A Critical Patent to Produce Covid-19 Vaccines (II)

choi

I do not know, but perhaps only biologists will be interested in this. However, Read (2) definitely to know what Barney Graham's patent is about.


Publishers make the research papers available to the public. Latest first.

(1) Wrapp D et al, Cryo-EM Structure of the 2019-nCoV Spike in the Prefusion Conformation. Science 367: 1260 (Mar 13, 2020: hard-copy date; online publication earlier)
https://pubmed.ncbi.nlm.nih.gov/32075877/
("a 3.5-angstrom-resolution structure of the 2019-nCoV trimeric spike protein by cryo–electron microscopy * * * The S protein is a trimeric class I fusion protein that exists in a metastable prefusion conformation that undergoes a substantial structural rearrangement to fuse the viral membrane with the host cell membrane. This process is triggered when the S1 subunit binds to a host cell receptor. Receptor binding destabilizes the prefusion trimer, resulting in shedding of the S1 subunit and transition of the S2 subunit to a stable postfusion conformation. To engage a host cell receptor, the receptor-binding domain (RBD) of S1 undergoes hinge-like conformational movements that transiently hide or expose the determinants of receptor binding") (citation omitted) ")

My comment:
(a)
(i) To the right of this Web page, you have two choices to read the article for free: from the magazine itself or from PMC (which was authorized to reproduce content).
(ii) The principal investigator and last author is Jason S McLellan, currently at University of Texas at Austin but was at National Institute of Health (NIH) in Maryland close to Washington, DC. His specialty is crystallography; with the exact protein structure, biological research (such as drug making) can be more targeted.
(iii) The author before him is Barney Graham, who is a virologist. Barney can be either a surname (from a place of the same name in Norfolk) or a pet form of Bernard. The Scottish and English surname Graham is from a place called Grantham in Lincolnshire, of uncertain origin and meaning. Both name items are from Dictionary of American Family Names, by Oxford University Press.
(iv) 1 angstrom = 0.1 nanometers

(b) A picture is worth a thousand words. So you need not read this article. Just follow me and take a look at figures. In fact, just view Figures 1 to 3, whose significance will be discussed next, in (2).
(i) First read the quotation from Science magazine. Type 1 means a protein with a single pass through cell membrane and N terminus on the outside and C terminus inside. Type 2 is reverse in orientation (with N terminus inside the cell and C terminus outside).

A trimer means S protein is made up of three identical (as here) protomers.
(ii) Figure 1: At the top (Fig 1A) is a horizontal scheme of a protomer, with N terminus on the left and C terminus on the right. (Two or more amino acids binding together always has a N terminus -- with amino group (-NH3) -- and a C terminus with carboxyl group (-COOH).  

For convenience, I will start from the C terminus on the right. You can see a stub of horizontal bar labeled with CT (cytoplasmic tail), with a vertical bar labeled with TM (transmembrane domain). There are three (3) more bars to the left of ™. These five (5) bars are white, because the legend of Figure 1 explained, "Domains that were excluded from the ectodomain expression construct or could not be visualized in the final map are colored white." These five bars will not show up in Figure 1B.
(iii) In Figure 1A, the extracellular domain is painted in rainbow color (blue near the N terminus to purple near the C terminus, with each bar painted with a different color). These painted bars are divided into two (S1 and S2 units; S because this protein is a S protein for spike). The boundary of S1 and S2 is marked with "S1/S2" (and S1 unit to the left of the boundary, and S2 unit to the right). This (Science) article went ahead and mentioned S1 and S2 without explanation what they are. S1 unit mainly contains the all-important (green-colored) RBD (receptor-binding domain) and, to its left, NTD (N-terminal domain).
(iv) Figure 1B show that a painted protomer in perfusion conformation, again from Blue through Green to Purple.
(v) Now return to the quotation, which says, "Receptor binding destabilizes the prefusion trimer, resulting in shedding of the S1 subunit."
(vi) Figure 3B jas a legend that said "ACE2 in blue and S protein protomers colored tan, pink, and green." The colors here (tan, pink, and green) do not correspond to those in Fig 1A; keep in mind that postfufion S1 unit is shed.

(c) The most important information for you is that the spike protein will hook up with ACE2 protein on the surface of human cells. Then the virus will be internalized into the cell, from which the virus wreaks havoc. Short for angiotensin-converting enzyme 2, ACE2 may be called the receptor for the virus.

choi

(3) Cryo–electron microscopy (cryo-EM) is an alternative to crystallography. Both can determine 3-d atomic structures, but cryo-EM need not purification or crystallography. The technique may be applied to molecules in their natural state (anchored to cellular membrane, for example, which crystallography dare not tread.
(a) Cheng Y, Single-Particle Cryo-EM -- How Did It Get Here and Where Will It Go. Science 361: 876 (Aug 31, 2018; review)
https://pubmed.ncbi.nlm.nih.gov/30166484/
("if one simply places a biological sample inside an electron microscope, vacuum-caused dehydration would destroy the sample's structural integrity")

Note: Decreasing air pressure causes boiling point to drop.

(b) The most difficult part of single-particle cryo-EM (the major branch of cryo-EM) is: what the hell is single particle. It turns out that the particle is not a particle in physics, but in biology. The en.wikipedia.org has a page for "single particle analysis," which is on themoney, it turns out, but poorly written.

Single particle analysis is a software, a computation method.

Sigworth FJ, Principles of cryo-EM Single-Particle Image Processing. Microscopy (official journal of the Japanese Society of Microscopy; publisher: Oxford University Press) 65: 57 (2016)
https://pubmed.ncbi.nlm.nih.gov/26705325/

Quote:

"cryo-EM single-particle reconstruction (SPR) might rightly be considered a very advanced technology. One starts with a set of perhaps 100 000 hopelessly noisy-looking images of single macromolecular 'particles,' and by a seemingly magical process —typically requiring thousands of CPU-hours on a computer cluster —the end result can be one or more 3D density maps from which atomic structures can be determined.

"The goal of this review article is to provide an overview of the processing that takes particle images to density maps.

"Figure  1 a shows one of the cryo-EM micrographs obtained by Liao et al [last author being Chen Y, a professor at Univ of California San Francisco; Nature 504 : 107 (2013)] in their pathbreaking work on the TRPV1 ion channel.

Note:
(a) Figure 1's legend read: "Cryo-EM micrograph and a particle image. (a) One quarter of a micrograph from the TRPV1 dataset of Liao et al. [ 6 ] with selected particles marked by boxes. (b), Boxed image (256 pixels on a side, pixel size 1.22 Å) of the particle marked with a thick box [thicker, whiter box at the 9 o'clock position] in (a). (c) Corresponding projection of the 3D map of the TRPV1 protein, computed according to the angles assigned to this particle image by the Relion reconstruction program [footnote 2]. TRPV1 is a membrane protein, here solubilized by amphipols, and the viewing direction is approximately in the membrane plane; the transmembrane region is at the lower right."
(b) TRPV1
https://en.wikipedia.org/wiki/TRPV1
(transient receptor potential cation channel subfamily V member 1; also known as the capsaicin receptor; section 4 Discovery)

Humans use this receptor to sense heat. But we all know that when chilly pepper is applied to our skin, we feel heat or burn, too, depending on how much is applied.

TRPV1 has 6 transmembrane domains. In other words, the protein passes through the cellular membranes six times.

choi

(2) Ryan Cross, The Tiny Tweak Behind COVID-19 Vaccines; Prepandemic coronavirus research by Jason McLellan and Barney Graham led to a trick for stabilizing the prefusion form of spike proteins. Chemical & Engineering News (c&en; "a weekly news magazine published by the American Chemical Society": en.wikipedia.org), Sept 29, 2020.
https://cen.acs.org/pharmaceutic ... ind-COVID-19/98/i38

Quote:

(a) "Scientists believe that for COVID-19 vaccines to be effective, our immune systems must develop antibodies that prevent this fusion [with neutralizing antibodies physically shrouding the part of S protein that would dock to the (cellular) receptor]. Such antibodies must target the spike protein in its aptly named prefusion conformation. Unfortunately for vaccine developers, spike proteins are liable to spring from their stubby prefusion shape into their elongated postfusion form on a hair trigger.

"Fortuitously, Graham and a former postdoc, Jason McLellan, devised a solution to this problem before the pandemic. Through a bit of structural biology [structural biology determine the 3-dimentional structure of a molecule] and persistent protein engineering, McLellan discovered that adding two prolines—the most rigid of the 20 amino acids—to a key joint of a vaccine's spike protein could stabilize the structure's prefusion shape. This 2P [two-proline] mutation worked in preclinical studies of Graham and Moderna's MERS vaccine, so they applied it to Moderna's COVID-19 vaccine.

(b) About Respiratory syncytial virus (RSV): " * * * There's still no vaccine available.

In 1966, a decade after RSV was discovered [in 1955], US National Institutes of Health researchers began testing an RSV vaccine made of a virus killed with formalin—an aqueous solution of formaldehyde. The trial was a disaster, McLellan says. Although infants who got the vaccine developed antibodies against the virus, they were not protected from infection. Instead, the vaccine seemed to make the disease worse. Some 80% of infants who got the shot were hospitalized after an RSV infection, compared with 5% of infants in the control group. Two vaccinated babies died from the infection. The tragedy tainted the RSV vaccine field for decades.

"Scientists now know that RSV infects and fuses with human cells using its F protein, which, like coronavirus spike proteins, shape-shifts during infection. The F protein is far more unstable than spike proteins, and in 2016 Graham reported that formalin inactivation leaves the viruses coated with postfusion F. Our immune systems can make antibodies against postfusion F, but they aren't very good ones.

(c) "McLellan joined NIAID [National Institute of Allergy and Infectious Diseases (whose head has been Anthony Fauci), a division of National Institute of Health (NIH)] as a postdoc in Peter Kwong's lab in 2008 to do that work [structural biology] with HIV, but he soon realized he was up against the hardest test case for these protein-engineering principles. [Barney] Graham, who, as the deputy director of NIAID's Vaccine Research Center, worked in the same building, encouraged McLellan to work with him [as Graham's postdoc] on RSV. If they could find a way to modify the F protein and keep it locked in its prefusion form, they might have a shot at creating a successful RSV vaccine.

"Figuring out exactly what the prefusion F protein looked like was their first challenge. McLellan and Graham decided to look for antibodies that neutralize RSV but don't bind postfusion F. Such antibodies were likely binding to prefusion F, they reasoned, and could thus be used to lock the protein down [which can mean to purify the prefusion F, or as here, to get 3-d of both prefusion F and antibody together (without separating the two first)]. Doing so would allow McLellan, a trained X-ray crystallographer, to capture a snapshot of the prefusion F and antibody bound together. The team found three highly potent neutralizing antibodies that fit the bill.

(d) "As McLellan was starting his own lab at Dartmouth College in 2013, the recently emerged MERS coronavirus was top of mind. No one had ever solved the structure of a full coronavirus spike protein. These proteins are similar to the F protein on RSV but a whopping 2.5 times as large.

"Despite many attempts, and even with outside help, the scientists couldn’t get the MERS spike protein to cooperate. So McLellan, Graham, and their collaborator at Scripps Research, Andrew Ward, turned to a different coronavirus, HKU1 [first discovered in Hong kong in 2004 but is found to be prevalent worldwide; I am clueless about U in HKU1]. The virus, which causes the common cold, was safer to work with and better at sitting still while its picture was being taken. In 2016, Ward's lab used a technique called cryogenic electron microscopy to capture the structure of the HKU1 spike.

(e) " * * * This 2P mutation enabled McLellan and Ward to solve the MERS prefusion spike structure in 2017 (Proc. Natl. Acad. Sci. U.S.A., DOI: 10.1073/pnas.1707304114). Graham began working with Moderna to make an mRNA vaccine for MERS using the 2P mutation that same year.

"Once the genetic sequence of SARS-CoV-2 was released this January [in 2020], Graham's lab, collaborating with McLellan's lab, was able to compare its genome with those of SARS and MERS, pinpoint the code corresponding to that bent spring [where 2P would be swapped], and then add the 2P mutation to lock the SARS-CoV-2 spike in its prefusion conformation.

(f) "One of the COVID-19 vaccine front-runners, produced by AstraZeneca, doesn't even use it [2P].


My comment:
(a) Regarding quotation (a).
(i) hair-trigger (n): "a gun trigger so adjusted as to permit the firearm to be fired by a very slight pressure"
https://www.merriam-webster.com/dictionary/hair-trigger
(ii) "prolines—the most rigid of the 20 amino acids"

proline
https://en.wikipedia.org/wiki/Proline

View structure in the table to the right and you will see that the proline can not turn around (there is no axis to turn, that is).

(b) Regarding quotation (b).
(i) respiratory syncytial virus
https://en.wikipedia.org/wiki/Respiratory_syncytial_virus  
("is the single most common cause of respiratory hospitalization in infants, reinfection remains common throughout the lifetime [meaning that antibodies, if any, are useless] * * * its name is derived from the large syncytia that form when infected cells fuse together. * * * Following inoculation of the nose or eyes, RSV infects ciliated columnar epithelial cells of the upper and lower airway")
(A) RSV is paramyxovirus (not coronavirus), whose members includes mumps, measle, and rubella
https://en.wikipedia.org/wiki/Rubella
(Rash "usually starts on the face and spreads to the rest of the body.  The rash is sometimes itchy and is not as bright as that of measles. * * * Only humans are infected. * * * The name "rubella" is from Latin and means little red. It was first described as a separate disease by German physicians in 1814 resulting in the name 'German measles' ")
(B) English dictionary:
* rubella (n; From Latin [adjective masculine] rubellus reddish, diminutive of [adjective masculine] ruber red)
https://en.wiktionary.org/wiki/rubella
The corresponding Latin noun (masculine) for red is rubor.
(C) RSV is not coronavirus. It just happens that RSV's extracellular domain undergoes pre- and post- fusion transformation and was used for study that phenomenon, which then paved the way for research of coronavirus's S protein.
(D) Griffiths CD et al, IGF1R Is an Entry Receptor for Respiratory Syncytial Virus. Nature 583: 615 (2020)
https://www.nature.com/articles/s41586-020-2369-7
(Abstract: "Nucleolin is an entry coreceptor for RSV [footnote 2 pointed to a 2011 Nature Medicine report] * * * These findings [reported here] reveal a mechanism of virus entry in which receptor engagement and signal transduction bring the coreceptor to viral particles at the cell surface")

The coreceptor is nucleolin, which usually stays inside nucleus.

The article is placed behind paywall. Here is the conceptual scheme (which is novel):
Carlos Castellanos, RSV and IGF1R: More Than Just a Scratch on the 'Cell' Surface. Breaking Down Biology, June 21, 2020
https://www.breakingdownbio.com/ ... he-cell-surfacenbsp
(iii) syncytium (n; plural syncytia (due to lts Latin root); etymology: New Latin, from syn- + cyt-)
https://www.merriam-webster.com/dictionary/syncytium
(pronunciation)

(c) Regarding quotation (c).

Peter Kwong, PhD  
Chief, Structural Biology Section[,] Vaccine Research Center[, NIAID]
https://www.niaid.nih.gov/resear ... ral-biology-section

(d) Regarding quotation (d).

"In 2016, Ward's lab used a technique called cryogenic electron microscopy to capture the structure of the HKU1 spike."

Kirchdoerfer RN et al, Pre-fusion structure of a human coronavirus spike protein. Nature 531: 118 (2016).
https://www.nature.com/articles/nature17200

There is no need to read it, because S protein of coronavirus family are all similar. Science published (1), only due to widespread interest in Covid-19.

(e) Regarding quotation (e).

"This 2P mutation enabled McLellan and Ward to solve the MERS prefusion spike structure in 2017"

The link connected:
Pallesen J et al (Jason S McLellan being the last author), Immunogenicity and Structures of a Rationally Designed Prefusion MERS-CoV Spike Antigen. Proc Nat Acad Sci 114: E7348 (Aug 29, 2017).
https://www.pnas.org/content/114/35/E7348

There is no need to read this article. What you have to know is that 2P was needed to study protein (here MERS perfusion S) structure, simply because crystallography was used, together with cryo-EM. Cryo-EM, when used alone, does not need 2P, as shown in both (1) and Bing Chen's article at (f)(i)(C) below.

(f) Now I am going to discuss the first two figures in (2).
(i) The top figure has a legend that read: "Harvard Medical School virologist Bing Chen determined prefusion and postfusion structures of the SARS-CoV-2 spike protein. The spike sheds a subunit and elongates during fusion with a human cell."
(A) "Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the virus that causes coronavirus disease 2019 (COVID-19)."  en.wikipedia.org for "severe acute respiratory syndrome coronavirus 2."
(B) The top figure demonstrates a trimer with each protomer painted a different color (red, green and blue).
(C) The top figure is from
Cai Y et al (Bing Chen is the last author), Distinct Conformational States of SARS-CoV-2 Spike Protein. Science 369: 1586 (Sept 25, 2020).
https://pubmed.ncbi.nlm.nih.gov/32694201/

There is no need to read this Science article.
(ii) The middle figure's legend reads, "The prefusion form of the F protein of respiratory syncytial virus revealed two sites, colored red and orange, that the most potent neutralizing antibodies bind to. These sites], having been shed,] are not found on postfusion F."