New platform using green algae tests for SARS-CoV-2 virus

Researchers at the University of California, San Diego, have demonstrated the potential of using green algae to make recombinant viral proteins that could be used in large scale assays to detect antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) - the agent that causes coronavirus disease 2019 (COVID-19).

The recombinant proteins could also be used as antigens for potential vaccines, says Stephen Mayfield and colleagues.

The team successfully produced recombinant SARS-CoV-2 spike receptor-binding domain (RBD) recombinant proteins in the microalgae Chlamydomonas reinhardtii.

The spike protein is the surface structure the virus uses to bind to and infect host cells. The RBD is the region on the spike that mediates this process through its interaction with the host cell receptor angiotensin-converting enzyme 2 (ACE2).

“Because algae can be grown at large scale very inexpensively, this recombinant protein may be a low-cost alternative to other expression platforms,” writes the team.

A pre-print version of the research paper is available on the bioRxiv* server, while the article undergoes peer review.

Chlamydomonas reinhardtii. Image Credit: Dartmouth Electron Microscope Facility, Dartmouth College
Chlamydomonas reinhardtii. Image Credit: Dartmouth Electron Microscope Facility, Dartmouth College

The challenges with current testing systems

Tests that detect SARS-CoV-2 RNA, such as reverse transcriptase quantitative polymerase chain reaction (RT-qPCR), have become the standard method for detecting infection.

However, serologic assays that measure antibody responses to the virus and indicate seroconversion are not yet widely available.

While these assays are not suitable for detecting acute infection (because antibodies are often produced after symptoms develop), they are among the best indicators of prior immune responses to viral antigens. They could be used to determine whether people are immune.

As well as its application as a reagent to detect antibodies, recombinant spike protein can also serve as a vaccine antigen. Almost all of the vaccines currently being deployed are based on the SARS-CoV-2 spike protein.

Since these vaccines are being rolled out at a population level, monitoring immune responses to the spike protein will be key in assessing vaccine efficacy, as well as potentially indicating when boosters might be required due to immune responses waning over time.

“To develop such antibody tests, it is critical to produce the viral protein antigens at extremely large scale and at an affordable price, so that antibody tests can become available around the world, and not just in the economically advantaged countries,” says Mayfield and colleagues.

Non-animal cell systems have been problematic

The production of functional SARS virus spike proteins using non-animal cell production systems has been challenging. For instance, the production of SARS-CoV-1 viral proteins using an E.coli-based system resulted in proteins that were poorly folded. Also, the use of fungal systems has produced coronavirus RBD proteins at orders of magnitude lower than the amounts previously observed with these systems for other recombinant proteins.

Studies have previously demonstrated the successful production of viral recombinant proteins using plant-based systems, but these platforms generate less biomass than microbial systems and involve technically demanding and time-consuming methods.

However, green microalgae platforms have been shown to generate correctly-folded recombinant proteins that can be directed to any subcellular structure, which allows for their rapid, large-scale, cost-effective production.

What did the researchers do?

The team examined whether the unicellular green microalgae Chlamydomonas reinhardtii could serve as a platform for the production of recombinant SARS-CoV-2 spike RBD protein.

The researchers tested three protein-targeting strategies. Proteins were targeted to be retained within the endoplasmic reticulum, to be secreted from the cell into culture media, or to accumulate within the chloroplast.

The viral protein was fused to a fluorescent mClover protein to enable high-throughput fluorescent screening and rapid identification of algae expressing enough protein for its accumulation and function to be tested.

ddd

(A) Diagram of the SARS-CoV-2 viral particle with crystal structure of the spike (S) protein highlighted with Subunits 1 and 2 indicated (S1, S2, respectively). The Receptor Binding Domain (RBD) is located in the more variable subunit 1. (B) Vector design and construction. The peptide structure of the spike protein indicating the N-Terminal Domain (NTD), Receptor Binding Domain (RBD), Fusion Peptide (FP, Homology region 1 and 2 (HR1, HR2), Transmembrane association domain (TA) and Intracellular Terminal (IT). A C. reinhardtii nuclear codon optimized version of the RBD-coding sequence was cloned in to a vector containing the AR1 promoter (PAR1) driving a transcriptional fusion of the Bleomycin resistance gene (BleR), FMDV Foot-and-mouth disease virus 2A (F2A) ribosomal-skip motif and 5’ mClover green fluorescent protein tag. A separate Beta-tubulin2 promoter driving Hygromycin resistance was used for secondary selection. Three different versions of the RBD were generated. A chloroplast-directed version through N-terminal fusion of the PsaE chloroplast transit sequence, a secreted version by the addition of the PHC2 secretion signal peptide, and an ER188 Golgi system retained version by the subsequent addition of a C-terminal KDEL Golgi retention sequence. (C) Schematic summarizing transformation process and timeline including drug selection, clone down selection through 96-well microtiter plates, and then flask-scale characterization of candidate RBD-expressing lines.
(A) Diagram of the SARS-CoV-2 viral particle with crystal structure of the spike (S) protein highlighted with Subunits 1 and 2 indicated (S1, S2, respectively). The Receptor Binding Domain (RBD) is located in the more variable subunit 1. (B) Vector design and construction. The peptide structure of the spike protein indicating the N-Terminal Domain (NTD), Receptor Binding Domain (RBD), Fusion Peptide (FP, Homology region 1 and 2 (HR1, HR2), Transmembrane association domain (TA) and Intracellular Terminal (IT). A C. reinhardtii nuclear codon-optimized version of the RBD-coding sequence was cloned in to a vector containing the AR1 promoter (PAR1) driving a transcriptional fusion of the Bleomycin resistance gene (BleR), FMDV Foot-and-mouth disease virus 2A (F2A) ribosomal-skip motif and 5’ mClover green fluorescent protein tag. A separate Beta-tubulin2 promoter driving Hygromycin resistance was used for secondary selection. Three different versions of the RBD were generated. A chloroplast-directed version through N-terminal fusion of the PsaE chloroplast transit sequence, a secreted version by the addition of the PHC2 secretion signal peptide, and an ER188 Golgi system retained version by the subsequent addition of a C-terminal KDEL Golgi retention sequence. (C) Schematic summarizing transformation process and timeline including drug selection, clone down selection through 96-well microtiter plates, and then flask-scale characterization of candidate RBD-expressing lines.

What did they find?

Recombinant RBD protein accumulated to a high level in the chloroplast but was truncated at the amine end and not recognized by anti-RBD antibodies in western blotting assays. However, proteins targeted to the endoplasmic reticulum or secreted out of the cell were of the correct size and amino acid sequence.

Next, the researchers showed that spike RBD protein purified from the endoplasmic reticulum specifically bound to recombinant ACE2 protein at a similar affinity to spike-RBD expressed in mammalian cells.

“Here we have demonstrated that production of a correctly folded and functional SARS-CoV-2 spike protein RBD is possible in the green alga C. reinhardtii,” writes Mayfield and colleagues.

What are the study implications?

The researchers say the study shows the potential of using eukaryotic algae as an efficient and scalable platform to make correctly-folded and functional spike RBD recombinant proteins that could be used in large-scale antibody assays or as potential vaccine antigens.

“Because algae can be grown at scale for a fraction of the cost of mammalian cell lines, this system offers the potential to produce this recombinant viral protein, for a variety of uses including as an antigen to detect serum antibodies against SARS-CoV-2 RBD or even as a potential viral antigen for vaccine development, rapidly and cost-effectively,” concludes the team.

*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.

Journal reference:
Sally Robertson

Written by

Sally Robertson

Sally first developed an interest in medical communications when she took on the role of Journal Development Editor for BioMed Central (BMC), after having graduated with a degree in biomedical science from Greenwich University.

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