Development of an ELISA-Based Potency Assay for Inactivated Influenza Vaccines Using Cross-Reactive Nanobodies.

Inactivated vaccines are the main influenza vaccines used today; these are usually presented as split (detergent-disrupted) or subunit vaccines, while whole-virus-inactivated influenza vaccines are rare. The single radial immune diffusion (SRD) assay has been used as the gold standard potency assay for inactivated influenza vaccines for decades; however, more recently, various alternative potency assays have been proposed. A new potency test should be able to measure the amount of functional antigen in the vaccine, which in the case of influenza vaccines is the haemagglutinin (HA) protein. Potency tests should also be able to detect the loss of potency caused by changes to the structural and functional integrity of HA. To detect such changes, most alternative potency tests proposed to date use antibodies that react with native HA. Due to the frequent changes in influenza vaccine composition, antibodies may need to be updated in line with changes in vaccine viruses. We have developed two ELISA-based potency assays for group 1 influenza A viruses using cross-reactive nanobodies. The nanobodies detect influenza viruses of subtype H1N1 spanning more than three decades, as well as H5N1 viruses, in ELISA. We found that the new ELISA potency assays are sensitive to the nature of the reference antigen (standard) used to quantify vaccine antigens; using standards matched in their presentation to the vaccine type improved correspondence between the ELISA and SRD assays.

Nanobodies mapped to cross-reactive and divergent epitopes on A(H7N9) influenza hemagglutinin using yeast display.

Influenza H7N9 virus continues to cause infections in humans and represents a significant pandemic risk. During the most recent 5th epidemic wave in 2016/17 two distinct lineages with increased human infections and wider geographical spread emerged. In preparation for any future adaptations, broadly reactive antibodies against H7N9 are required for surveillance, therapy and prophylaxis. In this study we have isolated a panel of nanobodies (Nbs) with broad reactivity across H7 influenza strains, including H7N9 strains between 2013 and 2017. We also describe Nbs capable of distinguishing between the most recent high and low pathogenicity Yangtze River Delta lineage H7N9 strains. Nanobodies were classified into 5 distinct groups based on their epitope footprint determined using yeast display and mutational scanning. The epitope footprint of Nbs capable of distinguishing high pathogenic (HP) A/Guangdong/17SF003/2016 from low pathogenic (LP) A/Hong Kong/125/2017 (H7N9) were correlated to natural sequence divergence in the head domain at lysine 164. Several Nbs binding to the head domain were capable of viral neutralisation. The potency of one nanobody NB7-14 could be increased over 1000-fold to 113 pM by linking two Nbs together. Nbs specific for distinct epitopes on H7N9 may be useful for surveillance or therapy in human or veterinary settings.

Cross-Reactive and Lineage-Specific Single Domain Antibodies against Influenza B Hemagglutinin.

Influenza B virus (IBV) circulates in the human population and causes considerable disease burden worldwide, each year. Current IBV vaccines can struggle to mount an effective cross-reactive immune response, as strains become mismatched, due to constant antigenic changes. Additional strategies which use monoclonal antibodies, with broad reactivity, are of considerable interest, both, as diagnostics and as immunotherapeutics. Alternatives to conventional monoclonal antibodies, such as single domain antibodies (NanobodiesTM) with well-documented advantages for applications in infectious disease, have been emerging. In this study we have isolated single domain antibodies (sdAbs), specific to IBV, using alpacas immunised with recombinant hemagglutinin (HA) from two representative viruses, B/Florida/04/2006 (B/Yamagata lineage) and B/Brisbane/60/2008 (B/Victoria lineage). Using phage display, we have isolated a panel of single domain antibodies (sdAbs), with both cross-reactive and lineage-specific binding. Several sdAbs recognise whole virus antigens, corresponding to influenza B strains included in vaccines spanning over 20 years, and were capable of neutralising IBV pseudotypes corresponding to prototype strains from both lineages. Lineage-specific sdAbs recognised the head domain, whereas, sdAbs identified as cross-reactive could be classified as either head binding or stem binding. Using yeast display, we were able to correlate lineage specificity with naturally occurring sequence divergence, at residue 122 in the highly variable 120 loop of the HA1 domain. The single domain antibodies described, might have applications in IBV diagnostics, vaccine potency testing and as immunotherapeutics.

Development of a high yielding E. coli periplasmic expression system for the production of humanized Fab' fragments.

Humanized Fab' fragments may be produced in the periplasm of Escherichia coli but can be subject to degradation by host cell proteases. In order to increase Fab' yield and reduce proteolysis we developed periplasmic protease deficient strains of E. coli. These strains lacked the protease activity of Tsp, protease III and DegP. High cell density fermentations indicated Tsp deficient strains increased productivity two fold but this increase was accompanied by premature cell lysis soon after the induction of Fab' expression. To overcome the reduction in cell viability we introduced suppressor mutations into the spr gene. The mutations partially restored the wild type phenotype of the cells. Furthermore, we coexpressed a range of periplasmic chaperone proteins with the Fab', DsbC had the most significant impact, increasing humanized Fab' production during high cell density fermentation. When DsbC coexpression was combined with a Tsp deficient spr strain we observed an increase in yield and essentially restored "wild type" cell viability. We achieved a final periplasmic yield of over 2.4g/L (final cell density OD600 105), 40 h post Fab' induction with minimal cell lysis.The data suggests that proteolysis, periplasm integrity, protein folding and disulphide bond formation are all potential limiting steps in the production of Fab' fragments in the periplasm of E. coli. In this body of work, we have addressed these limiting steps by utilizing stabilized protease deficient strains and chaperone coexpression. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:212-220, 2017.