Pan-HA antibodies for influenza detection and quantification

The influenza virus imposes a heavy burden for society in terms of health and economy. Influenza is an elusive enveloped virus due to antigenic shift and drift of two surface proteins: neuraminidase (NA) and hemagglutinin (HA). As a result, new strains emerge every year which require seasonal vaccination for protection. Furthermore, large vaccine quantities are urgently needed in case of pandemics. Theoretically, vaccines against a new strain can be manufactured in as little as three weeks with certain platforms and technologies. However, vaccine quantification and release are still relying on the use of the Single Radial Immunodiffusion (SRID) assay using a strain-specific antibody to calculate HA concentration. This is a major limitation because it can take up to three months to generate the reagents necessary to run the SRID assay, including the strain-specific antibody. Hence, one of the major hurdles in the process of influenza vaccine production is the quantification of HA which is critical to establish proper dosing. To circumvent the need for strain-specific antibodies, we have produced two monoclonal antibodies (F211-11H12-3 and F211-10A9-2) against a highly conserved peptide sequence found within the HA molecule (1). Multiple strains belonging to 13 different influenza A subtypes, as well as 6 strains belonging to B lineages were detected by Western blot and dot blot. Overall, mAb F211-11H12-3 recognizes preferentially influenza A subtype 1, while the mAb F211-10A9-2 has a higher affinity for influenza A subtype 2. Therefore, all strains tested could be detected when both mAb are combined and used as a cocktail. Next, we performed quantitative dot blots by generating a standard curve ranging from 160ng/ml to 20µg/ml HA. This method is simple, easy to implement and highly reproducible. In-process samples as well as purified material can be quantified by dot blot after denaturation with urea. Even though the SRID is the only assay approved by regulatory agencies, quantitative dot blots can be used during manufacturing to optimize and monitor the production process. Finally, ELISA is widely used for quantification and preliminary data demonstrates that samples can be quantified with the pan-HA mAbs. In conclusion, a pan-HA antibody cocktail was generated against a highly conserved peptide sequence of influenza. Viruses produced in eggs and mammalian cells from 40 different strains were detected by Western blot. Reproducible quantification was achieved by dot blot using the two mAbs and an appropriate calibrating standard. The combination of pan-HA antibodies with an immunoassay such as the dot blot assay could accelerate process development and help establish new generation quantification methods for influenza. As the field is looking for flexible and versatile solutions to shift away from the SRID assay and strain-specific antibodies, the development of broad-spectrum antibodies offers a long-awaited alternative. 1) Chun et al, Universal antibodies and their applications to the quantitative determination of virtually all subtypes of the influenza A viral hemagglutinins, Vaccine (26), pp 6068-6076, 2008

Accelerated mass production of influenza virus seed stocks in HEK-293 suspension cell cultures by reverse genetics

Despite major advances in developing capacities and alternative technologies to egg-based production of influenza vaccines, responsiveness to an influenza pandemic threat is limited by the time it takes to generate a Candidate Viral Vaccine (CVV) as reported by the 2015 WHO Informal Consultation report titled “Influenza Vaccine Response during the Start of a Pandemic”. In previous work, we have shown that HEK-293 cell culture in suspension and serum free medium is an efficient production platform for cell culture manufacturing of influenza candidate vaccines. This report, took advantage of, recombinant DNA technology using Reverse Genetics of influenza A/Puerto Rico/8/34 H1N1 strain, and advances in the large-scale transfection of suspension cultured HEK-293 cells. Transfection in shake flasks was performed using 1ug of total plasmid and 1x106 cells/mL. The supernatant was harvested after 48 hpt and used to infect a new shake flasks at 1x106 cells/mL for virus amplification. 3-L bioreactor was inoculated and transfected at 1x106 cells/mL with 1ug of total plasmid and harvested after 48hpt and the virus generated was amplified in shake flask. Quantification by TCID50, SRID, Dot-blot and TRPS were performed as well as characterization by TEM and HA and NA sequencing. Small-scale transfection in shake flasks generated 1.5x105 IVP/mL after 48 hpt and 1x107 IVP/mL after 96 hpi. For large-scale experiment a 3-L controlled stirred tank bioreactor resulted in supernatant (P0) virus titer of 5x104 IVP/mL and 2.8x107 IVP/mL after only one amplification (P1) in HEK-293 suspension cells. We demonstrate the efficent generation of H1N1 with the PR8 backbone reassortant under controlled bioreactor conditions in two sequential steps (transfection/rescue and infection/production). This approach could deliver a CVV for influenza vaccine manufacturing within two-weeks, starting from HA and NA pandemic sequences. Thus, this innovative approach is better suited to rationally design and mass produce the CVV within timelines dictated by pandemic situations and produce effective responsiveness than previous methodolog

Critical review of current and emerging quantification methods for the development of influenza vaccine candidates

Significant improvements in production and purification have been achieved since the first approved influenza vaccines were administered 75 years ago. Global surveillance and fast response have limited the impact of the last pandemic in 2009. In case of another pandemic, vaccines can be generated within three weeks with certain platforms. However, our Achilles heel is at the quantification level. Production of reagents for the quantification of new vaccines using the SRID, the main method formally approved by regulatory bodies, requires two to three months. The impact of such delays can be tragic for vulnerable populations. Therefore, efforts have been directed toward developing alternative quantification methods, which are sensitive, accurate, easy to implement and independent of the availability of specific reagents. The use of newly-developed antibodies against a conserved region of hemagglutinin (HA), a surface protein of influenza, holds great promises as they are able to recognize multiple subtypes of influenza; these new antibodies could be used in immunoassays such as ELISA and slot-blot analysis. HA concentration can also be determined using reversed-phase high performance liquid chromatography (RP-HPLC), which obviates the need for antibodies but still requires a reference standard. The number of viral particles can be evaluated using ion-exchange HPLC and techniques based on flow cytometry principles, but non-viral vesicles have to be taken into account with cellular production platforms. As new production systems are optimized, new quantification methods that are adapted to the type of vaccine produced are required. The nature of these new-generation vaccines might dictate which quantification method to use. In all cases, an alternative method will have to be validated against the current SRID assay. A consensus among the scientific community would have to be reached so that the adoption of new quantification methods would be harmonized between international laboratories.Peer reviewed: YesNRC publication: Ye

Particle quantification of influenza viruses by high performance liquid chromatography using CIM\uae anion exchange monolithic column

A high performance liquid chromatography (HPLC) method using a strong anion exchange monolithic column based on convective interaction media) CIM\uae technology was developed for the particle quantification of influenza viruses. The virus which was specifically detected by native fluorescence eluted in 5.55 min at 1.5 M NaCl gradient in 20 mM Tris-HCl + o.01% Zwittergent, pH 8.0 in a total analysis time of 13.5 min. The linearity of a curve was demonstrated for all inluenza virus investigated with a good correlation coefficient (R2) greater than 0.995. Among all the virus investigated, the detection limit of the method ranged between 2.07x108 and 4.35x109 while the quantification limit ranged between 6.90x108 and 1.45x1010 virus particle per ml (VP/ml), respectively. The intra- and inter-assay precision of the method were less than 5% and 10% respectively. The method which was developed using sucrose cushion purified influenza viruses was shown to be suitable in the analysis of cell culture supernatants making it an ideal in-process monitoring tool to facilitate the development of influenza vaccine manufacturing processes.posterNRC publication: Ye

Particle quantification of influenza viruses by high performance liquid chromatography using CIM\uae anion exchange monolithic column

A high performance liquid chromatography (HPLC) method using a strong anion exchange monolithic column based on convective interaction media) CIM\uae technology was developed for the particle quantification of influenza viruses. The virus which was specifically detected by native fluorescence eluted in 5.55 min at 1.5 M NaCl gradient in 20 mM Tris-HCl + o.01% Zwittergent, pH 8.0 in a total analysis time of 13.5 min. The linearity of a curve was demonstrated for all inluenza virus investigated with a good correlation coefficient (R2) greater than 0.995. Among all the virus investigated, the detection limit of the method ranged between 2.07x108 and 4.35x109 while the quantification limit ranged between 6.90x108 and 1.45x1010 virus particle per ml (VP/ml), respectively. The intra- and inter-assay precision of the method were less than 5% and 10% respectively. The method which was developed using sucrose cushion purified influenza viruses was shown to be suitable in the analysis of cell culture supernatants making it an ideal in-process monitoring tool to facilitate the development of influenza vaccine manufacturing processes.posterNRC publication: Ye

Concentrations obtained by dot blot on three different runs for two influenza samples (H1N1 A/Puerto Rico/8/34).

<p>Concentrations obtained by dot blot on three different runs for two influenza samples (H1N1 A/Puerto Rico/8/34).</p

KD values (M) measured by SPR for pan-HA antibodies produced in mouse hybridomas and CHO pools.

<p>KD values (M) measured by SPR for pan-HA antibodies produced in mouse hybridomas and CHO pools.</p