Validation of an in vitro assay for the determination of rabies antigen.

The modified antibody binding test, an in vitro method, was validated by a number of studies and was used as a reliable method for in process controls in the manufacture of rabies vaccines for man and animals over the last 6 years. A calculation was made showing the number of laboratory mice which were not needed for animal experiments because these were substituted by the in vitro method mentioned above.

Pitfalls of single radial immunodiffusion techniques used for the quantification of influenza haemagglutinin.

In the case of Tween-ether split vaccines the quantification of haemagglutinin is not achievable by the single radial immunodiffusion test alone. Aggregate formation of solubilized haemagglutinin frequently occurs when the applied detergent is removed and, therefore, a physico-chemical method including an effective disaggregation procedure like SDS treatment in combination with PAGE is recommended.

Quantification of haemagglutinin of influenza Tween-ether split vaccines by immunodiffusion.

The haemagglutinin content of monovalent influenza whole virus and Tween-ether split vaccines derived therefrom, were assayed comparatively using single radial immunodiffusion (SRID, the only test recommended for influenza vaccines by the European Pharmacopoeia Commission), quantitative SDS-polyacrylamide gel electrophoresis and immunization of guinea pigs. If SRID was performed with split vaccines, reduced haemagglutinin values were consistently recorded which were 50-25% of values obtained before disruption of virions. If, however, disruption was conducted in the presence of excess detergent thus preventing aggregate formation of solubilized haemagglutinin, test values comparable to those of whole virus vaccines were obtained. In agreement with these results, immunization experiments revealed that whole virus and the corresponding split vaccines exhibited comparable immunogenicity in guinea pigs. From SDS-polyacrylamide gel electrophoresis and densitometer tracings obtained by scanning the gels after staining with either Coomassie Blue or fluorescein isothiocyanate-labelled concanavalin A it was calculated that about 90% of whole virus HA2 was recovered in Tween-ether split vaccines. From our experiments we conclude that precise quantification of solubilized haemagglutinin is not achievable by the single radial immunodiffusion test alone. Aggregate formation of solubilized haemagglutinin frequently occurs when the applied detergent is removed and, therefore, a physico-chemical method including an effective disaggregation procedure like SDS treatment in combination with PAGE is recommended.

Purified chick embryo cell (PCEC) rabies vaccine for human use. Laboratory data.

A new inactivated rabies vaccine (Purified Chick Embryo Cell vaccine) for use in humans has been developed. The FLURY-LEP C25 strain of rabies virus was adapted to propagate in primary chick embryo cell cultures. The antigen was purified and concentrated by continuous density gradient centrifugation and inactivated by Betapropiolactone. The PCEC-rabies vaccine was tested for innocuity, tolerability and protective capacity in a series of laboratory tests and compared with Human Diploid Cell Strain rabies vaccine (HDCSV) of similar antigenicity. The results of the laboratory tests indicated that this new vaccine was excellently tolerated and that its protective activity met the high standard of HDCS-vaccine, conditions which were imposed on the PCEC-vaccine before clinical trials in man were initiated.

Clinical trials in healthy volunteers with the new purified chick embryo cell rabies vaccine for man.

Behringwerke has developed the new, safe and economical purified chick embryo cell (PCEC) rabies vaccine. Due to the purification by zonal centrifugation the compatibility of this vaccine is excellent. Among 933 vaccinations in 219 healthy volunteers the only side-effect was mild pain at the injection-site in 17 vaccinations (1.7 per cent). The sero-conversion of PCEC rabies vaccine was 100 per cent in the tested healthy volunteers. The kinetics of antibody induction after PCEC rabies vaccine is comparable to antibody induction after HDC rabies vaccine. PCEC rabies vaccine induces cellular immunity as measured in lymphocyte transformation test, but no interferon activity.

A new inactivated tissue culture rabies vaccine for use in man. Evaluation of PCEC-vaccine by laboratory tests.

A new inactivated rabies vaccine (purified chick embryo cell vaccine) has been developed using the Flury LEP-C 25 strain of rabies virus propagated in primary chick embryo cell cultures. The antigen was purified and concentrated by continuous density gradient centrifugation and inactivated by betapropiolactone. This vaccine was tested for innocuity, tolerability and protective capacity in a series of laboratory tests and compared with human diploid cell strain (HDC)-vaccines of similar antigenicity. The results indicated that this new vaccine was excellently tolerated and that its protective activity met the high standard of HDC-vaccine, conditions which were imposed on this vaccine before entering clinical trials in man.

The quantification of the haemagglutinin content of influenza whole virus and Tween-ether split vaccines.

Monovalent whole virus and Tween-ether split vaccines prepared from influenza A/Bangkok, A/Brazil and B/Singapore were assayed for haemagglutinin content using single radial immunodiffusion (SRID), quantitative sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunization of guinea pigs. When SRID was performed with split vaccines, haemagglutinin values were consistently recorded which were in the range of 50 to 25% of the values obtained before disruption of virions. When, however, disruption was conducted in the presence of excess detergent, thus preventing aggregate formation of solubilized haemagglutinin, test values comparable with those of whole virus vaccines were obtained. In agreement with these results, immunization experiments revealed that whole virus and corresponding split vaccines exhibited comparable immunogenicity in guinea pigs. Additionally it could be calculated from SDS-PAGE and densitometer tracings, obtained by scanning the gels after staining with either Coomassie blue or FITC-Con A, that 90 to 95% of whole virus HA2 was recovered in Tween-ether split vaccines. On the basis of these findings we conclude that precise quantification of Tween-ether split vaccines is not possible by the SRID test alone. As aggregate formation of solubilized haemagglutinin occurs, we suggest that either a physico-chemical method including a disaggregation procedure, such as SDS treatment, or immunological evaluation of the original whole virus preparation before disruption of virions should be applied as an additional criterion for quantification of influenza Tween-ether split vaccines.

Purification, concentration and evaluation of rabies virus antigen.

A number of concentration and purification procedures for rabies virus antigen have been developed during the last decades but only ultrafiltration and density gradient centrifugation have been found suitable for large scale production. The antibody binding test developed by Arko, Wiktor and Sikes was modified and was found useful for evaluation of inactivated antigen-concentrates. This test was used as an 'in process' control. Three examples of practical experience are reported and discussed.

The M protein of influenza viruses has no immunizing effect.

Influenza A virus M protein was prepared by electrophoresis in SDS polyacrylamide gel from virus particles which had been pretreated with octylglucoside to remove the surface glycoproteins; M antigens from the influenza virus strains A/Victoria/3/75 (H3N2), A/FPV/Rostock (Hav1N1) and A/chick/Germany/49 (Hav2Neq1) did not protect mice against a lethal challenge infection with the virulent Victoria strain.

On stability of oral poliovirus vaccines.

A stabilizer which prevents oral poliovirus from degradation is described. It contains mainly phosphate buffer 0.3 mol, at a pH of 6.7.

Rabies vaccine HDC: toxicological studies in laboratory animals.

A purified rabies vaccine prepared in human diploid cell cultures (Wistar 38 adapted Pitman-Moore strain) was tested in several animal species with single and repeated administration. Acute toxicity studies were performed in mice, rats, and guinea pigs with s.c. or i.m. injections of at least 1 human dose/animal. The results gave no evidence of toxic effects. Studies on local compatibility were carried out in white rabbits (i.c. and s.c.) and albino guinea pigs (i.m.). During the observation period of 1 or 2 weeks after inoculation no adverse reactions were seen. Pyrogen tests in rabbits with 6 lots of vaccines showed that rabies vaccine is pyrogenfree. Long-term studies with 4 injections into rats and dogs and observation over a 4-month period gave no alterations of hematological parameters and of the urinalysis. The behaviour of the animals was not altered. Short- and long-term experiments in animals showed the innocuity of this type of rabies vaccine.

A purified human diploid cell rabies vaccine.

Continuous-flow isopycnic banding of rabies virus in sucrose gradient was used to purify the antigen for the preparation of a human diploid cell rabies vaccine. Prior to the addition of the stabilizer, the average specific potency of several vaccine batches was 234 antigenic values per 1.0 mg of protein. Further data regarding potency and stability of this vaccine are presented.

[Epidemiological effect of influenza vaccination (author's transl)]. / Epidemiologische Wirkung der Influenza-Impfung.

No influenza-A virus epidemic occurred in the Federal Republic of Germany during 1971-1975. The neighbouring countries, however, reported up to three such epidemics. The vaccines used had differences: contrary to neighbouring countries, in the FRG largely those were used which had mineral adjuvants, and they more frequently had viral subgroups. Comparison between the USA and FRG with respect to influenza death-rates over 20 years revealed a strict correlation from 1956 up to 1965. But since 1966 the death-rate has decreased progressively in the FRG while it has remained unchanged in the USA. Immunisation methods in the two countries have differed since 1966 in that the Public Health Authorities of the two countries have recommended immunisation of different population groups: in the USA it has been only for patients at risk, while in the FRG the rest of the population has also been urged to be immunised. As a result, immunisation rates differ markedly between the two countries. Absence of an influenza epidemic, accompanied by a reduction in death-rate due to influenza, strongly suggests that the two phenomena are the result of a break in the infection chain. This seems to be more successful when both part of the total population and the risk groups are immunised.

Reactogenicity to primary and repeated vaccination with influenza split virus vaccine.

Influenza vaccines (split, adsorbed, low nitrogen) of two different antigen concentrations (commercial production) were administered to 1,111 adult persons of both sexes. Up to five previous vaccinations had been given to the population under observation. Their reactions both local and general were not different in primo-vaccinees or boostered persons. Increasing numbers of previous vaccinations were not reflected by increasing rates of complaints. Disregarding immunization history, the group given the higher concentrated vaccine showed a lower rate of side effects while the lower antigen concentration produced a higher rate. The relevance of the observation that no sensitizing effect occurred in persons with up to five vaccinations may be a characteristic of split adsorbed influenza vaccines of high purity.

A rapid microassay for detecting antibodies against poliovirus based on [14C]thymidine uptake of treated cell cultures.

DNA synthesis of mammalian cells propagated in microplates can easily be measured if cell cultures incubated with [14C]thymidine are harvested on the glass fibre filters by a semiautomatic harvesting technique. Soon after infection with poliovirus, [14C]thymidine uptake of U cells (established, human amniotic cell line) is inhibited. This inhibition can be prevented by previous virus neutralization with antibody. Based on this effect a rapid, precise assay method was set up to determine neutralizing antibody titres against poliovirus. There was a good correlation between titres obtained by this assay and those obtained by 50% end point titrations in cytopathogenic effect inhibition assays.

The use of new polymethylmethacrylate adjuvants for split influenza vaccines.

Split influenza virus vaccines with varying antigen content were adjuvated with polymethylmethacrylate particles, produced by polymerizing monomeric methylmethacrylate in the presence of the subunits, or by addition of the subunits to previously polymerized methacrylate particles. Both adjuvants yielded higher antibody titers than aluminum hydroxide or fluid vaccines. The character of the antigen-adjuvant conjugate of both types of polymer adjuvants is discussed.