Production of antigenically stable enterovirus A71 virus-like particles in Pichia pastoris as a vaccine candidate.

Enterovirus A71 (EVA71) causes widespread disease in young children with occasional fatal consequences. In common with other picornaviruses, both empty capsids (ECs) and infectious virions are produced during the viral lifecycle. While initially antigenically indistinguishable from virions, ECs readily convert to an expanded conformation at moderate temperatures. In the closely related poliovirus, these conformational changes result in loss of antigenic sites required to elicit protective immune responses. Whether this is true for EVA71 remains to be determined and is the subject of this investigation.We previously reported the selection of a thermally resistant EVA71 genogroup B2 population using successive rounds of heating and passage. The mutations found in the structural protein-coding region of the selected population conferred increased thermal stability to both virions and naturally produced ECs. Here, we introduced these mutations into a recombinant expression system to produce stabilized virus-like particles (VLPs) in Pichia pastoris.The stabilized VLPs retain the native virion-like antigenic conformation as determined by reactivity with a specific antibody. Structural studies suggest multiple potential mechanisms of antigenic stabilization, however, unlike poliovirus, both native and expanded EVA71 particles elicited antibodies able to directly neutralize virus in vitro. Therefore, anti-EVA71 neutralizing antibodies are elicited by sites which are not canonically associated with the native conformation, but whether antigenic sites specific to the native conformation provide additional protective responses in vivo remains unclear. VLPs are likely to provide cheaper and safer alternatives for vaccine production and these data show that VLP vaccines are comparable with inactivated virus vaccines at inducing neutralising antibodies.

The safety and immunogenicity of two novel live attenuated monovalent (serotype 2) oral poliovirus vaccines in healthy adults: a double-blind, single-centre phase 1 study.

BACKGROUND: Use of oral live-attenuated polio vaccines (OPV), and injected inactivated polio vaccines (IPV) has almost achieved global eradication of wild polio viruses. To address the goals of achieving and maintaining global eradication and minimising the risk of outbreaks of vaccine-derived polioviruses, we tested novel monovalent oral type-2 poliovirus (OPV2) vaccine candidates that are genetically more stable than existing OPVs, with a lower risk of reversion to neurovirulence. Our study represents the first in-human testing of these two novel OPV2 candidates. We aimed to evaluate the safety and immunogenicity of these vaccines, the presence and extent of faecal shedding, and the neurovirulence of shed virus. METHODS: In this double-blind, single-centre phase 1 trial, we isolated participants in a purpose-built containment facility at the University of Antwerp Hospital (Antwerp, Belgium), to minimise the risk of environmental release of the novel OPV2 candidates. Participants, who were recruited by local advertising, were adults (aged 18-50 years) in good health who had previously been vaccinated with IPV, and who would not have any contact with immunosuppressed or unvaccinated people for the duration of faecal shedding at the end of the study. The first participant randomly chose an envelope containing the name of a vaccine candidate, and this determined their allocation; the next 14 participants to be enrolled in the study were sequentially allocated to this group and received the same vaccine. The subsequent 15 participants enrolled after this group were allocated to receive the other vaccine. Participants and the study staff were masked to vaccine groups until the end of the study period. Participants each received a single dose of one vaccine candidate (candidate 1, S2/cre5/S15domV/rec1/hifi3; or candidate 2, S2/S15domV/CpG40), and they were monitored for adverse events, immune responses, and faecal shedding of the vaccine virus for 28 days. Shed virus isolates were tested for the genetic stability of attenuation. The primary outcomes were the incidence and type of serious and severe adverse events, the proportion of participants showing viral shedding in their stools, the time to cessation of viral shedding, the cell culture infective dose of shed virus in virus-positive stools, and a combined index of the prevalence, duration, and quantity of viral shedding in all participants. This study is registered with EudraCT, number 2017-000908-21 and ClinicalTrials.gov, number NCT03430349. FINDINGS: Between May 22 and Aug 22, 2017, 48 volunteers were screened, of whom 15 (31%) volunteers were excluded for reasons relating to the inclusion or exclusion criteria, three (6%) volunteers were not treated because of restrictions to the number of participants in each group, and 30 (63%) volunteers were sequentially allocated to groups (15 participants per group). Both novel OPV2 candidates were immunogenic and increased the median blood titre of serum neutralising antibodies; all participants were seroprotected after vaccination. Both candidates had acceptable tolerability, and no serious adverse events occurred during the study. However, severe events were reported in six (40%) participants receiving candidate 1 (eight events) and nine (60%) participants receiving candidate 2 (12 events); most of these events were increased blood creatinine phosphokinase but were not accompanied by clinical signs or symptoms. Vaccine virus was detected in the stools of 15 (100%) participants receiving vaccine candidate 1 and 13 (87%) participants receiving vaccine candidate 2. Vaccine poliovirus shedding stopped at a median of 23 days (IQR 15-36) after candidate 1 administration and 12 days (1-23) after candidate 2 administration. Total shedding, described by the estimated median shedding index (50% cell culture infective dose/g), was observed to be greater with candidate 1 than candidate 2 across all participants (2·8 [95% CI 1·8-3·5] vs 1·0 [0·7-1·6]). Reversion to neurovirulence, assessed as paralysis of transgenic mice, was low in isolates from those vaccinated with both candidates, and sequencing of shed virus indicated that there was no loss of attenuation in domain V of the 5'-untranslated region, the primary site of reversion in Sabin OPV. INTERPRETATION: We found that the novel OPV2 candidates were safe and immunogenic in IPV-immunised adults, and our data support the further development of these vaccines to potentially be used for maintaining global eradication of neurovirulent type-2 polioviruses. FUNDING: Bill & Melinda Gates Foundation.

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.

Pathogenic Events in a Nonhuman Primate Model of Oral Poliovirus Infection Leading to Paralytic Poliomyelitis.

Despite a great deal of prior research, the early pathogenic events in natural oral poliovirus infection remain poorly defined. To establish a model for study, we infected 39 macaques by feeding them single high doses of the virulent Mahoney strain of wild type 1 poliovirus. Doses ranging from 107 to 109 50% tissue culture infective doses (TCID50) consistently infected all the animals, and many monkeys receiving 108 or 109 TCID50 developed paralysis. There was no apparent difference in the susceptibilities of the three macaque species (rhesus, cynomolgus, and bonnet) used. Virus excretion in stool and nasopharynges was consistently observed, with occasional viremia, and virus was isolated from tonsils, gut mucosa, and draining lymph nodes. Viral replication proteins were detected in both epithelial and lymphoid cell populations expressing CD155 in the tonsil and intestine, as well as in spinal cord neurons. Necrosis was observed in these three cell types, and viral replication in the tonsil/gut was associated with histopathologic destruction and inflammation. The sustained response of neutralizing antibody correlated temporally with resolution of viremia and termination of virus shedding in oropharynges and feces. For the first time, this model demonstrates that early in the infectious process, poliovirus replication occurs in both epithelial cells (explaining virus shedding in the gastrointestinal tract) and lymphoid/monocytic cells in tonsils and Peyer's patches (explaining viremia), extending previous studies of poliovirus pathogenesis in humans. Because the model recapitulates human poliovirus infection and poliomyelitis, it can be used to study polio pathogenesis and to assess the efficacy of candidate antiviral drugs and new vaccines.IMPORTANCE Early pathogenic events of poliovirus infection remain largely undefined, and there is a lack of animal models mimicking natural oral human infection leading to paralytic poliomyelitis. All 39 macaques fed with single high doses ranging from 107 to 109 TCID50 Mahoney type 1 virus were infected, and many of the monkeys developed paralysis. Virus excretion in stool and nasopharynges was consistently observed, with occasional viremia; tonsil, mesentery lymph nodes, and intestinal mucosa served as major target sites of viral replication. For the first time, this model demonstrates that early in the infectious process, poliovirus replication occurs in both epithelial cells (explaining virus shedding in the gastrointestinal tract) and lymphoid/monocytic cells in tonsils and Peyer's patches (explaining viremia), thereby supplementing historical reconstructions of poliovirus pathogenesis. Because the model recapitulates human poliovirus infection and poliomyelitis, it can be used to study polio pathogenesis, candidate antiviral drugs, and the efficacy of new vaccines.

Continued use of poliovirus after eradication: hyper-attenuated strains as a safe alternative for release testing of human immunoglobulins.

BACKGROUND: Wild-type poliovirus may be eradicated soon and under WHO GAPIII guidance, laboratory use will be discontinued or subject to strict containment. Per US Code of Federal Regulations, however, immunoglobulin lot release testing will still require use of replicating poliovirus. The suitability of S19 hyper-attenuated and apathogenic poliovirus strains as alternatives to the currently used wild-type virus in such a release assay was investigated. STUDY DESIGN AND METHODS: S19 poliovirus strains were propagated in a commercial setting using good virological practices and maintenance of the S19 hyper-attenuated genotype was confirmed by massively parallel sequencing. RESULTS: The attenuated phenotype of the produced S19 stocks was confirmed in a highly sensitive mouse-model. Equivalency in performance was seen in the lot release assay for the S19 and wild-type polioviruses. CONCLUSION: The deployment of such hyper-attenuated and thoroughly characterized S19 stocks in these and other essential activities might reconcile final containment measures with continued safe use of poliovirus.

New Strains Intended for the Production of Inactivated Polio Vaccine at Low-Containment After Eradication.

Poliomyelitis has nearly been eradicated through the efforts of the World Health Organization's Global Eradication Initiative raising questions on containment of the virus after it has been eliminated in the wild. Most manufacture of inactivated polio vaccines currently requires the growth of large amounts of highly virulent poliovirus, and release from a production facility after eradication could be disastrous; WHO have therefore recommended the use of the attenuated Sabin strains for production as a safer option although it is recognised that they can revert to a transmissible paralytic form. We have exploited the understanding of the molecular virology of the Sabin vaccine strains to design viruses that are extremely genetically stable and hyperattenuated. The viruses are based on the type 3 Sabin vaccine strain and have been genetically modified in domain V of the 5' non-coding region by changing base pairs to produce a cassette into which capsid regions of other serotypes have been introduced. The viruses give satisfactory yields of antigenically and immunogenically correct viruses in culture, are without measurable neurovirulence and fail to infect non-human primates under conditions where the Sabin strains will do so.

Production of antigenically stable enterovirus A71 virus-like particles in Pichia pastoris as a vaccine candidate.

Enterovirus A71 (EVA71) causes widespread disease in young children with occasional fatal consequences. In common with other picornaviruses, both empty capsids (ECs) and infectious virions are produced during the viral lifecycle. While initially antigenically indistinguishable from virions, ECs readily convert to an expanded conformation at moderate temperatures. In the closely related poliovirus, these conformational changes result in loss of antigenic sites required to elicit protective immune responses. Whether this is true for EVA71 remains to be determined and is the subject of this investigation. We previously reported the selection of a thermally resistant EVA71 genogroup B2 population using successive rounds of heating and passage. The mutations found in the structural protein-coding region of the selected population conferred increased thermal stability to both virions and naturally produced ECs. Here, we introduced these mutations into a recombinant expression system to produce stabilised virus-like particles (VLPs) in Pichia pastoris . The stabilised VLPs retain the native virion-like antigenic conformation as determined by reactivity with a specific antibody. Structural studies suggest multiple potential mechanisms of antigenic stabilisation, however, unlike poliovirus, both native and expanded EVA71 particles elicited antibodies able to directly neutralise virus in vitro . Therefore, the anti-EVA71 neutralising antibodies are elicited by sites which are not canonically associated with the native conformation, but whether antigenic sites specific to the native conformation provide additional protective responses in vivo remains unclear. VLPs are likely to provide cheaper and safer alternatives for vaccine production and these data show that VLP vaccines are comparable with inactivated virus vaccines at inducing neutralising antibodies.

Development of an Enzyme-Linked Immunosorbent Assay for Detection of the Native Conformation of Enterovirus A71.

Enterovirus A71 (EVA71) is a medically important virus that is commonly associated with hand, foot, and mouth disease (HFMD). It is responsible for periodic outbreaks, resulting in significant economic impact and loss of life. Vaccination offers the potential to control future outbreaks, and vaccine development has been increasingly the focus of global research efforts. However, antigenic characterization of vaccine candidates is challenging because there are few tools to characterize the different antigenic forms of the virus. As with other picornaviruses, EVA71 virions exist in two antigenic states, native (NAg) and expanded (HAg). It is likely that the composition of vaccines, in terms of the proportions of NAg and HAg, will be important for vaccine efficacy and batch-to-batch consistency. This paper describes the development of a single-chain fused variable (scFv) domain fragment and the optimization of a sandwich enzyme-linked immunosorbent assay (ELISA) for the specific detection of the NAg conformation of EVA71. NAg specificity of the scFv was demonstrated using purified EVA71, and conversion of NAg to HAg by heating resulted in a loss of binding. We have thus developed an effective tool for characterization of the specific antigenic state of EVA71. IMPORTANCE EVA71 is a medically important virus that is commonly associated with HFMD, resulting in periodic outbreaks, significant economic impact, and loss of life. Vaccination offers the potential to curtail future outbreaks, and vaccine development has been increasingly the focus of global research efforts. However, antigenic characterization of vaccine candidates is challenging because there are very limited effective tools to characterize the different antigenic forms of EV71. As with other picornaviruses, EVA71 virions exist in two antigenic states, native and expanded. This paper describes the development of an scFv and the optimization of a sandwich ELISA for the specific detection of the native conformation of EVA71 as an effective tool for characterization of the specific antigenic state of EVA71.

Generated Randomly and Selected Functionally? The Nature of Enterovirus Recombination.

Genetic recombination in RNA viruses is an important evolutionary mechanism. It contributes to population diversity, host/tissue adaptation, and compromises vaccine efficacy. Both the molecular mechanism and initial products of recombination are relatively poorly understood. We used an established poliovirus-based in vitro recombination assay to investigate the roles of sequence identity and RNA structure, implicated or inferred from an analysis of circulating recombinant viruses, in the process. In addition, we used next-generation sequencing to investigate the early products of recombination after cellular coinfection with different poliovirus serotypes. In independent studies, we find no evidence for a role for RNA identity or structure in determining recombination junctions location. Instead, genome function and fitness are of greater importance in determining the identity of recombinant progeny. These studies provide further insights into this important evolutionary mechanism and emphasize the critical nature of the selection process on a mixed virus population.

A conserved glutathione binding site in poliovirus is a target for antivirals and vaccine stabilisation.

Strategies to prevent the recurrence of poliovirus (PV) after eradication may utilise non-infectious, recombinant virus-like particle (VLP) vaccines. Despite clear advantages over inactivated or attenuated virus vaccines, instability of VLPs can compromise their immunogenicity. Glutathione (GSH), an important cellular reducing agent, is a crucial co-factor for the morphogenesis of enteroviruses, including PV. We report cryo-EM structures of GSH bound to PV serotype 3 VLPs showing that it can enhance particle stability. GSH binds the positively charged pocket at the interprotomer interface shown recently to bind GSH in enterovirus F3 and putative antiviral benzene sulphonamide compounds in other enteroviruses. We show, using high-resolution cryo-EM, the binding of a benzene sulphonamide compound with a PV serotype 2 VLP, consistent with antiviral activity through over-stabilizing the interprotomer pocket, preventing the capsid rearrangements necessary for viral infection. Collectively, these results suggest GSH or an analogous tight-binding antiviral offers the potential for stabilizing VLP vaccines.

Mammalian expression of virus-like particles as a proof of principle for next generation polio vaccines.

Global vaccination programs using live-attenuated oral and inactivated polio vaccine (OPV and IPV) have almost eradicated poliovirus (PV) but these vaccines or their production pose significant risk in a polio-free world. Recombinant PV virus-like particles (VLPs), lacking the viral genome, represent safe next-generation vaccines, however their production requires optimisation. Here we present an efficient mammalian expression strategy producing good yields of wild-type PV VLPs for all three serotypes and a thermostabilised variant for PV3. Whilst the wild-type VLPs were predominantly in the non-native C-antigenic form, the thermostabilised PV3 VLPs adopted the native D-antigenic conformation eliciting neutralising antibody titres equivalent to the current IPV and were indistinguishable from natural empty particles by cryo-electron microscopy with a similar stabilising lipidic pocket-factor in the VP1 ß-barrel. This factor may not be available in alternative expression systems, which may require synthetic pocket-binding factors. VLPs equivalent to these mammalian expressed thermostabilized particles, represent safer non-infectious vaccine candidates for the post-eradication era.

Plant-made polio type 3 stabilized VLPs-a candidate synthetic polio vaccine.

Poliovirus (PV) is the causative agent of poliomyelitis, a crippling human disease known since antiquity. PV occurs in two distinct antigenic forms, D and C, of which only the D form elicits a robust neutralizing response. Developing a synthetically produced stabilized virus-like particle (sVLP)-based vaccine with D antigenicity, without the drawbacks of current vaccines, will be a major step towards the final eradication of poliovirus. Such a sVLP would retain the native antigenic conformation and the repetitive structure of the original virus particle, but lack infectious genomic material. In this study, we report the production of synthetically stabilized PV VLPs in plants. Mice carrying the gene for the human PV receptor are protected from wild-type PV when immunized with the plant-made PV sVLPs. Structural analysis of the stabilized mutant at 3.6 Å resolution by cryo-electron microscopy and single-particle reconstruction reveals a structure almost indistinguishable from wild-type PV3.Despite the success of current vaccination against poliomyelitis, safe, cheap and effective vaccines remain sought for continuing eradication effort. Here the authors use plants to express stabilized virus-like particles of type 3 poliovirus that can induce a protective immune response in mice transgenic for the human poliovirus receptor.

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.