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.     

Inactivated Eastern Equine Encephalomyelitis Vaccines Prepared in Monolayer and Concentrated Suspension Chick Embryo Cultures

Eastern equine encephalomyelitis vaccines were prepared with virus propagated in stationary monolayer cultures and in concentrated suspension cultures of primary chick embryo cells. Virus pools for vaccine preparation were inactivated by three different methods: 0.05% formalin, 41 C heat, and 0.16% beta-propiolactone. Heat-and beta-propiolactone-inactivated vaccines maintained high hemagglutinating titers in the fluid state for at least 10 months, whereas formalin-inactivated vaccines lost their hemagglutinating activity within a few hours after treatment. The hemagglutinin of beta-propiolactone-inactivated virus particles was more dense than the hemagglutinin of the parent virus particles, as determined by sucrose density gradient centrifugation. The increase in density may be due to the degradation or removal of the lipid from the virus envelope. When administered to guinea pigs, all three vaccines stimulated hemagglutination-inhibiting, complement-fixing, and neutralizing antibodies and afforded protection against a live virus challenge. Test results showed that vaccines prepared with virus propagated in concentrated suspension cultures were more immunogenic and stimulated greater serologic responses in guinea pigs than vaccines derived from monolayer-propagated virus. The beta-propiolactone-inactivated vaccine was most protective, the heat-inactivated (41 C) vaccine next, and the formalin-inactivated vaccine least potent.     

Stability-related studies on 17D yellow fever vaccine

Yellow fever (YF) vaccine using the 17D strain of YF attenuated virus has been produced at the Institut Pasteur in Dakar since 1962. Until now, the stabilised YF had an expiry date of utilization of two years from the end of the lot control process under storage at +4 degrees C. We conducted a stability study to assess the three full year validity of this preparation, when correctly stored at +4 degrees C to optimise the conditions of production, storage and availability of such a vaccine. The activity of 19 consecutive batches of vaccines kept for three years at +4 degrees C was compared to that of the same batches that were kept three years at -20 degrees C. Using the in vitro microculture method, we found that three-year storage at +4 degrees C induced a higher loss of activity than storage at -20 degrees C or than the accelerated degradation test of vaccines kept for 14 days at 37 degrees C. Whatever the conditions of storage, in all cases decreases in activity were below the WHO's requirements, i.e., < 1 log PFU/dose, and residual activity of the selected batches was over 1000 mouse LD50 per dose. We demonstrated that the 17D YF vaccine produced in Dakar has a shelf-life of three years and that its required potency was maintained at +4 degrees C, after reconstitution with saline diluent, following three-year storage at +4 degrees C.     

Quantitative Detection of the Foot-And-Mouth Disease Virus Serotype O 146S Antigen for Vaccine Production Using a Double-Antibody Sandwich ELISA and Nonlinear Standard Curves

The efficacy of an inactivated foot-and-mouth disease (FMD) vaccine is mainly dependent on the integrity of the foot-and-mouth disease virus (FMDV) particles. At present, the standard method to quantify the active component, the 146S antigen, of FMD vaccines is sucrose density gradient (SDG) analysis. However, this method is highly operator dependent and difficult to automate. In contrast, the enzyme-linked immunosorbent assay (ELISA) is a time-saving technique that provides greater simplicity and sensitivity. To establish a valid method to detect and quantify the 146S antigen of a serotype O FMD vaccine, a double-antibody sandwich (DAS) ELISA was compared with an SDG analysis. The DAS ELISA was highly correlated with the SDG method (R² = 0.9215, P<0.01). In contrast to the SDG method, the DAS ELISA was rapid, robust, repeatable and highly sensitive, with a minimum quantification limit of 0.06 μg/mL. This method can be used to determine the effective antigen yields in inactivated vaccines and thus represents an alternative for assessing the potency of FMD vaccines in vitro. But it still needs to be prospectively validated by analyzing a new vaccine preparation and determining the proper protective dose followed by an in vivo vaccination-challenge study to confirm the ELISA findings.     

Uptake of minute virus of mice into cultured rodent cells.

The uptake of minute virus of mice into cells in tissue culture was examined biochemically and by electron microscopy. Cell-virus complexes were formed at 4 degrees C, and uptake of virus was followed after the cells were shifted to 37 degrees C. The infectious particles appeared to enter cells at 37 degrees C by a two-step process. The first and rapid phase was measured by the resistance of cell-bound virus to elution by EDTA. The bulk of the bound virus particles became refractory to elution with EDTA within 30 min of incubation at 37 degrees C. The infectious particles became resistant to EDTA elution at the same rate. The second, slower phase of the uptake process was measured by the resistance of infectious particles to neutralization by antiserum. This process was complete within 2 h of incubation at 37 degrees C. During this 2-h period, labeled viral DNA became progressively associated with the nuclear fraction of disrupted cells. The uptake of infectious virus could occur during the G1 phase of the cell cycle and was not an S phase-specific event. The uptake process was not the cause of the S phase dependence of minute virus of mice replication. In electron micrographs, virus absorbed to any area of the cell surface appeared to be taken into the cell by pinocytosis.     

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.     

A群C群脑膜炎球菌结合疫苗稳定性

目的评价A群C群脑膜炎球菌结合疫苗原液和成品的稳定性。方法分别将A群、C群脑膜炎球菌结合疫苗原液及A群C群脑膜炎球菌结合疫苗各选取连续3批,分别放置于37℃、20~25℃和2~8℃3种温度下,在一定的时间取样进行主要项目测定,在关键时间点进行全面检测。结果 A群结合疫苗原液于2~8℃保存9个月,20~25℃保存4周,37℃保存4 d;C群结合疫苗原液于2~8℃保存9个月,20~25℃保存6个月,37℃保存4周;A群C群脑膜炎球菌结合疫苗于2~8℃保存2年3个月,20~25℃保存6个月,37℃可以保存9周;各项检测指标均符合质量标准的要求。结论在2~8℃条件下,A群、C群脑膜炎球菌结合疫苗原液存放6个月,A群C群脑膜炎球菌结合疫苗存放2年,其质量稳定。     

Virus content and effectiveness of foot-and-mouth disease vaccines.

(I) The amount of 22 nm particles in 26 batches of cattle tongue epithelium extract used for the preparation of C-type vaccine was determined with an improved 50% haemolytic and point complement fixation test after fluorocarbon precipitation of non-immunizing 7 nm particles. The total amount of 22 nm and 7 nm particles (alpha GN value) varied considerably from batch to batch, 22 nm components (alpha GF value) showing a maximum 5-fold difference. (ii) Effectiveness of vaccines with known virus content was tested in adult mice challenged with an adapted virus strain. In commercial C-type vaccines the complement-fixing activitiy of 22 nm particles and the potency of the vaccine showed a logarithmic regression (mouse index = -1.43 + 2.27 1g alpha GF).     

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.     

Formation of influenza virus particles lacking hemagglutinin on the viral envelope.

We investigated the intracellular block in the transport of hemagglutinin (HA) and the role of HA in virus particle formation by using temperature-sensitive (ts) mutants (ts134 and ts61S) of influenza virus A/WSN/33. We found that at the nonpermissive temperature (39.5 degrees C), the exit of ts HA from the rough endoplasmic reticulum to the Golgi complex was blocked and that no additional block was apparent in either the exit from the Golgi complex or post-Golgi complex transport. When MDBK cells were infected with these mutant viruses, they produced noninfectious virus particles at 39.5 degrees C. The efficiency of particle formation at 39.5 degrees C was essentially the same for both wild-type (wt) and ts virus-infected cells. When compared with the wt virus produced at either 33 or 39.5 degrees C or the ts virus formed at 33 degrees C, these noninfectious virus particles were lighter in density and lacked spikes on the envelope. However, they contained the full complement of genomic RNA as well as all of the structural polypeptides of influenza virus with the exception of HA. In these spikeless particles, HA could not be detected at the limit of 0.2% of the HA present in wt virions. In contrast, neuraminidase appeared to be present in a twofold excess over the amount present in ts virus formed at 33 degrees C. These observations suggest that the presence of HA is not an obligatory requirement for the assembly and budding of influenza virus particles from infected cells. The implications of these results and the possible role of other viral proteins in influenza virus morphogenesis are discussed.     

Studies on yellow fever vaccine. II--Stability of the reconstituted product

This work evaluated the stability of diluted yellow fever vaccine in order to determine conditions that maintain the minimum of 3 log10 of 17D virus per human dose as required by WHO. The vaccines were held at 0 degrees C or at 37 degrees C and were diluted either with distilled water, with 0.15 M saline or with 0.15 M PBS at pH 5.5, 7.2 and 8.0. In a next step, stabilizer substances such as gelatin and peptone were added to the vaccines. Dilution of the vaccines in distilled water maintained the virus titre for up to three hours at 37 degrees C and this diluent has been adopted for routine use in Brazil.     

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.     

Further field testing of the more heat-stable measles vaccines in Cameroon.

Two of the more heat-stable measles vaccines were field tested in Cameroon. Both maintained the minimum required infectivity titre and the ability to induce seroconversion after storage unreconstituted at 37 degrees C for 14 days. One of the vaccines, studied after reconstitution, maintained its ability to induce seroconversion after reconstitution and storage at 25 degrees C for 48 hours and at 37 degrees C for at least four hours. The increased heat stability of the studied vaccines will not eliminate the need for a well-monitored system of vaccine conservation and distribution but will ease the rigid cold-storage requirements of conventional measles vaccines.     

Mechanisms of inactivation of HSV-2 during storage in frozen and lyophilized forms

The structural integrity of herpes simplex virus 2 (HSV-2) during freezing, thawing, and lyophilization has been studied using scanning and transmission electron microscopy. Viral particles should be thawed quickly from -80 to 37 degrees C to avoid artifacts of thawing. To avoid freezing damage, the virus should be rapidly frozen (>20 K s(-1)) rather than slowly frozen as occurs on the shelf of a lyophilizer (<1 K s(-1)). Fast freezing and thawing allows six cycles of freeze thaw with no loss of viral titer TCID50. Viral particles were characterized using immunogold labeling methods. Freshly thawed virus had 19 +/- 4 polyclonal immunogold particles virus(-1); virus stored at -80 degrees C for at least 4 months had 17 +/- 3 particles virus(-1); virus stored for 1 week at 4 degrees C had 8 +/- 4 particles virus(-1). By bulk lyophilization the number of particles was 4 +/- 4, but by fast freezing and lyophilization the number of gold particles improved to 12 +/- 5. The loss of viral membrane was directly observed, and the in vitro loss was demonstrated to occur through three possible pathways, including (i) simultaneous release of tegument and membrane, (ii) sequential release of membrane and then tegument, and (iii) release like by in vivo infection. The capsids were not further degraded as indicated by the lack of free DNA, which was only released by boiling the viral samples with 1% SDS, followed by a dilution to 0.001% w/v SDS for the real-time PCR reaction.     

Innocuity testing of foot-and-mouth disease vaccines. II. Aziridine-inactivated antigen produced in baby hamster kidney cells

Methods for the testing of preparations of aziridine-inactivated foot-and-mouth disease virus for the absence of infective particles were studied. The system used for virus production, suspension cultures of baby hamster kidney cells, proved to be the most sensitive detection system for traces of infective virus as long as the 146S antigen concentration was below 1 microgram per 10(6) cells. Above this level interference may mask the presence of non-inactivated virus. Thus in a 1-1 suspension culture 1 mg of inactivated 146S antigen equivalent to at least 300 doses of vaccine could be tested. The kinetics of inactivation may be studied by the agar-cell suspension plaque assay which is nearly equal in sensitivity to the method described above. Antigen concentrations at which interference occurred were also estimated for this type of assay. Inactivation of polyethylene glycol-concentrated virus showed 'tailing-off' and such virus preparations should not be used in vaccine production. The data are discussed with reference to the recommendations for innocuity testing in the European Pharmacopoeia.     

Rabies vaccine standardization: International collaborative study for the characterization of the fifth international standard for rabies vaccine

A collaborative study was carried out to establish a replacement for the International Standard for Rabies Vaccine, the stocks of which are exhausted. Three rabies vaccines for human use derived from different rabies virus strains and prepared on different cell culture substrates were compared with the International Standard for Rabies Vaccine using in vivo and in vitro assay methods in a collaborative study involving 14 participants. The proposed fifth International Standard (PISRAV) which was derived from the same virus strain as the present international standard preparation, the Pitman Moore (PM) strain, was found to be approximately twice as potent relative to the International Standard in immunogenicity assays as in antigenicity assays. On the other hand another vaccine, derived from the LEP strain, was considerably more potent in antigenicity assays than in immunogenicity assays. The glycoprotein of the proposed replacement standard measured in antigenicity assays appeared to be stable at +37 degrees C for 245 days, whereas the immunogenicity of the proposed replacement vaccine was sensitive to this heat treatment and the vaccine lost 66% of its immunogenic potency. The results of this study indicate that the NIH protection test should continue to form the primary basis for potency assay of rabies vaccine as glycoprotein content does not appear to correlate with immunogenic potency for different types of vaccine. The vaccine coded PISRAV has been established as the fifth International Standard for Rabies Vaccine and a potency of 16 International Units of Rabies Vaccine (based on the immunogenicity assays) assigned to the contents of each ampoule. Each ampoule has also been assigned a unitage of 10 IU of PM Rabies Virus Glycoprotein and 135 IU of PM Rabies Virus Ribonucleoprotein.     

Effects of short-term cold storage on recovery of proteases from extracellular products of Aeromonas hydrophila.

Three protease-containing fractions were recovered by gel filtration from concentrated crude extracellular products produced by Aeromonas hydrophila grown in a defined medium. The recovery of a heat-stable protease was differentially prevented when the crude preparation was stored for 48 h at -20 degrees C but was unaffected by storage of the crude preparation at either 4 or -70 degrees C. Once fractionated, the heat-stable protease appeared to be unaffected by subsequent storage at 4, -20, or -70 degrees C.     

Effects of short-term cold storage on recovery of proteases from extracellular products of Aeromonas hydrophila

Three protease-containing fractions were recovered by gel filtration from concentrated crude extracellular products produced by Aeromonas hydrophila grown in a defined medium. The recovery of a heat-stable protease was differentially prevented when the crude preparation was stored for 48 h at -20 degrees C but was unaffected by storage of the crude preparation at either 4 or -70 degrees C. Once fractionated, the heat-stable protease appeared to be unaffected by subsequent storage at 4, -20, or -70 degrees C.     

Plant expression,lyophilisation and storage of HBV medium and large surface antigens for a prototype oral vaccine formulation

Current immunisation programmes against hepatitis B virus (HBV) increasingly often involve novel tri-component vaccines containing—together with the small (S-HBsAg)—also medium and large surface antigens of HBV (M- and L-HBsAg). Plants producing all HBsAg proteins can be a source of components for a potential oral ‘triple’ anti-HBV vaccine. The objective of the presented research was to study the potential of M/L-HBsAg expression in leaf tissue and conditions of its processing for a prototype oral vaccine. Tobacco and lettuce carrying M- or L-HBsAg genes and resistant to the herbicide glufosinate were engineered and integration of the transgenes was verified by PCR and Southern hybridizations. M- and L-HBsAg expression was confirmed by Western blot and assayed by ELISA at the level of micrograms per g of fresh weight. The antigens displayed a common S domain and characteristic domains preS2 and preS1 and were assembled into virus-like particles (VLPs). Leaf tissues containing M- and L-HBsAg were lyophilised to produce a starting material of an orally administered vaccine formula. The antigens were distinctly sensitive to freeze-drying conditions and storage temperature, in the aspect of stability of S and preS domains and formation of multimeric particles. Efficiency of lyophilisation and storage depended also on the initial antigen content in plant tissue, yet M-HBsAg appeared to be approximately 1.5–2 times more stable than L-HBsAg. The results of the study provide indications concerning the preparation of two other constituents, next to S-HBsAg, for a plant-derived prototype oral tri-component vaccine against hepatitis B.