key: cord-0014659-iqeirwsl
authors: Seefried, A. v.; Chun, J. H.; Grant, J. A.; Letvenuk, L.; Pearson, E. W.
title: Inactivated Poliovirus Vaccine and Test Development at Connaught Laboratories Ltd.
date: 1984-05-03
journal: Rev Infect Dis
DOI: 10.1093/clinids/6.supplement_2.s345
sha: f29662e1771790f09914a05ba0473337d486461c
doc_id: 14659
cord_uid: iqeirwsl

Trivalent inactivated poliovirus vaccine (lPV) (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.

A program was introduced at Connaught Laboratories in 1980 to develop a large-scale production method for inactivated poliovirus vaccine (IPV) of increased potency. Although the method of the Rijks Instituut in The Netherlands [1] was adopted, the use of primary monkey kidney tissue has many obvious disadvantages and was replaced by serially subcultivated MRC-5 human diploid cells. Preliminary studies of the use of these cells were presented at the polio vaccine symposium in Bilthoven, The Netherlands, three years ago [2] . The physiologic growth conditions were further standardized and are now applied to produce trial vaccines. This paper describes the results obtained with an experimental trivalent IPV produced in this way, together This study was supported by a grant from the Federal Government of Canada under the Industrial Research Assistance Programme (SALK 687).

The authors wish to express their gratitude to Mrs. J. Plavinskis, Mrs. S. Graham, and Miss A. Truscott for their excellent assistance and to Mr. J. B. Campbell for the neutralization tests.

The supply of the adjuvant, stearyl tyrosine, and the data on the effect of the adjuvant in monkeys weresupplied courtesy of Dr. S. Landi with findings from the analysis of different antigenic components in the vaccine.

Trial vaccine production. IPV (MK) had been prepared according to the formulation of the Rijks Instituut [1] , and this vaccine had been filled into type 1 glass ampules, which require modification as described in the text. The protocol for the production of IPV from MRC-5 cells was as described earlier [2] , and strict controls of pH 7.2 and 20% pOl during cell growth were applied. Medium CMRL-1969 was used with amino acid amplification towards the end of the growth cycle. The medium was supplemented with fetal bovine serum at the early passage levels and with 6070 adult bovine serum at the production level. The cells were grown in 2.5 g of Cytodex beads (Pharmacia) per liter. For virus growth the medium was replaced with medium 199 containing Earle's salts.

Potency tests. Radioimmunoassays (RIA) were carried out with IgG derived from rabbit sera after immunization with purified D antigen. The specificity of the poliovirus type 1 IgG was further increased by adsorption with heat-treated antigen. The IgG was used for coating tubes and for the lactoperoxidase-iodinated reagent. The quantitative measurements were based on comparisons 

Losses of poliovirus type 2 antigen after filling. Trivalent IPV (MK) was successfully prepared by the Rijks Instituut's method (1] . The vaccine concentrate was diluted for the final formulation in medium 199 and transferred into glass ampules. An immediate loss of the type 2-antigen component was noted. The antigen could be eluted from the ampule with diluent containing Tween 80 and human serum albumin, indicating that the loss was a result of adsorption to the glass. This problem was solved by changing the composition of the diluent. As shown in table 1, no further losses occurred after filling.

IPV produced from human diploid cells, MRC-5. Approximately three to four cell population doublings were consistently obtained in microcarrier cultures during the seven-day growth cycle. Experimental vaccine was produced from 20-liter cultures, but scaling up to 150-litercultures has been successfully carried out with similar Vaccine growth rates and final yields of~1 x 10 6 cells/ml. These cells have a tendency to clump the beads together, which in moderation does not interfere with virus propagation. An experimental vaccine was prepared from virus harvest fluids containing 112, 32, and 33 D-antigen units/ml for poliovirus types 1, 2, and 3, respectively, as shown in table 2. The harvest fluids were concentrated and were used for further processing. The percent recovery after processing and inactivation with formaldehyde and the total D-antigen yields are shown. The monovalent concentrates were diluted and pooled to prepare an IPV for testing in comparison with the Rijks' vaccine standard, PU 78-02.

The effect of the stearyl tyrosine adjuvant mixed with the vaccine was also studied. The D-antigen . units/ml were determined by RIA and the vaccine was tested at various dilutions in rats for in vivo potency, as shown in table 3 D, c: and DP particles. IPV consists of different poliovirus particles, and the vaccine composition may differ under certain conditions, making it difficult to assess its potency in vitro. Three major fractions were isolated by CsCI gradient centrifugation from a type 1 concentrate: the empty capsids (C particles at 1.32 g/ml); full particles (D-antigenic at 1.34g/ml); and dense par- ticles (DP at 1.42 g/ml). The D and DP have similar appearance and require CsCI for differentiation. As shown in table 8, at rv1 JAg of protein/ml, the RIA indicates high D-antigen values, whereas 21 days after the first injection, only the D fraction induced an antibody response. Also included in this experiment is the heated D fraction, with reduced D-antigen value and no primary immune response. However, as shown by the booster response after injecting 10 D-antigen units of a purified D fraction, the C and DP fractions had actually primed the immune response in the test animals. Similar results were obtained when the second dose contained the same fractions, as shown in table 9. 

The more potent Rijks Instituut's IPV [4, 5] was introduced and made at Connaught according to the protocol of van Wezel and colleagues [1] . This vaccine is now being evaluated in the Johns Hopkins' (Baltimore, Md.) clinical trial. Furthermore, an experimental lot of IPV produced from MRC-5 cells has been described and shown to yield poliovirus antigens equivalent to those in the monkey kidney-derived vaccine.

A very significant increase in antigenicity, approximately fourfold, was obtained by adding the adjuvant stearyl tyrosine. This adjuvant so far has been used only in animal potency tests, as described here. However, it is biodegradable, and preliminary studies have indicated that it is also nontoxic. Sensitization or reactions at the injection sites do not occur.

Quantitative measurements of poliovirus immunogenic protein depend on the specificity of the reagents used in any in vitro test. Antibody specificity is more easily achieved for serotype and strain differentiation than for the immunogens. Differences in antigenicity and cross-reactivities in in vitro tests have been shown for the C, D, and DP fractions in various poliovirus preparations [6] [7] [8] [9] [10] . The specific antigenicities of these fractions are not stable, i.e., changes in pH and temperature may cause reversible antigenic conversions [11] . Poliovirus type 1 is antigenically the least stable component of IPV, and the immune response in the rat potency test described here showed that the Dantigen determination by RIA could be misleading in predicting the primary dose response. However, a small booster dose resulted in a priming effect even with the so-called nonimmunogenic fractions. For the acceleration of antibody production, a second stimulus was required. Determination of vaccine composition may be useful in monitoring IPV production, but further studies are required to determine the importance of poliovirus antigens in various configurations for a long-lasting protection from the disease.

New approach to the production of concentrated and purified inactivated polio and rabies tissue culture vaccines

Serially subcultivated cells as substrates for poliovirus production for vaccine

Potency testing of killed polio vaccine in rats

Antigen content of inactivated poliovirus vaccine for use in a one-or two-dose regimen

Theoretical and practical considerations in the application of killed poliovirus vaccine for the control of paralytic poliomyelitis

The preparation of specific immune sera against type 3 poliovirus D-antigen and C-antigen and the demonstration of two C-antigenic components in vaccine strain populations

Studies on the complementfixing antigens of poliomyelitis. II. Preparation of typespecific anti-N and anti-H indicator sera

Antigenic structure of poliovirus

The modification of poliovirus antigens by heat and ultraviolet light

Poliovirus empty capsid morphogenesis: evidence for conformational differences between self-and extract-assembled empty capsids

A pH-dependent antigenic conversion of empty capsids of poliovirus studied with the aid of monoclonal antibodies to Nand H antigen