Poliovirus crystals within the endoplasmic reticulum of endothelial and mononuclear cells in the monkey spinal cord.

The lumbar motor columns of a cynomolgus monkey that had become tetraplegic after experimental infection with a highly virulent strain of type 3 poliovirus were examined by electron microscopy. Crystalline aggregates of poliovirus occurred within the endoplasmic reticulum of endothelial cells as well as of mononuclear inflammatory cells. This finding suggests that the endoplasmic reticulum might be much more involved in poliovirus multiplication than has been previously supposed.

Studies on the attenuation of the Sabin type 3 oral polio vaccine.

The genetic basis of attenuation of the poliovirus type 3 vaccine strain P3/Leon 12a1b has been investigated by comparing the nucleotide sequence of this strain with that of its neurovirulent progenitor P3/Leon/37 and by constructing recombinants between these two viruses using infectious cDNAs. Preliminary results suggest that attenuation is caused by just two point mutations, one occurring in the 5' non-coding region and the other causing an amino acid change in coat protein VP3.

Studies on the characteristics of poliovirus type 3. II. Characteristics of "hot" clones.

Markers d, IST, EA1(OH)3, rct (at sub- and supraoptimal temperatures) and neurovirulence were determined for clones isolated from two lots (S2 and S3) of vaccines containing poliovirus strain Leon 12a1b. Changes of markers rct, d and neurovirulence were observed in several clones isolated from S2 vaccine. No changes were observed in IST and EA1(OH)3 markers.

Studies on the characteristics of Poliovirus type 3. III. Strain characteristics after passage in man.

The markers d, IST, EA1(OH)3 and rct at sub- and supraoptimal temperatures as well as neurovirulence (PMic) for monkeys was determined for strains isolated from children vaccinated with Leon 12a1b vaccine, their contacts and from paralytic cases. The strains were isolated at early and late phases of excretion. The changes concerned mainly rct determined at supraoptimal temperatures, d and PMic markers, especially in strains isolated at the late phase of excretion. The passage through the human alimentary tract did not change such markers as IST and EA1(OH)3. Some degree of correlation was observed between the rct 40.3, d and PMic markers.

Availability of vaccines of an assured quality.

In establishing standards of quality for vaccines used in vaccination programmes, it is necessary to protect the recipients without demanding unnecessarily high specifications and standards of purity. Such standards reduce profitability, jeopardizing further vaccine supplies and future research, and also diminish the probability of vaccine manufacture outside the industrialized countries. Funds will be needed to encourage research and local manufacture, and internationally accepted procedures of quality assurance must be established.

On the role of the World Health Organization in the development of Sabin vaccines.

The World Health Organization has played a major part in the development, surveillance and distribution of attenuated poliovirus vaccines. At a time when most of the United States' efforts concerned the introduction of Salk-type vaccines, WHO initiated studies that set standards and permitted the large scale trials of Sabin and other attenuated vaccines. Independent expert review validated studies in countries such as the U.S.S.R. which helped lead to the adoption of Sabin vaccines for worldwide usage. Surveillance by WHO Collaborative Centres established the safety of Sabin vaccines and identified issues of reversion primarily concerning type 3 viruses, initiating studies which have elucidated the molecular mechanism of reversion. Efforts by the Biological Unit of the World Health Organization have ensured worldwide acceptable standards to control the safety and manufacture of vaccines. Revision of neurovirulence test methods has ensured adequate safety testing of vaccine lots, reduced the costs of such studies and the numbers of primates needed, important ethical and conservation issues. Finally, the World Health Organization has played a major part in the worldwide supply of vaccines at affordable prices and has been the repository of, and had the exclusive license, to Sabin vaccines since 1972.

Response of children to a single dose of oral or inactivated polio vaccine.

Approximately 200 children aged 8, 12 and 16 years with poliovirus neutralising antibody titres of less than 1/45 to at least one type were given a single dose of oral or inactivated polio vaccine. Almost all children given IPV responded with high levels of neutralising antibody to all 3 types. The response to the oral vaccine was less dramatic. The relationship between pre-existing antibody and the further response and the implications for susceptibility to reinfection will be discussed.

The standardization of infectivity titrations of poliovaccines--a WHO collaborative study.

The reproducibility of a method for infectivity titrations of live poliovaccines using microtitre plates was investigated in a WHO collaborative study involving eight laboratories. The large variation (up to 100-fold) in estimates of infectivity observed between laboratories using their local methods of assay was reduced to no more than fourfold when a common method was used. However, expressing infectivities relative to those of the monovalent reference viruses improved the agreement between the laboratories irrespective of the titration method employed. On the basis of these results, WHO adopted the common method used in the study as its recommended method for poliovirus titrations and established the preparations studied as international reference materials for the infectivity titrations of live poliovaccines.

A W.H.O. collaborative study of in vitro and in vivo methods for the assay of yellow fever vaccines.

An international collaborative study was carried out to compare the plaque assay in monolayer cell cultures with the mouse assay for the potency testing of yellow fever vaccines. Twelve laboratories assayed four preparations of yellow fever virus by both methods. Differences were found between estimates of infectivities obtained by different laboratories using the plaque assay. There was also large variation between the estimates of mouse LD50 obtained by different laboratories. However, expressing potencies of the preparations relative to a common preparation greatly reduced the variation between laboratories for both the plaque and the mouse tests; it also resulted in remarkably close agreement between the methods.

Conservation in vivo of protease cleavage sites in antigenic sites of poliovirus.

Polioviruses possess three major antigenic sites which have been located chemically and structurally on the particle. One of these sites, designated site 1, is strongly immunodominant for serotype 3, but highly immunorecessive for type 1. We report that monoclonal antibodies directed against site 1 of type 1 poliovirus may be isolated by an altered route of immunization of the donor mice. Site 1 is shown to be highly variable for type 1, but highly conserved for type 3 poliovirus, although the converse would be predicted from their immunodominance. The evidence presented suggests that the antigenic conservation is associated with a strong selective pressure for a proteolytic cleavage site within site 1 of type 3. As proteolytic cleavage results in the loss of the antigenicity of site 1 the presence of the cleavage site in a virus replicating in the gut in the presence of proteases would protect the virus from neutralizing antibodies directed against uncleaved site 1. The conservation of the site in type 3 is thus consistent with the view that site 1 is a significant target of a human as well as a murine immune response against type 3.

Immunity of children to diphtheria, tetanus, and poliomyelitis.

A survey of titres of diphtheria and tetanus antitoxins and of antibodies to polioviruses in the sera of 291 schoolchildren aged 15, 11, and 7 years showed that high immunisation rates can evoke protective concentrations of tetanus antitoxin in 98% of children and protective levels of the antibodies to diphtheria and all three types of poliomyelitis in 85% of children. Reinforcing immunisation at school entry appeared to be necessary to maintain adequate titres of diphtheria antitoxin in children up to 15 years of age, not essential to maintain adequate titres of tetanus antitoxin, and to have little effect on the titres of antibodies to poliomyelitis.

Antibody state to poliovirus in first year university students, 1984.

Circulating antibodies to poliovirus were estimated in a group of 300 British and 84 foreign first year students who registered at the health centre of Nottingham University in 1984. Detectable antibodies to all three poliovirus serotypes were found in 212 (71%) of the British students but in only 47 (56%) of those from abroad. Most of the British students (280; 93%) had been born in 1965 or 1966, when uptake of poliomyelitis vaccine was declining. Immunisation histories showed that 10 British and 29 foreign students (3% and 35%) had no record of any immunisation; only five British and two foreign students, however, were negative for all three poliovirus serotypes. These findings provide evidence that a high proportion of British born people aged 18-29 have adequate circulating poliovirus antibodies despite incomplete immunisation schedules. Though this is reassuring, the absence of antibodies in some students and the lack of previous immunisation against poliomyelitis in 39 suggest that reinforcing doses of vaccine at the time of leaving school or beginning further education are still warranted, particularly for students from other countries. The findings also emphasise the need for accurate immunisation records.

WHO collaborative study on the use of monoclonal antibodies for the intratypic differentiation of poliovirus strains.

An international collaborative study was carried out to identify monoclonal antibodies that could reliably discriminate between wild polioviruses and strains derived from Sabin vaccine viruses. For poliovirus types 2 and 3, monoclonal antibodies were identified that reacted specifically with type 2 or type 3 strains which gave T1-oligonucleotide maps similar to or indistinguishable from that of Sabin vaccine virus, thus indicating their vaccine origin. These monoclonal antibodies failed to react with strains which gave T1 maps unrelated to that of Sabin vaccine virus. However for type 1, five of the six antibodies examined in the study reacted only with strains with a T1 map indistinguishable from that of type 1 Sabin vaccine virus. In contrast, other monoclonal antibodies against poliovirus types 1, 2 and 3 reacted broadly within a serotype.

Antigenic analysis of poliovirus type 3 using monoclonal antibodies.

Hybridoma cell lines secreting monoclonal antibodies to type 3 poliovirus have been prepared and their reactivity with infectious virus ('D' antigen), empty particles ('C' antigen) and isolated virus capsid proteins examined. Eight antibodies reacted with epitopes common to 'D' and 'C' antigen and all of these possessed high titres of neutralizing activity. However only 12 out of 19 antibodies which reacted exclusively with 'D' antigen neutralized virus infectivity and some of these reacted only with strains of virus with T1 oligonucleotide maps identical or similar to that of Sabin vaccine virus. These antibodies will be of value in identifying strains of virus derived from Sabin vaccine. None of the 20 monoclonal antibodies which neutralized type 3 poliovirus strains reacted in immunoblot experiments with isolated virus capsid proteins. However, 6 of the 24 antibodies which reacted only with non-infectious 'C' antigen bound to VP1 and VP3 and 3 of these antibodies also reacted with proteins of types 1 or 2 poliovirus. The lack of reactivity of neutralizing monoclonal antibodies with isolated viral proteins suggests that the antigenic properties of proteins are determined by their arrangement in the virus.

Antigenic characterization of poliovirus type 3 using monoclonal antibodies.

Hybridoma cell lines secreting monoclonal antibodies to type 3 poliovirus were prepared, and their reactivity with infectious virus (D antigen), empty particles (C antigen), and isolated virion capsid proteins ( VPs ) were examined. Eight antibodies reacted with epitopes common to D and C antigens, and all of these possessed high titers of neutralizing activity. However, only 12 of 19 antibodies that reacted exclusively with D antigen neutralized virus infectivity, and some of these reacted only with strains of virus with T1-oligonucleotide maps identical or similar to that of Sabin vaccine polio virus. These antibodies will be of value in identifying strains of virus derived from Sabin vaccine. None of the 20 monoclonal antibodies that neutralized type 3 poliovirus strains reacted in immunoblot experiments with isolated virion capsid proteins. However, six of the 24 antibodies that reacted only with noninfectious C antigen bound to VP1 and VP3, and three of these antibodies also reacted with proteins of poliovirus types 1 or 2. The lack of reactivity of neutralizing monoclonal antibodies with isolated viral proteins suggests that the antigenic properties of proteins are determined by their arrangement in the virus and not simply by amino acid sequence.

Durability of immunity to diphtheria, tetanus and poliomyelitis after a three dose immunization schedule completed in the first eight months of life.

Blood samples were obtained from school entrants whose primary immunization schedule had consisted of three doses of DT or DTP vaccine and three doses of OPV all given before the age of 8 months. The sera were separated and assayed for diphtheria antitoxin, tetanus antitoxin and antibodies to the three serotypes of poliovirus. The results of the assays showed that the abbreviated three dose schedule induced satisfactory immunity to all five infections until school entry and that a reinforcing dose at 18 months was unnecessary.

Induction by synthetic peptides of broadly reactive, type-specific neutralizing antibody to poliovirus type 3.

A region of virus capsid protein VP1 located 89-100 amino acids from the N-terminus has been proposed to comprise a major antigenic site involved in the neutralization of poliovirus type 3. Synthetic peptides 10-18 amino acids in length, containing all or part of this sequence, were tested for their ability to induce antiviral antibodies. Rabbits, but not guinea pigs or mice, immunized with the most active peptide, developed hightitered, type-specific, neutralizing antibodies for a wide range of poliovirus type 3 strains. Consistent with the broad type specificity of the antibody response was the observation that amino acids 89-100 of VP1 are highly conserved among different poliovirus type 3 strains. This sequence thus appears to provide, at least in part, a molecular basis for serotype antigenic specificity. Individual amino acids from 93 to 98 within this sequence were shown to be important for the neutralization of virus by antipeptide sera by examination of the ability of the sera to neutralize laboratory-derived poliovirus type 3 mutants with known single amino acid substitutions in the proposed antigenic site.

Immunoassay of poliovirus antigens by single-radial-diffusion: development and characteristics of a sensitive autoradiographic zone size enhancement (ZE) technique.

The reactions of polioviruses in single-radial-immunodiffusion (SRD) tests were investigated with a view to developing accurate and sensitive antigen assay systems. In direct SRD tests, employing high concentrations of immune poliovirus serum in agarose gels, poliovirus D-antigens produced clear reaction zones demonstrated by protein staining. The reactions were type-specific for polioviruses of types 1, 2 and 3 but the tests were of low sensitivity, being applicable only to the assay of virus concentrates. A novel autoradiographic zone size enhancement (ZE) test was developed which increased the sensitivity of the SRD assay 40- to 100-fold. The ZE test was dependent upon the ability of unlabelled poliovirus to co-migrate with the radioactive marker virus and so enhance the zone size detected autoradiographically. The areas of the autoradiographic zones were directly proportional to the concentration of unlabelled antigen. The ZE test was capable of detecting poliovirus D antigens in diluted cell culture fluid harvests in amounts corresponding to 10(3.3) to 10(4.3) TCID50 of infectious virus. Studies with poliovirus type 3 strains indicated that the ZE test was narrowly strain-specific for the D-antigen of poliovirus type 3 strains when homologous type 3 D-antigen was used as radioactive marker, but broadly cross-reactive for the D-antigen of type 3 viruses when heterologous poliovirus type 3 D-antigen was used as marker.