Vaccines are one of the most effective means of preventing infectious diseases, and COVID-19 is no exception. Given the speed at which SARS-CoV-2 overwhelmed healthcare systems and the ensuing economic paralysis, the timely development of novel vaccines has become one of the most urgent public health priorities in a decade. At the time of writing, a handful of vaccines have gained emergency approval, while many more continue to undergo evaluation in clinical trials.1 Nearly all candidates seek to elicit immune responses against SARS-CoV-2’s immunodominant Spike protein as blocking Spike-ACE2 interactions has shown to be a viable means of preventing infection.2 However, to ensure these vaccines induce safe antibody responses, vaccine developers will need to develop a range of specific serological tools.
Knowing the unknowns
Following the emergence of SARS in 2002, numerous Spike vaccine candidates entered the pre-clinical pipeline.3 However, to the concern of many, some of the early studies found that vaccine-induced antibody responses enhanced subsequent SARS infections in animal models. Inoculation of rhesus macaques with a Spike-encoding vector, for example, did not protect from viral challenge while eliciting antibody responses that correlated with severe lung damage.4 Inactivated, subunit and virus-like particles (VLP) vaccines administered to ferrets and mice similarly caused distinctive lung pathologies.5
Follow-up studies implicated antibody-dependent enhancement (ADE) of infection, whereby vaccine-induced antibodies bind viruses and facilitate their entry to target cells instead of neutralizing them.1 Typically, ADE occurs when the Fc ends of IgG antibodies are bound by host Fc receptors on the surface of cells, which mediate viral uptake.6 Instead of being digested by intracellular enzymes, viruses are then able to establish an infection within the cell and proliferate. This results in a more severe infection, typically associated with adverse events. The factors influencing ADE vary considerably between diseases and hosts, though they are typically associated with stimulation of non-neutralizing antibodies, or neutralizing antibodies (nAbs) at sub-neutralizing titers.7
Image: Illustration of the FcγR-mediated mechanism of antibody-dependent enhancement of disease. Virus binds a non-neutralizing antibody, which binds an Fcγ receptor, which induces endocytosis of the viral particle. The virus then replicates within the host cell to produce progeny and stimulate the release of inflammatory cytokines.
Nevertheless, SARS disappeared as quickly as it emerged, and investment in vaccine R&D dried up. As such, no vaccines made it past Phase I trials, and potential ADE effects could not be investigated in large human cohort studies. A decade later, MERS emerged, but it was a similar story: vaccinated animals showed the same characteristic lung pathologies when exposed to live virus,8 with in vitro studies implicating enhancing antibody responses.9
Fast forward to 2020 and the emergence of SARS-CoV-2 brought forth fresh concerns about potential antibody-enhancing pathologies. Fortunately, the evidence for ADE in COVID-19 has so far been scant.10 Furthermore, given that late-stage vaccines have shown to consistently elicit highly neutralizing antibody titers, the risks of ADE so far seem small. However, as adverse events such as these are typically rare, finding them within trials of healthy individuals can be difficult. Therefore, continued post-market surveillance will be required to detect potential adverse events. Whole inactivated or attenuated vaccines, in particular, will require close attention, given that they present many non-neutralizing antigen targets.10
Reading the response
To identify potential ADE effects, individuals who experience vaccine-associated adverse events will require careful serological investigation. Doing so will require the development of highly specific and sensitive assays that can qualitatively and quantitatively assess patient antibody profiles. The insights gained from such data should, in turn, help to build a better picture of the correlates of protection vs. potential enhancement for at-risk groups.
When looking at the functional requirements, such assays should be able to measure indicators of ADE in vaccine recipients, including those who are subsequently infected, with pre- and post-measurements of anti-Spike/Nucleoprotein IgG, and the proportion of antibodies that are directed against ADE-associated epitopes.11 Multiplex assays that are able to characterize the ratio of useful nAbs to those that are enhancing, will prove instrumental in demonstrating safety. In addition, given the evidence of ADE at sub-neutralizing antibody titers12 and the likelihood of this in the context of vaccination, assays will also need to be highly sensitive for detection of nAbs at low levels.
To achieve assay specificity, there are multiple routes of investigation. Classical enzyme-linked immunosorbent assays (ELISAs) measure the binding of patient sera antibodies to antigens, immobilized in microtiter wells. However, given that coronavirus nucleoproteins contain cross-reacting epitopes, alternative designs should be considered. One approach is to coat anti-IgM onto a microtiter plate to capture IgM from patient sera and measure its reactivity with antigen (so-called μ-capture). However, such assays tend to be both complicated and time-consuming. More recent formats include blockade-of-binding assays in which antibodies compete to bind antigen, double antigen bridging assays that use a double antigen sandwich to increase specificity, and the use of quenching antigens to ‘absorb-out’ cross-reactive antibodies.13
Image: Four alternative ELISA formats: 1) μ-IgM capture assay: IgM antibodies in patient sera bind to well-bound anti-human IgM. Once bound, Spike and detection antibody bind; 2) Blockade of binding assay: In the absence of patient antibody sera, a specific monoclonal antibody binds well-bound Spike and produces a signal. In the presence of patient antibody sera, patient antibodies bind well-bound Spike and exclude the detection antibody, resulting in a negative signal; The monoclonal antibody utilized binds a highly specific Spike epitope, and thus is only blocked by CoV-specific patient sera and not cross-reactive CoV sera; 3) Double antigen bridging assay: Patient sera antibody binds well-bound Spike, followed by labelled detection Spike; This format inherently detects higher affinity antibodies with higher specificity; 4) Quenching assay: Non-specific CoV patient sera antibodies are able to bind cross-reactive Spike epitopes leading to false positive results. To prevent this, excess Spike is added in solution to quench cross-reacting antibodies. This allows only CoV-specific antibodies to produce a true-positive signal.
Undoubtedly, the most important consideration when designing serological diagnostics is the identification, selection, and presentation of virus-specific epitopes. Due to safety restrictions, most of the antigens used in studies are expressed in recombinant systems that aim to faithfully represent the structure of the native protein in question. However, the degree to which these biological reagents can vary is significant, and structural fidelity is not always ensured. To ensure reagents are suitably specific, expression in mammalian or insect systems is preferred to ensure proper folding and full glycosylation. Further improvements to reagent specificity can then be achieved through the screening of antigen arrays alongside mutation studies that aim to remove cross-reactive epitopes (among various other methods).
References
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2. Jiang, S. et al. Neutralizing Antibodies against SARS-CoV-2 and Other Human Coronaviruses. Trends in Immunology 41, 545 (2020).
3. World Health Organization. List of candidate vaccines developed against SARS-CoV (2020).
4. Liu, L. et al. Anti–spike IgG causes severe acute lung injury by skewing macrophage responses during acute SARS-CoV infection. JCI Insight 4, e123158 (2019).
5. Tseng, C-T. et al. Immunization with SARS Coronavirus Vaccines Leads to Pulmonary Immunopathology on Challenge with the SARS Virus. PLOS ONE 7, e35421 (2012).
6. Jaume, M. et al. Anti-severe acute respiratory syndrome coronavirus spike antibodies trigger infection of human immune cells via a pH- and cysteine protease-independent FcγR pathway. Journal of Virology 85, 10582-97 (2011).
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8. Houser, K.V. et al. Enhanced inflammation in New Zealand white rabbits when MERS-CoV reinfection occurs in the absence of neutralizing antibody. PLoS Pathogens 13, e1006565 (2017).
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10. Lee, W.S. et al. Antibody-dependent enhancement and SARS-CoV-2 vaccines and therapies. Nature Microbiology 5, 1185–1191 (2020).
11. Wang, J. & Zand, M. The potential for antibody-dependent enhancement of SARS-CoV-2 infection: Translational implications for vaccine development. Journal of Clinical and Translational Science, 1-4 (2020).
12. Wang. S.F. et al. Antibody-dependent SARS coronavirus infection is mediated by antibodies against spike proteins. Biochemical and Biophysical Research Communications 451, 208-14 (2014).
13. The Native Antigen Company. Paper Synopsis: ELISA formats and antibody quenching to reduce ZIKV cross-reactivity (2019).