Vaccine scenarios

The strategy of targeting the Spike protein so as to prevent its binding and fusion with human cells is the cornerstone of almost all the 100-odd vaccine development proposals across the world that are in various stages of development.

This photo provided by Imperial College London shows a COVID-19 vaccine candidate.

In an earlier issue of Frontline (May 22), we had discussed, based on the work of Nidhan Biswas and Partha P. Majumder of the National Institute of Biomedical Genomics (NIBG), Kalyani, West Bengal, how mutations accumulating in the original strain since the COVID-19 outbreak started in Wuhan, China, in December 2019, have resulted in 11 (phylogenetically) distinct types or clades circulating in different parts of the world. We also saw how, from among these, Type A2a had emerged as the dominant strain in virtually all geographic regions of the world, including India, replacing the ancestral Wuhan strain. The advance e-print of this peer-reviewed work, which is due to be published in Indian Journal of Medical Research (IJMR), appeared on April 28.

A few days after the Biswas-Majumder work, which was an analysis of annotated database of 3.636 genomes publicly available up to April 6, appeared, a similar analysis was carried out by B. Korber and associates—an American-British research group from the Los Alamos National Laboratory (LANL) and the Duke Human Vaccine Institute, United States, and the Sheffield COVID-19 Genomics Group, United Kingdom—with a somewhat larger database of 4,535 genomes (publicly available until April 13). This LANL-Duke-Sheffield work came to the same broad conclusion as Biswas-Majumder, that the type defined by the mutation D614G (which substitutes amino acid aspartic acid (D) to glycine (G) at the site 614 in the Spike protein of the virus), namely A2a, had emerged as the dominant type in nearly all parts of the world. This work was posted on the e-preprint repository bioRxiv on April 30.

Now, the Spike protein (S) is what enables the virus to gain entry into the human cells by binding with receptor ACE2 on the surface of human cells, fuse with the cells and use the cell machinery to replicate itself into multiple copies which go on to infect other cells. The S protein has two components S1 and S2. While the receptor binding domain (RBD) of the virus is part of S1, which is responsible for binding with the human ACE2 receptor, S2 performs the function of fusion of the virus membrane with the human cell. The crucial difference between SARS-CoV-1 and SARS-CoV-2 is the presence of a cleavage site at the S1/S2 boundary and the virus makes use of host enzymes like furin to do the job of splitting the S protein at the boundary. This makes its entry into, and fusion with, the cell far more efficient than in the case of SARS-CoV-1.

So, naturally, the strategy of targeting the S protein, so as to prevent its binding and fusion with human cells, is the cornerstone of almost all the 100-odd vaccine development proposals across the world that are in various stages of development. In fact, the LANL-Duke-Sheffield approach in studying the various mutations of the virus was from the perspective of knowing if there were any mutations that were gaining ground in the disease spread that could affect the strategies currently being pursued for vaccine development. As the group’s paper said, “We have developed an analysis pipeline to facilitate real-time mutation tracking in SARS-CoV-2, focussing initially on the Spike protein because it mediates infection of human cells and is the target of most vaccine strategies and antibody-based therapeutics.”

The authors further observed: “Although the observed diversity among pandemic SARS-CoV-2 sequences is low, its rapid global spread provides the virus with ample opportunity for natural selection to act upon rare but favorable mutations…. Antigenic drift in influenza, the accumulation of mutations [in the haemagglutinin protein] by the virus during an influenza season… is the primary reason we need to develop new influenza vaccines every few seasons…. SARS-CoV-2 is new to us; we do not yet know if it will wane seasonally…but our lack of pre-existing immunity and its high transmissibility relative to influenza are among the reasons it may not. If the pandemic fails to wane, this could exacerbate the potential for antigenic drift and the accumulation of immunologically relevant mutations in the population during the year or more it will take to deliver the first vaccine. Such a scenario is plausible, and by attending to this risk now, we may be able to avert missing important evolutionary transitions in the virus that if ignored could ultimately limit the effectiveness of the first vaccines to clinical use.”

Having noted that the mutation D614G, which characterises globally dominant Type A2a of the virus, was located in the S1/S2 junction near the furin recognition site for the cleavage of S protein, Biswas and Majumder observed: “It is unclear whether the derived allele producing glycine directly provides a selective/transmission advantage for the entry of the virion”.

Recently, two e-preprints have appeared on the bioRxiv repository, both of which have followed very similar experimental procedure of using pseudotyped viruses (with the envelope and S protein of SARS-CoV-2, mutated and unmutated) and have showed that the mutation D614G resulted in increased infectivity of the mutated virus as against the ancestral Wuhan strain. (Pseudotyped viruses or viral vectors are produced by combining a given virus with foreign envelope (E) proteins. In this case, other known viruses were combined with SARS-CoV-2’s E and (mutated and unmutated) S proteins).

On June 12, Lizhou Zhang and others from the Scripps Research Institute, U.S., posted their work in which they created retroviruses pseudotyped with the mutated and unmutated S protein of SARS-CoV-2 and found that mutated S protein infected ACE2-expressing cells markedly more efficiently than the unmutated version. This greater infectivity was correlated with less S1 shedding, which implied that the mutated S protein was more stable. This observation was consistent with epidemiological data suggesting that Type A2a viruses transmitted more efficiently, the paper said.

On June 15, Zharko Daniloski and associates from the New York Genome Centre and New York University posted a similar work, but they had used lentivirus for pseudotyping as against retroviruses by the Scripps group. They observed that in multiple cell lines, including human lung epithelial cells, lentivirus carrying the D614G mutation was up to eightfold more effective at transferring foreign genetic material into human cells than the wild-type lentivirus. “This,” the authors said, “provides functional evidence that the D614G mutation in the Spike protein increases transduction of human cells…. Given that several vaccines in development and in clinical trials are based on the initial [unmutated] Spike sequence, this result has important implications for the efficacy of these vaccines in protecting against this recent and highly-prevalent SARS-CoV-2 isolate.” They did, however, note as a caveat that the pseudotyped lentivirus model had a different pathway for assembling the entire virus [as it multiplies in the host] and it was unclear whether the number of Spike molecules on the pseudotyped lentivirus is comparable to that of the full SARS-CoV-2 virus.

So, is the D614G mutation likely to have serious implications for worldwide vaccine development?

Before we address that question, as an aside, we note here that, on May 31, scientists from the Council of Scientific and Industrial Research (CSIR) laboratories of the Centre for Cellular and Molecular Biology (CCMB), Hyderabad, and the Institute for Genomics and Integrative Biology (IGIB), New Delhi, posted on the e-preprint server bioRxiv an analysis of publicly available genomic data of 361 Indian isolates, an order of magnitude more than what Biswas-Majumder had access to. They found that, while Type A2a continues to be dominant in India, a new type, which they had christened A3i, characterised by four other mutations, had begun to appear with significant frequency in India.

However, as the authors of this work themselves note in the paper, the mutations that define this particular Type A3i all pertain to structural proteins, the Nucleocapsid (N) protein and the Envelope (E) while A2a had to do with amino acid substitution in the S protein and Membrane (M) protein, the critical infective components of the coronavirus SARS-COV-2. So mutations in Type A3i have no particular significance vis-a-vis vaccine development efforts.

Responding to a basic query, whether mutation D614G had any implication for vaccine development that is based on the original unmutated strain, Shahid Jameel, former virologist from the International Centre for Genetic Engineering and Biotechnology (ICGEB), New Delhi, and currently the chief of the Wellcome Trust-DBT India Alliance, said: “From a vaccine point of view… the most important region is the receptor binding domain (RBD) in the Spike protein that attaches to the ACE2 receptor on the surface of target cells. However, the RBD in the SARS-CoV-2 Spike protein is located between amino acids 319 to 529. This is away from the region of this mutation (amino acid 614). Therefore, this is unlikely to have any effect on the vaccines that are being developed using either the complete Spike protein or its RBD.”

This is certainly reassuring that the strategies and efforts currently under way are not going to be impacted by this mutation that seems to have given the virus a selective advantage over the other types. Its apparent increased infectivity and stability notwithstanding, any efficacious and viable vaccine targeting the S protein that is ready should, according to Jameel, be effective as a public health measure, which needs to be made available globally, sans politics and diplomacy, as an affordable prophylactic against COVID-19.