Poliovirus-binding inhibition ELISA for evaluation of immune response to oral poliovirus vaccine: a possible alternative to the neutralization test.

This study describes an ELISA variant (Binding Inhibition ELISA, BI ELISA) for the quantitative determination of neutralization-relevant antibodies to polioviruses. The test differs from previously described analogs by utilizing polyclonal immune reagents: capture antibodies and biotin-labeled detecting antibodies. The BI ELISA was compared with the conventional neutralization test (NT) by testing live Sabin and wild-type poliovirus strains. The comparison of 68 serum samples taken from Oral Poliovirus Vaccine (OPV) recipients showed 100% specificity and sensitivity for Sabin 1 and Sabin 2, and 100% sensitivity and 97% specificity for Sabin 3. Good correlations (r = 0.7, 0.77, 0.65 for Sabin 1, 2, 3, respectively) were demonstrated between the NT and BI ELISA results. These results indicate that the BI ELISA can be used as a reliable alternative to the NT for determination of neutralization-relevant antibodies to polioviruses in vaccinees and in large-scale sero-epidemiological studies.

Mutations in Sabin 2 strain of poliovirus and stability of attenuation phenotype.

In this study, we attempted to identify the molecular determinants in the genome of the attenuated Sabin 2 vaccine strain of poliovirus that may change during vaccine production and result in an increase in monkey neurovirulence. An extensive search for suitable vaccine lots identified six batches that had failed the monkey neurovirulence test (MNVT). On repeated tests, these batches were found to have acceptable levels of monkey neurovirulence. One of the batches was additionally passaged six times under conditions used in vaccine production, and the resulting high-passage sample was screened for the presence of mutations and tested in monkeys. In addition to the previously described A --> G reversion at nucleotide 481, high-passage stock also contained a mutation in the VP1-coding region (3364 = G --> A) that consistently accumulated in the course of passaging. However, despite the presence of substantial amounts of these mutations, high-passage stock passed the MNVT. Replication of Sabin 2 poliovirus in the central nervous system of transgenic mice susceptible to poliovirus or in cultures of mouse cells, resulted in another mutation (3363 = A --> G). Even though its presence correlated with paralysis in mice, the introduction of 3363-G into the Sabin 2 genome did not increase neurovirulence of the virus. Previous studies identified the 481-G mutation as an important determinant of monkey neurovirulence. We prepared virus samples with varying amounts of genetically defined single mutants at this nucleotide and tested them in monkeys. The results demonstrated that even a 100% substitution at this site introduced into Sabin 2 strain did not increase monkey neurovirulence. The determination of the nucleotide sequence of an alternative strain used for the production of type 2 OPV (Chung 2) showed that it contained 100% of the wild-type 481-G but possessed an extremely low level of neurovirulence. These results demonstrate the remarkable stability of the attenuated phenotype of the Sabin 2 strain and show that (1) no batch of OPV 2 has ever repeatedly failed the MNVT, (2) growing the virus beyond the passage level allowed in vaccine production did not result in increased neurovirulence in monkeys, (3) a test for neurovirulence in transgenic mice may be more sensitive than the MNVT, and (4) determination of the mutational profile of vaccine batches detects inconsistencies in vaccine manufacturing processing that would not be detected by the MNVT.

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.

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.

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.