi. The study design included: collection and scoring of vaccine vial monitor (VVM) status of the samples and testing for total oral polio virus concentration (TOPV) by standard WHO protocol. Ten samples each were exposed to 25 degrees C and 37 degrees C, and 10 samples as controls were kept at -20 degrees C. VVM were scored daily till they attained grade 4 and each sample was subsequently subjected to potency testing for individual polio serotypes 1, 2 and 3, and TOPV.Of the 674 samples tested it was observed that: samples from immunization clinics and district stores had an acceptable VVM score of grade 1 and 2; however the probable risk that a sub potent vaccine could have been administered was 2.15%. In 2.5% samples received from district stores vaccine had a VVM score of grade 3 (i.e., discard point), although vaccine when tested was found to be potent (i.e., leading to the vaccine wastage). With exposure to higher temperatures, VVM changed score to grade 2 and 3 when the vaccine was kept at 25 degrees C/37 degrees C, and the titres of individual serotypes 1, 2 and 3 and TOPV were beyond the acceptable limits.Important observations at the different levels of vaccine distribution network and correlation of VVM and potency status of OPV are discussed in the paper which will be of help to the EPI program managers at different transit levels.     

Inactivation of simian rotavirus SA11 by chlorine, chlorine dioxide, and monochloramine.

The kinetics of inactivation of simian rotavirus SA11 by chlorine, chlorine dioxide, and monochloramine were studied at 5 degrees C with a purified preparation of single virions and a preparation of cell-associated virions. Inactivation of the virus preparations with chlorine and chlorine dioxide was studied at pH 6 and 10. The monochloramine studies were done at pH 8. With 0.5 mg of chlorine per liter at pH 6, more than 4 logs (99.99%) of the single virions were inactivated in less than 15 s. Both virus preparations were inactivated more rapidly at pH 6 than at pH 10. With chlorine dioxide, however, the opposite was true. Both virus preparations were inactivated more rapidly at pH 10 than at pH 6. With 0.5 mg of chlorine dioxide per liter at pH 10, more than 4 logs of the single-virus preparation were inactivated in less than 15 s. The cell-associated virus was more resistant to inactivation by the three disinfectants than was the preparation of single virions. Chlorine and chlorine dioxide, each at a concentration of 0.5 mg/liter and at pH 6 and 10, respectively, inactivated 99% of both virus preparations within 4 min. Monochloramine at a concentration of 10 mg/liter and at pH 8 required more than 6 h for the same amount of inactivation.     

Protective effects of nasal immunization in mice with various kinds of inactivated Sendai virus vaccines

The immunoprophylactic effects of nasal vaccination with 13 different kinds of inactivated Sendai virus vaccines were compared by contact exposure to infector mice. Efficacies of the vaccines were evaluated on the basis of the presence of virus-infected cells by immunofluorescent examination of the entire respiratory tract, including the nasal mucosa. A single or double inoculations of B-propiolactone (0.5%)-vaccine promoted the infection in the respiratory tract, particularly in the nasal mucosa, whereas three inoculations of B-propiolactone (0.2%)-vaccine provided considerable protection throughout the respiratory tract with only slight development of serum HI titer. Formalin (0.1%)-vaccine and UV irradiated-vaccine strongly protected the nasal mucosa from infection, but did not sufficiently safeguard the lower respiratory tract even with three vaccinations despite adequate development of serum antibody. Nearly complete protection of the entire respiratory tract was induced with six to eight inoculations of a vaccine treated excessively with both UV rays and 1% formalin, without significant development of serum antibody. Out of eight thermal vaccines, five (inactivated at 23 C, 30 C, 37 C and 7 C, and 30 C and 7 C) provided strong protection against infection when inoculated three times. The others inactivated at higher temperatures (37 C, 50 C, or 60 C) were not so protective. High serum HI titers developed, on the whole, with the drop in the temperature required for inactivating the virus. In eight immune mouse groups in which infection was strongly suppressed in the entire respiratory tract, most of the mice harbored less than 50 viral antigen-positive cells in their nasal mucosa in the postexposure period. The number of the cells was assumed to be a useful criterion for evaluation of vaccine efficacy.     

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).