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. 2022 Sep 5;10(9):1473.
doi: 10.3390/vaccines10091473.

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

Chung Y Cheung  1 , Sitara Dubey  1 , Martina Hadrovic  1 , Christina R Ball  2 , Walter Ramage  2 , Jacqueline U McDonald  1 , Ruth Harvey  1 , Simon E Hufton  2 , Othmar G Engelhardt  1
Affiliations

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

Chung Y Cheung et al. Vaccines (Basel). .

Abstract

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.

Keywords: ELISA; cross-reactive; influenza; nanobodies; potency test; vaccine.

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Conflict of interest statement

O.G.E. reports funding from IFPMA outside the area of this work. The other authors declare no conflict of interest. One funder (BARDA) interacted regularly with the funded scientists during the period the research was carried out. The funders had no role in the writing of the manuscript.

Figures

Figure 1
Figure 1
Difference plot analysis of results from competitive ELISA using different nanobodies. The percent differences between assigned values (in μg/mL) and values estimated by competitive ELISA with nanobodies R1a-A5 (i), R1a-B6 (ii), R2a-G8 (iii), R2b-D9 (iv) and R2b-E8 (v) are shown for (A) A(H1N1)pdm09 antigen reagents, (B) A(H1N1) antigen reagents from 1976–2007 and (D) H5 subtype antigen reagents. (C) A dose–response curve is shown for nanobody R2b-E8 which only reacted with one antigen reagent out of the H1N1 reagents from 1976–2007 in competitive ELISA. Black dotted lines indicate 95% confidence intervals; red dotted lines indicate average percentage differences.
Figure 1
Figure 1
Difference plot analysis of results from competitive ELISA using different nanobodies. The percent differences between assigned values (in μg/mL) and values estimated by competitive ELISA with nanobodies R1a-A5 (i), R1a-B6 (ii), R2a-G8 (iii), R2b-D9 (iv) and R2b-E8 (v) are shown for (A) A(H1N1)pdm09 antigen reagents, (B) A(H1N1) antigen reagents from 1976–2007 and (D) H5 subtype antigen reagents. (C) A dose–response curve is shown for nanobody R2b-E8 which only reacted with one antigen reagent out of the H1N1 reagents from 1976–2007 in competitive ELISA. Black dotted lines indicate 95% confidence intervals; red dotted lines indicate average percentage differences.
Figure 1
Figure 1
Difference plot analysis of results from competitive ELISA using different nanobodies. The percent differences between assigned values (in μg/mL) and values estimated by competitive ELISA with nanobodies R1a-A5 (i), R1a-B6 (ii), R2a-G8 (iii), R2b-D9 (iv) and R2b-E8 (v) are shown for (A) A(H1N1)pdm09 antigen reagents, (B) A(H1N1) antigen reagents from 1976–2007 and (D) H5 subtype antigen reagents. (C) A dose–response curve is shown for nanobody R2b-E8 which only reacted with one antigen reagent out of the H1N1 reagents from 1976–2007 in competitive ELISA. Black dotted lines indicate 95% confidence intervals; red dotted lines indicate average percentage differences.
Figure 2
Figure 2
Sandwich ELISA. (A) Absorbance signal is shown after pre-treatment of antigen with or without 1% Zwittergent 3–14 detergent. (B) Difference plot analysis of results from sandwich ELISA on A(H1N1) antigen reagents from 1976–2007, (C) A(H1N1)pdm09 antigen reagents and (D) H5 subtype antigen reagents. Assigned potency is in μg/mL; black dotted lines indicate 95% confidence intervals; red dotted lines indicate average percentage differences.
Figure 3
Figure 3
Forced degradation. (A) After treatment of antigen reagent (i) at 56 °C, (ii) by deamidation and (iii) by acid treatment, SRD precipitation zones were only present at timepoint 0 (i) or in untreated controls (ii,iii) in SRD assays. (B) Estimated potency graphs from ELISA assays after forced degradation at 56 °C or by deamidation.
Figure 4
Figure 4
Dose–response of two-fold dilution series of antigen reagents in (A) the competitive and (B) sandwich ELISA using antigen reagents 13/164 (H1N1)pdm09, 14/254 (H3N2), 14/252 (influenza B) as well as a mix of all three reagents.
Figure 5
Figure 5
Detection limit and linearity. (A) Antigen reagent 09/196 was diluted in 2-fold steps in triplicate, and each dilution was independently analysed by competitive ELISA using R2b-D9 and (B) sandwich ELISA using R1a-B6 as capture antibody and R2b-D9 as primary detection antibody. Estimated potency for each dilution is plotted against expected concentration.
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References

    1. Minor P.D. Assaying the Potency of Influenza Vaccines. Vaccines. 2015;3:90–104. doi: 10.3390/vaccines3010090. - DOI - PMC - PubMed
    1. Wood J.M., Schild G.C., Newman R.W., Seagroatt V. Application of an improved single-Radial-Immunodiffusion technique for the assay of haemagglutinin antigen content of whole virus and subunit influenza vaccines. Dev. Biol. Stand. 1977;39:193–200. - PubMed
    1. Wood J., Schild G., Newman R., Seagroatt V. An improved single-Radial-Immunodiffusion technique for the assay of influenza haemagglutinin antigen: Application for potency determinations of inactivated whole virus and subunit vaccines. J. Biol. Stand. 1977;5:237–247. doi: 10.1016/S0092-1157(77)80008-5. - DOI - PubMed
    1. Wood J.M., Weir J.P. Standardisation of inactivated influenza vaccines-Learning from history. Influenza Other Respir. Viruses. 2018;12:195–201. doi: 10.1111/irv.12543. - DOI - PMC - PubMed
    1. Hardy S., Eichelberger M., Griffiths E., Weir J.P., Wood D., Alfonso C. Confronting the next pandemic-Workshop on lessons learned from potency testing of pandemic (H1N1) 2009 influenza vaccines and considerations for future potency tests, Ottawa, Canada, July 27–29, 2010. Influenza Other Respir. Viruses. 2011;5:438–442. doi: 10.1111/j.1750-2659.2011.00250.x. - DOI - PMC - PubMed

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