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Premas Biotech’s VLP-ELISA for SARS-CoV-2 Diagnosis
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Over the past 20 years, the development of high-throughput technologies for genomic and proteomic analysis has ushered in a novel era of biomarker discovery, bearing a notable impact on disease diagnosis and therapy. On 30th January 2020 following the recommendations of the Emergency Committee, the outbreak of Coronavirus Disease 2019 (COVID-19) was declared a Public Health Emergency of International Concern and a pandemic on 11th March 2020 by the WHO Director-General.

Background:

Virus neutralization remains the gold standard for determining antibody efficacy. In view of the urgency of this outbreak, a high-throughput assay to measure SARS-CoV-2 neutralizing antibodies is urgently needed for COVID-19 serodiagnosis and vaccine development. Assays such as PRNT and neutralization are not suited for large-scale high throughput surveys because they are time and bench work-demanding, especially if two antigens are used. Therefore, serological tests, such as ELISA and Luminex microsphere assay, can be an important complementary approach since they are positively associated with the true antibody concentration.

Problem Statement:

However, serological tests with one antigen have been found to generate false-negative data in about 8-10% of the populations tested. Depending on the isotypes tested, false-negative data can sometimes rise to 23%. There is thus a need for alternative methods for screening in the context of epidemiological surveys.

World’s first triple-antigen VLP:

Unlike single-protein vaccines currently under trial worldwide, Premas Biotech has adopted a three-pronged approach to developing a multi-subunit vaccine. The world’s first triple-antigen VLP Covid-19 vaccine candidate, PRAK-03202, has produced a neutralizing immune response in animal studies. It expresses simultaneously the spike, envelope, and membrane antigens of SARS-CoV-2 to induce the formation of non-infectious VLP aggregates that mimics the antigenic structure of authentic SARS-CoV-2 particles.

Using the triple antigenic VLPs, we developed an ELISA (VLP-ELISA) to detect SARS-CoV-2 serum antibodies and evaluated the test using convalescent patients’ sera. Virus-like particles (VLPs) resemble authentic virus particles antigenically and morphologically but lack infectious genetic material. For this reason, they have been used as a highly effective type of alternative antigen in several viral diseases as well as to provide subunit vaccines.

PRAK-03202 was developed by cloning and transforming SARS-CoV-2 gene segments into a highly characterized S. cerevisiae-based D-Crypt™ platform. Due to its triple antigenic (S, E, and M protein) nature, it can deliver a result that can have high benefits, as seen by a small 80 convalescent RT-PCR positive patient data that has been generated. This interesting data on a relatively small sample of patients allows us the opportunity to build VLP-ELISA tests for serological surveillance studies and would be able to bring down the false-negative data in a greater population.

VLP-ELISA for SARS-CoV-2 diagnosis:

The median duration between COVID-19 symptoms onset and sample collection is between <6 to >22 days. Here, we developed a proof-of-concept ELISA assay to detect IgG antibodies in patients’ sera against PRAK-03202.

ELISA data shows the binding of the IgG present in the patients’ sera to PRAK-03202(Figure-I). We used the (Mean+2xSD) formula to calculate the cut-off by calculating the mean of OD of the COVID-19 negative samples for each of the recombinant proteins tested. We added to the value obtained two times the standard deviation. The result obtained was considered as the cut-off (0.85) for the molecule. Mean OD in the COVID-19 and negative groups were 1.7 ± 0.34 and 0.7 ± 0.07, respectively. Results showed that the IgG response of patient’s samples was more than two-fold higher than cut-off values. The sensitivity and specificity of the assay were 80% and 90%, respectively.

Conclusion:

This interesting data on a relatively small sample of patients allows us the opportunity to build VLP-ELISA tests for serological surveillance studies and would be able to bring down the false-negative data in a greater population.


Figure 1


By Reeshu Gupta
Lead-Content Generation

14 April 2021

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