RESUMO
BACKGROUND: Type 2 circulating vaccine-derived polioviruses (cVDPV2) from Sabin oral poliovirus vaccines (OPVs) are the leading cause of poliomyelitis. A novel type 2 OPV (nOPV2) has been developed to be more genetically stable with similar tolerability and immunogenicity to that of Sabin type 2 vaccines to mitigate the risk of cVDPV2. We aimed to assess these aspects of nOPV2 in poliovirus vaccine-naive newborn infants. METHODS: In this randomised, double-blind, controlled, phase 2 trial we enrolled newborn infants at the Matlab Health Research Centre, Chandpur, Bangladesh. We included infants who were healthy and were a single birth after at least 37 weeks' gestation. Infants were randomly assigned (2:1) to receive either two doses of nOPV2 or placebo, administered at age 0-3 days and at 4 weeks. Exclusion criteria included receipt of rotavirus or any other poliovirus vaccine, any infection or illness at the time of enrolment (vomiting, diarrhoea, or intolerance to liquids), diagnosis or suspicion of any immunodeficiency disorder in the infant or a close family member, or any contraindication for venipuncture. The primary safety outcome was safety and tolerability after one and two doses of nOPV2, given 4 weeks apart in poliovirus vaccine-naive newborn infants and the primary immunogenicity outcome was the seroconversion rate for neutralising antibodies against type 2 poliovirus, measured 28 days after the first and second vaccinations with nOPV2. Study staff recorded solicited and unsolicited adverse events after each dose during daily home visits for 7 days. Poliovirus neutralising antibody responses were measured in sera drawn at birth and at age 4 weeks and 8 weeks. This study is registered on ClinicalTrials.gov, NCT04693286. FINDINGS: Between Sept 21, 2020, and Aug 16, 2021, we screened 334 newborn infants, of whom three (<1%) were found to be ineligible and one (<1%) was withdrawn by the parents; the remaining 330 (99%) infants were assigned to receive nOPV2 (n=220 [67%]) or placebo (n=110 [33%]). nOPV2 was well tolerated; 154 (70%) of 220 newborn infants in the nOPV2 group and 78 (71%) of 110 in the placebo group had solicited adverse events, which were all mild or moderate in severity. Severe unsolicited adverse events in 11 (5%) vaccine recipients and five (5%) placebo recipients were considered unrelated to vaccination. 306 (93%) of 330 infants had seroprotective maternal antibodies against type 2 poliovirus at birth, decreasing to 58 (56%) of 104 in the placebo group at 8 weeks. In the nOPV2 group 196 (90%) of 217 infants seroconverted by week 8 after two doses, when 214 (99%) had seroprotective antibodies. INTERPRETATION: nOPV2 was well tolerated and immunogenic in newborn infants, with two doses, at birth and 4 weeks, resulting in almost 99% of infants having protective neutralising antibodies. FUNDING: Bill & Melinda Gates Foundation.
Assuntos
Poliomielite , Poliovirus , Recém-Nascido , Humanos , Lactente , Pré-Escolar , Bangladesh , Anticorpos Antivirais , Vacina Antipólio Oral , Poliomielite/prevenção & controle , Anticorpos Neutralizantes , Método Duplo-CegoRESUMO
Enormous progress has been made in global efforts to eradicate poliovirus, using live-attenuated Sabin oral poliovirus vaccine (OPV). However, as the incidence of disease due to wild poliovirus has declined, vaccine-derived poliovirus (VDPV) has emerged in areas of low-vaccine coverage. Coordinated global cessation of routine, type 2 Sabin OPV (OPV2) use has not resulted in fewer VDPV outbreaks, and continued OPV use in outbreak-response campaigns has seeded new emergences in low-coverage areas. The limitations of existing vaccines and current eradication challenges warranted development of more genetically stable OPV strains, most urgently for OPV2. Here, we report using codon deoptimization to further attenuate Sabin OPV2 by changing preferred codons across the capsid to non-preferred, synonymous codons. Additional modifications to the 5' untranslated region stabilized known virulence determinants. Testing of this codon-deoptimized new OPV2 candidate (nOPV2-CD) in cell and animal models demonstrated that nOPV2-CD is highly attenuated, grows sufficiently for vaccine manufacture, is antigenically indistinguishable from Sabin OPV2, induces neutralizing antibodies as effectively as Sabin OPV2, and unlike Sabin OPV2 is genetically stable and maintains an attenuation phenotype. In-human clinical trials of nOPV2-CD are ongoing, with potential for nOPV strains to serve as critical vaccine tools for achieving and maintaining polio eradication.
RESUMO
Since 2005, a large poliomyelitis outbreak associated with type 2 circulating vaccine-derived poliovirus (cVDPV2) has occurred in northern Nigeria, where immunization coverage with trivalent oral poliovirus vaccine (tOPV) has been low. Phylogenetic analysis of P1/capsid region sequences of isolates from each of the 403 cases reported in 2005 to 2011 resolved the outbreak into 23 independent type 2 vaccine-derived poliovirus (VDPV2) emergences, at least 7 of which established circulating lineage groups. Virus from one emergence (lineage group 2005-8; 361 isolates) was estimated to have circulated for over 6 years. The population of the major cVDPV2 lineage group expanded rapidly in early 2009, fell sharply after two tOPV rounds in mid-2009, and gradually expanded again through 2011. The two major determinants of attenuation of the Sabin 2 oral poliovirus vaccine strain (A481 in the 5'-untranslated region [5'-UTR] and VP1-Ile143) had been replaced in all VDPV2 isolates; most A481 5'-UTR replacements occurred by recombination with other enteroviruses. cVDPV2 isolates representing different lineage groups had biological properties indistinguishable from those of wild polioviruses, including efficient growth in neuron-derived HEK293 cells, the capacity to cause paralytic disease in both humans and PVR-Tg21 transgenic mice, loss of the temperature-sensitive phenotype, and the capacity for sustained person-to-person transmission. We estimate from the poliomyelitis case count and the paralytic case-to-infection ratio for type 2 wild poliovirus infections that â¼700,000 cVDPV2 infections have occurred during the outbreak. The detection of multiple concurrent cVDPV2 outbreaks in northern Nigeria highlights the risks of cVDPV emergence accompanying tOPV use at low rates of coverage in developing countries.
Assuntos
Poliomielite/epidemiologia , Vacina Antipólio Oral/efeitos adversos , Vacinas contra Poliovirus/efeitos adversos , Poliovirus/fisiologia , Animais , Proteínas do Capsídeo/genética , Proteínas do Capsídeo/imunologia , Surtos de Doenças , Feminino , Humanos , Masculino , Camundongos , Dados de Sequência Molecular , Nigéria/epidemiologia , Filogenia , Poliomielite/virologia , Poliovirus/classificação , Poliovirus/genética , Poliovirus/imunologia , Vacina Antipólio Oral/administração & dosagem , Vacinas contra Poliovirus/genética , Vacinas contra Poliovirus/imunologiaRESUMO
Replicative fitness of poliovirus can be modulated systematically by replacement of preferred capsid region codons with synonymous unpreferred codons. To determine the key genetic contributors to fitness reduction, we introduced different sets of synonymous codons into the capsid coding region of an infectious clone derived from the type 2 prototype strain MEF-1. Replicative fitness in HeLa cells, measured by plaque areas and virus yields in single-step growth experiments, decreased sharply with increased frequencies of the dinucleotides CpG (suppressed in higher eukaryotes and most RNA viruses) and UpA (suppressed nearly universally). Replacement of MEF-1 capsid codons with the corresponding codons from another type 2 prototype strain (Lansing), a randomization of MEF-1 synonymous codons, increased the %G+C without increasing CpG, and reductions in the effective number of codons used had much smaller individual effects on fitness. Poliovirus fitness was reduced to the threshold of viability when CpG and UpA dinucleotides were saturated within and across synonymous codons of a capsid region interval representing only approximately 9% of the total genome. Codon replacements were associated with moderate decreases in total virion production but large decreases in the specific infectivities of intact poliovirions and viral RNAs. Replication of codon replacement viruses, but not MEF-1, was temperature sensitive at 39.5 degrees C. Synthesis and processing of viral intracellular proteins were largely unaltered in most codon replacement constructs. Replacement of natural codons with synonymous codons with increased frequencies of CpG and UpA dinucleotides may offer a general approach to the development of attenuated vaccines with well-defined antigenicities and very high genetic stabilities.
Assuntos
Capsídeo/metabolismo , Ilhas de CpG/genética , Fosfatos de Dinucleosídeos/genética , Nucleotídeos/genética , Poliovirus/genética , Poliovirus/patogenicidade , Animais , Códon , DNA Complementar/metabolismo , Técnicas Genéticas , Células HeLa , Humanos , Cinética , Camundongos , Fenótipo , Poliovirus/metabolismo , RNA Viral/metabolismoRESUMO
We have adapted our previously described poliovirus diagnostic reverse transcription-PCR (RT-PCR) assays to a real-time RT-PCR (rRT-PCR) format. Our highly specific assays and rRT-PCR reagents are designed for use in the WHO Global Polio Laboratory Network for rapid and large-scale identification of poliovirus field isolates.
Assuntos
Primers do DNA/genética , Sondas de Oligonucleotídeos/genética , Poliovirus/classificação , Poliovirus/isolamento & purificação , RNA Viral/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Humanos , Sensibilidade e EspecificidadeRESUMO
We replaced degenerate codons for nine amino acids within the capsid region of the Sabin type 2 oral poliovirus vaccine strain with corresponding nonpreferred synonymous codons. Codon replacements were introduced into four contiguous intervals spanning 97% of the capsid region. In the capsid region of the most highly modified virus construct, the effective number of codons used (N(C)) fell from 56.2 to 29.8, the number of CG dinucleotides rose from 97 to 302, and the G+C content increased from 48.4% to 56.4%. Replicative fitness in HeLa cells, measured by plaque areas and virus yields in single-step growth experiments, decreased in proportion to the number of replacement codons. Plaque areas decreased over an approximately 10-fold range, and virus yields decreased over an approximately 65-fold range. Perhaps unexpectedly, the synthesis and processing of viral proteins appeared to be largely unaltered by the restriction in codon usage. In contrast, total yields of viral RNA in infected cells were reduced approximately 3-fold and specific infectivities of purified virions (measured by particle/PFU ratios) decreased approximately 18-fold in the most highly modified virus. The replicative fitness of both codon replacement viruses and unmodified viruses increased with the passage number in HeLa cells. After 25 serial passages (approximately 50 replication cycles), most codon replacements were retained, and the relative fitness of the modified viruses remained well below that of the unmodified virus. The increased replicative fitness of high-passage modified virus was associated with the elimination of several CG dinucleotides. Potential applications for the systematic modulation of poliovirus replicative fitness by deoptimization of codon usage are discussed.
Assuntos
Capsídeo/metabolismo , Códon , Poliovirus/genética , Poliovirus/fisiologia , Replicação Viral , Sequência de Bases , Células HeLa , Humanos , Mutagênese Sítio-Dirigida , Poliovirus/crescimento & desenvolvimento , Vacina Antipólio Oral/genética , RNA Viral/genética , Ensaio de Placa Viral , Vírion/crescimento & desenvolvimentoRESUMO
SER virus, a paramyxovirus closely related to simian virus 5, induces no syncytium formation. The SER virus F protein has a long cytoplasmic tail (CT), and truncation or mutations of the CT result in enhanced syncytium formation (S. Seth, A. Vincent, and R. W. Compans, J. Virol. 77:167-178, 2003; S. Tong, M. Li, A. Vincent, R. W. Compans, E. Fritsch, R. Beier, C. Klenk, M. Ohuchi, and H.-D. Klenk, Virology 301:322-333, 2002). We hypothesized that the presence of the long CT serves to stabilize the metastable conformation of the F protein. We observed that the hemifusion, cytoplasmic content mixing, and syncytium formation ability of the wild-type SER virus F coexpressed with the SER virus hemagglutinin-neuraminidase (HN) protein was enhanced, both qualitatively and quantitatively, at elevated temperatures. We also observed enhanced hemifusion, content mixing, and syncytium formation in SER virus F- and HN-expressing cells at reduced pH conditions ranging between 4.8 and 6.2. We have obtained evidence that in contrast to other paramyxoviruses, entry of SER virus into cells occurs by a low-pH-dependent process, indicating that the conversion to the fusion-active state for SER virus F is triggered by exposure to reduced pH.
Assuntos
Fusão de Membrana , Respirovirus/patogenicidade , Proteínas Virais de Fusão/metabolismo , Animais , Linhagem Celular , Cricetinae , Regulação Viral da Expressão Gênica , Células Gigantes/fisiologia , Cobaias , Concentração de Íons de Hidrogênio , Respirovirus/genética , Respirovirus/fisiologia , Temperatura , Proteínas Virais de Fusão/genéticaRESUMO
SER virus is closely related to the paramyxovirus simian virus 5 (SV5) but is defective in syncytium formation. The SER virus F protein has a long cytoplasmic tail (CT) domain that has been shown to inhibit membrane fusion, and this inhibitory effect could be eliminated by truncation of the C-terminal sequence (S. Tong, M. Li, A. Vincent, R. W. Compans, E. Fritsch, R. Beier, C. Klenk, M. Ohuchi, and H.-D. Klenk, Virology 301:322-333, 2002). To study the sequence requirements for regulation of fusion, codons for SER virus F protein residues spanning amino acids 535 to 542 and 548 were mutated singly to alanines, and the two leucine residues at positions 539 and 548 were mutated doubly to alanines. We found that leu-539 and leu-548 in the CT domain played a critical role in the inhibition of fusion, as mutation of the two leucines singly to alanines partially rescued fusion, and the double mutation L539, 548A completely rescued syncytium formation. Mutation of charged residues to alanines had little effect on the suppression of fusion activity, whereas the mutation of serine residues to alanines enhanced fusion activity significantly. The L539, 548A mutant also showed extensive syncytium formation when expressed without the SER virus HN protein. By constructing a chimeric SV5-SER virus F CT protein, we also found that the inhibitory effect of the long CT of the SER virus F protein could be partially transferred to the SV5 F protein. These results demonstrate that an elongated CT of a paramyxovirus F protein interferes with membrane fusion in a sequence-dependent manner.