A human monoclonal antibody binds within the poliovirus receptor-binding site to neutralize all three serotypes.

Global eradication of poliovirus remains elusive, and it is critical to develop next generation vaccines and antivirals. In support of this goal, we map the epitope of human monoclonal antibody 9H2 which is able to neutralize the three serotypes of poliovirus. Using cryo-EM we solve the near-atomic structures of 9H2 fragments (Fab) bound to capsids of poliovirus serotypes 1, 2, and 3. The Fab-virus complexes show that Fab interacts with the same binding mode for each serotype and at the same angle of interaction relative to the capsid surface. For each of the Fab-virus complexes, we find that the binding site overlaps with the poliovirus receptor (PVR) binding site and maps across and into a depression in the capsid called the canyon. No conformational changes to the capsid are induced by Fab binding for any complex. Competition binding experiments between 9H2 and PVR reveal that 9H2 impedes receptor binding. Thus, 9H2 outcompetes the receptor to neutralize poliovirus. The ability to neutralize all three serotypes, coupled with the critical importance of the conserved receptor binding site make 9H2 an attractive antiviral candidate for future development.

Safety, tolerability, and immunogenicity of inactivated poliovirus vaccine with or without E.coli double mutant heat-labile toxin (dmLT) adjuvant in healthy adults; a phase 1 randomized study.

BACKGROUND: Inactivated trivalent poliovirus vaccine (IPV) induces humoral immunity, which protects against paralytic poliomyelitis but does not induce sufficient mucosal immunity to block intestinal infection. We assessed the intestinal immunity in healthy adults in Belgium conferred by a co-formulation of IPV with the mucosal adjuvant double mutant Labile Toxin (dmLT) derived from Escherichia coli. METHODS: Healthy fully IPV-vaccinated 18-45-year-olds were randomly allocated to three groups: on Day 1 two groups received one full dose of IPV (n = 30) or IPV + dmLT (n = 30) in a blinded manner, and the third received an open-label dose of bivalent live oral polio vaccine (bOPV types 1 and 3, n = 20). All groups received a challenge dose of bOPV on Day 29. Participants reported solicited and unsolicited adverse events (AE) using study diaries. Mucosal immune responses were measured by fecal neutralization and IgA on Days 29 and 43, with fecal shedding of challenge viruses measured for 28 days. Humoral responses were measured by serum neutralizing antibody (NAb). RESULTS: Solicited and unsolicited AEs were mainly mild-to-moderate and transient in all groups, with no meaningful differences in rates between groups. Fecal shedding of challenge viruses in both IPV groups exceeded that of the bOPV group but was not different between IPV and IPV + dmLT groups. High serum NAb responses were observed in both IPV groups, alongside modest levels of fecal neutralization and IgA. CONCLUSIONS: Addition of dmLT to IPV administered intramuscularly neither affected humoral nor intestinal immunity nor decreased fecal virus shedding following bOPV challenge. The tolerability of the dose of dmLT used in this study may allow higher doses to be investigated for impact on mucosal immunity. Registered on ClinicalTrials.gov - NCT04232943.

VaxArray immunoassay for the multiplexed quantification of poliovirus D-antigen.

Next generation poliovirus vaccines are critical to reaching global poliovirus eradication goals. Recent efforts have focused on creating inactivated vaccines using attenuated Sabin strains that maintain patient safety benefits and immunogenicity of conventional inactivated vaccines while increasing manufacturing safety and lowering production costs, and on developing novel oral vaccines using modified Sabin strains that provide critical mucosal immunity but are further attenuated to minimize risk of reversion to neurovirulence. In addition, there is a push to improve the analytical tools for poliovirus vaccine characterization. Conventional and Sabin inactivated poliovirus vaccines typically rely on standard plate-based ELISA as in vitro D-antigen potency assays in combination with WHO international standards as calibrants. While widely utilized, the current D-antigen ELISA assays have a long time to result (up to 72 h), can suffer from lab-to-lab inconsistency due to non-standardized protocols and reagents, and are inherently singleplex. For D-antigen quantitation, we have developed the VaxArray Polio Assay Kit, a multiplexed, microarray-based immunoassay that uses poliovirus-specific human monoclonal antibodies currently under consideration as standardized reagents for characterizing inactivated Sabin and Salk vaccines. The VaxArray assay can simultaneously quantify all 3 poliovirus serotypes with a time to result of less than 3 h. Here we demonstrate that the assay has limits of quantification suitable for both bioprocess samples and final vaccines, excellent reproducibility and precision, and improved accuracy over an analogous plate-based ELISA. The assay is suitable for adjuvanted combination vaccines, as common vaccine additives and crude matrices do not interfere with quantification, and is intended as a high throughput, standardized quantitation tool to aid inactivated poliovirus vaccine manufacturers in streamlining vaccine development and manufacturing, aiding the global polio eradication effort.

Evaluation of manufacturing feasibility and safety of an MDCK cell-based live attenuated influenza vaccine (LAIV) platform.

Cell culture based live attenuated influenza vaccines (LAIV) as an alternative to egg-based LAIV have been explored because of lack of easy access to SPF eggs for large scale production. In this study, feasibility of MDCK platform was assessed by including multiple LAIV strains covering both type A (H1 and H3) and type B seasonal strains as well as the candidate pandemic potential strains like A/H5 and A/H7 for the growth in MDCK cells. A risk assessment study was conducted on the cell banks to evaluate safety concerns related to tumorigenicity with a regulatory perspective. Tumorigenic potential of the MDCK cells was evaluated in nude mice (107cells/mouse) model system. The 50% tumor producing dose (TPD50) of MDCK cells was studied in SCID mice to determine the amount of cells required for induction of tumors. Further, we conducted an oncogenicity study in three sensitive rodent species as per the requirements specified in the WHO guidelines. We determined TPD50 value of 1.9 X 104 cells/mice through subcutaneous route. Our results suggest that, the intranasal route of administration of the cell culture based LAIV pose minimal to no risk of tumorigenicity associated with the host cells. Also, non-oncogenic nature of MDCK cells was demonstrated. Host cell DNA in the vaccine formulations was < 10 ng/dose which ensures vaccine safety. Production efficiency and consistency were characterized and the observed titer values of the viral harvest and the processed bulk were comparable to the expansion in embryonated eggs. The present study clearly establishes the suitability of MDCK cells as a substrate for the manufacture of a safe and viable LAIV.

Immunogenicity and efficacy comparison of MDCK cell-based and egg-based live attenuated influenza vaccines of H5 and H7 subtypes in ferrets.

During a pandemic, the availability of specific pathogen free chicken eggs is a major bottleneck for up-scaling response to the demand for influenza vaccine. This has led us to explore the use of Madin-Darby Canine Kidney (MDCK) cells for the manufacture of live attenuated influenza vaccine (LAIV) that provides production flexibility and speed. The present study reports the comparison of the immunogenicity and efficacy of two MDCK-based LAIVs against two egg-based LAIVs prepared from the same pandemic potential strains of H5 and H7 subtypes after a single dose of the vaccine followed by a challenge with a homologous wild type strain. The vaccine strains have been generated by classical method of reassortment using the A/Leningrad/134/17/57 master donor strain. Additionally, a prime-boost regimen of the MDCK-based vaccine followed by a challenge with a homologous wild type strain for H5 and H7 immunized ferrets and also a heterologous wild type strain for the H5 immunized animals was studied. No difference in the hemagglutination inhibition and virus neutralization antibody titers against the homologous virus was observed following a single dose of either egg-based or MDCK-based H5 and H7 LAIV vaccine. A second dose of MDCK-based vaccine significantly boosted antibody titers in the vaccinated animals. Both a single dose or two doses of LAIV provided complete protection from lower respiratory tract infection and resulted in a significant reduction in the virus titers recovered from the throat, nasal turbinates and lungs after challenge with the homologous wild type strain. Protection from a challenge with a heterologous strain of H5 was also observed after two doses of the MDCK-based LAIVs. This data strongly supports the use of MDCK as a substrate for the manufacture of LAIV which ensures reliable quality, safety, production flexibility, speed and breadth of protection, features that are highly critical during a pandemic.

Immunogenicity and efficacy of the monovalent, trivalent and quadrivalent intranasal live attenuated influenza vaccines containing different pdmH1N1 strains.

A ferret challenge study was conducted to address the efficacy of the egg-based and Madin-Darby canine kidney (MDCK)-based live attenuated influenza vaccine (LAIV) strains. Vaccines derived as 6:2 reassortants from the A/Leningrad/134/17/57 master donor strain and the HA and NA components from the A/California/07/2009 (A/Cal)- and A/Michigan/45/2015 (A/Mich)-like strains of type A H1N1 influenza virus were used in the study. Monovalent, trivalent and quadrivalent formulations of the LAIV containing either of the two H1N1 strains were analysed. A total of ten groups of six animals each were immunised intranasally (i.n.) with a single dose of 0.5-ml vaccine formulation or placebo and challenged on day 28 with the homologous wild-type A/Cal or A/Mich strain. Immune response post immunisation and virus replication post challenge were studied. Both the strains derived from embryonated eggs or MDCK cells, irrespective of the vaccine valency, were capable of rendering complete protection from virus replication in the lung. The A/Mich vaccine strain showed higher immune titres and efficacy than the A/Cal vaccine strain in all the vaccine formulations. The haemagglutination inhibition and virus neutralisation antibody titres were induced, and the reduction in the virus load in the respiratory tract was observed to be higher in animals treated with the monovalent formulation compared to the trivalent and quadrivalent formulations. Overall, it appears that the monovalent formulations render better protection from infection and would therefore be the best candidate during a pandemic.

A multi-laboratory study of diverse RSV neutralization assays indicates feasibility for harmonization with an international standard.

A current barrier to the standardized evaluation of respiratory syncytial virus (RSV) vaccine candidates is the wide variety of virus neutralization assay formats currently in use for assessing immunogenicity. Assay formats vary widely in labor intensiveness, duration, and sample throughput. Furthermore, the cell lines and virus strains used are not consistent among formats. The purpose of this multi-laboratory study was to assess the variability across a diverse array of assay formats that quantitate RSV neutralizing antibodies. Using a common specimen panel, the degree of overall agreement among existing assays was evaluated to inform on the need for harmonization of assay results. A total of 12 laboratories participated in the blinded survey study by testing a panel comprised of 57 samples chosen to span the reportable titer range of the assays. An independent statistical analysis was conducted to measure overall agreement of assay results. This analysis showed that precision was consistently high, whereas agreement varied widely among assays. To examine whether agreement could be improved, we conducted a harmonization exercise using a variety of sample types as pseudo standards. The results showed that the level of agreement could be improved, and provided information on the suitability of samples for developing an international standard.

Development of a stable liquid formulation of live attenuated influenza vaccine.

Vaccination is the most effective means of preventing influenza. However, the cost of producing annual seasonal influenza vaccines puts them out of reach for most developing countries. While live attenuated influenza vaccines are among the most efficacious and can be manufactured at low cost, they may require lyophilization to be stable enough for developing-country use, which adds a significant cost burden. The development of a liquid live attenuated seasonal influenza vaccine that is stable for around a year-the duration of an annual influenza season-would significantly improve not only the production output but also the use and accessibility of influenza vaccines in low-resource settings. In this study, potential stabilizing excipients were screened and optimized using the least stable influenza vaccine strain presently known, H1N1 (A/California/07/2009), as a model. The stability-conferring properties of the lead formulations were also tested with a Type B strain of influenza virus (B/Brisbane/60/2008). Stability was also evaluated with higher titers of influenza virus and exposure to agitation and freeze-thaw stresses to further confirm the stability of the lead formulations. Through this process, we identified a liquid formulation consisting of sucrose phosphate glutamate buffer with 1% arginine and 0.5% recombinant human serum albumin that provided storage stability of one year at 2-8°C for the influenza A and B strains tested.

Hexavalent IPV-based combination vaccines for public-sector markets of low-resource countries.

In anticipation of the successful eradication of wild polio virus, alternative vaccination strategies for public-sector markets of low-resource countries are extremely important, but are still under development. Following polio eradication, inactivated polio vaccine (IPV) would be the only polio vaccine available, and would be needed for early childhood immunization for several years, as maintenance of herd immunity will be important for sustaining polio eradication. Low-cost combination vaccines containing IPV could provide reliable and continuous immunization in the post-polio eradication period. Combination vaccines can potentially simplify complex pediatric routine immunization schedules, improve compliance, and reduce costs. Hexavalent vaccines containing Diphtheria (D), Tetanus (T), whole cell pertussis (wP), Hepatitis B (HBV), Haemophilus b (Hib) and the three IPV serotype antigens have been considered as the ultimate combination vaccine for routine immunization. This product review evaluates potential hexavalent vaccine candidates by composition, probable time to market, expected cost of goods, presentation, and technical feasibility and offers suggestions for development of low-cost hexavalent combination vaccines. Because there are significant technical challenges facing wP-based hexavalent vaccine development, this review also discusses other alternative approaches to hexavalent that could also ensure a timely and reliable supply of low-cost IPV based combination vaccines.

A trivalent virus-like particle vaccine elicits protective immune responses against seasonal influenza strains in mice and ferrets.

There is need for improved human influenza vaccines, particularly for older adults who are at greatest risk for severe disease, as well as to address the continuous antigenic drift within circulating human subtypes of influenza virus. We have engineered an influenza virus-like particle (VLP) as a new generation vaccine candidate purified from the supernatants of Sf9 insect cells following infection by recombinant baculoviruses to express three influenza virus proteins, hemagglutinin (HA), neuraminidase (NA), and matrix 1 (M1). In this study, a seasonal trivalent VLP vaccine (TVV) formulation, composed of influenza A H1N1 and H3N2 and influenza B VLPs, was evaluated in mice and ferrets for the ability to elicit antigen-specific immune responses. Animals vaccinated with the TVV formulation had hemagglutination-inhibition (HAI) antibody titers against all three homologous influenza virus strains, as well as HAI antibodies against a panel of heterologous influenza viruses. HAI titers elicited by the TVV were statistically similar to HAI titers elicited in animals vaccinated with the corresponding monovalent VLP. Mice vaccinated with the TVV had higher level of influenza specific CD8+ T cell responses than a commercial trivalent inactivated vaccine (TIV). Ferrets vaccinated with the highest dose of the VLP vaccine and then challenged with the homologous H3N2 virus had the lowest titers of replicating virus in nasal washes and showed no signs of disease. Overall, a trivalent VLP vaccine elicits a broad array of immunity and can protect against influenza virus challenge.