Inactivated poliovirus vaccine protects transgenic poliovirus receptor mice against type 3 poliovirus challenge.

Transgenic (Tg) mice expressing the human poliovirus receptor (PVR) were vaccinated with inactivated poliovirus vaccine (IPV) and evaluated for induced immunity against type 3 poliomyelitis. One injection of monovalent type 3 IPV elicited protective immunity against wild-type poliovirus. In contrast, 2 injections of trivalent IPV were required for protection. Neutralizing antibody response and protection were vaccine dose-dependent. Administration of polio-immune mouse plasma protected unimmunized mice, demonstrating that neutralizing antibody was sufficient for immunity. IPV heated to remove its D antigen component did not induce protection in Tg PVR mice. IPV derived from a wild-type poliovirus strain gave better protection against wild-type viral challenge than IPV derived from an attenuated poliovirus strain. The newly developed Tg PVR mouse-protection test may be useful in evaluating existing IPV potency tests and for attempts to improve formulations of trivalent IPV or combined vaccines for childhood immunization schedules.

Succession of mutations in the Sabin strain of type 3 poliovirus replicating in the central nervous system of monkeys.

Sabin strains of oral poliovirus vaccine (OPV) undergo limited genetic changes during replication in cell cultures, the gastrointestinal tract of vaccinees, and the central nervous system of monkeys. Some of these changes are associated with loss of attenuation markers. Here we report the dynamics of mutant accumulation in the Sabin strain of poliovirus type 3 inoculated intraspinally into monkeys. Thr --> lle reversion in amino acid 6 of VP1 (2493 C --> U) occurred within the first few days postinoculation (p.i.), but decreased on later days and completely disappeared by Day 17 p.i. 472 U --> C reversion in the 5'-untranslated region appeared to accumulate slower and by Day 17 completely substituted for the vaccine-type nucleotide at this site. These results indicate that experimental infection of the central nervous system of monkeys consists of early and late phases in which a different genetic constitution of the virus is favored. In several isolates one additional neurovirulent revertant was found: a Phe --> Ser at amino acid 91 of VP3 (2034 U --> C). Since this mutation was never detected in vaccine lots and is strongly selected against in cell cultures at temperatures below 38.5 degrees, it does not threaten the safety of OPV.

Limited genetic changes in the Sabin 1 strain of poliovirus occurring in the central nervous system of monkeys.

Replication of attenuated poliovirus strains results in their partial deattenuation. Recently we identified mutations accumulating in the Sabin 1 poliovirus in cell cultures. Here we report genetic changes occurring in this virus during replication in the central nervous system (CNS) of monkeys. Viruses isolated from different parts of the CNS of rhesus monkeys (inoculated into the spinal cord) were screened for sequence heterogeneities and newly identified mutations were independently confirmed and quantified using mutant analysis by PCR and restriction enzyme cleavage (MAPREC). All consistently accumulating mutations identified in this study were located in untranslated regions: GU-->AU or GU-->GC substitution at a complementary pair formed by nucleotides 480 and 525, U-->C substitution at nucleotide 612, and GU-->AU or GU-->GC substitution of a base pair formed by the nucleotides 7427/7441 immediately preceding the poly(A) tract. All these mutations except one (7427) were previously identified in cell culture passages or stool isolates from vaccinees. Sequencing of 11 CNS isolates also identified a few random silent mutations that accumulated as neutral 'passengers', passively co-selected with genuinely selectable mutations present on the same RNA molecule. One isolate also contained the wild-type base at nucleotide 2741 (Ala88-->Thr in VP1). Our results demonstrate a remarkable genetic stability of the Sabin 1 poliovirus in the CNS of monkeys, suggesting that deattenuation is determined by a very limited number of mutations. These mutations can be assayed by MAPREC to monitor the consistency of oral poliovirus vaccine (OPV) production.

Microevolution of type 3 Sabin strain of poliovirus in cell cultures and its implications for oral poliovirus vaccine quality control.

Screening for sequence heterogeneities in Sabin Type 3 strains of attenuated poliovirus demonstrated mutations that consistently accumulate to significant levels following 10 passages in cultures of primary African green monkey kidney (AGMK) cells or continuous cultures of Vero cells. Fourteen newly identified mutations were quantified by mutant analysis by PCR and restriction enzyme cleavage in passages and in batches of commercial vaccines made in AGMK and Vero cells from the Sabin original (SO) seed virus and from a seed virus rederived by RNA plaque purification (RSO or "Pfizer" seed). Nine of the 14 mutations were reproducibly observed in more than one series of passages. Although 5 other mutations were observed in only one set of passages each, their content gradually increased to a high percentage, suggesting that all the mutations that we found accumulated consistently. SO-derived samples accumulated more mutations than did RSO-derived ones, and the number of mutations and the rates of their accumulation were higher in Vero than in AGMK cells. While the rates of accumulation of most mutations were higher when passaging was performed at 37 degrees, a U-->C transition at nucleotide 5832 occurred faster at 34 degrees, the temperature used for vaccine production. Analysis of Type 3 oral poliovirus vaccine (OPV) monopools made by six manufacturers found only 5 of these newly identified mutations in vaccine batches (nucleotides 3956, 4935, 5357, 5788, and 5832). Some of the mutations were found in trace amounts (less than 0.1%) while others were present at up to 1.8% levels. The pattern of these mutations was characteristic for the type of seed virus and the cell substrate but demonstrated no correlation with results of the monkey neurovirulence test. Therefore the only mutation occurring in Type 3 OPV which contributed to neurovirulence in monkeys was the previously described reversion at nucleotide 472. Quantitation of reversion at nucleotide 472 can be utilized for assessment of acceptability of vaccine lots, while other mutations can be used for monitoring the consistency of vaccine production.

Genetic stability and mutant selection in Sabin 2 strain of oral poliovirus vaccine grown under different cell culture conditions.

Mutations that consistently accumulated in the attenuated Sabin 2 strain of poliovirus during propagation in cell cultures were identified by sequence heterogeneity assay and quantified by mutant analysis by PCR and restriction enzyme cleavage (MAPREC). Eight additional sites previously identified in stool isolates were also examined by MAPREC in the virus passages. The pattern of selectable mutations and the rate of their accumulation depended on the type and confluence of the cell culture and the temperature of virus growth. Five unstable genomic sites were identified in Sabin 2 virus passaged 10 times at 34 degrees in African green monkey kidney (AGMK) cells, with the mutations accumulating in the range 1 to 24%. Accumulation of these mutations did not appear to result in a loss of attenuated phenotype since the virus passaged under these conditions passed the monkey neurovirulence test (MNVT). The content of the 481-G revertant known to be related to neurovirulence in monkeys did not increase. Thus, our results suggest that upon growth of Sabin 2 virus in AGMK cells at 34 degrees, the key determinant(s) of attenuation remained stable, and the mutations that occurred did not affect monkey neurovirulence. In virus passaged 10 times at 37 degrees in AGMK cells, 4 unstable genomic sites were identified, in some of them accumulating up to 12% of the mutants. This virus sample severely failed the MNVT. Virus passaged in Vero cells at 34 and 37 degrees accumulated mutants at 7 and 14 genomic sites, respectively, including 481-G in both cases, with almost complete substitution of the original nucleotides at some of the sites. We tested 44 commercial monopools of Type 2 OPV and found out that all of them contained 481-G revertants in the range 0.4-1.1%. An increase in the 481-G revertants in passaged viruses to the level of 4% and above correlated with failure of these samples by the MNVT. Since the pattern of selectable mutations differed in viruses grown in the two cell cultures used in this study, specific mutation profiles should be determined for each cell substrate used for vaccine production to assess manufacturing consistency.

Microevolution of Sabin 1 strain in vitro and genetic stability of oral poliovirus vaccine.

Mutants consistently accumulating in Sabin 1 poliovirus during serial passaging in vitro were identified by sequence heterogeneity assay and quantitated using mutant analysis by PCR and restriction enzyme cleavage (MAPREC). Only four unstable genomic sites were identified in virus passaged 10 times in African green monkey kidney (AGMK) cells, and eight sites in virus passaged in Vero cells. Mutations accumulated both in untranslated regions of RNA (nucleotides 480, 525 and 7441) and in coding sequences, as missense (nucleotides 1449, 4944, and 6203) or silent (nucleotides 1123 and 1141) mutations. The most prominent selectable mutations were found at complementary nucleotides 480 and 525 of the 5'-untranslated region (5'-UTR) of the Sabin strain, changing the G:U pair in F-domain to either A:U or G:C variants. These two variants have been shown previously to have an increased neurovirulence in monkeys. The G:C variant accumulated during passage in Vero cells, while A:U variant accumulated in CV-1 cells. Virus passaged in AGMK cells accumulated both variants. Higher temperature (37 instead of 34 degrees) strongly favored selection of mutants in Vero cells, had a smaller effect on mutant accumulation in AGMK cells, and had no effect in CV-1 cells. Monopools of type 1 oral poliovirus vaccine (OPV) made by seven manufacturers were found to contain both 480-A and 525-C revertants at a combined level of 1.1-2.7%. Viral samples with increased amounts of these revertants had higher neurovirulence in monkeys. Our results suggest that quantitation of these reversions by MAPREC may be prognostic for results of the monkey neurovirulence test (MNVT) and can be used for monitoring type 1 OPV consistency.

Consistent selection of mutations in the 5'-untranslated region of oral poliovirus vaccine upon passaging in vitro.

We have previously found that upon passaging type 3 oral poliovirus vaccine (OPV) in cell cultures the proportion of revertants at nucleotide 472 rapidly increases [Chumakov et al.: Proceedings of the National Academy of Sciences of the United States of America 88:199-203 1991]. Systematic study on the accumulation of these revertants showed that it was dependent on the multiplicity of infection and the temperature at which virus was grown. Revertants at position 472 of type 3 OPV accumulated faster in vaccines derived from Sabin Original (SO) substrain than from RNA-plaque purified (RSO) substrain. The rate of accumulation of 472-C revertants differed among cell lines and was higher in overgrown cell cultures suggesting that host factors are involved in the selection of mutants. We also found that accumulation of mutants occurred in vitro at position 480 in type 1 and position 481 in type 2 OPV, making the selection for revertants in domain F of the 5'-noncoding region a general phenomenon for all three Sabin strains. Assessment of the abundance of these mutants may be used for evaluation of the quality of OPV lots.

RNA sequence variants in live poliovirus vaccine and their relation to neurovirulence.

Mutant analysis by polymerase chain reaction and restriction enzyme cleavage (MAPREC) was used to study sequence heterogeneity and stability in attenuated poliovirus type 3 at positions in which the vaccine virus differs from its wild-type progenitor. Of seven genomic positions tested, only two (positions 472 and 2493) show nucleotide heterogeneity. Propagation of the vaccine virus in cell cultures leads to rapid selection of virus with reversions at these two positions of the genome. The relative abundance of reversions at position 472 correlates with the results of monkey neurovirulence tests, while the mutation at position 2493 is not directly associated with neurovirulence of the virus in monkeys. Instead, the abundance of mutations at the latter position correlates with the source of the seed virus and its passage level. These results further indicate that MAPREC at position 472 can be used to assess the quality of poliovirus type 3 vaccine.

Experimental evaluation of antitoxic protective effect of new cholera vaccines in mice.

Intraperitoneal immunization of mice and subsequent challenge with purified cholera toxin (CT) were employed to evaluate the anti-cholera toxin protective effect of two new oral cholera vaccines, live CVD 103-HgR and killed B subunit-whole cell (BS-WC). CVD 103-HgR vaccine demonstrated 100% protection of mice against 2.25 LD50 and 70% against 3 LD50 of CT. Mice immunized with BS-WC vaccine were protected against 2.25 and 3 LD50 of CT in 88 and 62% of cases, respectively. All three killed parenteral vaccines failed to protect against CT. We suggest this mouse system for preliminary evaluation of the antitoxic protective activity of cholera vaccines.

Propagation and properties of Kaposi's sarcoma-derived cell lines obtained from patients with AIDS: similarity of cultured cells to smooth muscle cells.

Cells derived from Kaposi's sarcoma (KS) were propagated in vitro using conditions which resulted in elimination of contaminating fibroblasts and the emergence of homogeneous cell populations which morphologically resembled smooth muscle cells and had neoplastic characteristics. In long-term culture, they differentiated into large ribbon-like cells with longitudinal fibrillarity of their cytoplasm. These fibrils stained red by Masson trichrome staining, and were reactive with antibodies to desmin. Dense bodies typical of myoblasts were observed in some cells by electron microscopy. The cells did not form capillary structures like endothelial cells, they lacked Weible-Palade bodies, and did not express the blood-clotting Factor VIII-related antigen or receptors for the lectin Ulex europaeus agglutinin I. They did express four other antigens, however, in common with endothelial cells. The cells did not form tumors in athymic nude mice; however, they formed colonies in soft agar, manifested tumor-like growth on muscle organ cultures, and were invasive in an artificial basement membrane invasion assay. The results indicate that a component of KS is closely related to leiomyoblasts and and has neoplastic properties.

Correlation between amount of virus with altered nucleotide sequence and the monkey test for acceptability of oral poliovirus vaccine.

Production of live attenuated oral poliomyelitis vaccine (OPV) requires rigorous neurovirulence safety testing of each vaccine lot, currently carried out in monkeys. It has been reported that a change from 472-U to 472-C in the type 3 OPV RNA is associated with an increased histologic lesion score produced upon intraspinal inoculation of the mutant virus in monkeys. We have developed a method, based on polymerase chain reaction, for measuring the relative abundance of these mutant sequences directly in vaccine preparations and used this method to evaluate the proportion of 472-C in 40 different lots of type 3 OPV. Six vaccine lots that had failed the intraspinal monkey neurovirulence test contained a higher proportion of 472-C than all other lots that had passed this test. OPV type 3 virus containing 472-C was rapidly selected during serial passages in African green monkey kidney cells that are used for manufacturing of the vaccine. We have also found that the wild-type poliovirus type 3 strain Leon/37, from which the vaccine strain was originally derived, contained a mixture of 472-U and 472-C sequences. No other mutations in OPV type 3 RNA have been detected by similar assays at position 2034, also associated with attenuation, or at several other positions reported to be altered in some vaccine preparations. Our results suggest that molecular diagnostics may provide a supplement or a potential alternative to animal testing of live attenuated vaccines.

In vitro characterization of Salmonella typhi mutant strains for live oral vaccines.

Several Salmonella typhi attenuated mutant strains, suggested as candidates for live oral vaccine, were examined for their characteristics in vitro in comparison with parental strains Ty2 and CDC10-80. Three methods were used: interaction of bacteria with the human monocyte-macrophage U937 cell line evaluated by microscopic examination, bacterial growth in the cell culture medium estimated by absorbance and bacterial resistance to human plasma assessed by the viable count technique. The most informative data were obtained in the test with U937 cells. Ty2 penetrated almost 100% of the cells, multiplied rapidly and caused death of the cells. CDC10-80 infected about 30% of the cells, multiplied slightly and did not kill the cells. The Ty2 mutant galE via EX462 behaved like CDC10-80. Bacteria of the galE Ty21a, Vi + Ty21a, 541 Ty and 543 Ty, found in only 3-4% of the cells, did not multiply within the cells and decreased in number with time. These findings correlate with the reported virulence of these strains for humans. With the second method, the rate of bacterial growth in cell culture medium did not differentiate Ty2, CDC10-80 and EX462. They grew at the same rate and faster than the remaining mutants. The plasma resistance test did not discriminate between EX462 and other mutants. These tests did not reveal any difference between Vi + Ty21a and Vi-Ty21a.

Salmonella typhi vaccine strain in vitro; low infectivity in human cell line U937.

Salmonella typhi strain Ty21a has been used for live oral vaccine. The infectivity of Ty21a, in comparison with S. typhi Ty2, was evaluated using the human monocyte-macrophage cell line U937. Assays were performed by quantitative microscopy and viable count technique. Ty2 infected approximately 100% of the cells, multiplied extensively within these cells and caused cell death. The same dose of Ty21a infected only about 15% of the cells, resulting in a low number of intracellular bacilli and cell survival. The use of gentamicin in the test confirmed intracellular multiplication of Ty2 but not Ty21a. The system described may be suitable as a test system for characterization of the degree of virulence of Ty21a and other live, oral typhoid vaccines.

The monkey safety test for neurovirulence of yellow fever vaccines: the utility of quantitative clinical evaluation and histological examination.

Two different derivatives of the 17D strain of yellow fever (YF) vaccine virus, i.e. ALV-free seed virus 6676 and three consecutive vaccine lots, A, B and C, obtained from another seed, were compared in monkey neurovirulence tests using rhesus and cynomolgus monkeys. In addition the ALV-contaminated seed lot AB 237 was safety tested in rhesus monkeys. According to WHO clinical criteria for acceptability, lots A, B, C and lot AB 237 consistently passed, while lot 6676 passed some tests and failed others. In the same tests, quantitative clinical evaluation and histological examination of CNS gave more definitive and consistent data on the higher degree of neurovirulence of lot 6676. Especially differentiating histological findings were obtained from some anatomical structures of the CNS ('discriminator areas') and almost no difference was found between the products in the structures apparently most susceptible to YF virus ('target areas'). Although some of the target and discriminator areas were different, cynomolgus monkeys were not less susceptible than rhesus monkeys to YF vaccine virus. The use of a quantitative method of scoring specific lesions in CNS of monkeys and the comparison of the average scores of a YF vaccine lot under study with a 'reference' vaccine, which has proven to be safe and effective in humans, should provide for a more reproducible method of assessing vaccine neurovirulence.

Early passage primate cell immortality is independent of tumorigenicity.

Seven continuous primate cell lines were tested in three systems (nude mice, muscle organ culture, and soft agarose) for their ability to express characteristics usually associated with malignant cell lines. Five of the seven cell lines failed to produce tumors in nude mice, failed to show a tumor-like pattern of growth in muscle organ culture, and failed to produce colonies in soft agarose. The remaining two cell lines showed different degrees of tumorigenicity in nude mice, and gave frankly positive results in the two in vitro assays. In addition, one of these lines appeared to progress from potential to overt tumorigenicity. We conclude that acquisition of infinite life in primate cell lines is not invariably equivalent to the ability to form tumors.

Rhesus diploid rabies vaccine (adsorbed): neurological safety in guinea pigs and Lewis rats.

Rabies vaccine produced in rhesus diploid cells (RDRV) and adsorbed on aluminium phosphate was evaluated for its neurological safety in guinea pigs and Lewis rats. The vaccine (as well as aluminium phosphate itself) in combination with myelin basic protein did not induce experimental allergic encephalomyelitis (EAE) when injected into either species. RDRV combined with complete Freund's adjuvant still failed to induce any signs of EAE. In contrast, basic protein combined with complete Freund's adjuvant induced severe EAE in both guinea pigs and rats. No experimental evidence was obtained to indicate adverse neuroimmunological activity of RDRV.

Sensitization induced in guinea pigs with beta-propiolactone-treated serum albumin: experimental evidence for the cause of allergic reactions in humans receiving human diploid cell rabies vaccines.

beta-Propiolactone-treated (BPL-T) homologous serum albumin caused anaphylaxis in guinea pigs with a frequency and severity equal to that of guinea pigs inoculated with human albumin. Untreated guinea pig serum albumin did not cause any reactions in these animals. Some recipients of current rabies vaccine produced in human diploid cells available in the USA develop systemic allergic reactions, usually following booster immunization. The BPL-T human albumin component of the vaccines was believed to be the cause of the complications. Our studies support this conclusion.

Tumorigenicity testing of primate cell lines in nude mice, muscle organ culture and for colony formation in soft agarose.

Primate neoplastic and finite cell lines were tested in one in vivo and two in vitro test systems: adult nude mice, muscle organ culture (MOC) and soft agarose (SA). Comparison of the sensitivity of the systems indicated that nude mice were inferior to either in vitro system: WI-38 VA13 (an SV40 transformed cell line) did not cause tumours in these animals yet it behaved as if it were neoplastic in MOC and formed colonies in SA. There was complete correlation between results obtained in MOC and SA. All cell lines which produced tumors in vivo were positive in both in vitro test systems. None of the lines which showed normal patterns in MOC and in SA was tumorigenic in nude mice. Since testing in vitro is simpler, faster, and is thought to be reliable, we recommend SA followed by MOC as the initial assays for determining tumorigenicity of cells.

Tumorigenicity of Vero cells.

One of the current criteria for evaluating the acceptability of cell lines for use in vaccine production is lack of tumorigenicity. Vero cells represent an example of a class of cells known as continuous cell lines. They were derived from African green monkey kidney, and their growth properties and culture characteristics have many advantages over other cell substrates for use in vaccine production. We have tested Vero cells for tumorigenicity in nude mice and in a human muscle organ culture system, and found a significant increase in their tumorigenic potential with increasing passage numbers. Cells at passage 232 and higher produced nodules in all nude mice inoculated. Histologically the nodules were well defined, anaplastic tumors, which exhibited some of the characteristics of renal adenocarcinomas. In about 6 to 8 days all of the nodules began to regress. Data were obtained that suggested an immune mechanism was the basis for the regression phenomenon.