Connect with us


Plasma cell lines clonally grown on SARS-CoV-2 produce specific neutralizing antibodies




The immune response to infection with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), which causes ongoing coronavirus disease 2019 (COVID-19), consists of both humoral and cellular responses. I am. Serum antibodies against the virus neutralize the virus by preventing it from entering cells and successfully establishing an infection. Other antibodies help get rid of the virus from the body.

New preprint research treatise submitted to bioRxiv* The server gains a better understanding of aspects of humoral immunity to the virus, plasma cells that infuse a flood of specific antibodies that target various epitopes of the pathogen.

Spike antigen

The major viral antigen is a spike glycoprotein composed of S1 and S2 subunits. The S1 subunit contains a receptor binding domain (RBD) that mediates the attachment of the virus to the angiotensin converting enzyme 2 (ACE2) receptor on the host cell. The S2 subunit is involved in viral fusion to the cell membrane, following further processing by the host protease TMPRSS2 and viral internalization.

S1 is the target of many immunoglobulin antibodies, and antibodies to RBD are often neutralized because they interact with the viral interface and ACE2 receptors and can block the virus’s entry into cells. TMPRSS2 inhibitors can also interfere with virus priming and prevent virus invasion.

Plasma cells for SARS-CoV-2 infection

Convalescent plasma Including high titer of Neutralizing antibody Then, several therapeutic monoclonal antibodies directed against RBD and isolated from B cells of COVID-19 patients have been used to treat COVID-19. These include gamlanivimab and Eli Lilly, many others in advanced clinical trials.

Memory B cells have a B cell receptor (BCR) on their surface, so it is relatively easy to screen for virus-specific antibodies. Soluble forms of certain antigens to which these BCRs are homologous, such as spikes and RBDs, can be used to label the required subset of antigen-binding cells and classify by flow cytometry.

However, antigen-specific B cell clones make up less than one-thousandth of all memory B cells in COVID-19 patients, and single cells need to be classified and cloned, further reducing yields.

Importantly, plasma cells (PCs) produce antibodies detected in serum rather than memory B cells. Recent studies have shown that many antibodies closely associated with SARS-CoV-2 exhibit low somatic hypermutation rates, similar to germline antibodies.

Therefore, early antigen-specific plasma cells in peripheral blood may encode antibodies that are specific and neutralize the virus, even though they are clearly immature.

Transitional plasma cells come in many varieties, from plasmablasts to short-lived PCs, and are found slightly in the blood before they migrate to the bone marrow to form long-lived PCs. Transitional PCs make up only 5% of B cells in peripheral blood, but COVID-19 increases by almost one-fifth. However, memory B cells make up 40-50% of all B cells both before and after SARS-CoV-2 infection.

Clonely expanded PCs in people exposed to the antigen have been associated with specific binding to the target antigen, but the association of this finding with COVID-19 patients has not yet been tested.

Details of the study

Current research began with the development of a protocol for selecting SARS-CoV-2 spike antigen-specific plasma cells. This was necessary because they depend on the expression of the BCR of various immunoglobulins on the cell surface, and therefore it is not possible to select a PC that produces a particular antibody using available enrichment methods.

An integrated workflow for examining the antibody specificity of PCs from COVID-19 patients. Serum and peripheral blood mononuclear cells (PBMC) are collected from convalescent COVID-19 patients (verified PCR positive test). Serum is assayed by IgA and IgG ELISA, as well as POCT. From a subset of 16 patients, magnetic cell sorting isolates PCs from PBMCs, followed by gel encapsulation and bar coding for single-cell sequences of heavy and light chain transcripts of the antibody. Antibody repertoire analysis was performed to identify extended plasma cell clonal lines, which were then reformatted into a single ORF full-length synthetic antibody gene containing homologous arms, and a single-step cloning-free genome editing. Will be possible. The resulting mammalian display library undergoes high-throughput screening for SARS-CoV-2 binding by flow cytometry and deep sequencing to restore the identity of the corresponding clonal lineage. The supernatant is used to determine the cross-reactivity of the antibody in the library with the coronavirus antigen.

Researchers first used the protocol to sequence the antibody repertoire on a single PC. They were able to identify extended clones of the PC line using variable heavy and variable light chain sequences (VH and VL, respectively). The total antibody repertoire, and extended clones, showed the use of a wide range of germline genes and sequences.

They identified 45 antibodies with 80% similarity in the amino acid sequences of the complementarity determining regions (CDRs) H3 and formed clonal lines.

They quickly discovered that synthesizing thousands of synthetic antibody genes corresponding to the extended antibody sequence was not feasible and restricted the use of this tool. Nonetheless, they sought to answer the question as to whether PCs with high clonal growth produced COVID-19-specific and neutralizing antibodies.

They selected 132 extended PC clones with the most abundant clonal lineage. These showed a wide range of sequence diversity. As expected, immunoglobulin class switch antibody sequences averaged more than 94% identity to germline genes, 26 of which were 100% identity.

These were inserted into the mammalian cell antibody surface display screening system by genome editing using the CRISPR-Cas9 platform. In this way, they introduced the synthetic antibody gene into the endogenous IGHV locus on the genome.

Specific antibody

They found 37 unique antibodies specifically directed against the SARS-CoV-2 antigen resulting from unique extended clones from 11 patients. Of these, 11 were identical to the germline. Therefore, most COVID-19 patients had PCs from highly expanded clones and produced virus-specific antibodies. The two patients had no anti-S antibodies.

Researchers have found that two of the monoclonal antibodies identified here have been reported in previous studies.

Not all patients have shown comparable specificity for the S1 or S2 subunits because antibody levels against the S protein cannot be detected. Also, due to the low depth of the single cell sequence, it was sometimes not possible to identify the clone expansion.

The two antibody libraries created by the Display Platform contained cross-reactive antibodies that bind to other human seasons. Coronavirus, But not highly pathogenic SARS-CoV or MERS (Middle East Respiratory Syndrome) -CoV. This is in contrast to the broader cross-reactivity found in other studies.

Antibodies specific to the SARS-CoV-2 antigen fail to show a familiar repertoire or uniform sequence, showing a broad spectrum of antibody repertoire in this infection. However, researchers have found sequence similarities to those reported by previous researchers. This indicates that germline genes promote the antibody response to SARS-CoV-2 infection.

Again, only anti-S antibodies were screened, except for those that bind to the nucleocapsid protein. Again, changing the format of the antibody construct can affect antibody affinity, stability, and expression, resulting in loss of reactive sequences.

The researchers commented:The total number of antibodies identified as specific for SARS-CoV-2 in this study may be underestimated because not all of the pooled candidates were individually tested... “

Neutralizing antibody

Three of these antibodies were found to have strong neutralizing activity against RBD of SARS-CoV-2 using a pseudoviral assay. Two of them have been previously explained in other studies. Again, two of the three were 100% identical to germline antibodies, but were RBD-specific.

Two of these seemed to bind to overlapping epitopes and the third seemed to bind to distinct epitopes.

What is the impact?

“”An integrated workflow of single-cell sequencing and mammalian display screening can demonstrate that convalescent COVID-19 patients produce highly expanded PCs with specific neutralizing antibodies to SARS-CoV-2. .... “

Although limited, these findings serve as a proof of concept, using a single-cell sequencing approach in peripheral blood PCs to identify specific antibodies with high binding affinity and strong neutralizing ability. Shows the ability of the line.

Such an approach is very useful in the case of a pandemic, or when the infection needs to be diagnosed early when the antigen is not available for testing, or when the number of memory B cells for a particular antigen is low. PCs in the peripheral blood are also the only source of serum antibodies, from which therapeutic antibodies can be obtained.

Improvements to the current selection process for clonally expanded strains may allow the use of this technique to identify more antigen-specific antibodies.

*Important Notices

bioRxiv Publish preliminary scientific reports that should not be considered definitive as they are not peer-reviewed, guide clinical practice / health-related behaviors, and should not be treated as established information.


What Are The Main Benefits Of Comparing Car Insurance Quotes Online

LOS ANGELES, CA / ACCESSWIRE / June 24, 2020, / Compare-autoinsurance.Org has launched a new blog post that presents the main benefits of comparing multiple car insurance quotes. For more info and free online quotes, please visit https://compare-autoinsurance.Org/the-advantages-of-comparing-prices-with-car-insurance-quotes-online/ The modern society has numerous technological advantages. One important advantage is the speed at which information is sent and received. With the help of the internet, the shopping habits of many persons have drastically changed. The car insurance industry hasn't remained untouched by these changes. On the internet, drivers can compare insurance prices and find out which sellers have the best offers. View photos The advantages of comparing online car insurance quotes are the following: Online quotes can be obtained from anywhere and at any time. Unlike physical insurance agencies, websites don't have a specific schedule and they are available at any time. Drivers that have busy working schedules, can compare quotes from anywhere and at any time, even at midnight. Multiple choices. Almost all insurance providers, no matter if they are well-known brands or just local insurers, have an online presence. Online quotes will allow policyholders the chance to discover multiple insurance companies and check their prices. Drivers are no longer required to get quotes from just a few known insurance companies. Also, local and regional insurers can provide lower insurance rates for the same services. Accurate insurance estimates. Online quotes can only be accurate if the customers provide accurate and real info about their car models and driving history. Lying about past driving incidents can make the price estimates to be lower, but when dealing with an insurance company lying to them is useless. Usually, insurance companies will do research about a potential customer before granting him coverage. Online quotes can be sorted easily. Although drivers are recommended to not choose a policy just based on its price, drivers can easily sort quotes by insurance price. Using brokerage websites will allow drivers to get quotes from multiple insurers, thus making the comparison faster and easier. For additional info, money-saving tips, and free car insurance quotes, visit https://compare-autoinsurance.Org/ Compare-autoinsurance.Org is an online provider of life, home, health, and auto insurance quotes. This website is unique because it does not simply stick to one kind of insurance provider, but brings the clients the best deals from many different online insurance carriers. In this way, clients have access to offers from multiple carriers all in one place: this website. On this site, customers have access to quotes for insurance plans from various agencies, such as local or nationwide agencies, brand names insurance companies, etc. "Online quotes can easily help drivers obtain better car insurance deals. All they have to do is to complete an online form with accurate and real info, then compare prices", said Russell Rabichev, Marketing Director of Internet Marketing Company. CONTACT: Company Name: Internet Marketing CompanyPerson for contact Name: Gurgu CPhone Number: (818) 359-3898Email: [email protected]: https://compare-autoinsurance.Org/ SOURCE: Compare-autoinsurance.Org View source version on accesswire.Com:https://www.Accesswire.Com/595055/What-Are-The-Main-Benefits-Of-Comparing-Car-Insurance-Quotes-Online View photos

Pictures Credit


to request, modification Contact us at Here or [email protected]