Polio eradication: Addressing the hurdles on the last mile.

1988, the World Health Assembly committed to eradicate poliomyelitis, a viral disease that can cause permanent paralysis. Today, only type 1 of the three wild poliovirus types remains circulating in limited geographic areas following widespread use of different poliovirus vaccines. While we are close to zero new cases of wild polio, it is a fragile situation, and there are many remaining and new hurdles to overcome. Here, experts discuss how to address them.

A defective viral genome strategy elicits broad protective immunity against respiratory viruses.

RNA viruses generate defective viral genomes (DVGs) that can interfere with replication of the parental wild-type virus. To examine their therapeutic potential, we created a DVG by deleting the capsid-coding region of poliovirus. Strikingly, intraperitoneal or intranasal administration of this genome, which we termed eTIP1, elicits an antiviral response, inhibits replication, and protects mice from several RNA viruses, including enteroviruses, influenza, and SARS-CoV-2. While eTIP1 replication following intranasal administration is limited to the nasal cavity, its antiviral action extends non-cell-autonomously to the lungs. eTIP1 broad-spectrum antiviral effects are mediated by both local and distal type I interferon responses. Importantly, while a single eTIP1 dose protects animals from SARS-CoV-2 infection, it also stimulates production of SARS-CoV-2 neutralizing antibodies that afford long-lasting protection from SARS-CoV-2 reinfection. Thus, eTIP1 is a safe and effective broad-spectrum antiviral generating short- and long-term protection against SARS-CoV-2 and other respiratory infections in animal models.

Synthetic Genomes.

DNA synthesis technology has progressed to the point that it is now practical to synthesize entire genomes. Quite a variety of methods have been developed, first to synthesize single genes but ultimately to massively edit or write from scratch entire genomes. Synthetic genomes can essentially be clones of native sequences, but this approach does not teach us much new biology. The ability to endow genomes with novel properties offers special promise for addressing questions not easily approachable with conventional gene-at-a-time methods. These include questions about evolution and about how genomes are fundamentally wired informationally, metabolically, and genetically. The techniques and technologies relating to how to design, build, and deliver big DNA at the genome scale are reviewed here. A fuller understanding of these principles may someday lead to the ability to truly design genomes from scratch.

Experimental Evolution to Study Virus Emergence.

Understanding how viruses adapt to new environments and acquire new phenotypes is critical for developing comprehensive responses to outbreaks. By studying the emergence of vaccine-derived poliovirus outbreaks, Stern et al. describe how a combination of sequence analysis and experimental evolution can be used to reveal adaptive pathways.

The Evolutionary Pathway to Virulence of an RNA Virus.

Paralytic polio once afflicted almost half a million children each year. The attenuated oral polio vaccine (OPV) has enabled world-wide vaccination efforts, which resulted in nearly complete control of the disease. However, poliovirus eradication is hampered globally by epidemics of vaccine-derived polio. Here, we describe a combined theoretical and experimental strategy that describes the molecular events leading from OPV to virulent strains. We discover that similar evolutionary events occur in most epidemics. The mutations and the evolutionary trajectories driving these epidemics are replicated using a simple cell-based experimental setup where the rate of evolution is intentionally accelerated. Furthermore, mutations accumulating during epidemics increase the replication fitness of the virus in cell culture and increase virulence in an animal model. Our study uncovers the evolutionary strategies by which vaccine strains become pathogenic and provides a powerful framework for rational design of safer vaccine strains and for forecasting virulence of viruses. VIDEO ABSTRACT.

Phosphatidylserine vesicles enable efficient en bloc transmission of enteroviruses.

A central paradigm within virology is that each viral particle largely behaves as an independent infectious unit. Here, we demonstrate that clusters of enteroviral particles are packaged within phosphatidylserine (PS) lipid-enriched vesicles that are non-lytically released from cells and provide greater infection efficiency than free single viral particles. We show that vesicular PS lipids are co-factors to the relevant enterovirus receptors in mediating subsequent infectivity and transmission, in particular to primary human macrophages. We demonstrate that clustered packaging of viral particles within vesicles enables multiple viral RNA genomes to be collectively transferred into single cells. This study reveals a novel mode of viral transmission, where enteroviral genomes are transmitted from cell-to-cell en bloc in membrane-bound PS vesicles instead of as single independent genomes. This has implications for facilitating genetic cooperativity among viral quasispecies as well as enhancing viral replication.

Genetic stabilization of attenuated oral vaccines against poliovirus types 1 and 3.

Vaccination with Sabin, a live attenuated oral polio vaccine (OPV), results in robust intestinal and humoral immunity and has been key to controlling poliomyelitis. As with any RNA virus, OPV evolves rapidly to lose attenuating determinants critical to the reacquisition of virulence1-3 resulting in vaccine-derived, virulent poliovirus variants. Circulation of these variants within underimmunized populations leads to further evolution of circulating, vaccine-derived poliovirus with higher transmission capacity, representing a significant risk of polio re-emergence. A new type 2 OPV (nOPV2), with promising clinical data on genetic stability and immunogenicity, recently received authorization from the World Health Organization for use in response to circulating, vaccine-derived poliovirus outbreaks. Here we report the development of two additional live attenuated vaccine candidates against type 1 and 3 polioviruses. The candidates were generated by replacing the capsid coding region of nOPV2 with that from Sabin 1 or 3. These chimeric viruses show growth phenotypes similar to nOPV2 and immunogenicity comparable to their parental Sabin strains, but are more attenuated. Our experiments in mice and deep sequencing analysis confirmed that the candidates remain attenuated and preserve all the documented nOPV2 characteristics concerning genetic stability following accelerated virus evolution. Importantly, these vaccine candidates are highly immunogenic in mice as monovalent and multivalent formulations and may contribute to poliovirus eradication.

Cotranslational prolyl hydroxylation is essential for flavivirus biogenesis.

Viral pathogens are an ongoing threat to public health worldwide. Analysing their dependence on host biosynthetic pathways could lead to effective antiviral therapies1. Here we integrate proteomic analyses of polysomes with functional genomics and pharmacological interventions to define how enteroviruses and flaviviruses remodel host polysomes to synthesize viral proteins and disable host protein production. We find that infection with polio, dengue or Zika virus markedly modifies polysome composition, without major changes to core ribosome stoichiometry. These viruses use different strategies to evict a common set of translation initiation and RNA surveillance factors from polysomes while recruiting host machineries that are specifically required for viral biogenesis. Targeting these specialized viral polysomes could provide a new approach for antiviral interventions. For example, we find that both Zika and dengue use the collagen proline hydroxylation machinery to mediate cotranslational modification of conserved proline residues in the viral polyprotein. Genetic or pharmacological inhibition of proline hydroxylation impairs nascent viral polyprotein folding and induces its aggregation and degradation. Notably, such interventions prevent viral polysome remodelling and lower virus production. Our findings delineate the modular nature of polysome specialization at the virus-host interface and establish a powerful strategy to identify targets for selective antiviral interventions.

In vitro reconstitution reveals membrane clustering and RNA recruitment by the enteroviral AAA+ ATPase 2C.

Enteroviruses are a vast genus of positive-sense RNA viruses that cause diseases ranging from common cold to poliomyelitis and viral myocarditis. They encode a membrane-bound AAA+ ATPase, 2C, that has been suggested to serve several roles in virus replication, e.g. as an RNA helicase and capsid assembly factor. Here, we report the reconstitution of full-length, poliovirus 2C's association with membranes. We show that the N-terminal membrane-binding domain of 2C contains a conserved glycine, which is suggested by structure predictions to divide the domain into two amphipathic helix regions, which we name AH1 and AH2. AH2 is the main mediator of 2C oligomerization, and is necessary and sufficient for its membrane binding. AH1 is the main mediator of a novel function of 2C: clustering of membranes. Cryo-electron tomography reveal that several 2C copies mediate this function by localizing to vesicle-vesicle interfaces. 2C-mediated clustering is partially outcompeted by RNA, suggesting a way by which 2C can switch from an early role in coalescing replication organelles and lipid droplets, to a later role where 2C assists RNA replication and particle assembly. 2C is sufficient to recruit RNA to membranes, with a preference for double-stranded RNA (the replicating form of the viral genome). Finally, the in vitro reconstitution revealed that full-length, membrane-bound 2C has ATPase activity and ATP-independent, single-strand ribonuclease activity, but no detectable helicase activity. Together, this study suggests novel roles for 2C in membrane clustering, RNA membrane recruitment and cleavage, and calls into question a role of 2C as an RNA helicase. The reconstitution of functional, 2C-decorated vesicles provides a platform for further biochemical studies into this protein and its roles in enterovirus replication.

Enterovirus virus-like-particle and inactivated poliovirus vaccines do not elicit substantive cross-reactive antibody responses.

Human enteroviruses are the most common human pathogen with over 300 distinct genotypes. Previous work with poliovirus has suggested that it is possible to generate antibody responses in humans and animals that can recognize members of multiple enterovirus species. However, cross protective immunity across multiple enteroviruses is not observed epidemiologically in humans. Here we investigated whether immunization of mice or baboons with inactivated poliovirus or enterovirus virus-like-particles (VLPs) vaccines generates antibody responses that can recognize enterovirus D68 or A71. We found that mice only generated antibodies specific for the antigen they were immunized with, and repeated immunization failed to generate cross-reactive antibody responses as measured by both ELISA and neutralization assay. Immunization of baboons with IPV failed to generate neutralizing antibody responses against enterovirus D68 or A71. These results suggest that a multivalent approach to enterovirus vaccination is necessary to protect against enterovirus disease in vulnerable populations.

Safety of the novel oral poliovirus vaccine type 2 (nOPV2) in infants and young children aged 1 to <5 years and lot-to-lot consistency of the immune response to nOPV2 in infants in The Gambia: a phase 3, double-blind, randomised controlled trial.

BACKGROUND: Novel oral poliovirus vaccine type 2 (nOPV2) has been engineered to improve the genetic stability of Sabin oral poliovirus vaccine (OPV) and reduce the emergence of circulating vaccine-derived polioviruses. This trial aimed to provide key safety and immunogenicity data required for nOPV2 licensure and WHO prequalification. METHODS: This phase 3 trial recruited infants aged 18 to <52 weeks and young children aged 1 to <5 years in The Gambia. Infants randomly assigned to receive one or two doses of one of three lots of nOPV2 or one lot of bivalent OPV (bOPV). Young children were randomised to receive two doses of nOPV2 lot 1 or bOPV. The primary immunogenicity objective was to assess lot-to-lot equivalence of the three nOPV2 lots based on one-dose type 2 poliovirus neutralising antibody seroconversion rates in infants. Equivalence was declared if the 95% CI for the three pairwise rate differences was within the -10% to 10% equivalence margin. Tolerability and safety were assessed based on the rates of solicited adverse events to 7 days, unsolicited adverse events to 28 days, and serious adverse events to 3 months post-dose. Stool poliovirus excretion was examined. The trial was registered as PACTR202010705577776 and is completed. FINDINGS: Between February and October, 2021, 2345 infants and 600 young children were vaccinated. 2272 (96·9%) were eligible for inclusion in the post-dose one per-protocol population. Seroconversion rates ranged from 48·9% to 49·2% across the three lots. The minimum lower bound of the 95% CIs for the pairwise differences in seroconversion rates between lots was -5·8%. The maximum upper bound was 5·4%. Equivalence was therefore shown. Of those seronegative at baseline, 143 (85·6%) of 167 (95% CI 79·4-90·6) infants and 54 (83·1%) of 65 (71·7-91·2) young children seroconverted over the two-dose nOPV2 schedule. The post-two-dose seroprotection rates, including participants who were both seronegative and seropositive at baseline, were 604 (92·9%) of 650 (95% CI 90·7-94·8) in infants and 276 (95·5%) of 289 (92·4-97·6) in young children. No safety concerns were identified. 7 days post-dose one, 78 (41·7%) of 187 (95% CI 34·6-49·1) infants were excreting the type 2 poliovirus. INTERPRETATION: nOPV2 was immunogenic and safe in infants and young children in The Gambia. The data support the licensure and WHO prequalification of nOPV2. FUNDING: Bill & Melinda Gates Foundation.

An efficient trans complementation system for in vivo replication of defective poliovirus mutants.

The picornavirus genome encodes a large, single polyprotein that is processed by viral proteases to form an active replication complex. The replication complex is formed with the viral genome, host proteins, and viral proteins that are produced/translated directly from each of the viral genomes (viral proteins provided in cis). Efficient complementation in vivo of replication complex formation by viral proteins provided in trans, thus exogenous or ectopically expressed viral proteins, remains to be demonstrated. Here, we report an efficient trans complementation system for the replication of defective poliovirus (PV) mutants by a viral polyprotein precursor in HEK293 cells. Viral 3AB in the polyprotein, but not 2BC, was processed exclusively in cis. Replication of a defective PV replicon mutant, with a disrupted cleavage site for viral 3Cpro protease between 3Cpro and 3Dpol (3C/D[A/G] mutant) could be rescued by a viral polyprotein provided in trans. Only a defect of 3Dpol activity of the replicon could be rescued in trans; inactivating mutations in 2CATPase/hel, 3B, and 3Cpro of the replicon completely abrogated the trans-rescued replication. An intact N-terminus of the 3Cpro domain of the 3CDpro provided in trans was essential for the trans-active function. By using this trans complementation system, a high-titer defective PV pseudovirus (PVpv) (>107 infectious units per mL) could be produced with the defective mutants, whose replication was completely dependent on trans complementation. This work reveals potential roles of exogenous viral proteins in PV replication and offers insights into protein/protein interaction during picornavirus infection. IMPORTANCE: Viral polyprotein processing is an elaborately controlled step by viral proteases encoded in the polyprotein; fully processed proteins and processing intermediates need to be correctly produced for replication, which can be detrimentally affected even by a small modification of the polyprotein. Purified/isolated viral proteins can retain their enzymatic activities required for viral replication, such as protease, helicase, polymerase, etc. However, when these proteins of picornavirus are exogenously provided (provided in trans) to the viral replication complex with a defective viral genome, replication is generally not rescued/complemented, suggesting the importance of viral proteins endogenously provided (provided in cis) to the replication complex. In this study, I discovered that only the viral polymerase activity of poliovirus (PV) (the typical member of picornavirus family) could be efficiently rescued by exogenously expressed viral proteins. The current study reveals potential roles for exogenous viral proteins in viral replication and offers insights into interactions during picornavirus infection.

The development of resistance to an inhibitor of a cellular protein reveals a critical interaction between the enterovirus protein 2C and a small GTPase Arf1.

The cellular protein GBF1, an activator of Arf GTPases (ArfGEF: Arf guanine nucleotide exchange factor), is recruited to the replication organelles of enteroviruses through interaction with the viral protein 3A, and its ArfGEF activity is required for viral replication, however how GBF1-dependent Arf activation supports the infection remains enigmatic. Here, we investigated the development of resistance of poliovirus, a prototype enterovirus, to increasing concentrations of brefeldin A (BFA), an inhibitor of GBF1. High level of resistance required a gradual accumulation of multiple mutations in the viral protein 2C. The 2C mutations conferred BFA resistance even in the context of a 3A mutant previously shown to be defective in the recruitment of GBF1 to replication organelles, and in cells depleted of GBF1, suggesting a GBF1-independent replication mechanism. Still, activated Arfs accumulated on the replication organelles of this mutant even in the presence of BFA, its replication was inhibited by a pan-ArfGEF inhibitor LM11, and the BFA-resistant phenotype was compromised in Arf1-knockout cells. Importantly, the mutations strongly increased the interaction of 2C with the activated form of Arf1. Analysis of other enteroviruses revealed a particularly strong interaction of 2C of human rhinovirus 1A with activated Arf1. Accordingly, the replication of this virus was significantly less sensitive to BFA than that of poliovirus. Thus, our data demonstrate that enterovirus 2Cs may behave like Arf1 effector proteins and that GBF1 but not Arf activation can be dispensable for enterovirus replication. These findings have important implications for the development of host-targeted anti-viral therapeutics.

Structural basis for cloverleaf RNA-initiated viral genome replication.

The genomes of positive-strand RNA viruses serve as a template for both protein translation and genome replication. In enteroviruses, a cloverleaf RNA structure at the 5' end of the genome functions as a switch to transition from viral translation to replication by interacting with host poly(C)-binding protein 2 (PCBP2) and the viral 3CDpro protein. We determined the structures of cloverleaf RNA from coxsackievirus and poliovirus. Cloverleaf RNA folds into an H-type four-way junction and is stabilized by a unique adenosine-cytidine-uridine (A•C-U) base triple involving the conserved pyrimidine mismatch region. The two PCBP2 binding sites are spatially proximal and are located on the opposite end from the 3CDpro binding site on cloverleaf. We determined that the A•C-U base triple restricts the flexibility of the cloverleaf stem-loops resulting in partial occlusion of the PCBP2 binding site, and elimination of the A•C-U base triple increases the binding affinity of PCBP2 to the cloverleaf RNA. Based on the cloverleaf structures and biophysical assays, we propose a new mechanistic model by which enteroviruses use the cloverleaf structure as a molecular switch to transition from viral protein translation to genome replication.

Two-Year Duration of Immunity of Inactivated Poliovirus Vaccine: A Follow-up Study in Pakistan in 2020.

This was a follow-up study conducted in 2020 assessing changes in levels of type 2 poliovirus-neutralizing antibodies 2 years postimmunization in children who received inactivated poliovirus vaccine (IPV) in Karachi, Pakistan. Unexpectedly, the findings revealed an increase in seroprevalence of type 2 antibodies from 73.1% to 81.6% 1 year and 2 years after IPV, respectively. The increase in type 2 immunity could result from the intensive transmission of circulating vaccine-derived poliovirus type 2 (cVDPV2) in Karachi during the second year of IPV administration. This study suggests that the cVDPV2 outbreak detected in Pakistan infected large proportions of children in Karachi. Clinical Trials Registration . NCT03286803.

Modeling Poliovirus Transmission and Responses in New York State.

BACKGROUND: In July 2022, New York State (NYS) reported a case of paralytic polio in an unvaccinated young adult, and subsequent wastewater surveillance confirmed sustained local transmission of type 2 vaccine-derived poliovirus (VDPV2) in NYS with genetic linkage to the paralyzed patient. METHODS: We adapted an established poliovirus transmission and oral poliovirus vaccine evolution model to characterize dynamics of poliovirus transmission in NYS, including consideration of the immunization activities performed as part of the declared state of emergency. RESULTS: Despite sustained transmission of imported VDPV2 in NYS involving potentially thousands of individuals (depending on seasonality, population structure, and mixing assumptions) in 2022, the expected number of additional paralytic cases in years 2023 and beyond is small (less than 0.5). However, continued transmission and/or reintroduction of poliovirus into NYS and other populations remains a possible risk in communities that do not achieve and maintain high immunization coverage. CONCLUSIONS: In countries such as the United States that use only inactivated poliovirus vaccine, even with high average immunization coverage, imported polioviruses may circulate and pose a small but nonzero risk of causing paralysis in nonimmune individuals.

Impact of Supplementary Immunization Activities using Novel Oral Polio Vaccine Type 2 during a Large outbreak of Circulating Vaccine-Derived Poliovirus in Nigeria.

BACKGROUND: Novel oral poliovirus vaccine (OPV) type 2 (nOPV2) has been made available for outbreak response under an emergency use listing authorization based on supportive clinical trial data. Since 2021 more than 350 million doses of nOPV2 were used for control of a large outbreak of circulating vaccine-derived poliovirus type 2 (cVDPV2) in Nigeria. METHODS: Using a bayesian time-series susceptible-infectious-recovered model, we evaluate the field effectiveness of nOPV2 immunization campaigns in Nigeria compared with campaigns using monovalent OPV type 2 (mOPV2). RESULTS: We found that both nOPV2 and mOPV2 campaigns were highly effective in reducing transmission of cVDPV2, on average reducing the susceptible population by 42% (95% confidence interval, 28-54%) and 38% (20-51%) per campaign, respectively, which were indistinguishable from each other in this analysis (relative effect, 1.1 [.7-1.9]). Impact was found to vary across areas and between immunization campaigns. CONCLUSIONS: These results are consistent with the comparable individual immunogenicity of nOPV2 and mOPV2 found in clinical trials but also suggest that outbreak response campaigns may have small impacts in some areas requiring more campaigns than are suggested in current outbreak response procedures.

Differences in Oligomerization of the SARS-CoV-2 Envelope Protein, Poliovirus VP4, and HIV Vpu.

Viroporins constitute a class of viral membrane proteins with diverse roles in the viral life cycle. They can self-assemble and form pores within the bilayer that transport substrates, such as ions and genetic material, that are critical to the viral infection cycle. However, there is little known about the oligomeric state of most viroporins. Here, we use native mass spectrometry in detergent micelles to uncover the patterns of oligomerization of the full-length SARS-CoV-2 envelope (E) protein, poliovirus VP4, and HIV Vpu. Our data suggest that the E protein is a specific dimer, VP4 is exclusively monomeric, and Vpu assembles into a polydisperse mixture of oligomers under these conditions. Overall, these results revealed the diversity in the oligomerization of viroporins, which has implications for the mechanisms of their biological functions as well as their potential as therapeutic targets.

Co-folding and RNA activation of poliovirus 3Cpro polyprotein precursors.

Positive-strand RNA viruses use long open reading frames to express large polyproteins that are processed into individual proteins by viral proteases. Polyprotein processing is highly regulated and yields intermediate species with different functions than the fully processed proteins, increasing the biochemical diversity of the compact viral genome while also presenting challenges in that proteins must remain stably folded in multiple contexts. We have used circular dichroism spectroscopy and single molecule microscopy to examine the solution structure and self-association of the poliovirus P3 region protein composed of membrane binding 3A, RNA priming 3B (VPg), 3Cpro protease, and 3Dpol RNA-dependent RNA polymerase proteins. Our data indicate that co-folding interactions within the 3ABC segment stabilize the conformational state of the 3C protease region, and this stabilization requires the full-length 3A and 3B proteins. Enzymatic activity assays show that 3ABC is also an active protease, and it cleaves peptide substrates at rates comparable to 3Cpro. The cleavage of a larger polyprotein substrate is stimulated by the addition of RNA, and 3ABCpro becomes 20-fold more active than 3Cpro in the presence of stoichiometric amounts of viral cre RNA. The data suggest that co-folding within the 3ABC region results in a protease that can be highly activated toward certain cleavage sites by localization to specific RNA elements within the viral replication center, providing a mechanism for regulating viral polyprotein processing.

Novel Oral Poliovirus Vaccine 2 Safety Evaluation during Nationwide Supplemental Immunization Activity, Uganda, 2022.

Given its enhanced genetic stability, novel oral poliovirus vaccine type 2 was deployed for type 2 poliovirus outbreak responses under World Health Organization Emergency Use Listing. We evaluated the safety profile of this vaccine. No safety signals were identified using a multipronged approach of passive and active surveillance.