Cross reactivity of serological response to SARS-CoV-2 vaccination with viral variants of concern detected by lateral flow immunoassays
Authors: Daniel Ebanks, Sian Faustini, Adrian Shields, Helen Parry, Paul Moss, Tim Plant, Alex Richter, Mark Drayson
Journal: Journal of Infection (2021)

•IgG anti-SARS-CoV-2 spike glycoprotein antibody levels were greatest after vaccination and lowest after natural non-hospitalised infection.•Using lateral flow tests incorporating wildtype (Wuhan) spike glycoprotein, 40 serum samples post second vaccine dose were positive.•Changing the spike glycoprotein in the lateral flow tests to that from Alpha or Beta or Gamma variants of concern resulted for all three variants, in 40/40 positive results albeit at lower levels.•Changing the spike glycoprotein in the lateral flow tests to that from the Delta variant of concern (VOC) resulted in 37/40 positive results.•The WHO standard NIBSC 20/136 had reactivity to Alpha, Beta and Gamma VOC down to 7 IU/ml and for the Delta VOC to 15 IU/ml. The Coronavirus 2 (SARS-CoV-2) pandemic continues to affect almost every country in the world (1Cavic M. Krivokuca A. Boljevic I. Spasic J. Mihajlovic M. Pavlovic M. et al.Exploring the real-world effect of the SARS-CoV-2 pandemic on the molecular diagnostics for cancer patients and high-risk individuals.Expert Rev Mol Diagn. 2021; 21 ([accessed 7 Jun 2021] Available from:): 101-107https://www.tandfonline.com/action/journalInformation?journalCode=iero20Crossref PubMed Scopus (13) Google Scholar). In the UK, two main vaccines have formed the basis of the national immunisation strategy: the AstraZeneca ChAdOx1 nCoV-19 vaccine (AZ) (2Voysey M. Clemens S.A.C. Madhi S.A. Weckx L.Y. Folegatti P.M. Aley P.K. et al.Safety and efficacy of the ChAdOx1 nCoV-19 vaccine (AZD1222) against SARS-CoV-2: an interim analysis of four randomised controlled trials in Brazil, South Africa, and the UK.Lancet. 2021 Jan 9; 397 ([accessed 7 Jun 2021] Available from:): 99-111https://pubmed.ncbi.nlm.nih.gov/33306989/Abstract Full Text Full Text PDF PubMed Scopus (3210) Google Scholar) and the Pfizer-BioNtech COVID-19 vaccine (PFZ)(3). Both elicit immune responses targeting the viral spike protein and both have been shown to reduce the incidence of severe disease in early trials (2Voysey M. Clemens S.A.C. Madhi S.A. Weckx L.Y. Folegatti P.M. Aley P.K. et al.Safety and efficacy of the ChAdOx1 nCoV-19 vaccine (AZD1222) against SARS-CoV-2: an interim analysis of four randomised controlled trials in Brazil, South Africa, and the UK.Lancet. 2021 Jan 9; 397 ([accessed 7 Jun 2021] Available from:): 99-111https://pubmed.ncbi.nlm.nih.gov/33306989/Abstract Full Text Full Text PDF PubMed Scopus (3210) Google Scholar,3Polack F.P. Thomas S.J. Kitchin N. Absalon J. Gurtman A. Lockhart S. et al.Safety and efficacy of the BNT162b2 mRNA Covid-19 vaccine.N Engl J Med. 2020 Dec 31; 383 ([accessed 7 Jun 2021] Available from:): 2603-2615https://pubmed.ncbi.nlm.nih.gov/33301246/Crossref PubMed Scopus (9296) Google Scholar). With the emergence of several variants of concern (VOC) such as the B.1.1.7 (United Kingdom - Alpha), that have increased transmissibility (4Davies N.G. Abbott S. Barnard R.C. Jarvis C.I. Kucharski A.J. Munday J.D. et al.Estimated transmissibility and impact of SARS-CoV-2 lineage B.1.1.7 in England.Science (80-). 2021; https://doi.org/10.1126/science.abg3055originallyCrossref Scopus (1499) Google Scholar,5WHO World Health Organisation. Tracking SARS-CoV-2 variants. ONLINE. 2021. [accessed 17 Jun 2021] Available from: https://www.who.int/en/activities/tracking-SARS-CoV-2-variants/Google Scholar), it is necessary to understand whether current vaccines will provide protection against VOC. Detection of serum IgG against SARS-CoV-2 spike protein correlates with future protection against COVID-19 infection (6Shields A.M. Faustini S.E. Kristunas C.A. Cook A.M. Backhouse C. Dunbar L. et al.COVID-19: seroprevalence and vaccine responses in UK dental care professionals.J Dent Res. 2021 Jun 2; (220345211020270. [accessed 16 Jun 2021] Available from:)http://www.ncbi.nlm.nih.gov/pubmed/34077690Crossref PubMed Scopus (41) Google Scholar, 7Lumley S.F. O'Donnell D. Stoesser N.E. Matthews P.C. Howarth A. Hatch S.B. et al.Antibody status and incidence of SARS-CoV-2 infection in health care workers.N Engl J Med. 2021 Feb 11; 384 ([accessed 16 Jun 2021] Available from:): 533-540https://pubmed.ncbi.nlm.nih.gov/33369366/Crossref PubMed Scopus (623) Google Scholar) but does IgG induced by the wildtype spike protein interact with the spike protein from VOC and give protection against infection by VOC? Immunodiagnostic tests to measure vaccine responses are available in specialised laboratories whilst lateral flow tests (LFTs) can also be made available at point of care and easily deployed in resource poor settings. Here we have used LFTs to examine IgG antibody levels against spike protein from the SARS-CoV-2 wildtype (Wuhan) and from four VOC: B.1.1.7 (United Kingdom - Alpha), B.1.351 (South Africa – Beta), P.1. (Brazil - Gamma) and B.1.617.2 (Indian – Delta). We have used serum from donors with ELISA confirmed anti-spike antibody induced by natural infection (NI) and from donors who had received two doses of the AZ or PFZ vaccines. LFTs targeting SARS-CoV-2 wildtype (AbC-19™) (8Robertson L.J. Moore J.S. Blighe K. Ng K.Y. Quinn N. Jennings F. Warnock G. et al.Evaluation of the IgG antibody response to SARS CoV-2 infection and performance of a lateral flow immunoassay: cross-sectional and longitudinal analysis over 11 months.BMJ Open. 2021 Jun 29; 11 (Available from)e048142https://bmjopen.bmj.com/content/11/6/e048142.fullCrossref PubMed Scopus (14) Google Scholar) and the four VOC, Alpha, Beta, Gamma and Delta (prototype LFTs) were provided by Abingdon Health for use in this analysis. The five different SARS-CoV-2 spike antigens sprayed onto the nitrocellulose test lines were stabilised full trimeric spike protein made by mammalian cell culture using HEK cells. The LFTs were identical except for the nature of the spike protein and that the AbC-19™ (Wildtype) LFT was CE marked and the variant LFTs were prototypes. All LFTs used the same colloidal gold-labelled anti-IgG signal and run buffer and all were used according to manufacturer instructions. Each assay was scored after 20 min using a line-intensity score – an ordinal, semi-quantitative assessment of reactivity - by three independent observers; scores of 0–10 with 0 negative, 1–3 weak and 4 and above as medium to strong (Supplementary figure 1). An enzyme-linked immunosorbent assay (ELISA) measured total antibody response against the spike glycoprotein (MK654; The Binding Site). Serial dilutions at 1 in 2 (1000 – 7.8 IU/mL) of the WHO standard NIBSC 20/136 (9NIBSC NI for BS andControlsWHO international standard first WHO international standard for anti-SARS-CoV-2 immunoglobulin (human) NIBSC code: 20/136 instructions for use. Potters Bar, Hertfordshire, EN6 3QG2020http://www.nibsc.org/standardisation/international_standards.aspxGoogle Scholar), were made into pre-2019 negative control serum. Antibody responses from 3 groups were assessed: 10 otherwise healthy individuals (median age 49 yrs, range 28–60) bled 4–12 weeks after a non-hospitalised COVID-19 infection (NI) with a positive PCR result via swab test during Spring 2020 when the Wuhan strain was prevalent; 20 individuals (median age 47 yrs, range 28–60) post second dose of PFZ (median time between vaccination and sampling 30.5 days, range 25-47 ) (London-Camden & Kings Cross Research Ethics Committee (20/HRA/1817) 20 individuals (median age 72 yrs, range 64–81) post second dose of AZ (median time between vaccination and sampling = 27.8 days, 4 - 37) (North West ethics committee, Preston CIA UPH IRAS approval (REC 20\NW\0240)). Two-way ANOVA was performed on the data to analyse the statistical relationship between each group and the intrinsic differences between variants. 100% of Pfizer vaccinated individuals showed pan specificity to all spike variants tested here (20/20). 85% of Astra Zeneca vaccinated individuals (17/20) and 40% of individuals whom have recovered from first wave infection (4/10) also displayed pan specificity to the five spike variants used here. Reactivity correlated with measurement of total IgG, IgA and IgM ant-Wuhan spike protein by laboratory ELISA test (10Cook A.M. Faustini S.E. Williams L.J. Cunningham A.F. Drayson M.T. Shields A.M. et al.Validation of a combined ELISA to detect IgG, IgA and IgM antibody responses to SARS-CoV-2 in mild or moderate non-hospitalised patients.J Immunol Methods. 2021 Jul 1; 494113046https://doi.org/10.1016/j.jim.2021.113046Crossref PubMed Scopus (29) Google Scholar) with the exception of two NI samples that were ELISA positive and negative on all four virus variant LFTs. All 20 PFZ group sera reacted against each VOC; and mean scores from the PFZ group were Wuhan 7.4, Alpha 7.3, Beta 6.1, Gamma 6.5 and Delta 6.1. These scores were greater than both the 20 AZ group sera that were Wuhan 3.8, Alpha 3.3, Beta 2.7, Gamma 2.7 and Delta 2.3 and the 10 NI group sera that were Wuhan 2.5, Alpha 2.0, Beta 1.3, Gamma 1.1 and Delta 0.5 (p<0.0001) (Fig. 1). Titration of the WHO standard NIBSC 20/136 sera showed positive results to all variants down to 15 IU/ml and at 7 IU/ml only failed to detect the Delta variant (Fig. 2, Supplementary figure 1).Fig. 2Lateral flow score against each variant: Wuhan (wild type WT), Alpha, Beta, Gamma, Delta for a titration of the WHO standard NIBSC 20/136.View Large Image Figure ViewerDownload Hi-res image Download (PPT) We have shown the use of the AbC-19™ rapid IgG LFT (Abingdon Health) can successfully determine seropositivity for SARS-CoV-2 in all 40 individuals aged 28–81 years of age tested following second dose of vaccine, and 8/10 health care workers following natural infection. IgG antibody levels were greatest after PFZ vaccination (median age 47 years) and lowest after natural infection (median age 49 years). PFZ induced greater antibody levels than AZ vaccination but donors within the AZ cohort were older (median 71 years) which may have impacted on antibody response. The IgG anti-spike antibody in these three groups had been induced by the Wuhan spike protein. We assessed its cross-reactivity with spike protein from the Alpha, Beta, Gamma and Delta variants using spike protein from each variant in LFTs otherwise identical to the AbC-19™ LFT. All 48 serum samples that were positive in the AbC-19™ LFT were positive against three variant LFTs (Alpha, Beta and Gamma) albeit at lower levels; taking the mean of all the variant LFT scores these means were a percentage of the mean Wuhan score 88%, 71% and 49% respectively for PFZ, AZ and NI. In the Pfizer group 100% (20/20) of individuals showed specificity to the delta variant, whereas 85% of AZ and only 40% of NI group showed reactivity to the Delta variant. The WHO standard NIBSC 20/136 derived from hospitalised donors most likely infected by the Wuhan virus had reactivity to all VOC down to 15 IU/ml. London-Camden & Kings Cross Research Ethics Committee (20/HRA/1817) North West ethics committee, Preston CIA UPH IRAS approval (REC 20\NW\0240) Not applicable. Not applicable. None.