How does the SARS-CoV-2 Delta variant’s spike drive membrane fusion and immunity evasion?

A fascinating and timely new study shows how the Delta variant of the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) achieved higher transmissibility and resistance to neutralization.

A preprint version of the study is available on the bioRxiv* server, while the article undergoes peer review.

Study: Membrane fusion and immune evasion by the spike protein of SARS-CoV-2 Delta variant. Image Credit: Design_Cells / Shutterstock

Background  

The current coronavirus disease 2019 (COVID-19) pandemic was triggered by SARS-CoV-2. Despite advances in its management, new variants keep emerging, often showing partial resistance to pre-existing antibodies elicited by vaccines or natural infection.

The Delta variant B.1.617.2 is a variant of the virus that emerged first in India but has rapidly spread and become dominant over the course of a few months. It is a variant of concern (VOC) because it has twice the transmissibility potential of the reference Wuhan strain.

Earlier research suggests that it has a shorter incubation period, while the viral load is a thousand times greater compared to that achieved by earlier lineages. It has caused breakthrough infections after full vaccination. This makes it important to understand the underlying mechanisms that make it so different so that appropriate intervention strategies can be developed.

The virus spike is a surface envelope glycoprotein that mediates the attachment and entry of the virus into the host cell. Found in nature as a trimer, it binds to the viral receptor angiotensin-converting enzyme 2 (ACE2).

The spike has two domains, the S1 and S2, which mediates receptor engagement and membrane fusion, respectively. Cleavage into these two fragments is with the help of a host furin-like protease.

Receptor binding is followed by spike cleavage via the host enzyme TMPRSS2, or cathepsins B and L. This causes the S1 domain to fall away, while S2 undergoes a cascade of events that causes the virus to fuse to the cell membrane and adjacent cell membranes to fuse together.

These promote viral entry into the cell as well as propagation of the infection to neighboring cells via syncytia formation.

The S1 domain has an NTD (N-terminal domain), RBD (receptor-binding domain), and two CTDs (C-terminal domains), all surrounding a bundle of helices that comprises the prefusion S2 domain. The RBDs may be in the ‘up’ or ‘down’ conformation, when it is accessible for receptor binding or not, respectively.

This movement at the RBD prevents the host immune response from targeting this functionally important site on the virus.

What did this study show?

The researchers found that the Delta spike fuses the membranes of adjacent cells more efficiently, increasing over time. When replicated using a pseudovirus with an engineered spike that promotes incorporation into viral particles, the Delta variant was observed to infect the cells much faster than any other variant, over one hour.

All variants reached their maximum level of infection over eight hours. Comparing the Gamma, Kappa and Delta spike variants, they found the prefusion spike trimer made up <40% of the whole, for the Gamma spike, with inefficient spike cleavage by furin.

In contrast, the Delta variant formed a single prefusion spike peak, indicating that it is very stable in the cleaved S1/S2 complex state, similar to the G614 and beta spike variants.

Receptor binding was stronger for the Gamma spike relative to G614 because of the K417T, E484K and N501Y mutations in the RBD. The Delta had an intermediate affinity, perhaps because receptor binding causes the S1 subunit to dissociate, especially with dimeric ACE2. However, spike dissociation from the receptor was comparable for all three variants.

The G614 trimer bound to antibodies in convalescent plasma directed against the spike protein, either the NTD or the RBD, but the Gamma mutant did not bind to the RBD antibodies and one of the NTD antibodies. For the other NTD antibody, it had reduced affinity.

The Delta variant did not bind the NTD antibodies, but retained binding to the others. Binding affinity was related to the neutralization capacity for almost all antibodies. The mutations affected sensitivity to antibody-mediated neutralization for the Gamma variant more than the Delta.

Structure of spike trimers

The cryo-electron microscopic structures of the spike trimers were examined, showing no major structural changes have occurred in the different variants compared to the G614 parent. The Delta trimer is the most stable among the spike variants, while the Gamma prefusion trimeric spike tends to dissociate.

When the Delta spike trimer was superposed onto the G614 parent trimer in the closed RBD conformation, focusing on the S2 region, the differences were most apparent in the NTD, with its three mutations and one two-residue deletion.

When the NTDs are aligned, the loop between residues 143-154 is seen to take on a different shape. This makes it face away from the virus membrane. Simultaneously, the mutations reshape the N-terminal segment and another loop between residues 173-187. This changes the shape of the antigen at the NTD-1 group of epitopes in the NTD.

Such changes help explain why NTD-1 antibodies fail to bind and neutralize the Delta variant as efficiently. Meanwhile, the two Delta RBD mutations L452R and T478K fail to cause structural changes and are not on the ACE2 interface. They do not make up part of a neutralizing epitope either, as they do not alter either binding or neutralization.

Another mutation is the S2 D950N, which may change the local electrostatic state.

What are the implications?

Explanations are being sought for the increased transmissibility of the Delta variant over the Alpha VOC, itself much more infectious than the Wuhan strain. It is possible that the viral replication process for the Delta variant is itself the subject of unique mutations that speed up genomic replication.

Many other steps are also key to assembling viral particles. However, to explain how the viral load in the infected cell is a thousand times higher for this variant. While ACE2 binding by this variant is comparable to that of earlier variants, and spike cleavage remains similar, the current study reveals two other factors that may contribute to its unusual speed of transmission and propagation.

One is the increased fusion efficiency with high Delta spike expression on the cell surface even when the ACE2 levels are low, compared to any other variant. Secondly, the fusion step is optimized to allow entry into the cell even at low ACE2 levels.

This optimization may explain why the Delta variant can transmit upon relatively brief exposure and infect many more host cells rapidly, leading to a short incubation period and greater viral load during the infection,” write the researchers.

Further studies will be required to confirm this, using authentic viruses rather than the spike trimer or RBD construct used in this experiment.

Structural changes appear inadequate to explain the increased fusogenicity. The D950N mutation, found only in the Delta variant, removes one negative charge from each of the protomers in the trimeric spike. Its location near a possible control unit of the spike protein may destabilize the prefusion S2 subunit by electrostatic mechanisms.

Such loss of stability cannot be too great, as it could cause the spike trimer to change its conformation too soon and thus become inactive before membrane fusion occurs.

Intriguingly, while the RBD conserves its structure and function among all variants, with mutations occurring at only a few specific sites, the NTD seems to allow rearrangement of its surface loops, central Beta-strands and a few N-linked glycans while retaining infectivity but evading the host immune response.

The important take-home from this is that therapeutic antibodies should avoid targeting the NTD as it is easily able to evade them. New-generation vaccines will depend on such studies of structure, antigenicity and function to choose the most effective antigens that elicit antibodies against the most highly conserved epitopes.

*Important notice

bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.

Written by

Dr. Liji Thomas

Dr. Liji Thomas is an OB-GYN, who graduated from the Government Medical College, University of Calicut, Kerala, in 2001. Liji practiced as a full-time consultant in obstetrics/gynecology in a private hospital for a few years following her graduation. She has counseled hundreds of patients facing issues from pregnancy-related problems and infertility, and has been in charge of over 2,000 deliveries, striving always to achieve a normal delivery rather than operative.