Polio vaccine and retroviruses.

In this paper we consider the main steps in the process of manufacture of oral polio vaccine and assess the probable clearance factor for HIV retrovirus at each step. We conclude that the processes employed would have eliminated retrovirus contamination for all practical purposes.

Karyology and tumorigenicity testing requirements: past, present and future.

At the present time, karyology and tumorigenicity are applied to primary, diploid, and continuous cell systems in an uneven and inappropriate manner, largely for historical reasons. It is a significant anomaly that such rigorous requirements are applied only to diploid cells when, of all three cell types, they represent the category with the least potential problems. During the 1992 presidential campaign in the U.S., a very direct and telling slogan was used to the advantage of Mr. Clinton: <>. That message was meant to have the effect of focusing on the real issue that was of concern to the electorate. The other topics were of secondary significance and tended to act as distractions from the central issue. In a very similar sense, one could say: <>. In other words, we should be focussing our time and attention on the characteristics of the biological product manufactured in a given cell system that has been well characterized rather than continue to belabour the issue of cell substrates. To a large degree, this has already been initiated with the recommendations of various groups already mentioned. However, current diploid cell quality control regulations stand out as a peculiar throwback to an earlier era. HDCs should be treated on a par with primary and continuous cells. There are three basic questions related to the routine use karyology that need to be addressed: (i) is the original rationale for requiring cytogenetic analysis of a diploid cell substrate still valid; (ii) is there a new rationale that would warrant its continued use; and (iii) if there is a continuing need for karyology, is it unique to diploid cell cultures or does it need to be extended to all types of cell systems? The original rationale was that karyology provided evidence of the normal character of WI-38 cells and therefore supported its acceptability as a cell substrate for vaccine production. Karyology essentially has remained as a legacy of the intense debate that led to the acceptance of WI-38 cells. There is no new information over the past 30 years to suggest that there is a new rationale for instituting chromosomal analysis of cell substrates. If there were, however, it is difficult to imagine why it should not be applied to all types of cell substrates (primary, diploid, and continuous). Taking all the above into consideration, there would seem to be no rationale for continuing to single out diploid cell cultures as the only cell type for which karyology is required on a routine basis. As stated above, the initial characterization of a new diploid cell line should include karyology. Like karyology, tumorigenicity testing was incorporated into the assessment of diploid cells in an attempt to persuade regulatory authorities that the cells were normal and acceptable. Again, after 30 years of testing, there has never been an instance of normal diploid fibroblasts generating a tumour in any in vitro or in vivo assays. The futility of continuing to do these tests is obvious. A description of the tumorigenic potential of a cell substrate should be an element in the characterization of a new cell line; but it has little if any value as a routine test. If history teaches us anything at all about risk, it is that we need to focus serious attention on contaminants rather than be diverted to remote theoretical issues that may be interesting to discuss and argue about, but which pale in the face of the potential impact on public health of viral and viral-like contaminants of biological products. One has only to recall the transmission of SV-40 from primary monkey kidney cells that were used to produce polio vaccine, or more recently the transmission of Creutzfeld-Jacob disease to recipients of human growth hormone derived from human pituitaries and of HIV to recipients of blood and blood derivatives. (ABSTRACT TRUNCATED)

Viral safety of blood derivatives: an overview.

The causes of past accidental viral transmissions associated with blood derivatives are reviewed briefly, as are the safety procedures instituted in Europe on the basis of viral validation studies. A comprehensive five-class classification system for delineating viral risk according to source material and comparative viral safety is presented. Newly emerging viruses are briefly discussed in view of the viral safety of blood products.

Viral vaccines and residual cellular DNA.

The acceptability of viral vaccines manufactured in culture of continuous cell lines opened the way to a new technology of vaccine preparation. The large scale cultivation of continuous cell lines contributed greatly to the improvement of the safety and the consistency of viral vaccines. Experimental studies concerning residual cellular DNA and its oncogenic potential showed clearly that this material is not able to induce tumours. The small amounts of cellular DNA present in the final product, and the deleterious effect of the vaccine manufacturing process on the integrity and biological activity of DNA, both contribute to regarding residual cellular DNA as an issue of no practical importance. There is no evidence that limits on the concentration of residual cellular DNA in the final product is scientifically justified.

Viral iatrogenic contamination and safety of biologicals.

The use of biologicals such as bacterial or viral vaccines have an inert risk as a result of their preparation. In the last century it was difficult to determine the correlation of the clinical accidents until the characteristic viral genome became available. Generally, the frequency of accidents provoked by viral vaccines has diminished due to (1) the improvements of the methods used for viral inactivation and purification, (2) the introduction of good manufacturing practices and rigorous regulatory policy and (3) the progress in the post-clinical surveillance of adverse reactions after vaccine administration, allowing a better risk-benefit analysis.

A view on regulatory aspects of live viral vaccine safety.

Regulatory requirements on live viral vaccines take into consideration their value in the improvement of public health in countries that have used them in large vaccination campaigns. However, since they have also been involved in clinical accidents, a careful assessment of their safety is necessary to establish the risk-benefit balance. From this point of view, live recombinant vaccines represent a new category of products and their future use in the field should be preceded by scientific debate concerning their safety and potency.

Albert B. Sabin and the development of oral poliovaccine.

There are many reasons for the modern interest in viral vaccines, but there is no doubt that the key role played by viral vaccines in public health is the major factor since other prophylactic or therapeutic anti-viral products simply do not exist. Viral vaccines have a long history that has been marked by successful events and by tragic accidents. Live viral vaccines are an extraordinary category of biologicals since, despite their reputed efficacy, they were developed by empirical experiments and patient epidemiological observation. From this point of view oral polio vaccine should be considered a 'miracle' since it became a major tool for public health in the 20th century, before we were able to understand the molecular basis of polio virus neurovirulence attenuation. The first evidence that polio virus can be attenuated was provided in the early 1940s by Max Theiler, but it was Hilary Koprowsky who demonstrated further in 1952, that a rodent adapted strain was safe and able to immunise a limited number of volunteers. Koprowsky studies were confirmed later during a mass field trial in Africa. However it is undeniable that the patient and systematic work of Albert B. Sabin was primordial in developing live oral attenuated poliovaccine. The excellence of Sabin's testing of poliovirus neurovirulence in the accurate studies that he developed, enabled him to select, after the cloning of viral populations by plaque assay, the best attenuated variants.(ABSTRACT TRUNCATED AT 250 WORDS)

A mutation in the RNA polymerase of poliovirus type 1 contributes to attenuation in mice.

The attenuated Sabin strain of poliovirus type 1 (PV-1) differs from the neurovirulent PV-1 Mahoney strain by 55 nucleotide mutations. Only one of these mutations (A-480-->G, in the 5' noncoding (5' NC) region of the genome, is well characterized, and it confers a strong attenuating effect. We attempted to identify genetic attenuation determinants in the 3'-terminal part of the Sabin 1 genome including the 3D polymerase (3Dpol) gene and the 3' NC region. Previous studies suggested that some of the 11 mutations in this region of the Sabin 1 genome, and in particular a mutation in the polymerase gene (U-6203-->C, Tyr-73-->His), are involved to some extent in the attenuation of PV-1. We analyzed the attenuating effect in the mouse model by using the mouse-adapted PV-1/PV-2 chimeric strain v510 (a Mahoney strain carrying nine amino acids of the VP1 capsid protein from the Lansing strain of PV-2). Mutagenesis of locus 6203 was performed on the original v510 (U-6203-->C) and also on a hybrid v510/Sabin 1 (C-6203-->U) carrying the downstream 1,840 nucleotides of the Sabin 1 genome including the 3Dpol and 3' NC regions. Statistical analysis of disease incidence and time to disease onset in numerous mice inoculated with these strains strongly suggested that nucleotide C-6203 is involved in the attenuation of the Sabin 1 strain. Results also suggested that, among the mutations located in the 3Dpol and 3' NC regions, nucleotide C-6203 may be the principal or the only one to be involved in attenuation in this mouse model. We also found that the effect of C-6203 was weaker than that of nucleotide G-480; the two nucleotides acted independently and may have a cumulative effect on attenuation. The U-6203-->C substitution also appeared to contribute to the thermosensitivity of the Sabin 1 strain.

Molecular characterization of mouse-virulent poliovirus type 1 Mahoney mutants: involvement of residues of polypeptides VP1 and VP2 located on the inner surface of the capsid protein shell.

Most poliovirus (PV) strains, including PV PV-1/Mahoney, are unable to cause paralysis in mice. Determinants for restriction of PV-1/Mahoney in mice have been identified by manipulating PV-1 cDNA and located on the viral capsid protein VP1. These determinants consist of a highly exposed amino acid sequence on the capsid surface corresponding to the B-C loop (M. Murray, J. Bradley, X. Yang, E. Wimmer, E. Moss, and V. Racaniello, Science 241:213-215, 1988; A. Martin, C. Wychowski, T. Couderc, R. Crainic, J. Hogle, and M. Girard, EMBO J. 7:2839-2847, 1988) and of residues belonging to the N-terminal sequence located on the inner surface of the protein shell (E. Moss and V. Racaniello, EMBO J. 10:1067-1074, 1991). Using an in vivo approach, we isolated two mouse-neurovirulent PV-1 mutants in the mouse central nervous system after a single passage of PV-1/Mahoney inoculated by the intracerebral route. Both mutants were subjected to two additional passages in mice, plaque purified, and subsequently characterized. The two cloned mutants, Mah-NK13 and Mah-NL32, retained phenotypic characteristics of the parental PV-1/Mahoney, including epitope map, heat lability, and temperature sensitivity. Mah-NK13 exhibited slightly smaller plaques than did the parental virus. The nucleotide sequences of the mutant genomes were determined, and mutations were identified. Mutations were independently introduced into the parental PV-1/Mahoney genome by single-site mutagenesis. Mutated PV-1/Mahoney viruses were then tested for their neurovirulence in mice. A single amino acid substitution in the capsid proteins VP1 (Thr-22-->Ile) and VP2 (Ser-31-->Thr) identified in the Mah-NK13 and Mah-NL32 genomes, respectively, conferred the mouse-virulent phenotype to the mouse-avirulent PV-1/Mahoney. Ile-22 in VP1 was responsible for the small-plaque phenotype of Mah-NK13. Both mutations arose during the first passage in the mouse central nervous system. We thus identified a new mouse adaptation determinant on capsid protein VP1, and we showed that at least one other capsid protein, VP2, could also express a mouse adaptation determinant. Both determinants are located in the inside of the three-dimensional structure of the viral capsid. They may be involved in the early steps of mouse nerve cell infection subsequent to receptor attachment.

Analysis of neutralization-escape mutants selected from a mouse virulent type 1/type 2 chimeric poliovirus: identification of a type 1 poliovirus with antigenic site 1 deleted.

A chimeric type 1/type 2 poliovirus (v510), in which the antigenic site 1 (Ag1) of poliovirus type 1 (PV-1) Mahoney was replaced by the corresponding site of poliovirus type 2 (PV-2) Lansing, is known to be neurovirulent for mice and neutralized by both type 1 and type 2 monoclonal antibodies. Neutralization-escape mutants to monoclonal antibodies specifically recognizing the PV-2 sequence were obtained from v510. The nucleotide sequence and the mouse neurovirulence of mutants were determined. Amino acid substitutions obtained inside the replaced sequence, at positions 95 and 99, and outside this site, at positions 93 or 104, rendered the virus attenuated for mice. One of the escape mutants harboured a deletion of the entire substituted nonapeptide sequence in v510. This particular virus, which is a PV-1 Mahoney lacking the natural Ag1 loop, does not react with PV-2-specific monoclonal antibodies, has a ts phenotype, is heat-labile and is devoid of neurovirulence for mice.