Genetically Thermo-Stabilised, Immunogenic Poliovirus Empty Capsids; a Strategy for Non-replicating Vaccines.

While wild type polio has been nearly eradicated there will be a need to continue immunisation programmes for some time because of the possibility of re-emergence and the existence of long term excreters of poliovirus. All vaccines in current use depend on growth of virus and most of the non-replicating (inactivated) vaccines involve wild type viruses known to cause poliomyelitis. The attenuated vaccine strains involved in the eradication programme have been used to develop new inactivated vaccines as production is thought safer. However it is known that the Sabin vaccine strains are genetically unstable and can revert to a virulent transmissible form. A possible solution to the need for virus growth would be to generate empty viral capsids by recombinant technology, but hitherto such particles are so unstable as to be unusable. We report here the genetic manipulation of the virus to generate stable empty capsids for all three serotypes. The particles are shown to be extremely stable and to generate high levels of protective antibodies in animal models

Stabilisation of virus particles reduces infectivity.

<p>Different numbers and combinations of the stabilising mutations described in Tables <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006117#ppat.1006117.t001" target="_blank">1</a> & <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1006117#ppat.1006117.t003" target="_blank">3</a>, as well as others identified in similar ways, were introduced into capsid-coding sequences of infectious clones. HEp2c cells were transfected with infectious RNA transcripts and incubated at 37°C until 100% cytopathic effect (CPE) was observed, or frozen after 7 days if no CPE was apparent; clarified supernatants of these cell cultures were blind passaged into fresh HEp2c cells and cells incubated at 37°C until 100% CPE was observed or for a further 7 days. (A) type 1 Mahoney capsid mutants, (B) type 2 MEF-1 capsid mutants, (C) type 3 Leon capsid mutants. * no CPE observed after blind passage.</p

Mutations that suppress the effect of the capsid destabilising mutation VP3 91F in the Sabin type 3 strain.

<p>Mutations that suppress the effect of the capsid destabilising mutation VP3 91F in the Sabin type 3 strain.</p

Thermostability of type 3 Leon particles.

<p>Reactivity of (A) virus and (B) empty capsid aliquots with MAb 520 and MAb 517 in ELISA after incubation at different temperatures for 10’. MAb 520 is specific for D Antigen and MAb 517 is specific for C Antigen.</p

Mutations that destabilise capsids in super-optimally stable Leon mutants and are also present in wild type Saukett.

<p>Mutations that destabilise capsids in super-optimally stable Leon mutants and are also present in wild type Saukett.</p

Seroconversion and protection against challenge induced by VLPs.

<p>Transgenic mice expressing the human poliovirus receptor were immunised intraperitoneally once or twice (x2) with PBS or 0.5 human dose equivalents of IPV or VLPs (A, B—type 1; C, D—type 2; E, F—type 3) then challenged intramuscularly with 25 PD₅₀ of homologous wild type virus. Graphs A, C & E show neutralising antibody titres against homologous serotype viruses in blood samples taken the day prior to challenge. Graphs B, D & F show survival rates following challenge with (B) type 1 Mahoney, (D) type 2 MEF-1 and (F) type 3 Saukett. Bars (A, C & E) indicate 95% CI of the geometric mean titre.</p

Thermostability of empty capsid (VLP) preparations.

<p>Thermostability of empty capsid (VLP) preparations.</p

Long-term stability of virus and empty capsid preparations compared to the IPV reference.

<p>Proportion of D antigen reactivity remaining after incubation at 37°C relative to incubation at 4°C. Aliquots of IPV, virus and empty capsid samples were incubated at 37°C and samples were removed at intervals and analysed by D Antigen ELISA; reactivity is expressed relative to samples incubated at 4°C for the same period. (A) Type 1, (B) Type 2, (C) Type 3.</p

Mutations included in capsid stabilised mutants for further study: Mahoney-SC7, MEF-SC5a and Saukett-SC8.

<p>Mutations included in capsid stabilised mutants for further study: Mahoney-SC7, MEF-SC5a and Saukett-SC8.</p

Rational Design of Genetically Stable, Live-Attenuated Poliovirus Vaccines of All Three Serotypes: Relevance to Poliomyelitis Eradication

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