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.

Genetic and phenotypic stability of poliovirus shed from infants who received novel type 2 or Sabin type 2 oral poliovirus vaccines in Panama: an analysis of two clinical trials.

BACKGROUND: Sabin strains used in oral poliovirus vaccines (OPV) can revert to virulence and, in rare instances, cause disease or generate vaccine-derived strains leading to outbreaks in areas of low immunisation coverage. A novel OPV2 (nOPV2) was designed to stabilise the viral genome against reversion and reduce recombination events that might lead to virulent strains. In this study, we evaluated the genetic and phenotypic stability of shed poliovirus following administration of one dose of monovalent OPV2 (mOPV2) or nOPV2 to infants aged 18-22 weeks. METHODS: In two similarly designed clinical trials (NCT02521974 and NCT03554798) conducted in Panama, infants aged 18-22-weeks, after immunisation with three doses of bivalent OPV (types 1 and 3) and one dose of inactivated poliovirus vaccine, were administered one or two doses of mOPV2 or nOPV2. In this analysis of two clinical trials, faecally shed polioviruses following one dose of mOPV2 or nOPV2 were isolated from stools meeting predetermined criteria related to sample timing and viral presence and quantity and assessed for nucleotide polymorphisms using next-generation sequencing. A transgenic mouse neurovirulence test was adapted to assess the effect of the possible phenotypic reversion of shed mOPV2 and nOPV2 with a logistic regression model. FINDINGS: Of the 91 eligible samples, 86 were able to be sequenced, with 72 evaluated in the transgenic mouse assay. Sabin-2 poliovirus reverts rapidly at nucleotide 481, the primary attenuation site in domain V of the 5' untranslated region of the genome. There was no evidence of neurovirulence-increasing polymorphisms in domain V of shed nOPV2. Reversion of shed Sabin-2 virus corresponded with unadjusted paralysis rates of 47·6% at the 4 log10 50% cell culture infectious dose (CCID50) and 76·7% at the 5 log10 CCID50 inoculum levels, with rates of 2·8% for 4 log10 CCID50 and 11·8% for 5 log10 CCID50 observed for shed nOPV2 samples. The estimated adjusted odds ratio at 4·5 log10 of 0·007 (95% CI 0·002-0·023; p<0·0001) indicates significantly reduced odds of mouse paralysis from virus obtained from nOPV2 recipients compared with mOPV2 recipients. INTERPRETATION: The data indicate increased genetic stability of domain V of nOPV2 relative to mOPV2, with significantly lower neurovirulence of shed nOPV2 virus compared with shed mOPV2. While this vaccine is currently being deployed under an emergency use listing, the data on the genetic stability of nOPV2 will support further regulatory and policy decision-making regarding use of nOPV2 in outbreak responses. FUNDING: Bill & Melinda Gates Foundation.

Evaluating stability of attenuated Sabin and two novel type 2 oral poliovirus vaccines in children.

Novel oral poliovirus vaccine type 2 (nOPV2) is being developed to reduce the rare occurrence of disease and outbreaks associated with the genetic instability of the Sabin vaccine strains. Children aged 1 to 5 years were enrolled in two related clinical studies to assess safety, immunogenicity, shedding rates and properties of the shed virus following vaccination with nOPV2 (two candidates) versus traditional Sabin OPV type 2 (mOPV2). The anticipated pattern of reversion and increased virulence was observed for shed Sabin-2 virus, as assessed using a mouse model of poliovirus neurovirulence. In contrast, there were significantly reduced odds of mouse paralysis for shed virus for both nOPV2 candidates when compared to shed Sabin-2 virus. Next-generation sequencing of shed viral genomes was consistent with and further supportive of the observed neurovirulence associated with shed Sabin-2 virus, as well as the reduced reversion to virulence of shed candidate viruses. While shed Sabin-2 showed anticipated A481G reversion in the primary attenuation site in domain V in the 5' untranslated region to be associated with increased mouse paralysis, the stabilized domain V in the candidate viruses did not show polymorphisms consistent with reversion to neurovirulence. The available data from a key target age group for outbreak response confirm the superior genetic and phenotypic stability of shed nOPV2 strains compared to shed Sabin-2 and suggest that nOPV2 should be associated with less paralytic disease and potentially a lower risk of seeding new outbreaks.

Evaluation and validation of next-generation sequencing to support lot release for a novel type 2 oral poliovirus vaccine.

A novel, genetically-stabilized type 2 oral polio vaccine (nOPV2), developed to assist in the global polio eradication program, was recently the first-ever vaccine granted Emergency Use Listing by the WHO. Lot release tests for this vaccine included-for the first time to our knowledge-the assessment of genetic heterogeneity using next-generation sequencing (NGS). NGS ensures that the genetically-modified regions of the vaccine virus genome remain as designed and that levels of polymorphisms which may impact safety or efficacy are controlled during routine production. The variants present in nOPV2 lots were first assessed for temperature sensitivity and neurovirulence using molecular clones to inform which polymorphisms warranted formal evaluation during lot release. The novel use of NGS as a lot release test required formal validation of the method. Analysis of an nOPV2 lot spiked with the parental Sabin-2 strain enabled performance characteristics of the method to be assessed simultaneously at over 40 positions in the genome. These characteristics included repeatability and intermediate precision of polymorphism measurement, linearity of both spike-induced and nOPV2 lot-specific polymorphisms, and the limit-of-detection of spike-induced polymorphisms. The performance characteristics of the method met pre-defined criteria for 34 spike-induced polymorphic sites and 8 polymorphisms associated with the nOPV2 preparation; these sites collectively spanned most of the viral genome. Finally, the co-location of variants of interest on genomes was evaluated, with implications for the interpretation of NGS discussed.

Scaleable purification of adenovirus vectors.

Adenovirus vectors currently are being evaluated in gene delivery studies ranging from prophylactic vaccination to therapeutic gene therapy. The quantity of purified virus required for these studies necessitate that purification methods must shift from classical density gradient ultracentrifugation to scaleable approaches. A methodology is described herein using batch centrifugation, tangential flow ultrafiltration, and chromatography to purify adenovirus particles at a scale of approximately 10(13) viral particles. This method has been demonstrated to easily scale an additional 40-fold. While purification of human adenovirus type 5 is exemplified, modifications are suggested for the purification of other serotypes.

Sterilizing filtration of plasmid DNA: effects of plasmid concentration, molecular weight, and conformation.

Plasmid DNA purification development has been driven by the increased need for large quantities of highly purified, sterile plasmid DNA for clinical studies. Detailed characterization and development of the terminal sterile filtration process step is often limited due to time constraints and the scarcity of sufficient quantities of purified plasmid. However, the large size of the plasmid molecule and variations in conformation can lead to significant yield losses if this process step is not optimized. In this work, the gradual pore-plugging model of flow decay was found to be valid for plasmid DNA by using an ultra scaledown apparatus (1-4 cm(2) filter area). Filtration capacity was found to be insensitive to pressure. Multiple filter types were screened and both source and composition of materials were found to affect filter capacity dramatically. The filter capacity for plasmid was improved by increasing plasmid concentrations as well as by modifying buffer conditions to reduce the apparent size of the plasmid. Filtration capacities varied over a greater than 2 log range when plasmids with sizes ranging from 5.5 to 11 kb and supercoiled plasmid content of 55-95% were explored. Larger plasmids and feeds with lower supercoiled contents led to reduced capacities. These results can be used to define conditions for scale-up of plasmid sterile filtration, as evidenced by processing a 30 g lot using a filtration area of 1,000 cm(2), with a 96% yield, based on filtration capacity data from 4 cm(2) test filters.

Serotype specificity of adenovirus purification using anion-exchange chromatography.

Recombinant adenoviruses continue to be a leading vector choice for gene transfer applications, with growing interest in the use of less prevalent serotypes, and of chimeras. As a result, the development of scaleable purification processes for alternative serotypes is needed. Anion-exchange chromatography is routinely used for scaleable adenovirus type 5 purification; however, retention varies for other serotypes because of differences in the exposed capsid proteins. Understanding how the viral surface influences retention behavior can provide a rational basis for chromatography development and optimization. In this work, chimeric vectors were used to show that the hexon protein is responsible for retention differences in anion-exchange chromatography. Next, the relative retention of eight serotypes from three subgroups was studied. Although all serotypes bound to the anion-exchange resin, the sodium chloride required to elute the virus varied over a 2- fold range, from 270 to 490 mM. Retention was accurately correlated to the electrostatic properties of the hexon protein, with an average error in sodium chloride concentration required to elute of only 14 mM. This correlation enables preparative chromatography gradients for alternative serotypes to be established with minimal effort.

Development of a purification process for adenovirus: controlling virus aggregation to improve the clearance of host cell DNA.

The clearance of host cell DNA is a critical goal for purification process development for recombinant Ad5 (rAd5) based vaccines and gene therapy products. We have evaluated the clearance of DNA by a rAd5 purification process utilizing nuclease digestion, ultrafiltration, and anion exchange (AEX) chromatography and found residual host cell DNA to consistently reach a limiting value of about 100 pg/10(11) rAd5 particles. Characterization of the purified rAd5 product using serial AEX chromatography, hydroxyapatite chromatography, or nuclease treatment with and without particle disruption showed that the residual DNA was associated with virus particles. Using a variety of additional physical characterization methods, a population of rAd5 virus in an aggregated state was detected. Aggregation was eliminated using nonionic detergents to attenuate hydrophobic interactions and sodium chloride to attenuate electrostatic interactions. After implementation of these modifications, the process was able to consistently reduce host cell DNA to levels at or below 5 pg/10(11) rAd5 particles, suggesting that molecular interactions between cellular DNA and rAd5 are important determinants of process DNA clearance capability and that the co-purifying DNA was not encapsidated.

Monitoring of RNA clearance in a novel plasmid DNA purification process.

As the field of plasmid DNA-based vaccines and therapeutics matures, improved methods for impurity clearance monitoring are increasingly valuable for process development and scale-up. Residual host-cell RNA is a major impurity in current large-scale separation processes for the production of clinical-grade plasmid DNA. Current RNA detection technologies include quantitative rtPCR, HPLC, and fluorescent dye-based assays. However, these methodologies are difficult to employ as in-process tests primarily as a result of impurity and buffer interferences. To address the need for a method of measuring RNA levels in various process intermediates, a sample pretreatment strategy has been developed that utilizes spermidine affinity precipitation to eliminate a majority of solution impurities, followed by a quantitative precipitation with alcohol to concentrate RNA and allow detection at lower concentrations. RNA concentrations as low as 80 ng/mL have been measured using detection with gel electrophoresis and 20 ng/mL if microplate-based detection with Ribogreen fluorescent dye is used. The assay procedure has been utilized to troubleshoot RNA clearance issues encountered during scale-up of a novel, non-chromatographic purification process for plasmid DNA. Assay results identified residual liquor removal inadequacies as the source of elevated RNA levels, enabling process modifications in a timely fashion.

Production and formulation of adenovirus vectors.

Adenovirus vectors have attracted considerable interest over the past decade, with ongoing clinical development programs for applications ranging from replacement therapy for protein deficiencies to cancer therapeutics to prophylactic vaccines. Consequently, considerable product, process, analytical, and formulation development has been undertaken to support these programs. For example, "gutless" vectors have been developed in order to improve gene transfer capacity and durability of expression; new cell lines have been developed to minimize recombination events; production conditions have been optimized to improve volumetric productivities; analytical techniques and scaleable purification processes have advanced towards the goal of purified adenovirus becoming a "well-characterized biological"; and liquid formulations have been developed which maintain virus infectivity at 2-8 degrees C for over 18 months. These and other advances in the production of adenovirus vectors are discussed in detail in this review. In addition, the needs for the next decade are highlighted.

Development of a novel adenovirus purification process utilizing selective precipitation of cellular DNA.

The use of recombinant adenoviral vectors for vaccination and gene therapy requires the development of purification processes that are cost-effective, scalable, and capable of robust host cell DNA clearance. An adenovirus purification process was developed which incorporates selective precipitation of host cell DNA, enabling a reduction in the use of costly nucleases and chromatographic resins while substantially improving DNA and protein clearance capabilities. In this work, three cationic detergents were evaluated for their capacity to selectively precipitate DNA from adenovirus-containing cell lysate. Parameters including pH, sodium chloride concentration, nonionic surfactant concentration, and cell density were investigated during development of the precipitation step. In a novel application, the cationic detergent domiphen bromide was found to have superior selectivity for host cell DNA. In addition, domiphen bromide-induced precipitation of adenovirus was shown to be reversible, which reduces the importance of mixing. Precipitation of DNA in the cell lysate coupled with primary clarification resulted in 3 logs of DNA clearance and improved impurity clearance in the subsequent ultrafiltration step. As a result, nuclease treatment and/or anion exchange chromatography can be eliminated, or included exclusively to improve process robustness.