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Keeping track of COVID-19 variants through genomic surveillance and assessing the impact of the mutated residues on vaccine efficacy

Despite the gallant effort by the global scientific community to halt the progression of pandemic caused by COVID-19, the inevitable emergence of novel circulating variants may paint a more realistic scenario in the making.  The evolution of variants is not unexpected given the mutation-prone nature of RNA viruses like the coronavirus.  The mutations occur due to a combined effect of errors introduced during replication and recombination.  Despite the proofreading capacity provided by Nsp14 (exoribonuclease which lowers the rate of replication error), recombination may still contribute to the genetic diversity (Rausch et al., 2020).  As a result, its impact on the current ability to diagnose or treat with vaccines is being examined with urgency.

To date, numerous variants of COVID-19 have been documented.  Of significance are the specific mutations that confer greater infectivity potential, thus providing a vantage point for its transmission.  The genomic surveillance of viral sequences has identified mutations occurring at distinct genetic loci.  D614G initially identified in Germany and spread globally refers to a point mutation converting the residue #614 in the spike (S) protein of COVID-19 from aspartic acid to glycine (not located in receptor binding domain). Though this mutation may allow greater infectivity, the infected host was able to neutralize the variant (Hou et al., 2020;  Yurkovetskiy et al., 2021).

The D614G mutation (plus N501Y in the receptor binding domain) was present in the B.1.1.7 variant (harbors 23 mutations including 3 amino acid deletions plus 7 missense mutations in S protein), which rapidly expanded in the United Kingdom.  It is interesting to note that N501Y represents mutation in one of six residues (L455, F486, Q493, S494, N501 and Y505) implicated in binding to the ACE2 receptor.  Nevertheless, the immunity rendered by the mRNA vaccine (BNT162b2) was able to neutralize (albeit less effectively) the B.1.1.7 variant (Muik et al., 2021)

 This was followed by the emergence of the B.1.1.298 variant in Denmark (harbors 2 amino acid deletion and 4 missense mutations, ex. Y453F), which led to the destruction of 17 million minks to avoid cross-infectivity to humans, and the B.1.429 variant in California (USA) containing 4 missense mutations in the spike protein, ex. L452R in the receptor binding domain).  Their impact on the vaccine efficacy has not been determined.

Of concern are P.1 (12 missense mutation) and P.2 (3 missense mutation) variants emerged in Brazil (derived from B.1.1.28 lineage) harboring the mutations E484K along with K417T and N501Y in the receptor binding domain of S protein, which may evade antibody-based immunity (Nonaka et al., 2021). 

Of greater concern is B.1.351 variant emerged in South Africa and has spread internationally (contains K417N, E484K, and N501Y plus other mutations in S protein along with other mutations outside the S gene) as it may escape neutralization by the antibodies (Wang et al, 2021).  

                    

In general, the lower the genetic diversity, the higher is the probability of developing a successful vaccine.  Hence, for mumps or measles virus displaying low degree of genetic diversity, vaccines are already available.  In contrast, for HIV-1 or hepatitis C virus exhibiting high level of genetic diversity, no vaccines are currently available.  In the case of COVID-19, its genetic diversity falls below that of mumps or measles.

 Nevertheless, to assess the impact of variants on vaccine-induced humoral (B cell based; antibody) immunity, the investigators at the Harvard University (USA) constructed pseudo-COVID-19 virus expressing the specific mutations found in the variants.  Next, they asked whether the serum from individuals vaccinated by mRNA vaccines (Pfizer–BioNTech or Moderna vaccine issued under Emergency Use Authorization by FDA) could neutralize the pseudovirus (Garcia-Beltran et al. 2021).  Of the 10 globally circulating COVID-19 variants examined, 5 variants (P.1, P.2, B.1.351(v1, v2, v3) were resistant to neutralization by the serum.  As these variants share only several mutated residues (K417N/T, E484K, and N501Y), it suggested that a small number of mutations might be sufficient to escape the vaccine-induced antibody response. 

 Despite the above, there has been reports (not published or independently reviewed) suggesting that current vaccines may still prevent disease progression in the B.1.351 infected individuals.  To investigate if the purported protection could result from cellular (cytotoxic T cell) response, the investigators at the Johns Hopkins University (USA) compared the site of mutations in the variants B.1.1.7, B.1.351, and B.1.1.248 (this Brazilian variant was later re-classified as B.1.1.28 that gave rise to P.1 and P.2) with epitopes recognized by T cells.  Intriguingly, of the 52 mutations, 51 did not disrupt the epitopes (Redd et al., 2021).  Though the experimental data for T cells response (against COVID-19 variants) was lacking, it raises a possibility that vaccine-induced cytotoxic T cells may be able to recognize the variants. 

In seeking to stay ahead of the harmful COVID-19 variants, the current U. S. Administration is planning on massive genomic surveillance via genomic sequencing (COVID-19 positive specimens) as well as bioinformatics (to track virus mutation and spreading) by investing $1.7B.  For COVID-19, CDC's Advance Molecular Detection program established SPHERE (SARS-CoV-2 Sequencing for Public Health Emergency Response, Epidemiology and Surveillance), which is comprised of numerous public health laboratories (ex. NIH) state public health labs, academic institutions (ex. Baylor Univ), and private companies (ex. Qiagen).  As for diagnosing B.1.1.7, Thermo Fisher (TaqPath) kit detects N or ORF1ab but not S gene (due to a deletion); whereas, Applied DNA Sciences' kit detects one of two regions in S gene.  The latter may be more reliable (though it represents a negative data).  Other assays (not authorized by U.S. FDA for clinical use) that can directly diagnose the mutations are Roche's kit (detects N501Y, del 69-70 in B1.1.7; E484K in P.1) and Seegen's kit (detects all of the above plus P681H in B.1.1.7 and K417T in P.1). 

The key to preventing epidemic is the ability to diagnose the infected early to preempt further propagation.  For this, Bio-Synthesis, Inc. provides primers and probes (as well as synthetic RNA control) for COVID-19 diagnosis via RT-PCR assay.  It specializes in oligonucleotide modification and provides an extensive array of chemically modified nucleoside analogues (over ~200) including bridged nucleic acid (BNA).  A number of options are available to label oligonucleotides (DNA or RNA) with fluorophores either terminally or internally as well as to conjugate to peptides or antibodies.  It recently acquired a license from BNA Inc. of Osaka, Japan, for the manufacturing and distribution of BNANC, a third generation of BNA oligonucleotides.  To meet the demands of therapeutic application, its oligonucleotide products are approaching GMP grade.  Bio-Synthesis, Inc. has recently entered into collaborative agreement with Bind Therapeutics, Inc. to synthesize miR-21 blocker using BNA for triple negative breast cancer.  The BNA technology provides superior, unequalled advantages in base stacking, binding affinity, aqueous solubility and nuclease resistance.  It also improves the formation of duplexes and triplexes by reducing the repulsion between the negatively charged phosphates of the oligonucleotide backbone.  Its single-mismatch discriminating power is especially useful for diagnosis (ex. FISH using DNA probe).  For clinical application, BNA oligonucleotide exhibits lesser toxicity than other modified nucleotides. 

 

https://www.biosyn.com/oligo-flourescent-labeling.aspx

https://www.biosyn.com/tew/Speed-up-Identification-of-COVID19.aspx

https://www.biosyn.com/covid-19.aspx

https://www.biosyn.com/tew/Mutations-in-the-SARS-CoV-2-Spike-Protein.aspx

 

References

Garcia-Beltran WF, Lam EC, et al. Multiple SARS-CoV-2 variants escape neutralization by vaccine-induced humoral immunity.  Cell. 2021 Mar 12:S0092-8674(21)00298-1.  PMID: 33743213

Hou YJ, Chiba S, Halfmann P, et al. SARS-CoV-2 D614G variant exhibits efficient replication ex vivo and transmission in vivo.  Science. 370:1464-1468 (2020).  PMID: 33184236

Muik A, Wallisch AK, et al. Neutralization of SARS-CoV-2 lineage B.1.1.7 pseudovirus by BNT162b2 vaccine-elicited human sera.  Science  371:1152-1153 (2021).  PMID: 33514629

Nonaka CKV, Franco MM, et al. Genomic Evidence of SARS-CoV-2 Reinfection Involving E484K Spike Mutation, Brazil.  Emerg Infect Dis.  27:1522-1524 (2021).  PMID: 33605869

Rausch JW, Capoferri AA, et al.  Low genetic diversity may be an Achilles heel of SARS-CoV-2.   Proc Natl Acad Sci U S A. 117:24614-24616 (2020). PMID: 32958678

Redd AD, Nardin A, et al. CD8+ T cell responses in COVID-19 convalescent individuals target conserved epitopes from multiple prominent SARS-CoV-2 circulating variants.  medRxiv. 2021 Feb 12:2021.02.11.21251585.  PMID: 33594378

Wang P, Nair MS, et al. Increased Resistance of SARS-CoV-2 Variants B.1.351 and B.1.1.7 to Antibody Neutralization.  bioRxiv. 2021 Jan 26:2021.01.25.428137.    PMID: 33532778

Yurkovetskiy L, Wang X, et al. Structural and Functional Analysis of the D614G SARS-CoV-2 Spike Protein Variant.  Cell.  183:739-751.e8 (2020).  PMID: 32991842