A point-of-care lateral flow assay for neutralising antibodies against SARS-CoV-2

Background: As vaccines against SARS-CoV-2 are now being rolled out, a better understanding of immunity to the virus, whether from infection, or passive or active immunisation, and the durability of this protection is required. This will benefit from the ability to measure antibody-based protection to SARS-CoV-2, ideally with rapid turnaround and without the need for laboratory-based testing. Methods: We have developed a lateral flow POC test that can measure levels of RBD-ACE2 neutralising antibody (NAb) from whole blood, with a result that can be determined by eye or quantitatively on a small instrument. We compared our lateral flow test with the gold-standard microneutralisation assay, using samples from convalescent and vaccinated donors, as well as immunised macaques. Findings: We show a high correlation between our lateral flow test with conventional neutralisation and that this test is applicable with animal samples. We also show that this assay is readily adaptable to test for protection to newly emerging SARS-CoV-2 variants, including the beta variant which revealed a marked reduction in NAb activity. Lastly, using a cohort of vaccinated humans, we demonstrate that our whole-blood test correlates closely with microneutralisation assay data (specificity 100% and sensitivity 96% at a microneutralisation cutoff of 1:40) and that fingerprick whole blood samples are sufficient for this test. Interpretation: Taken together, the COVID-19 NAb-testTM device described here provides a rapid readout of NAb based protection to SARS-CoV-2 at the point of care

Anti-HIV-1 ADCC antibodies following latency reversal and treatment interruption

There is growing interest in utilizing antibody-dependent cellular cytotoxicity (ADCC) to eliminate infected cells following reactivation from HIV-1 latency. A potential barrier is that HIV-1-specific ADCC antibodies decline in patients on long-term antiretroviral therapy (ART) and may not be sufficient to eliminate reactivated latently infected cells. It is not known whether reactivation from latency with latency-reversing agents (LRAs) could provide sufficient antigenic stimulus to boost HIV-1-specific ADCC. We found that treatment with the LRA panobinostat or a short analytical treatment interruption (ATI), 21 to 59 days, was not sufficient to stimulate an increase in ADCC-competent antibodies, despite viral rebound in all subjects who underwent the short ATI. In contrast, a longer ATI, 2 to 12 months, among subjects enrolled in the Strategies for Management of Antiretroviral Therapy (SMART) trial robustly boosted HIV-1 gp120-specific Fc receptor-binding antibodies and ADCC against HIV-1-infected cells in vitro. These results show that there is a lag between viral recrudescence and the boosting of ADCC antibodies, which has implications for strategies toward eliminating latently infected cells

Systems serology detects functionally distinct coronavirus antibody features in children and elderly

The hallmarks of COVID-19 are higher pathogenicity and mortality in the elderly compared to children. Examining baseline SARS-CoV-2 cross-reactive immunological responses, induced by circulating human coronaviruses (hCoVs), is needed to understand such divergent clinical outcomes. Here we show analysis of coronavirus antibody responses of pre-pandemic healthy children (n = 89), adults (n = 98), elderly (n = 57), and COVID-19 patients (n = 50) by systems serology. Moderate levels of cross-reactive, but non-neutralizing, SARS-CoV-2 antibodies are detected in pre-pandemic healthy individuals. SARS-CoV-2 antigen-specific Fcγ receptor binding accurately distinguishes COVID-19 patients from healthy individuals, suggesting that SARS-CoV-2 infection induces qualitative changes to antibody Fc, enhancing Fcγ receptor engagement. Higher cross-reactive SARS-CoV-2 IgA and IgG are observed in healthy elderly, while healthy children display elevated SARS-CoV-2 IgM, suggesting that children have fewer hCoV exposures, resulting in less-experienced but more polyreactive humoral immunity. Age-dependent analysis of COVID-19 patients, confirms elevated class-switched antibodies in elderly, while children have stronger Fc responses which we demonstrate are functionally different. These insights will inform COVID-19 vaccination strategies, improved serological diagnostics and therapeutics

The immunoglobulin G binding site of complement protein C1q.

The involvement of Clq lysine residues in the binding of human Clq to rabbit IgG was assessed by chemical modification. Modification on of Clq lysyls with acetic anhydride was complicated by the lack of selectivity of the reagent, as loss of Clq functional activity, due to the modification of tyrosine residues, occurred bcfore any effect due to lysyl modification was apparent. Treatment of Clq with trinitrobenzene sulfonate or fluorescein isothiocyanate resulted in loss of binding activity but was also associated with a Clq concentration dependent precipitation of Clq suggesting structural alteration of the protein. N-(iodoacetylaminoethyl) -8 naphthylamine-l-sulphonate was found to be a non-covalent inhibitor of the interaction between Clq and antigen-aggregated IgG. Crosslink ing of human Clq to antigen-aggregated IgG by treatment with dimethyl -3,3'-di thiobispropionimidate (Lysyl-Lysyl crosslinking), 1,5difluoro- 2,4—dinitrobenzene (Lysyl—, Tyrosyl-, Histidyl-) or l-ethyl-S- (Bdimethyl aminopropyl) carbodiimide (Lysyl-Carboxyl) was found to involve the disulfide-linked AB and CC chains of Clq. As the DTBP crosslinking involved linking lysyl groups, the distribution of the lysine residues restricted the possible sites for crosslinking. Possible candidate lysyls in the globular head region of the C chain are limited to four residues flanking the interchain disulfide bond, which are thus likely to be close in space and may constitute a single contiguous contact site for IgG. This region is of high identity between the C and B chains. 1—ethyl-3 -(3-dimethylaminopropyl) carbodiimide (EDC) crosslinking was also determined to involve lysyl groups of Clq. This strengthened the assignment of the proposed lysine residues as points of contact with IgG as the chemistry of carbodiimide crosslinking is a " zero-length" process introducing no additional atoms between the participating sidechain s. This crosslinking method also indicated that iodination of Clq may inactivate the Ig binding activity associated with the C chain into which the 125I-label is incorporated. All three crosslinking methods gave evidence for an altered conformation of Clq bound to antigen-aggregated IgG. Only when C1q bound to immune complexes was treated with crosslinkers was crosslinking within the disulfide-linked AB dimer apparent by the appearance, on Western blotting of non-reduced SDS-PAG E samples, of a more rapidly migrating band with A/ B chain reactivity. Direct identification of the crosslinking sites by proteolysis of the covalent complex formed by DTBP-treatment of Clq and aggregated IgG and purification of the fragment containing crosslinks was unsuccessful. Two anti-Clq monoclonal antibodies, BUS—1 and BUS-2, which inhibited the binding of Clq to antigen aggregated IgG were produced. These mAbs competitively inhibited the binding of each other to Clq and so probably recognised the same or overlapping epitopes. Western blotting analysis showed BUS-1 to have weak reactivity with both the disulfide-linked AB and CC chains, but not with the reduced and alkylated free A, B or C chains. Reduction of the accessible disulfides of 125I—labeled C1q, under non-denaturing conditions, diminished the binding of Clq to immobilized BUS-1. These data were consistent with the BUS—1 epitope being discontinuous and occurring in the homologous regions of both the C and B chains flanking the intrachain disulfides. This interpretation was supportive of the conclusions of the crosslinking study from which it was also proposed these regions of Clq contact IgG. The modification of tyrosine residues of Clq decreased both the IgG-binding activity of the protein and its reactivity with the BUS-1 mAb. This was consistent with the contention that the mAb binds to the Ig recognition site. The loss of both BUS-1 reactivity and IgG binding activity on the cleavage of the accessible cysteines of Clq also indicates some degree of identity between the Ig recognition site and the BUS—1 epitope. A functionally active Fab/c fragment of IgG (i.e. consisting of one antigen-binding Fab and one complement interactive Fc) was produced by the use of cyanocysteine chemistry to achieve cleavage of the heavy chain polypeptide in the hinge region. This fragment is of value as a potential therapeutic agent for the activation of effector functions in situ as it is not subject to antigenic modulation. The physicochemical basis of the enhancement of Clq binding to immune complexes in the presence of polyethylene glycol was shown to be an excluded volume effect. This is of interest as Clq binding activity in the presence polyethylene glycol forms the basis of some assays for circulating immune complexes

Immunization with inactivated whole virus particle influenza virus vaccines improves the humoral response landscape in cynomolgus macaques

Although antibody-inducing split virus vaccines (SV) are currently the most effective way to combat seasonal influenza, their efficacy can be modest, especially in immunologically-naive individuals. We investigated immune responses towards inactivated whole influenza virus particle vaccine (WPV) formulations, predicated to be more immunogenic, in a non-human primate model, as an important step towards clinical testing in humans. Comprehensive analyses were used to capture 46 immune parameters to profile how WPV-induced responses differed to those elicited by antigenically-similar SV formulations. Naive cynomolgus macaques vaccinated with either monovalent or quadrivalent WPV consistently induced stronger antibody responses and hemagglutination inhibition (HI) antibody titres against vaccine-matched viruses compared to SV formulations, while acute reactogenic effects were similar. Responses in WPV-primed animals were further increased by boosting with the same formulation, conversely to modest responses after priming and boosting with SV. 28-parameter multiplex bead array defined key antibody features and showed that while both WPV and SV induced elevated IgG responses against A/H1N1 nucleoprotein, only WPV increased IgG responses against A/H1N1 hemagglutinin (HA) and HA-Stem, and higher IgA responses to A/H1N1-HA after each vaccine dose. Antibodies to A/H1N1-HA and HA-Stem that could engage Fc gamma R2a and Fc gamma R3a were also present at higher levels after one dose of WPV compared to SV and remained elevated after the second dose. Furthermore, WPV-enhanced antibody responses were associated with higher frequencies of HA-specific B-cells and IFN-gamma-producing CD4(+) T-cell responses. Our data additionally demonstrate stronger boosting of HI titres by WPV following prior infection and support WPV administered as a priming dose irrespective of the follow up vaccine for the second dose. Our findings thus show that compared to SV vaccination, WPV-induced humoral responses are significantly increased in scope and magnitude, advocating WPV vaccination regimens for priming immunologically-naive individuals and also in the event of a pandemic outbreak

Source data file.

Raw data for all the figures and supplementary figures. (XLSX)</p

Flow cytometry panel for the analysis of influenza-specific B cells.

(DOCX)</p