Rational design of genetically stable, live-attenuated poliovirus vaccines of all three serotypes: relevance to poliomyelitis eradication

Abstract

The global eradication of poliomyelitis caused by wild-type virus is likely to be completed within the next few years, despite immense logistic and political difficulties, and may ultimately be followed by the cessation of vaccination. However, the existing live-attenuated vaccines have the potential to revert to virulence, causing occasional disease, and viruses can be shed by immunocompromised individuals for prolonged periods of time. Moreover, several outbreaks of poliomyelitis have been shown to be caused by viruses derived from the Sabin vaccine strains. The appearance of such strains depends on the prevailing circumstances but poses a severe obstacle to strategies for stopping vaccination. Vaccine strains that are incapable of reversion at a measurable rate would provide a possible solution. Here, we describe the constructions of strains of type 3 poliovirus that are stabilized by the introduction of four mutations in the 5' noncoding region compared to the present vaccine. The strains are genetically and phenotypically stable under conditions where the present vaccine loses the attenuating mutation in the 5' noncoding region completely. Type 1 and type 2 strains in which the entire 5' noncoding regions of Sabin 1 and Sabin 2 were replaced exactly with that of one of the type 3 strains were also constructed. The genetic stability of 5' noncoding regions of these viruses matched that of the type 3 strains, but significant phenotypic reversion occurred, illustrating the potential limitations of a rational approach to the genetic stabilization of live RNA virus vaccines.

FIG. 1.

Predicted RNA secondary structure of domain V (nucleotides 471 to 538) of the type 3 vaccine parent, P3/Leon. Arrows show attenuating nucleotide changes found in the three Sabin vaccine strains (attenuating bases are in boldface type, followed by the relevant serotype in parentheses). Due to slight differences in lengths of the 5′ noncoding regions, nucleotide 480 in Sabin 1 is at position 483 in this figure, and nucleotide 481 in Sabin 2 is at position 484. The base-paired stem regions are designated a, b, and c.

FIG. 2.

Correlation of base pair strength of nucleotides 472/537 with temperature sensitivity. Leon and site-directed mutants, which differed from Leon only at nucleotides 472 and 537, were assayed by plaque formation at different temperatures in L20B cells. Curves represent the reductions in numbers of plaques compared to the numbers of plaques formed at 35°C as a function of temperature. Symbols: ⧫, Leon; •, Leon/UA; ▴, Leon/UG.

FIG. 3.

Temperature sensitivities of type 1 and type 2 strains in different cells. Viruses were assayed by plaque formation at different temperatures in L20B cells (filled symbols) and Hep2C cells (open symbols), and results were plotted on graphs showing the reduction in the numbers of PFU at each temperature compared to the numbers of PFU at 35°C. (A) Triangles, Sabin 1; squares, S15/1. (B) Triangles, Sabin 2; squares, S15/2.

FIG. 4.

Stability of 472U in Sabin 3 upon passage in different cell lines. Sabin 3 was passaged 10 times in different cells at 37°C, and the 472C content was then measured by PCR and restriction endonuclease digestion (MAPREC). Symbols: ⧫, L20B cells; •, Vero cells; ▴ MRC-5 cells. L20B cells are mouse L cells expressing the human poliovirus receptor. Vero cells and MRC-5 cells are used for vaccine production.

FIG. 5.

Stability of attenuating mutations in domain V of Sabin 2 and Sabin 1 upon passage in different cell lines. Viruses were passaged 10 times in different cells at 37°C, and mutant proportions were then measured by PCR and restriction endonuclease digestion (MAPREC). (A) Mutation at nucleotide 481 in Sabin 2 during passage in L20B cells (▴) and Vero cells (▪). (B) Mutation in Sabin 1 during passage in Vero cells at nucleotide 480 or 525 (▴) and at nucleotide 476 (▪). (C) Mutation in Sabin 1 during passage in L20B cells at nucleotide 480 or 525 (▴) and at nucleotide 476 (▪).