Information

How do scientists discover a new antigen and its epitope?


I've found some database on the internet that list all discovered antigens and their epitopes. So how do scientists discover a new antigen? Do they try to inject them into the body to see if it causes an immune response or not? And finally if it's an antigen, how do they know their epitopes?


Okay, you have a few questions building on top of each other (rephrased the questions for clarity):

How do scientists discover a new antigen?

There are multiple ways to this, which are applied for different purposes.

In order to discover a natural antigen, which is recognised by an antibody normally produced by the immune system, one would first have get hold of this specific antibody. This would be an approach to reproduce or understand the effective response of the immune system against a pathogen (e.g. a virus).

In order to get hold of the antibody (and ideally the coding DNA sequence for it), researchers have to use the pathogen, or parts of it and find the antibodies (or B cells) that bind to it. To do this the pathogen (particles) could be fixed to a column and after running blood through the column the respective antibodies and B cells will remain in the column. Then the DNA of these cells can be sequences, or the antibody can be further purified.

In most cases however, scientists want to find a not-yet existing antibody against an antigen, which leads us to:

Do scientists try to inject antigens to the body to see if it causes an immune response or not?

Yes, this is done - but only with animals (mostly rats, rabbits & goats). Polyclonal antibodies can be extracted directly from the blood of these animals, but most often monoclonal antibodies are needed for research or medical purposes. The process required for these is much more complicated, but leads to a cell lines which can then be used to produce the desired antibody in greater quantities.

How do scientists know the corresponding epitope to an antibody?

There are multiple ways to find the exact epitope of an antibody, all of which are commonly described as epitope mapping.

The most common methods are based either on site-directed mutagenesis of the antigen to see which positions (amino acids) are crucial for binding of the antibody, or on peptide fragments of the antigen, which can still be bound by the antibody.


I would like to just add to Nicolai answer.

What is an antigen ?

First, and Nicolai said this, but I just want to make it clear, an antigen is anything that antibodies bind. That is distinct from and immunogen which is a type of antigen that causes your immune system to produce antibodies. But an antigen does not necessarily have to be an immunogen.

Peptides, sugars, nucleic acids and lipids are all common antigens, but peptides are probably what you would be most familiar with. Usually, most sugars and lipids have a hard time producing an immune response on their own and generally are coupled with a protein (peptide). This makes sense because your body naturally produces lots of sugars and lipids and if you made antibodies to all of them, it would end up attacking itself.

How is an antigen discovered?

There are two ways to approach this.

  1. Antibody First Approach

Let's say a person get's infected with some unknown virus but ends up surviving. The next step would be to do two things:

  • Culture the virus so scientist can study it in the lab. You need to culture most virus so you can grow an unlimited amount in vitro. It would be a really tough task to go out and naturally find all the virus you needed to study.

  • Genotype the virus. It is necessary to know the viral genome so we can manipulate the genes and see what their effect is.

We can start making changes of the virus changing various components of the genome. We could see how that changes binding to the patient's serum.Since the serum contains antibodies that will bind the virus, the serum will often neutralise or test positive for binding antibodies. Now if we change gene A from the virus and the serum still binds/neutralises, we can assume gene A is not the antigen which made an immune response in the patient. If gene B is mutated and the serum stops binding, we can assume gene B is the antigen of the unknown virus.

  1. Antigen first approach

If we only have the virus and don't have an infected patient, then we would have to inject it into model organisms to find out the antigen. However, since we know an incredible amount of viral species we can probably make a best guess on what the antigen component of the virus is.

For instance, in 2012, when a patient fell ill, they sequenced a virus in them which was found to be a close relative to Coronavirus. Since they know that Coronavirus primary antigen is its spike protein, they rightly assumed that the gene that was closely related to the spike protein of the unknown virus was also the primary antigen. That virus turned out to be Middle East respiratory syndrome coronavirus which the main antigen is the spike protein.


Scripps Research Institute scientists find new point of attack on HIV for vaccine development

IMAGE: A team at The Scripps Research Institute has discovered a new vulnerable site on the HIV virus. Shown here is an electron microscopic reconstruction of the HIV-1 envelope glycoprotein trimer. view more

Credit: Image by Christina Corbaci, courtesy of The Scripps Research Institute.

LA JOLLA, CA-- April 24, 2014 --A team led by scientists at The Scripps Research Institute (TSRI) working with the International AIDS Vaccine Initiative (IAVI) has discovered a new vulnerable site on the HIV virus. The newly identified site can be attacked by human antibodies in a way that neutralizes the infectivity of a wide variety of HIV strains.

"HIV has very few known sites of vulnerability, but in this work we've described a new one, and we expect it will be useful in developing a vaccine," said Dennis R. Burton, professor in TSRI's Department of Immunology and Microbial Science and scientific director of the IAVI Neutralizing Antibody Center (NAC) and of the National Institutes of Health's Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery (CHAVI-ID) on TSRI's La Jolla campus.

"It's very exciting that we're still finding new vulnerable sites on this virus," said Ian A. Wilson, Hansen Professor of Structural Biology, chair of the Department of Integrative Structural and Computational Biology and member of the Skaggs Institute for Chemical Biology at TSRI and member of the NAC and CHAVI-ID.

The findings were reported in two papers--one led by Burton and the second led by TSRI Assistant Professor Andrew B. Ward, also a member of NAC and CHAVI-ID, and Wilson--appearing in the May issue of the journal Immunity.

The discovery is part of a large, IAVI- and NIH-sponsored effort to develop an effective vaccine against HIV. Such a vaccine would work by eliciting a strong and long-lasting immune response against vulnerable conserved sites on the virus--sites that don't vary much from strain to strain, and that, when grabbed by an antibody, leave the virus unable to infect cells.

HIV generally conceals these vulnerable conserved sites under a dense layer of difficult-to-grasp sugars and fast-mutating parts of the virus surface. Much of the antibody response to infection is directed against the fast-mutating parts and thus is only transiently effective.

Prior to the new findings, scientists had been able to identify only a few different sets of "broadly neutralizing" antibodies, capable of reaching four conserved vulnerable sites on the virus. All these sites are on HIV's only exposed surface antigen, the flower-like envelope (Env) protein (gp140) that sprouts from the viral membrane and is designed to grab and penetrate host cells.

The identification of the new vulnerable site on the virus began with tests of blood samples from IAVI Protocol G, in which IAVI and its NAC partnered with clinical research centers in Africa, India, Thailand, Australia, the United Kingdom and the United States to collect blood samples from more than 1,800 healthy, HIV-positive volunteers to look for rare, broadly neutralizing antibodies. The serum from a small set of the samples indeed turned out to block the infectivity, in test cells, of a wide range of HIV isolates, suggesting the presence of broadly neutralizing antibodies. In 2009, scientists from IAVI, TSRI and Theraclone Sciences succeeded in isolating and characterizing the first new broadly neutralizing antibodies to HIV seen in a decade.

Emilia Falkowska, a research associate in the Burton laboratory who was a key author of the first paper, and colleagues soon found a set of eight closely related antibodies that accounted for most of one of the sample's HIV neutralizing activity. The scientists determined that the two broadest neutralizers among these antibodies, PGT151 and PGT152, could block the infectivity of about two-thirds of a large panel of HIV strains found in patients worldwide.

Curiously, despite their broad neutralizing ability, these antibodies did not bind to any previously described vulnerable sites, or epitopes, on Env--and indeed failed to bind tightly anywhere on purified copies of gp120 or gp41, the two protein subunits of Env. Most previously described broadly neutralizing HIV antibodies bind to one or the other Env subunit. The researchers eventually determined, however, that PGT151 and PGT152 attach not just to gp120 or gp41 but to bits of both.

In fact, gp120 and gp41 assemble into an Env structure not as one gp120-gp41 combination but as three intertwined ones--a trimer, in biologists' parlance. PGT151 and 152 (which are nearly identical) turned out to have a binding site that occurs only on this mature and properly assembled Env trimer structure.

"These are the first HIV neutralizing antibodies we've found that unequivocally distinguish mature Env trimer from all other forms of Env," said Falkowska. "That's important because this is the form of Env that the virus uses to infect cells."

The second of the two new studies was an initial structural analysis of the new vulnerable epitope.

Using an integrative approach that combined electron microscopy on the Env trimer complex with PGT151 (led by the Ward lab) with the structure of the PGT151 Fab by x-ray crystallography (led by the Wilson lab), the scientists were able to visualize the location of the PGT151-series binding site on the Env trimer--which includes a spot on one gp41 protein with two associated sugars (glycans), a patch on the gp120 protein and even a piece of the adjacent gp41 within the trimer structure--"a very complex epitope," said Claudia Blattner, a research associate in the Wilson laboratory at TSRI and member of the IAVI Neutralizing Antibody Center who, along with graduate student Jeong Hyun Lee, was a first author of the second paper.

A surprise finding was that the PGT151-series antibodies bind to the Env trimer in a way that stabilizes its otherwise fragile structure. "Typically when you try to purify the native Env trimer, it falls apart, which has made it very hard to study," said Ward. "It was a key breakthrough to find an antibody that stabilizes it."

Although the PGT151 site is valuable in itself as an attack point for an HIV vaccine, its discovery also hints at the existence of other similar complex and vulnerable epitopes on HIV.

In addition to the scientists named above, the contributors to the first paper, "Broadly neutralizing HIV antibodies define a novel glycan-dependent epitope on the pre-fusion conformation of gp41 on cleaved Envelope trimers," were Alejandra Ramos, Jeong Hyun Lee, Chi-Hui Liang and Pascal Poignard, all from TSRI and IAVI Neutralizing Antibody Center Alejandro Ramirez, Ryan McBride, Michael B. Zwick and James C. Paulson from TSRI Katie J. Doores from King's College London School of Medicine Ronald Derking, Marit J. van Gils and Rogier W. Sanders from the Academic Medical Center, Amsterdam Sachin S. Shivatare, Chung-Yi Wu and Chi-Huey Wong of Academia Sinica, Taipei, Taiwan Po-Ying Chan-Hui and Kristine Swiderek of Theraclone Sciences, Inc., Seattle Yan Liu and Ten Feizi of Imperial College London Michael S. Seaman of Beth Israel Deaconess Medical Center in Boston John P. Moore of Weill Medical College of Cornell University and Wayne C. Koff of IAVI in New York City.

The contributors to the second paper, "Structural delineation of a quaternary, cleavage-dependent epitope at the gp41-gp120 interface on intact HIV-1 Env trimers," included Kwinten Sliepen, Ronald Derking, Alba Torrents de la Peña, Marit van Gils and Rogier W. Sanders from the Academic Medical Center, Amsterdam Albert Cupo and John P. Moore of Weill Medical College of Cornell University Jean-Philippe Julien and Pascal Poignard of TSRI and IAVI Neutralizing Antibody Center and Wenjie Peng and James C. Paulson of TSRI.

Funding for the first study came from IAVI the National Institutes for Health (grant AI33232, HIVRAD P01 AI82362) the NIH-funded Center for HIV/AIDS Vaccine Immunology and Immunogen Discovery (CHAVI-ID) (grant UM1AI100663) The Ragon Institute of Massachusetts General Hospital, MIT and Harvard and the Aids Fonds Netherlands (grants #2011032, #2012041).

Funding for the second study was provided by IAVI, CHAVI-ID (UM1 AI100663) NIH (P30AI036214, HIVRAD P01 AI082362 and R01 AI084817) the University of California, San Diego Center for AIDS Research The California HIV/AIDS Research Program Aids Fonds Netherlands (grant #2011032) the Netherlands Organization for Scientific Research the European Research Council and the German Academic Exchange Service.

IAVI's funding of this work arose in part from the United States Agency for International Development (USAID). USAID administers the foreign assistance program providing economic and humanitarian assistance in more than 120 countries worldwide.

About The Scripps Research Institute

The Scripps Research Institute (TSRI) is one of the world's largest independent, not-for-profit organizations focusing on research in the biomedical sciences. TSRI is internationally recognized for its contributions to science and health, including its role in laying the foundation for new treatments for cancer, rheumatoid arthritis, hemophilia, and other diseases. An institution that evolved from the Scripps Metabolic Clinic founded by philanthropist Ellen Browning Scripps in 1924, the institute now employs about 3,000 people on its campuses in La Jolla, CA, and Jupiter, FL, where its renowned scientists--including three Nobel laureates--work toward their next discoveries. The institute's graduate program, which awards PhD degrees in biology and chemistry, ranks among the top ten of its kind in the nation. For more information, see http://www. scripps. edu.

The International AIDS Vaccine Initiative (IAVI) is a global not-for-profit organization whose mission is to ensure the development of safe, effective, accessible, preventive HIV vaccines for use throughout the world. Founded in 1996, IAVI works with private companies, academics, and civil society partners in 25 countries to research, design, and develop AIDS vaccine candidates. In addition, IAVI conducts policy analyses and serves as an advocate for the AIDS vaccine field. IAVI supports a comprehensive approach to addressing HIV and AIDS that balances the expansion and strengthening of existing HIV-prevention and treatment programs with targeted investments in the design and development of new tools to prevent HIV. IAVI is dedicated to ensuring that a future AIDS vaccine will be available and accessible to all who need it.

IAVI's work is made possible by generous support from many donors including: the Bill & Melinda Gates Foundation the Ministry of Foreign Affairs of Denmark Irish Aid the Ministry of Finance of Japan the Ministry of Foreign Affairs of the Netherlands the Norwegian Agency for Development Cooperation (NORAD) the United Kingdom Department for International Development (DFID), and the United States Agency for International Development (USAID). The full list of IAVI donors is available at http://www. iavi. org. These studies were made possible in part by the generous support of the American people through USAID. The contents are the responsibility of the authors and do not necessarily reflect the views of USAID or the United States Government.

Disclaimer: AAAS and EurekAlert! are not responsible for the accuracy of news releases posted to EurekAlert! by contributing institutions or for the use of any information through the EurekAlert system.


At a glance

Antigens of the Rh blood group

Number of antigens 49: D, C, E, c, and e are among the most significant
Antigen specificity Protein
The sequence of amino acids determines the specificity of most of the Rh antigens.
Antigen-carrying molecules Proteins with unknown function
The RhD and RhCE proteins are both transmembrane, multipass proteins that are integral to the RBC membrane. The RhCE protein encodes the C/c antigen (in the 2nd extracellular loop) and the E/e antigen (in the 4th extracellular loop), plus many other Rh antigens e.g., C w , C x .
Unlike most cell surface molecules, the Rh proteins are not glycosylated (they do not contain oligosaccharides) but they are closely associated with a RBC membrane glycoprotein called RhAG. The function of the Rh-RhAG complex might involve transporting ammonium or carbon dioxide. The RhD protein encodes the D antigen.
Molecular basis Two genes, RHD and RHCE, encode the Rh antigens.
The Rh genes are 97% identical, and they are located next to each other on chromosome 1. The D/d polymorphism most commonly arises from a deletion of the entire RHD gene. The C/c polymorphism arises from four SNPs that cause four amino acid changes, one of which (S103P) determines the C or c antigen specificity. The E/e polymorphism arises from a single SNP (676G𡤬) that causes a single amino acid change (A226P).
Frequency of Rh antigensD: 85% Caucasians, 92% Blacks, 99% Asians
C: 68% Caucasians, 27% Blacks, 93% Asians
E: 29% Caucasians, 22% Blacks, 39% Asians
c: 80% Caucasians, 96% Blacks, 47% Asians
e: 98% Caucasians, 98% Blacks, 96% Asians (1)
Frequency of Rh phenotypesRh haplotype DCe: most common in Caucasians (42%), Native Americans (44%), and Asians (70%)
Rh haplotype Dce: most common in Blacks (44%)
Rh D-negative phenotype: most common in Caucasians (15%), less common in Blacks (8%), and rare in Asians (1%) (1)

Antibodies produced against Rh antigens

Antibody type Mainly IgG, some IgM
The majority of Rh antibodies are of the IgG type.
Antibody reactivity Capable of hemolysis
Rh antibodies rarely activate complement. They bind to RBCs and mark them up for destruction in the spleen (extravascular hemolysis).
Transfusion reaction Yes—typically delayed hemolytic transfusion reactions
Anti-D, anti-C, anti-e, and anti-c can cause severe hemolytic transfusion reactions. Hemolysis is typically extravascular (1).
Hemolytic disease of the newborn Yes—the most common cause of HDN.
The D antigen accounts for 50% of maternal alloimmunization (2).
Anti-D and anti-c can cause severe disease.
Anti-C, anti-E, and anti-e can cause mild to moderate disease.

T Cell Receptors Are Antibodylike Heterodimers

Because T cell responses depend on direct contact with an antigen-presenting cell or a target cell, the antigen receptors made by T cells, unlike antibodies made by B cells, exist only in membrane-bound form and are not secreted. For this reason, T cell receptors were difficult to isolate, and it was not until the 1980s that they were first identified biochemically. On both cytotoxic and helper T cells, the receptors are similar to antibodies. They are composed of two disulfide-linked polypeptide chains (called α and β), each of which contains two Ig-like domains, one variable and one constant (Figure 24-42A). Moreover, the three-dimensional structure of the extracellular part of a T cell receptor has been determined by x-ray diffraction, and it looks very much like one arm of a Y-shaped antibody molecule (Figure 24-42B).

Figure 24-42

A T cell receptor heterodimer. (A) Schematic drawing showing that the receptor is composed of an α and a β polypeptide chain. Each chain is about 280 amino acids long and has a large extracellular part that is folded into two Ig-like domains—one (more. )

The pools of gene segments that encode the α and β chains are located on different chromosomes. Like antibody heavy-chain pools, the T cell receptor pools contain separate V, D, and J gene segments, which are brought together by site-specific recombination during T cell development in the thymus. With one exception, all the mechanisms used by B cells to generate antibody diversity are also used by T cells to generate T cell receptor diversity. Indeed, the same V(D)J recombinase is used, including the RAG proteins discussed earlier. The mechanism that does not operate in T cell receptor diversification is antigen-driven somatic hypermutation. Thus, the affinity of the receptors remains low (Ka

10 5 -10 7 liters/mole), even late in an immune response. We discuss later how various co-receptors and cell-cell adhesion mechanisms greatly strengthen the binding of a T cell to an antigen-presenting cell or a target cell, helping to compensate for the low affinity of the T cell receptors.

A small minority of T cells, instead of making α and β chains, make a different but related type of receptor heterodimer, composed of γ and δ chains. These cells arise early in development and are found mainly in epithelia (in the skin and gut, for example). Their functions are uncertain, and we shall not discuss them further.

As with antigen receptors on B cells, the T cell receptors are tightly associated in the plasma membrane with a number of invariant membrane-bound proteins that are involved in passing the signal from an antigen-activated receptor to the cell interior. We discuss these proteins in more detail later. First, however, we need to consider how cytotoxic and helper T cells function and the special ways in which they recognize foreign antigen.


Powerful Run on Enzolytics, Inc. (ENZC) Biotech Producing anti-SARS-CoV-2 Monoclonal Antibodies

Enzolytics , Inc. (ENZC) is rocketing up the charts on a powerful surge of 10s of millions of dollar volume daily since a brief dip below the .25 mark on Thursday. ENZC is a major league runner and powerhouse stock over the past few months ENZC has seen a legendary run to recent highs of 0.958 per share as it completes the historic merger between BioClonetics and Enzolytics the new biotech is getting noticed as its technology for producing fully human monoclonal antibodies is currently being employed to produce anti-SARS-CoV-2 ( CoronaVirus ) monoclonal antibodies for treating COVID-19.

It is becoming increasingly evident that Coronavirus vaccines are not as effective against newly discovered variant of the virus as scientists had hoped. With each day of progression of the Coronavirus pandemic, the dire need for multiple active therapeutics becomes more evident. ENZC is a pioneer in using monoclonal antibodies for treating COVID-19. Recently ENZC has identified eleven conserved, expectedly immutable sites (epitopes) on the Coronavirus against which it is producing targeted anti-SARS-CoV-2 monoclonal antibodies. Using computer analysis (Artificial Intelligence [AI]), the Company’s genetics and molecular biology data science team has now screened more than 50,512 Coronavirus isolates currently known and has identified conserved sites which expectedly are immutable. The 11 conserved sequences identified on the virus isolates curated have been identified on the basis that they are 98.71% to 99.29% conserved over the entirety of the 50,512 Coronavirus isolates analyzed. The Company has filed a comprehensive patent application covering these discoveries.

Enzolytics , Inc. is a drug development company committed to the commercialization of its proprietary proteins for the treatment of debilitating infectious diseases. ENZC has been rapidly building up an intellectual property portfolio filing numerous patents last year. Most recently, last month, the Company reported it has received the official filing receipt from the U.S. Patent Office confirming the filing of its patent application for “Nuclear Proteins Isolated from Mammalian Spinal Cord Immune Factor – Pharmaceutical Composition for Treatment.” Since than the ENZC has filed a number of new applications. The Company recently merged with BioClonetics Immunotherapeutics, Inc., now a wholly owned subsidiary of Enzolytics a Dallas and College Station, Texas biotech company with proprietary technology for producing fully human monoclonal antibodies ( mAbs ) against infectious diseases.

Microcapdaily has been reporting on the ENZC BioColnetics merger since the beginning recenlty stating: “ Enzolytics Inc. ENZC: is making a highly explosive move up the charts recently surpassing .50 per share and regularly topping $25 million USD per day in dollar volume ENZC has transformed into a major league runner in small caps. Enzolytics and its new subsidiary BioClonetics own licensing rights of the Irreversible Pepsin Fraction peptide molecule for the treatment of HIV/AIDS a market expected to be worth $30 billion plus by 2025. The Company produces targeted (nontoxic) monoclonal antibodies and is now advancing two separate but complementary therapy platforms for treating infectious diseases, targeting HIV and the CoronaVirus . Enzolytics has attracted a major league level management team behind it and has expanded its lab capabilities on the Texas A&M University campus at the Institute for Pre-clinical Studies, where it is producing both addition monoclonal antibodies against HIV and the against covid-19. This expansion allows Enzolytics to complete the production of monoclonal antibodies against both the HIV virus and the coronavirus and collaborate with the biopharma experts on the campus. Microcapdaily first reported on ENZC the day after the merger was announced in our article : “ BioClonetics LOI Sparks Enzolytics Inc (OTCMKTS: ENZC)” on September 16 when ENZC was trading for well under .01

Enzolytics has quickly attracted a power house team behind it which speaks of big things to come here. They recently appointed Ronald Moss, M.D., to the Medical Advisory Board. Mr. Moss has been an executive with numerous biotech’s over the past 25 years. He has extensive clinical and regulatory management expertise in guiding programs through Phase I, II, and III clinical trials, including IND and NDA experience. The Company’s Chief Science Officer, Mr. Henry Zhabilov has managed several clinical trials utilizing therapeutic proteins. He is the inventor of several U.S. patents related to the immunotherapy of HIV and cancer and an immune enhancer based on the company’s IPF platform.

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The newly combined Companies are led by CEO and majority shareholder Charles S. Cotropia, a well-known intellectual property attorney who has litigated over 200 patents in his career and served as lead counsel in several landmark patent disputes litigated in Federal Courts and the US Patent and Trademark Office. Mr. Cotropia founded BioCLonetics along with his brother Dr. Joseph Cotropia, MD, who pioneered BioCLonetics proprietary method for creating human cell lines that produce human antibodies directed against many infectious diseases. One cell (designated as CLONE 3) has been demonstrated in multiple tests and 5 Independent studies to neutralize the HIV virus in 98% of all known varieties world-wide.

Several weeks ago, the Company executed Articles of Association to form International Medical Partners (“IMPL”) a Bulgarian Limited Liability Company of which the Company is 50% owner. The Company’s partners in IMBL are a group of successful Bulgarian businessmen who will fund the cost of the Clinical trials under the European Medicine Agency (the “EMA”) standards and the application cost for the EMA permit for the Company’s ITV-1 patented therapeutics for treating HIV. Under the Mutual Recognition Agreement (the “MRA”) between the EMA and the United States Federal Drug Administration (the “FDA”), the company believes that issuance of the EMA permit for the ITV-1 compound should qualify ENZC’s treatment for recognition by the FDA. IMBL has entered negotiations to engage Clinic Design to begin the clinical trials that may be required under EMA standards. As the Company progresses in its efforts to commercialize all the current and yet to be discovered opportunities of its licensed and patented treatments the addition of IMBL and the benefits of obtaining an EMA permit has opened up new and exciting avenues for growth of the ENZC and the associated potential increase in value to its shareholders.

The Audits of the Company’s current and prior year Financial Statements are in process and the application for OTCQB is being prepared for submission upon issuance of the Audited Statements. The Company plans to complete the two-year audit as quickly as possible but will file the December 31, 2021 Annual Report Financial Statements pursuant to the OTC Markets Pink Basic Disclosure Guidelines. The Company anticipates filing the financial statements under the Basic Disclosure Guidelines for December 31, 2020 in the coming weeks before the filing deadline of March 31, 2021.

ENZC recently identified eleven conserved, expectedly immutable sites (epitopes) on the Coronavirus against which it is producing targeted anti-SARS-CoV-2 monoclonal antibodies. Using computer analysis (Artificial Intelligence [AI]), the Company’s genetics and molecular biology data science team has now screened more than 50,512 Coronavirus isolates currently known and has identified conserved sites which expectedly are immutable. The 11 conserved sequences identified on the virus isolates curated have been identified on the basis that they are 98.71% to 99.29% conserved over the entirety of the 50,512 Coronavirus isolates analyzed.

ENZC has filed a comprehensive patent application covering these discoveries. This initial application has been filed in the U.S. and will be extended to claim international patent coverage through the International Patent Cooperation Treaty (PCT) to which 153 countries subscribe. The patent coverage sought includes patent claims on the discovered epitope/antigens, vaccine claims, antibody claims, and related prophylactic/therapeutic method claims relating to the epitope/antigens.

Before completing the Artificial Intelligence analysis of the 50,512 SARS-CoV-2 isolates to identify conserved epitopes, the Company’s scientists predicted a specific target epitope that is correlative in structure to the site on the HIV virus to which the Company has produced a monoclonal antibody that has been shown to neutralize the HIV virus. The prediction was that this site would be conserved as is the correlative site on the HIV virus. The AI analysis of the 50,512 SARS-CoV-2 isolates identified this predicted site on the virus as 99% conserved across all 50,512 isolates. This primary site on the SARS-CoV-2 virus has also been confirmed as existing (100%) in the U.S. SARS-CoV-2 virus and the virus variants which have surfaced in United Kingdom, Brazil and South Africa, which are now in the U.S. This epitope on the SARS-Cov-2 virus is included in the first being targeted by the Company in its production of epitope specific monoclonal antibodies. The Company’s focus is on producing monoclonal antibodies that target immutable sites to avoid “virus escape”.

In addition to patenting Company’s findings of conserved sites on the SARS-CoV-2 (Coronavirus), the Company is also filing patent applications covering the conserved sites on the HIV virus. Filings will be made in the U.S. Patent Office and then extended for international coverage through the PCT covering 153 countries.

As the Company has previously reported, it is also curating (analyzing) the amino acid sequences of other major viruses and will file patent applications claiming the identified antigens/epitopes and associated therapeutics. Using AI analysis, the Company is now identifying and will claim the conserved epitopes/antigens on the infectious diseases caused by HIV-2, Influenza A and B, H1N1 influenza, Respiratory syncytial virus (RSV), Small-Pox, Ebola Virus, Tetanus, Diphtheria, HTLV-1/2, Rabies, Herpes zoster, Varicella zoster, Anthrax, Mason-Pfizer monkey virus (MPMV), Visna virus (VISNA) and mouse mammary tumor virus (MMTV). Patent applications will be filed claiming the inventive findings. Patent claims will cover the discovered epitope/antigens, with proposed vaccine claims, antibody claims, and related prophylactic/therapeutic method claims relating to these identified epitope/antigens.

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Enzolytics , Inc. (ENZC) is rocketing up the charts on a powerful surge of 10s of millions of dollar volume daily since a brief dip below the .25 mark on Thursday. ENZC is a major league runner and powerhouse stock over the past few months ENZC has seen a legendary run to recent highs of 0.958 per share as it completes the historic merger between BioClonetics and Enzolytics the new biotech is getting noticed as its technology for producing fully human monoclonal antibodies is currently being employed to produce anti-SARS-CoV-2 ( CoronaVirus ) monoclonal antibodies for treating COVID-19. It is becoming increasingly evident that Coronavirus vaccines are not as effective against newly discovered variant of the virus as scientists had hoped. With each day of progression of the Coronavirus pandemic, the dire need for multiple active therapeutics becomes more evident. ENZC is a pioneer in using monoclonal antibodies for treating COVID-19. Recently ENZC has identified eleven conserved, expectedly immutable sites (epitopes) on the Coronavirus against which it is producing targeted anti-SARS-CoV-2 monoclonal antibodies. Using computer analysis (Artificial Intelligence [AI]), the Company’s genetics and molecular biology data science team has now screened more than 50,512 Coronavirus isolates currently known and has identified conserved sites which expectedly are immutable. The 11 conserved sequences identified on the virus isolates curated have been identified on the basis that they are 98.71% to 99.29% conserved over the entirety of the 50,512 Coronavirus isolates analyzed. The Company has filed a comprehensive patent application covering these discoveries. Since a brief dip Investors are looking for a powerhouse move back to recent highs a break over .91 and its an all-out blue-sky breakout We will be updating on ENZC when more details emerge so make sure you are subscribed to Microcapdaily so you know what’s going on with ENZC.


Detection

Detection is typically achieved using one of two methods: (a) colorimetric or enzyme-mediated detection and (b) fluorescence-based detection.

In the colorimetric method, the bound primary or secondary antibody is conjugated to a substrate which yields a precipitating product when converted by an enzyme. This precipitate is visible as colored staining when viewed by light microscopy.

In the fluorescence-based detection method, antibody bound to the antigen of interest in the tissue is directly or indirectly conjugated to a fluorophore (also sometimes called a fluorochrome), a molecule that fluoresces in the presence of light of a specific wavelength.


Vaccine Ingredients

Injecting something into your body can be concerning for some, especially when you're unsure of what's inside the needle. We're here to take the mystery out of a vaccine's ingredients.

A vaccine contains a part of a germ (bacteria or virus) that is called an antigen. The antigen has already been killed or disabled before it's used to make the vaccine, so it can't make you sick. Antigens are substances, often a protein, that stimulate the body to produce an immune response to protect itself against attacks from future actual disease exposure. In addition, vaccines contain other ingredients that make them safer and more effective, including preservatives, adjuvants, additives and residuals of the vaccine production process. Because specific ingredients are necessary to make a vaccine, even though they are eventually removed, trace amounts can still remain. These residuals can include small amounts of antibiotics and egg or yeast protein. The American Academy of Pediatrics also provides a good explanation about what's inside the vaccine needle.

If you're a parent concerned that your child may be exposed to too many antigens, there's no need to worry: Today's vaccines contain far less antigens than in the past, thanks to advances in biomedical science. Additionally, children's bodies are well equipped to handle many antigens at the same time. A healthy baby can accommodate multiple vaccinations because vaccines, and the antigens they contain are designed for babies' immune systems. In fact, babies can handle significantly more antigens than those that are found in vaccines.

A few years ago, much attention was placed on thimerosal, an organic form of mercury (also called ethylmercury) that prevents vaccines from being contaminated. This form of mercury is different from methylmercury, which can damage the nervous system. Although thimerosal has been shown to be safe, now all routine childhood vaccines are produced in thimerosal-free form. This includes the flu vaccine.


How are Antibodies Produced?

How are Antibodies Produced?
Although detailed mechanics of the immune response are beyond the scope of this site, it is useful, in the context of developing a custom antibody, to have an overview of how antibodies are produced by the immune system.

When an organism’s immune system encounters a foreign molecule (typically a protein) for the first time, specialized cells such as macrophages and dendritic cells capture the molecule and begin breaking it down so that it can present these antigens to B cell lymphocytes.

Once Antigen Presentation to the B cell lymphocytes has occurred, a process known as Somatic Hypermutation allows the B cell to begin coding for a new antibody that will contain a unique Antigen Binding Site in the variable region that is capable of binding specifically to an epitope from the antigen.

Each B cell lymphocyte produces one unique antibody against one unique epitope.

Once antibodies with sufficient specificity to the epitope can be encoded, the B cell begins to release antibodies into the bloodstream. These antibodies then bind specifically with the foreign molecule and allow the immune system to eliminate the molecule from the system.

In some cases, these antibodies can disable pathogens such as viruses directly due to the binding action. In other cases, such as with bacterial pathogens, these antibodies bind to surface proteins on the bacterium’s surface, thereby signaling to the rest of the immune system that the pathogen should be destroyed.

After the foreign molecule has been eliminated, B cells remain in the bloodstream ready to produce antibodies if the antigen is encountered again.

From the perspective of developing a custom antibody against a protein antigen, the immune system captures the protein, breaks it down into individual epitopes and presents these epitopes to the B cells so that development of antibodies specific to those epitopes can begin. These antibodies can then be collected directly in the serum or by isolating the individual B cells that produce antibody against the epitope of interest. With a full-length protein antigen, there will typically be multiple B cells generating antibodies against multiple epitopes from different regions of the protein.


2. MATERIALS AND METHODS

2.1. Sequence alignment of 66 epitopes in IEDB database to SARS𠄌oV𠄂 spike protein

We downloaded the spike protein amino acid sequence of SARS𠄌oV𠄂 isolate Wuhan‐Hu𠄁 from GenBank (GenBank ID: <"type":"entrez-protein","attrs":<"text":"QHD43416.1","term_id":"1791269090","term_text":"QHD43416.1">> QHD43416.1). The sequences of the 66 epitopes containing pentapeptides of SARS𠄌oV𠄂 spike protein were from Lucchese G's report and checked in the IEDB database. 4 Then, the sequences of these epitopes were aligned with the amino acid sequence of SARS𠄌oV𠄂 spike protein to obtain 66 peptides at the corresponding sequence position of SARS𠄌oV𠄂 spike protein, which might be candidate epitopes of a vaccine.

2.2. Detection of nonsynonymous mutation sites of SARS𠄌oV𠄂 spike protein

As nonsynonymous mutation sites in the viral amino acid sequence may affect the recognition of vaccine antigens, vaccine candidate antigens are generally more inclined to choose conservative sequences. 7 , 8 Therefore, the inclusion of mutation sites in candidate epitopes of SARS𠄌oV𠄂 should be avoided as much as possible. We searched the 2019 Novel Coronavirus Resource (2019nCoVR, https://bigd.big.ac.cn/ncov) from the China National Center for Bioinformation (CNCB) to obtain high‐quality genomic data of SARS𠄌oV𠄂 clinical isolates. A total of 1218 isolates from 34 countries around the world sampled from June 1, 2020 to June 30, 2020 were selected for analysis. The detailed countries are shown in Table S1. We focused on counting nonsynonymous mutations that cause amino acid changes in spike protein single‐nucleotide polymorphism (SNPs). The amino acid sites with nonsynonymous mutations that appeared twice or more in 1218 isolates were considered to be easily mutated. The obtained 66 peptides of SARS𠄌oV𠄂 spike protein were checked for the presence of the easily mutated amino acid sites, and peptides containing the easily mutated sites should be noted in subsequent screening.

2.3. Screening candidate vaccine epitopes in spike protein

The immune protective antigens in the peptides of SARS𠄌oV𠄂 spike protein were predicted using immunoinformatics tool Vaxijen v2.0, 9 the toxic peptides were predicted using ToxinPred 10 and the allergenic peptides were predicted using AllergenFP v.1.0. 11 The ability of the epitopes to induce interferon‐γ (IFN‐γ), interleukin𠄄 (IL𠄄), and IL� secretion was predicted using IFNepitope, 12 IL4Pred, 13 and IL�Pred, 14 respectively. The peptides with nonantigenic protection, toxicity, or allergenicity were removed, and the remaining peptides were used as antigen epitopes for subsequent screening. The solvent accessibility of each amino acid of spike protein (template 6xr8.1 15 ) was predicted by SWISS‐MODEL 16 to screen the epitopes that were more likely to be exposed on the surface of the spike protein. ABCpred 17 and IEDB Bepipred Linear Epitope Prediction 2.0 18 were used to predict B�ll epitopes. NetMHC 4.0 Sever, 19 Rankpep, 20 ਊnd SYFPEITHI 21 were used to predict T�ll epitopes and HLA molecules. As different HLA types are expressed at dramatically different frequencies in different ethnicities, 22 after obtaining the results of HLA class I and class II molecules recognized by these epitopes, we predicted the coverage rate of each epitope in different populations using Population Coverage in IEDB Analysis Resource. 22 Although some epitopes contained easily mutated sites, some of them might be strong neutralizing epitopes which might induce strong protections and should also be considered in vaccine design. Therefore, according to the above analysis, the selected vaccine candidate epitopes for SARS𠄌oV𠄂 were predicted to be relatively conservative, immunoprotective, nontoxic, and nonallergenic,ਊnd਌ould promote the secretion of cytokines and more likely to be exposed on the surface of the spike protein. They were both B‐ and T�ll epitopes, which could identify a certain number of HLA molecules and had high coverage rates in different populations.

2.4. Acquisition, analysis, and screening of vaccine candidate sequences

The selected vaccine candidate epitopes were connected by different linkers (no linker, GGGGS, GGGSGGG, EAAAK, GPGPG, AAY, and KK, respectively) to obtain vaccine candidate sequences. Bioinformatics tools were used to analyze and screen the vaccine candidate sequences. PredictProtein was used to predict the amino acid composition, secondary structure composition, solvent accessibility, and gene ontology terms of the candidate sequences. 23 The flexibility and antigenic index of the candidate sequences were predicted using DNAStar software. 24 Expasy ProtParam tool was used to predict the half‐life and stability of the candidate proteins. 25 Finally, through a comprehensive analysis, the best candidate vaccine sequences were selected and will be prepared into vaccines and their immune effects verfied through animal experiments.


NIH Scientists Identify Atomic Structure of Novel Coronavirus Protein

The atomic-level structure of the SARS-CoV-2 spike protein in its prefusion conformation. The receptor binding domain, the part of the spike that binds to the host cell, is colored green.

The atomic-level structure of the SARS-CoV-2 spike protein in its prefusion conformation. The receptor binding domain, the part of the spike that binds to the host cell, is colored green.

NIAID scientists working with investigators from the University of Texas at Austin (UT) identified the atomic structure of an important protein on the surface of the novel coronavirus (SARS-CoV-2, formerly called 2019-nCoV). The findings appear in the peer-reviewed journal Science. The authors note that the findings will aid in the design of candidate vaccines and the development of treatments for COVID-19, the disease caused by the new virus, which was first identified in China in December 2019.

Like other coronaviruses, SARS-CoV-2 particles are spherical and have mushroom-shaped proteins called spikes protruding from their surface, giving the particles a crown-like appearance. The spike binds and fuses to human cells, allowing the virus to gain entry. However, coronavirus infection can be prevented or slowed if this process is disrupted.

Scientists in China shared the genome of a SARS-CoV-2 virus isolate to a global database, which NIAID and UT experts used to start their work determining the spike structure. The spike undergoes a massive rearrangement as it fuses the virus and cell membranes. The researchers confirmed that the original spike stabilized in its prefusion conformation is more likely to preserve targets for infection-blocking antibodies induced by a vaccine.

Importantly, the new data supports NIAID’s approach to a gene-based vaccine for COVID-19 and will also be useful in other vaccine approaches including protein-based vaccines and other nucleic acid or vector-based delivery approaches. NIAID scientists designed the stabilized spike antigen based on previous knowledge obtained from studying other coronavirus spike structures. NIAID and the biotechnology company Moderna, based in Cambridge, Massachusetts, are developing a messenger RNA (mRNA) vaccine, which directs the body’s cells to express the spike in its prefusion conformation to elicit an immune response.

The new research also confirms that the structure of the SARS-CoV-2 spike is very similar to that of the coronavirus responsible for the global outbreak of severe acute respiratory syndrome in 2003 that was eventually contained (known as SARS-CoV). However, despite the similarities, the paper shows that some monoclonal antibodies developed to target SARS-CoV do not bind to the new coronavirus, indicating that antibodies that recognize the SARS-CoV from 2003 will not necessarily be effective in preventing or treating COVID-19, the disease caused by the new virus.

Recent reports show that the novel virus and SARS-CoV also bind to the same receptor on the host cell. However, NIAID and UT scientists determined that SARS-CoV-2 binds more easily to this receptor as compared to SARS-CoV, which could potentially explain why SARS-CoV-2 appears to spread more efficiently from human-to-human. However, more data is needed to investigate this possibility, the authors note.

This research was supported by the NIAID Intramural Research Program and a NIAID grant to the University of Texas at Austin (R01-AI127521).