Isolation of sabin-like polioviruses from wastewater in a country using inactivated polio vaccine.

From 2001 to 2004, Switzerland switched from routine vaccination with oral polio vaccine (OPV) to inactivated polio vaccine (IPV), using both vaccines in the intervening period. Since IPV is less effective at inducing mucosal immunity than OPV, this change might allow imported poliovirus to circulate undetected more easily in an increasingly IPV-immunized population. Environmental monitoring is a recognized tool for identifying polioviruses in a community. To look for evidence of poliovirus circulation following cessation of OPV use, two sewage treatment plants located in the Zurich area were sampled from 2004 to 2006. Following virus isolation using either RD or L20B cells, enteroviruses and polioviruses were identified by reverse transcription-PCR. A total of 20 out of 174 wastewater samples were positive for 62 Sabin-like isolates. One isolate from each poliovirus-positive sample was analyzed in more detail. Sequencing the complete viral protein 1 (VP1) capsid coding region, as well as intratypic differentiation (ITD), identified 3 Sabin type 1, 13 Sabin type 2, and 4 Sabin type 3 strains. One serotype 1 strain showed a discordant result in the ITD. Three-quarters of the strains showed mutations within the 5' untranslated region and VP1, known to be associated with reversion to virulence. Moreover, three strains showed heterotypic recombination (S2/S1 and S3/S2/S3). The low number of synonymous mutations and the partial temperature sensitivity are not consistent with extended circulation of these Sabin virus strains. Nevertheless, the continuous introduction of polioviruses into the community emphasizes the necessity for uninterrupted child vaccination to maintain high herd immunity.

Titration of poliovaccines: improvement of end-point microassays by means of dose-response curves.

The W.H.O.'s titration method for attenuated poliovaccines, using two-fold dilution steps, was assayed concurrently with an alternative method, using four-fold dilution steps. For the two-fold dilution method, the dispersion of the observed proportions of response was very wide, inducing biased estimations for titers and their standard-error by means of simple usual calculation procedures. On the other hand, the use of the four-fold dilution method constantly provides more accurate estimates for the titers and their confidence limits and thus increases the reliability of the titration results. In addition, replicate poliovirus titrations were performed in three cell lines (HEp-2, LLC-MK2 and Vero), applying a restricted experimental design, in order to characterize statistically their sensitivity for poliovaccine titrations, and their consistency for infectivity titers. Means of improving the reliability of live poliovaccine potency estimates are suggested.

A comparison of three methods for titration of poliovirus vaccines.

Two methods for in vitro endpoint titration of poliovirus--the roller tube and the microtitration assay--were compared with each other and with the plaque assay, using secondary vervet monkey kidney cells and Vero cells as indicators. The roller tube method is the most reliable under difficult working conditions, but is otherwise cumbersome and expensive. The microtitre method is the most economical and the plaque assay the most sensitive. By suspending freshly trypsinized indicator cells with the virus dilutions before planting, it was possible to simplify the microtitre method considerably. The sensitivity of the plaque assay was improved for Vero cells by absorbing the virus onto freshly planted monolayers. The method was scaled down to a semi-micro level by using 24-well cell culture trays. The slower rate of plaque development under a low calcium overlay medium facilitated a more accurate plaque count.

Sera collected before introduction of contaminated polio vaccine contain antibodies against SV40.

Anti-SV40 IgG antibodies were found by ELISA test in 6 out of 51 sera collected in 1952, i.e. before introduction of the polio vaccine. This indicates that the presence of SV40 in the human population whose footprints can be found in a fraction of human cerebral tumours need not be the consequence of contamination of early batches of polio vaccine with SV40.

[Cytokines in vaccines and interferon preparations]. / Tsitokiny v vaktsinakh i preparatakh interferona.

Virus vaccines prepared on the basis of cells of mammals (rabies, poliomyelitis, measles and hepatitis A vaccines) contain cytokines (IL-1 beta, IL-6, TNF-alpha), whose concentration depends on the kind of the vaccine. Cell lines (green monkey kidney cells, VERO, 4647), used for the preparation of commercial and experimental vaccines, do not produce spontaneously any of the above cytokines. Cell line L-68, used for the manufacture of experimental measles vaccine, is capable of the spontaneous synthesis of IL-6. In Russian and foreign preparations of interferon the presence of IL-1 beta and TNF-alpha has been detected; the content of these cytokines is determined by the specific features of the methods used manufacturing these preparations.

Analysis of antigenic profiles of inactivated poliovirus vaccine and vaccine-derived polioviruses by block-ELISA method.

A new block-ELISA test for quantitative evaluation of relative reactivity of antigenic sites was developed and used to reveal the detailed epitope structure of inactivated poliovirus vaccines (IPV) and live poliovirus strains. Poliovirus was captured on ELISA plates coated with rabbit anti-poliovirus IgG and blocked by monoclonal antibodies (Mabs) specific to individual epitopes before the remaining reactive antigenic sites were quantified by polyclonal anti-poliovirus IgG conjugate. The decrease of conjugate binding by the pre-treatment with a Mab reflects its contribution to the overall reactivity of poliovirus antigen. The level of block activity of Mabs for a given antigen can be expressed as a percent of reduction of antigenic reactivity as determined by ELISA test. It can be normalized by expressing this value as a ratio to the block activity of a reference sample. The data on the blocking-activity of a panel of monoclonal antibodies specific to different antigenic sites represents the epitope composition (antigenic profile) of a sample. Quantitative differences in epitope composition were determined for nine samples of inactivated poliovirus vaccine (IPV) and compared with the International Reference Reagent. This method could be used for monitoring consistency of IPV production, comparison of vaccines made by different manufacturers, and for the analysis of antigenically modified strains of attenuated poliovirus. Antigenic structures of two isolates of type 1 vaccine-derived poliovirus (VDPV) were compared with the structures of parental Sabin 1 and wild-type Mahoney strains using 17 monoclonal antibodies and revealed significant differences, suggesting that the method can be used for screening of field isolates and rapid identification of antigenically divergent VDPV strains.

Improved ELISA test for determination of potency of Inactivated Poliovirus Vaccine (IPV).

An improved ELISA test for determination of potency of Inactivated Poliovirus Vaccine (IPV) is proposed. The method is based on the use of IgG purified from immune rabbit serum conjugated with biotin. Optimized and validated materials for the test can be stored for a long time in the form of ready-to-use kits. Optimization included selection of anti-poliovirus rabbit antibody batches with the best specificity to D-antigen as well as finding the most efficient parameters for all steps of ELISA protocol. The assay is based on direct ("sandwich") ELISA scheme, in which antigens are captured on ELISA plates coated with purified rabbit polyclonal D-antigen specific IgG raised against wild polioviruses of three serotypes. D-antigen specificity of the IgG was at least 10 times higher than to H-antigen (heat-inactivated virus). The presence of antigen was detected using biotin-conjugated IgG from the same source. Eight-point dose-response curves were obtained for each sample and the reference vaccine. The protocol ensured low background (less than 0.2 OD), linear response over the entire range of optical density measurements (up to 3.0 OD), and high precision of data (assay variability was about 3%). The quantitative results and the validity of the test were determined by two numerical approaches, linear regression and a new analysis procedure called the local interpolation method. For the first approach we also proposed a new method for testing of parallelism of regression lines. The ELISA protocol for all three types of poliovirus is based on standard off-the-shelf reagents, and is highly reproducible and reliable. An in-house Reference Reagent was formulated and calibrated against the International Reference for IPV.

The evaluation of a physical method for the quantification of inactivated poliovirus particles and its relationship to D-antigenicity and potency testing in rats.

The use of a density gradient procedure for the quantification of intact, inactivated poliovirus particles in vaccine preparations is described. The procedure is both sensitive and highly reproducible and the results correlate with those of potency tests in rats and with D-antigen content as measured by ELISA. Because of the occasional ambiguity observed with D-antigen assays, it is suggested that the density gradient procedure will provide valuable additional information for the in vitro assessment of inactivated poliovirus preparations.

The monitoring of antigen levels during inactivated poliovirus vaccine production: evaluation of filtration techniques.

The use of ELISA to estimate poliovirus antigen concentration has permitted an evaluation of the methodology used in vaccine production and allowed exploration of less wasteful filtration-techniques. The replacement of Seitz-EKS-1B filtration with either Seitz-Supra-EKS or Pall-filtration in the preparation of the vaccine could make a large saving in the total antigen yield, but the results in the safety test excluded their possible use in our process as it stands at the moment.

Presence and quantification of cell substrate DNA in inactivated poliovirus vaccine.

In order to follow the fate of cell substrate DNA in inactivated poliovirus vaccine (IPV), experiments simulating different steps in IPV preparation were performed. For this purpose, 3H-thymidine-labeled HeLa cell DNA released during lytic infection with Mahoney type 1 poliovirus strain was used as tracer. Low speed centrifugation (1500 xg for 15 min.) of crude virus suspension removed on the average 98.5% of the total labeled cell substrate DNA. Repeated freezing and thawing of the crude virus suspension before centrifugation did not increase the residual 1.5% radioactivity found in the supernatant. This level of contamination was not significantly influenced by filtration of the supernatant through a 0.22 micrometer pore size membrane filter. DEAE-Sepharose CL-6B chromatography of poliovirus suspensions containing large amounts of 3H-thymidine labeled HeLa cell extracts completely removed the contaminating substrate DNA. These data showed that IPV free of cell substrate DNA can easily be obtained by a usual purification procedure.

Capillary electrophoresis: separation and quantitation of reverse transcriptase polymerase chain reaction products from polio virus.

In the present study reverse transcriptase (RT) polymerase chain reaction (PCR) products were generated from the RNA of polio virus. The products of the RT-PCR were analyzed by slab-gel electrophoresis (SGE) on 4% agarose gels, and capillary electrophoresis (CE). CE separations were performed in a coated capillary containing a linear polyacrylamide. Samples were injected hydrodynamically or electrokinetically. Detection of the RT-PCR products on CE was by UV absorbance at (254 nm) or by laser-induced fluorescence (LIF). While SGE resulted in adequate separation of 163 and 97 base pair RT-PCR products, separation of the 97, 71 and 53 base pair products was minimal. CE separations showed baseline resolution for all the above PCR products. Finally, it was possible to quantitate the amount of RT-PCR product by developing a standard curve showing a linear relationship between the amount of RNA used in the RT-PCR and the amount of product formed in the RT-PCR. These results suggest the greater resolution and enhanced sensitivity observed, together with the ease of quantitation, make CE a powerful alternative to SGE for the separation and quantitation of PCR products.

Molecular epidemiology of polioviruses.

Poliovirus isolates can be identified according to their genotypes with use of the technique of oligonucleotide fingerprinting. Fingerprint analysis is performed by cleaving the viral RNA genome with ribonuclease T1 and separating the fragments (oligonucleotides) in two dimensions. The larger, structurally unique oligonucleotides distribute into patterns ("fingerprints") highly characteristic of a specific overall RNA sequence. Isolates from the same epidemic have very similar fingerprints. Isolates from distinct epidemics have very different fingerprints, a consequence of the rapid evolution of polioviruses during replication in humans. Similarity in the fingerprints of case isolates provides independent evidence for epidemiologic linkage. Fingerprinting can readily distinguish vaccine-related isolates from wild strains. Contemporary vaccine-related isolates are very probably vaccine-derived because their fingerprints contain characteristic vaccine-strain oligonucleotide spots (types 1 and 3) and because their wild-type parents are unlikely to have survived largely unaltered in the natural environment. Some examples of applications of this technique within different epidemiologic settings are described.

Inactivated poliovirus vaccine and test development at Connaught Laboratories Ltd.

Trivalent inactivated poliovirus vaccine (IPV) (MK) was made, purified, and inactivated according to the amended Rijks Instituut (The Netherlands) protocol from primary monkey kidney tissue grown on microcarrier cultures. Losses of the type 2 component due to adsorption to the glass ampule occurred with the purified vaccine preparation. This problem was solved by changing the diluent, and the vaccine was submitted for evaluation in clinical trials at Johns Hopkins (Baltimore, Md.). Phase 2 of the development was to standardize production of IPV from MRC-5 (human diploid) cells on microcarriers and otherwise follow the Rijks Instituut 's method. Results of experimental trivalent vaccine production and testing showed that the number of effective doses harvested from MRC-5 cell cultures compared favorably with vaccine derived from monkey kidney. The yields could be further increased with stearyl tyrosine as adjuvant. Large-scale production using 200-liter fermenters is in progress. Poliovirus particles of various densities in cesium chloride can be found in any IPV preparation and give rise to different immunogenic responses. As shown in this paper, some of these virus fractions produce a low primary humoral antibody response but appear to be important for memory induction.

Rat immunogenicity assay of inactivated poliovirus.

A rat immunogenicity assay for IPV potency was validated and applied to routine vaccine testing as a potential alternative to the CFR Monkey Potency Assay. Potencies of pure trivalent polio, various combinations and experimental vaccines were tested with a view of producing a single dilution assay.

Validation of the toxin-binding inhibition (ToBI) test for the estimation of the potency of the tetanus toxoïd component in vaccines.

A validation study has been performed to determine the suitability of the toxin binding inhibition (ToBI) test for the serological estimation of the potency of the tetanus toxoïd component in vaccines. 37 Murine serum pools over a wide range of antibody levels were titrated in both toxin neutralization (TN) and ToBI test. A good correlation was found between both assays. Sixteen DPT-polio, twelve DT-polio and seven T vaccines were tested in the mouse lethal challenge test and the in vitro serological test, using the ToBI test for determining vaccine-induced tetanus antibodies. For all three types of vaccine a statistically valid correlation between both assays was found. However, for two batches of DPT-polio vaccine an "overestimation" of the tetanus potency was observed in the serological assay compared to the challenge assay. This phenomenon could not be explained by the difference in immunization period nor by misinterpretation of the ToBI test of DPT-polio-induced antibodies. In the LPF test high LPF activity was observed for the deviating DPT-polio vaccines. Therefore, the effect of pertussis toxin (PT) on the potency of the tetanus component in the serological assay was examined. The addition of 2 micrograms of PT to a "normal" DPT-polio vaccine resulted in a nearly twofold increase of the tetanus potency. It was concluded that pertussis toxin has a vaccine dose-dependent adjuvant effect on the potency of tetanus toxoïd resulting in high potency values when determined by ToBI procedure. It is unclear how these findings should be interpreted with respect to the behaviour of such vaccines in man.

Quantitation of cell DNA in the evaluation of heteroploid cells as substrate for the preparation of killed viral vaccines.

To evaluate the risk of using heteroploid cell lines as substrates for viral vaccine production, the presence of cell DNA in poliovirus suspensions was examined. The time course of [3H]-thymidine-labeled HeLa cell DNA release during lytic infection with type 1 poliovirus was investigated. More DNA was found in filtered supernatants of poliovirus-inoculated cultures than in control cell supernatants. DNA concentration increased with time, paralleling virus release, but did not exceed 1.5% of the total DNA content of the culture. Only about 10% of this cell DNA was resistant to DNase treatment. By both ion-exchange chromatography and rate-zonal centrifugation it was possible to remove practically all cell DNA contaminating filtered poliovirus suspensions. Results obtained in this study permitted quantitative evaluation of cell substrate DNA present in poliovirus suspensions during successive steps of killed poliovirus vaccine preparation. Based on the sensitivity of our method, the amount of residual DNA was estimated at less than 0.02 pg per dose of purified vaccine.