French Study Claims That ADE Is Occurring In Delta Variant Infections


A new study by French researchers from Aix-Marseille Université has alarmingly found that ADE or antibody dependent enhancement is indeed occurring in infections with the SARS-CoV-2 Delta variant.

The study findings were peer reviewed and published in the Journal of Infections.
ADE or antibody dependent enhancement (ADE) of infection is a safety concern for vaccine strategies.
A misleading earlier study reported that infection-enhancing antibodies directed against the N-terminal domain (NTD) of the SARS-CoV-2 spike protein facilitate virus infection in vitro, but not in vivo.
This study however was performed with the original Wuhan/D614G strain.
Importantly since the COVID-19 pandemic is now dominated with Delta variants, the study team analyzed the interaction of facilitating antibodies with the NTD of these variants.
Utilizing molecular modeling approaches, the team showed that enhancing antibodies have a higher affinity for Delta variants than for Wuhan/D614G NTDs.
The study team demonstrated that enhancing antibodies reinforce the binding of the spike trimer to the host cell membrane by clamping the NTD to lipid raft microdomains. This stabilizing mechanism may facilitate the conformational change that induces the de-masking of the receptor binding domain.

As the NTD is also targeted by neutralizing antibodies, the study data suggest that the balance between neutralizing and facilitating antibodies in vaccinated individuals is in favor of neutralization for the original Wuhan/D614G strain.
Alarmingly, in the case of the Delta variant, neutralizing antibodies have a decreased affinity for the spike protein, whereas facilitating antibodies display a strikingly increased affinity.
Hence antibody dependent enhancement or ADE may be a concern for people receiving vaccines based on the original Wuhan strain spike sequence (either mRNA or viral vectors). Under these circumstances, second generation vaccines with spike protein formulations lacking structurally-conserved ADE-related epitopes should be considered.
The objective of the present study was to evaluate the recognition of SARS-CoV-2 Delta variants by infection enhancing antibodies directed against the NTD. The antibody studied is 1054 (pdb file #7LAB) which has been isolated from a symptomatic COVID-19 patient.
Molecular modeling simulations were performed as previously described
2. Two currently circulating Delta variants were investigated, with the following mutational patterns in the NTD:
– G142D/E154K (B.1.617.1)
– T19R/E156G/del157/del158/A222V (B.1.617.2)
Each mutational pattern was introduced in the original Wuhan/D614G strain, submitted to energy minimization, and then tested for antibody binding.
The energy of inte raction (ΔG) of the reference pdb file #7LAB (Wuhan/D614G strain) in the NTD region was estimated to -229 kJ/mol−1. In the case of Delta variants, the energy of interaction was raised to -272 kJ.mol−1 (B.1.617.1) and -246 kJ.mol−1 (B.1.617.2).
Hence it was found that these infection enhancing antibodies not only still recognize Delta variants but even display a higher affinity for those variants than for the original SARS-CoV-2 strain.
As expected, the facilitating antibody bound to the NTD is located behind the contact surface so that it does not interfere with virus-cell attachment. Indeed, a preformed antibody-NTD complex could perfectly bind to the host cell membrane. The interaction between the NTD and a lipid raft and a whole raft-spike-antibody complex.
Interestingly, a small part of the antibody was found to interact with the lipid raft. More precisely, two distinct loops of the heavy chain of the antibody encompassing amino acid residues 28-31 and 72-74, stabilize the complex through a direct interaction with the edge of lipid raft.
Overall, the energy of interaction of the NTD-raft complex was raised from -399 kJ.mol−1 in absence of the antibody to -457 kJ.mol−1 with the antibody. By clamping the NTD and the lipid raft, the antibody reinforces the attachment of the spike protein to the cell surface and thus facilitates the conformational change of the RBD which is the next step of the virus infection process.
This notion of a dual NTD-raft recognition by an infection enhancing antibody may represent a new type of ADE that could be operative with other viruses. Incidentally, the study data provide a mechanistic explanation of the FcR-independent enhancement of infection induced by the 1054 antibody.
The model the study team proposes, which links for the first time lipid rafts to ADE of SARS-CoV-2, is in line with previous data showing that intact lipid rafts are required for ADE of dengue virus infection.
Neutralizing antibodies directed against the NTD have also been detected in Covid-19 patients.
The 4A8 antibody is a major representant of such antibodies. The epitope recognized by this antibody on the flat NTD surface is dramatically affected in the NTD of Delta variants, suggesting a significant loss of activity in vaccinated people exposed to Delta variants. More generally, it can be reasonably assumed that the balance between neutralizing and facilitating antibodies may greatly differ according to the virus strain.
It should be noted that all current Covid-19 vaccines (either mRNA or viral vectors) are based on the original Wuhan spike sequence. In as much as neutralizing antibodies overwhelm facilitating antibodies, ADE is not a concern. However, the emergence of SARS-CoV-2 variants may tip the scales in favor of infection enhancement. The study’s structural and modeling data suggest that it might be indeed the case for Delta variants.
The study team concludes and warns that ADE may occur in individuals receiving vaccines based on the original Wuhan strain spike sequence (either mRNA or viral vectors) and then exposed to a Delta variant.
Despite this potential risk has been cleverly anticipated before the massive use of Covid-19 vaccines, the ability of SARS-CoV-2 antibodies to mediate infection enhancement in vivo has never been formally demonstrated. However, although the results obtained so far have been rather reassuring, to the best of our knowledge ADE of Delta variants has not been specifically assessed.
Importantly as the study data indicate that Delta variants are especially well recognized by infection enhancing antibodies targeting the NTD, the possibility of ADE should be further investigated as it may represent a potential risk for mass vaccination during the current Delta variant pandemic.
The study team stresses that in this respect, second generation vaccines
with spike protein formulations lacking structurally-conserved ADE-related epitopes should be considered urgently.

COVID-19 (SARS-CoV-2) disease severity and stages varies from asymptomatic, mild flu-like symptoms, moderate, severe, critical, and chronic disease. COVID-19 disease progression include lymphopenia, elevated proinflammatory cytokines and chemokines, accumulation of macrophages and neutrophils in lungs, immune dysregulation, cytokine storms, acute respiratory distress syndrome (ARDS), etc.

Development of vaccines to severe acute respiratory syndrome (SARS), Middle East Respiratory Syndrome coronavirus (MERS-CoV), and other coronavirus has been difficult to create due to vaccine induced enhanced disease responses in animal models.

Multiple betacoronaviruses including SARS-CoV-2 and SARS-CoV-1 expand cellular tropism by infecting some phagocytic cells (immature macrophages and dendritic cells) via antibody bound Fc receptor uptake of virus.

Antibody-dependent enhancement (ADE) may be involved in the clinical observation of increased severity of symptoms associated with early high levels of SARS-CoV-2 antibodies in patients. Infants with multisystem inflammatory syndrome in children (MIS-C) associated with COVID-19 may also have ADE caused by maternally acquired SARS-CoV-2 antibodies bound to mast cells.

ADE risks associated with SARS-CoV-2 has implications for COVID-19 and MIS-C treatments, B-cell vaccines, SARS-CoV-2 antibody therapy, and convalescent plasma therapy for patients. SARS-CoV-2 antibodies bound to mast cells may be involved in MIS-C and multisystem inflammatory syndrome in adults (MIS-A) following initial COVID-19 infection. SARS-CoV-2 antibodies bound to Fc receptors on macrophages and mast cells may represent two different mechanisms for ADE in patients.

These two different ADE risks have possible implications for SARS-CoV-2 B-cell vaccines for subsets of populations based on age, cross-reactive antibodies, variabilities in antibody levels over time, and pregnancy. These models place increased emphasis on the importance of developing safe SARS-CoV-2 T cell vaccines that are not dependent upon antibodies.

Antibody-Dependent Enhancement (ADE) of Coronaviruses

Antibody-dependent enhancement (ADE) may develop via more than one molecular mechanism. One model suggestions that antibody/Fc-receptor complex functionally mimics viral receptor enabling expanded host cell trophism of some phagocytic cells (54). Wan et al. (54) illustrate an antibody dosage effect for enhancing disease or inhibiting the virus dependent upon the antibody dosage. It is well-established that antibodies to one strain of a virus may be subneutralizing or non-neutralizing for viral infections of different strains (55–57). Infection of cells expressing Fc-gamma was shown for SARS-CoV-1 (58).

A possible case of ADE was observed in a patient with a second SARS-CoV-2 infection (59). Early vaccine results show significant antibody responses by day 14 (60) which represents memory B-cell responses (i.e., original antigenic sin) with cross-reactivity antibodies from likely other coronavirus strain(s).

Early high antibody responses are correlated with increased disease severity for both SARS (61) and COVID-19 (62–67). Wu et al. demonstrated that antibodies from COVID-19 patients enabled SARS-CoV-2 infections of Raji cells (lymphoma cells derived from B lymphocytes), K562 cells (derived from monocytes), and primary B cells (68). SARS-CoV-2 infection of some phagocytic cells (i.e., macrophages) may be a key gate step in disease progression for some patients.

Mast Cells Risks for ADE and Multisystem Inflammatory Syndromes (MIS-C & MIS-A)

Mast cells can degranulated by both IgE and IgG antibodies bound to Fc receptors (69). Cardiac injury is a common condition among hospitalized COVID-19 patients and is associated with higher risk of mortality (70). However, pathological manifestations of heart tissues found only scarce interstitial mononuclear inflammatory infiltrates without substantial myocardial damage (42). Myocardial injury significantly correlates with fatal outcome for COVID-19 (71).

Multisystem inflammatory syndrome in children (MIS-C) and adults (MIS-A) associated with COVID-19 has appeared in areas following SARS-CoV-2 outbreaks. A model of MIS-C has been proposed where activation and degranulation of mast cells with Fc receptor-bound SARS-CoV-2 antibodies leads to increased histamine levels (26). This model is consistent with MIS-C in infants with maternally transferred antibodies (matAbs) (37–40) to SARS-CoV-2. SARS-CoV-2 nucleocapsid binding to PTGS2 prompter resulting in upregulated prostaglandin E2 (PGE2) in COVID-19 patients (4).

Elevated PGE2 may be driving hyper-activated mast cells as an alternative mechanism driving increased histamine levels in older children and adults. These increased histamine levels are predicted to impede blood flow through cardiac capillaries due to constricted pericytes with increased risk for cardiac pathology due to cell death by anoxia and coronary artery aneurysms due to increased blood pressure (26). An instance of a 12 years old child with a previous asymptomatic COVID-19 infection developing MIS-C on likely second infection has been reported (72).

Vaccine Risks for Antibody-Dependent Enhancement (ADE)

Virus vaccines can use live-attenuated virus strains, inactivated (killed) virus, protein subunit, messenger ribonucleic acid (mRNA), or deoxyribonucleic acid (DNA) vaccine. Antibodies induced by vaccines can be neutralizing or non-neutralizing. Non-neutralizing antibodies can contribute to anti-viral activities with mechanisms including antibody-medicated complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP) [reviewed (73)].

The yearly influenza vaccine induces both neutralizing and non-neutralizing antibodies that provide projection against the strains in the vaccine and closely related strains. Vaccine-associated enhanced disease (VAED) can result when there are multiple circularizing serotypes of virus [e.g., Dengue fever (55–57)] or when the virus uses antibodies for expanded host cell trophism of phagocytic immune cells.

Many of the viruses associated with ADE have cell membrane fusion mechanisms (38). For influenza A H1N1, vaccine-induced cross-reactive anti-HA2 antibodies in a swine model promote virus fusion causing vaccine-associated enhanced respiratory disease (VAERD) (74). ADE was observed for the respiratory syncytial virus (RSV) in the Bonnet monkey model (37).

Van Erp et al. (37) recommends avoidance of induction of respiratory syncytial virus (RSV) non-neutralizing antibodies or subneutralizing antibodies to avoid ADE. ADE has been observed in multiple SARS-CoV-1 animal models. In a mouse model, attempts to create vaccines for SARS-CoV-1 lead to pulmonary immunopathology upon challenge with SARS-CoV-1 (75, 76); these vaccines included inactivated whole viruses, inactivated viruses with adjuvant, and a recombinant DNA spike (S) protein vaccine in a virus-like particle (VLP) vaccine. Severe pneumonia was observed in mice vaccinated with nucleocapsid protein after challenge with SARS-CoV-1 (77).

Enhanced hepatitis was observed in a ferret model with a vaccine with recombinant modified vaccinia virus Ankara (rMVA) expressing the SARS-CoV-1 Spike protein (78). ADE was observed for rhesus macaques with SARS-CoV-1 vaccine (79). SARS-CoV-1 ADE is mediated by spike protein antibodies (80). Antibodies to the SARS-CoV-1 spike protein can mediate viral entry via Fc receptor-expressing cells in a dose-dependent manner (54). Jaume et al. (34) point out the potential pitfalls associated with immunizations against SARS-CoV-1 Spike protein due to Fc mediate infection of immune cells.

This leads to the prediction that new attempts to create either SARS-CoV-1 vaccines, MERS-CoV vaccines (81), or SARS-CoV-2 vaccines have potentially higher risks for inducing ADE in humans facilitated by antibody infection of phagocytic immune cells. This potential ADE risk is independent of the vaccine technology (82) or targeting strategy selected due to predicted phagocytic immune cell infections upon antibody uptake.

For MERS patients, the seroconversion rate increased with disease severity (83). Severe clinical worsening for SARS patients occurs concurrently with timing of IgG seroconversion (84). Clinical evidence of early high IgG responses in SARS patients is correlated with disease progression (85) and severity (62–67). Antibody treatments for critically ill COVID-19 patients have been halted due to a potential safety signal and unfavorable risk-benefit profile (86). Current SARS-CoV-2 vaccines appear to be providing protection with high antibody titers; the possibility of ADE risks associated with waning titers of antibodies over time remains unknown.

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