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  • gsgs
    concerning the waning effecitveness of the current COVID-vaccines,
    I found this nice review published 2021-04-01: (see p.12)
    (after checking keywords wane,waning in the first 20 out of 172 papers at google-scholar
    found by keywords
    waning covid vaccine novavax moderna mrna adenovirus

    cited by 6 :,5&hl=de

    Nano-Enabled COVID-19 Vaccines: Meeting the Challenges of Durable Antibody Plus Cellular Immunity and Immune Escape
    André E. Nel* and Jeff F. Miller

    The neutralizing antibody response to seasonal
    (“cold”) coronaviruses is of transient duration, allowing the
    occurrence of reinfections.[33] In contrast, the protective
    antibody responses to SARS-CoV-1 and MERS lasted a
    minimum of 2−3 years after recovery.

    ...half-life of anti-RBD antibody decline to be ∼36 days;135

    ...people with milder infections generate lower antibody titers
    that decline more rapidly.[138]

    ... 8 months {followup study}.139

    { neutralizing IgG antibody titers against the
    spike protein and RBD remained relatively stable, with only a
    modest decline over 6−8 months }

    memory CD4+ and CD8+T-cells {have} half-lives of 3−5 months.

    The decay kinetics of memory T-cell responses after
    COVID-19 are similar to the vaccination response to the
    yellow fever virus, which is known to confer long-lasting

    there is a critical requirement for TFH
    cooperation with germinal center B-cells in the development
    of durable immunity to polio, smallpox, and other

    a promising approach for augmenting memory B-cell
    responses in COVID-19 could be to develop vaccinating
    nanoparticles ...
    Last edited by sharon sanders; October 6, 2021, 03:10 PM. Reason: added title of paper

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  • Vibrant62
    commented on 's reply
    Interestingly, a few new papers that expand on the potential risks here. I would suggest a MERS vaccination for camels asap would be prudent. Cats would seem to be less of a problem for us than we are to them.


  • Emily
    commented on 's reply
    You have a wealth of information about the immune system organized here. Even though I don't grasp this as well as you, my sense is that you are right about the variant threat - there isn't a biological basis for fear at this point.
    I don't fear a threat from other species as intermediates. I would think that if a human origin virus adapts to another species, it would lose its ability to spread easily among humans. That seems to be the usual pattern with farmers getting swine flu's. It is a risk we take to benefit financially or socially from relationships with other animal species.
    Chinese scientists are doing some cruel experiments with dogs and flu, justifying this by claiming dogs might be mixing vessels.

  • JJackson
    commented on 's reply
    gs I do not think there is a list of the type you want but the antigenic sites and the mAbs that react with them in the table in post #9 were selected because these are the serum antibodies that are most common. There are 12 in the table which would be a good start to your list. The IgG is about 150kDa where as the Spike is nearer 800kDa and, as it is a glycoprotein, you would need to add the weight of the glycans (just to get a sense of scale). I do not know if it will form clumps with SARS-CoV-2, as you would see in a agglutination assay, where the two branches of the IgG bind to spikes on two virions which then clump with others using more IgG bridges until you get a raft in which none of the virions able to infect a cell. The exposed Fc portion of the antibody will also bind other immune cell receptors which can then induce clumping by a similar bridging mechanism. This is not an uncommon mechanism.

  • gsgs
    commented on 's reply
    > the most important 15 antibodies

    is there a list ? I'd like to have the sequences

    Evolution of antibody immunity to SARS-CoV-2
    received Nov3 , published Jan18 , Nature , 30 pages .pdf
    they test 122 ("monoclonal"?) antibodies
    they assign Cxxx numbers to them, e.g. C144

    V367F,S477N,N439K,V483A,N440K,RBD ,R346S,A475V,E484K,Q493R

    figure 3a) 122 selected monoclonal antibodies
    figure 3b) 52 antibodies
    figure 3e) 26 antibody clonal pairs
    C098,C099 ; C202,C542 ; C032,C080 ; C132,C512 ; C108,C573
    C564,C546 ; C151,C062 ; C148,C060 ; C091,C092 ; C044,C045
    C548,C549 ; C114,C571 ; C005,C043 ; C143,C055 ; C164,C055
    C089,C090 ; C058,C059 ; C058,C057 ; C085,C086 ; C144,C051
    C021,C097 ; C554,C555 ; C002,C095 ; C144,C052 ; C144,C050


    comprehensive mapping of mutations to the SARS-CoV-2 receptor-binding
    domain that affect recognition py polyclonal human serum antibodies
    Jesse D.Bloom , Jan04, BioRxiv , 35 pages .pdf
    most important is E484
    figure 6B)
    E484K,K417N,S494P,L452R,G446V,F490S,L452M,L455F,E4 84Q,F486L,G485R
    zero hits for 144 or monoclonal (mAb)

    definitions :

    a monoclonal antibody (mAb or moAb) is an antibody made by cloning a
    unique white parent blood cell
    polyclonal antibodies (pAbs) are antibodies that are secreted by
    different B cell lineages within the body. They are a collection of
    immunoglobulin molecules that rreact against a specific antigen,
    each identifying a different epitope.
    an antibody (Ab) also known as an immunoglobulin (Ig) is a
    large Y-shaped protein used by the immune system to identify and
    neutralize foreign objects such as pathogenic bacteria and viruses.
    The antibody recognizes a unique molecule of the pathogen called an antigen.
    an antigen (Ag) is a molecule or molecular structure such as may be
    present on the outside of a pathogen (spike?), that can be bound by an
    antigen-specific antibody or B-cell antigen receptor.
    (# I assume the antibody binds to one of the spikes and the whole virus
    together with its other 20-100 spikes hangs at it)
    Last edited by gsgs; February 20, 2021, 12:38 AM.

  • bertrand789
    commented on 's reply
    il y a ceci :

    et puis cela


    ce serait bien pour paraphraser Sharon de bosser cela , non ?

  • JJackson
    commented on 's reply
    The one I am probably most concerned about are deer mice in the US. They are very common and frequently live around humans so are a perfect intermediate host if it gets established in this susceptible population.

  • Vibrant62
    Thank you for your interesting post. In terms of the final paragraph above, the study linked below provides some interesting (and concerning) insights. If this study is correct, it is not just mink and cats we need to be concerned about, but pigs. See The linkage to C. Dromedar is also concerning, in view of MERS

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  • JJackson
    Having looked a little at the Spike?s antigenic sites and the antibody interactions with it this post is going to look at the B cells that produce these antibodies.
    Again I will start with some graphics which will help with the discussion.

    Click image for larger version  Name:	B cell maturation.JPG Views:	9 Size:	199.0 KB ID:	907704

    In the diagram above we can see the 8 step process that takes a stem cell in the bone marrow through to the mature B cell in the Lymph nodes. This is not the main focus of this post but I will run through it briefly. The first 6 steps occur in the bone marrow and the last two in the Lymph nodes. From left to right the stromal cell binds to a receptor on the B cell causing it to produce a second receptor type which also binds causing IL7 release from the stromal cell initiating the production of the membrane bound antibody in the pre cell. This is further refined in the Immature cell which then travels to the secondary lymph nodes to complete the process.

    Before we get to the antigen dependent part of the process it is worth looking a little at these last two steps in which the Ig light and heavy chains are added. Complex multicellular organisms, like us, can?t evolve on the same time scale as viruses so need a mechanism to quickly react to novel pathogens with which they have no prior experience ? like SARS-2. Nature?s solution is to make antibodies which have a fairly common fc portion, which is the trunk of the ?Y? shaped receptor in the diagram, and a variable short light and heavy chains. It achieves this by having thousands of these short gene sequences in our DNA for both the light and heavy chains the combinations of which can produce millions of different antibodies. Each B cell produces one of these millions most of which will die without ever having encountered a matched antigenic site. In an individual who has not suffered any recent antigenic challenge they still have about 80% of the number of these random B cells as someone fighting an infection. All these are just circulating waiting for their chance to interact with their matched antigen. In the unlikely event that this happens they then mature and start to rapidly divide and produce and release the antibodies which have the same heavy & light chain combination they presented on their surface. The first dose in a prime boost vaccine initiates this process and the second dose is timed to repeat it. The second dose instead of only having one in a million chance of interacting with a matched antibody the rapid multiplication of the original matched B cell has produced thousands more matched cells. All of these are ready to be activated and start the production of vast quantities of matched antibodies and yet more B cells to produce them.
    The rest of this post is going to look at how that original light chain / heavy chain combination, which is close enough to the antigenic site to bind but probably not very strongly, is refined to give a better and better match. Again some graphics depicting some important players and sites.

    Click image for larger version  Name:	dendrictic cell.JPG Views:	9 Size:	85.6 KB ID:	907703

    The dendritic cells are a key player but only the green half of the image is relevant to our discussion. They are the body?s garbage collectors and are attracted to any sites of infection. Their octopus like shape allows them to squirm between tissues and on their surface they have MHC 1 & 2 receptors. They collect any bits of protein or nucleic acid they encounter and display them on their MHC cells. The Dendritic cells will then return to the lymph nodes and present the collected fragments to B and T cells. If their surface antibodies are a match for the presented antigens they will start to divide. T cells have a very similar heavy and light chain to the B cells and fall into two main groups, depending on their surface receptors, those with CD8 receptors target and kill infected cells presenting peptides that match their antibodies. The CD4 presenting cells are more relevant to the processes being discussed as they can become follicular helpers cells (Tfh). Most cells do have MHC1 receptors on their surface, which present very short pathogen peptides, due to their length they are not particularly specific and the matched CD8+ cytotoxic T cell?s will bind to them and kill the cells but upon infection they will also start making MHC2 receptors which present a slightly longer and more specific peptide presenting a higher bar to recognition. It is these that the CD4+ T cells recognise and dendritic cells always have MHC2, as well as MHC class 1, this allows them to become follicular helper cells which can then regulate the process of somatic hypermutation in the lymph node germinal centres. While the body may not care too much about CD8+ T cells killing a few cells that did not have exactly the right peptide on their MHC1?s it is fussier about making miss-targeted Tfh?s as it does not want to ramp up production of miss-matched antibodies ? hence the slightly higher bar set by the longer peptides presented by MHC2 cells.

    The next two graphics show the lymphatic system and the germinal centres which are found within them.

    Click image for larger version  Name:	Lypnatic system.JPG Views:	9 Size:	112.0 KB ID:	907705Click image for larger version  Name:	Germinal center.JPG Views:	9 Size:	147.1 KB ID:	907702

    The germinal centres have two zones, the light and the dark, in the light zone the follicular dendritic cell (FDC) can be seen with the red dots representing the MHC sites and the collected peptides with a B cell?s light and heavy chains bound to them. To the right of the B cell is a Tfh CD4+ cell which must also have the correct activation site. If both conditions are met the B cell can enter the dark zone where an enzyme is added that causes rapid mutation (hyper-mutation) of the light and heavy chains sequences which are then re-tested against the Tfh. Any with a low binding affinity die but those which have improved their bind go on to be reproduced. This process is repeated again and again against whatever peptides the dendritic cells have found and presented.

    If we consider how this relates to a natural SARS-CoV-2 infection a cell is infected and presents viral peptides on the cell?s MHC1 surface protein. Millions of B and T cells interact with it until that one in a million match finds it and binds and becomes activated. The cell also begins MHC2 production which can bind and start the CD4 T cell activation turning it into a Tfh. The B cells will start antibody production but of relatively poorly matched IgM antibodies. The B cells then refine the antibody in the germinal centres and switch to a better targeted IgG antibody which is then fine-tuned in the dark zone until an even better binding strength is achieved. At this point the B cell can take two paths either it turns in to a very long lived Memory B cell or it turns into an effector cell, or plasma cell. The effector cells produce antibodies during the acute phase of the disease and then die off. The plasma cell becomes much larger and is packed with rough endoplasmic reticulum which produces vast quantities of protein for export from the cell. These proteins are the matched IgG antibodies and they have so much rough ER they can churn out 2000 IgG per second. As long as there is some viral fragments left somewhere in the body the dendritic cells will find them and present it in the lymph nodes to help the B and Tfh cell refine the IgG affinity.

    If we now consider what would happen if you had been vaccinated, or had a natural infection, to one strain of the virus but then get challenged at a later date by a variant of that virus. This is only likely to have a few changes some of which reduce the binding affinity as we saw in the earlier post looking at weakened Mab to RBD binding. This time most of the antibodies work fine but some may not be as good as they were. The unaffected ones will give you a good level of protection while the process of somatic hyper mutation and Tfh matching for high affinity fits will start to produce new better fits to the changed antigenic sites. You are likely not to be symptomatic while this is occurring as the unchanged antigenic site / antibody interaction across all the unaffected sites give adequate protection while the germinal centres go about refining the one or two sites that had become less well paired.

    Finally I will get much more speculative and give some thoughts on what I think is going to happen next in the battle between SARS-2 and us. With the Northern hemisphere winter we have seen clear evidence that this respiratory virus has a strong seasonality. As we head out of this season cases should drop which will be further strengthened by the increasing level of herd immunity induced by the combination of already infected plus those vaccinated. Sadly this will highlight the discrepancy between rich and poor nations as only the former will initially have much of a vaccine component. Over the past year there has been a continuous process of single nucleotide mutation (SNPs) most of which have had little impact on spread and either died out or continued to circulate at low levels. As more people have some level of immunity changes that were neutral, and at low levels, now convey a selective advantage if they weaken the neutralising effect of important antibodies. This is going to cause these changes to suddenly begin to take off and become dominant. This has nothing to do with increased virulence or transmissibility ? although if any of those changes do change either then it may impact them either up or down ? but that is a secondary effect.

    The hypermutation can cope with gradual change but not massive jumps that cause many of the key antibodies to stop working effectively. In the 2009 flu pandemic, although many of us had antibodies to seasonal H1N1 (H & N being the two spikes which carry the antigenic sites to which the neutralising antibodies bind), the new strain had entirely new H1 & N1?s from another species which were sufficiently different for our immune system to view it as a new virus. This is likely to also happen for SARS. Although corona viruses only have one RNA strand and cannot therefore achieve this by recombination there is a reservoir in bats which is likely to cause a SARS 3 & 4 at some stage. The fact that SARS-2 has been transported all over the world by humans allowing it to interact with many new species like mink, dogs, cats etc. it is quite likely it will also set up shop in a novel species where it can adapt to that host for a while attaining enough changes to be viewed as new if it jumps back into us. This is the bigger risk in my opinion as these prospective host species have more interactions with humans and are physiologically closer to us than bats so a host optimised mink strain is likely to be better intermediate host as it started from a human optimised form and would probably retain a respiratory infection path rather than oral/fecal.

    This was long but is a simplified explanation and only covers a small part of the whole immune response but it is important in understanding why the variants are not the doomsday scenario the MSM has been depicting.
    Again if you see errors please point them out so I can make corrections.
    Last edited by JJackson; May 21, 2021, 07:22 AM.

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  • Emily
    commented on 's reply
    MIS kids are very low in antibodies for common coronaviruses.

    Here's the study:

  • JJackson

    In the previous two post I looked at the S protein and some of the RBD antigenic sites and variant changes and how they affect binding.

    This time I aim to think a little bit about these sites and the immune system.

    If you look at the antibodies in sera from different individuals, post natural infection, you will find a lot of different antibodies and significant differences in their relative abundance between individuals. There are however a number of points along the RBD, and spike more generally, that are over represented generally both in quantity in one serum and frequency across all the sera. They are concentrated in more neutralising antibodies and surprise, surprise they closely correlate with those areas targeted by the MAbs which showed the most reductions in neutralisation in the table in post #9 e.g. S 417, 484 & 501.

    What I am building up to is the hypothesis that these are occurring now because they are immune escape changes that effect a change at key antibody binding sites. If this is the case then the change would need to have a functional effect and it should become more prevalent as the number of potential hosts with pre-existing antibodies increases. What data is there and does it support this hypothesis?

    If we take the E484K change, found both in South Africa and South America, the side chain changes its charge from – to +. If we look at how it has changed over time The graphics below show firstly how K has grown over time and then the full tree – green being E484 and yellow K484 - and finally recorded case of SARS-CoV-2 globally.

    Click image for larger version  Name:	E484K OVER TIME.JPG Views:	1 Size:	57.6 KB ID:	906789
    Click image for larger version  Name:	E484K TREE.JPG Views:	1 Size:	340.7 KB ID:	906790
    Click image for larger version  Name:	cases.JPG Views:	1 Size:	75.5 KB ID:	906791

    I have attempted to line up the dates on the X axis so they correspond.

    The first will be hard to read but it should be possible to see some yellow by October but a closer examination shows it reached 1% by the first week in July and is now 14% (There were about 4.5m active cases in July so 1% would give 45,000 active infections carrying K484).
    The tree in image 2 will not be readable but the things to note are the yellow clump near the top (S America) and the one nearer the bottom (S Africa) the smaller intermediate clump is mainly Nigeria. Also note they are all independent emergences in different clades and have been happening for a while but have only taken off since October. A closer examination of the top branch shows the first case in Brazil on the 9th of October but that the calculated date for the route of that branch is late July. On the S African branch the route is dated as early July with the first sequenced case on the 28th of October.
    Moving to the bottom graph Mid July shows about 15 to 20 million cases which climbs to nearer 40 million by the first sequenced cases and then on to over a hundred million.

    Brazil and South Africa have the most cases on their respective continents so will have a relatively high sero-prevalence rate. In Brazil they have about 5% of the population as PCR confirmed cases with that actual number probably 3 times that. In SA the percentage is 2.5% but its undercount is probably even greater. These numbers will be beginning to challenge the virus at any critical points that are no longer doing what the virus needs them to due to antibody interference. Any change at these points that does not carry too big a fitness penalty will now begin to establish itself as it now has a selective advantage at least in the previously infected. As the percentage of the population that have antibodies to E484 increases the K484 variant’s advantage will grow.
    I will not go through all the others in detail but N501Y makes it more hydrophobic, there are 3 main unrelated branches with no sequences until late Oct. but routed back in the spring.

    The sera from vaccinated individuals is much the same across the Spike protein but lacks all the other antibodies generated against the rest of genome. While these are not likely to induce many neutralising antibodies they will react to a subsequent challenge activating the their B and T cells which can release cytokines and, in the case of CD8+ cells, kill infected cells displaying the peptides they can recognise. This may not be as effective as stopping cell fusion but will stimulate the immune response generally. Only recognising antigenic sites on S means they can target the antibody response to the most effective sites but on the down side that is the least conserved part of the least conserved protein. Any change in these site will impact it disproportionally, compared to a natural infection, as it does not have any other antibodies to fall back on.

    Single AA changes at any one point will only effect one or two of the most important 15 antibodies so without a radical change across many of these (which would probably render the RBD non-fuctional) it should give most of the protection against a variant you would have got to the strain the vaccine was based on. This protection should contain the virus to non-severe infection giving your immune system time to generates new antibodies to the changed antigens and boost all of the ones that had not changed. Under the scenario I have outlined the variants we have encountered to date are not a major concern even if they show reduced protection as long as they buy the patient time to redress the balance. I view the decline as much the same as the decline you are going to get anyway as time elapses from your last infection or vaccination.
    Last edited by JJackson; February 10, 2021, 06:48 PM.

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  • JJackson
    Thanks to you both but just to be clear I am not a scientist just an interested layman.

    I wish I had listened to TWiV 714 & 715 before writing the above post as they cover exactly this topic and I have learnt a lot of new information which has generated new questions.
    Firstly it appears in addition to the pathway I described above there is another which is similar to that used by HIV. From the first post you may recall that HIV also employs Class 1 fusion but instead of the whole virion entering in an endosome, which it has to get out of as an additional step, the capsid releases its payload directly through the pore into the cytosol - SARS-2 can perform the same trick but SARS-1 couldn't or at least to the same degree. This finding comes out of research into why Hydroxychloroquine worked well in cell culture but not in a clinical settings. The answer lies in the transmembrane host protease TMPRSS2 which is a major cleaver of the furin S1/S2 site in SARS-2 but SARS-1 did not have a furin accessible site at this point. SARS-1 was susceptible to HCQ as were the vero cells used in cell culture which had the human ACE2 receptor but very few TMPRESS2. If you re-engineer the vero cells to display plenty of TMPRSS2 then the HCQ stops working. This and a raft of confirmatory experiments showed TMPRESS2 cleavage of the furin site at S1/S2 prior to S2' cleavage enables direct entry into the cytoplasm by bypassing the endosome. The graphic below shows both routes of entry are available.

    Click image for larger version  Name:	image_33228.jpg Views:	40 Size:	174.6 KB ID:	906531

    In the first post I had said the S1/S2 cleavage was optional but helped, which is true as far as getting into the endosome, but it is obligatory at some point. For SARS-1 this happens in the endosome by another protease cathepsin-L. SARS-2 can use either route. Yet more experiments show HCQ is acting on the endosomal pathway and did not work with SARS-2 because it largely bypassed it.

    The next image relates more to the first post but shows the positioning of the all the areas I discussed along the S chain although their proximity in the tertiary protein structure has little to do with how close they are here.

    Click image for larger version  Name:	image_33227.jpg Views:	41 Size:	50.1 KB ID:	906530

    I forgot the links.
    714 is a conversation with Jason McLellan relating to the action of the Spike trimer and its cleavage sites.
    715 covers the HCQ inactivation and links it to an additional entry pathway avoiding HCQ's antiviral activity.
    The paper being discussed can be found here.
    Last edited by JJackson; February 8, 2021, 04:01 PM.

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  • senegal1
    Even for those of us who are not scientists this was understandable and interesting. Thank you.

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  • mscox
    JJackson, it both helps and is very much appreciated. Thanks

    I found these summaries extremely useful and approachable as guides into the more detailed references.

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  • JJackson
    In this post I aim to look in a little more detail at the role of the spike protein and SARS-CoV-2 viral fusion from a structural virology perspective and how that may impact antibody efficiency in some of the circulating variants.

    The problem the virus faces is the need to penetrate the animal cell wall which is a phospholipid bi-layer in a stable energy state. To puncture this layer it needs energy which it cannot generate as it is inert. Nature's solution is to provide this energy by ‘spring loading’ the S protein at assembly so it is in a stable state but has lower energy states available if it can overcome an energy hump (like a loaded mouse trap it is happy where it is but a small nudge and it releases lots of energy and adopts a lower energy very stable form). This occurs in a number of steps.

    The virus is a Class 1 enveloped virus (along with Influenza, HIV, Ebola and many others). All of these have use a ‘spring loaded’ homotrimeric trans-membrane proteins with a RBD and use the stored energy to force an opening in the cell wall.

    The cutaway image below shows the viral structure. In the centre there is the nucleocapsid (+ssRNA in a nuclear protein (N) coat). Around which is the capsid protein (M) and the viral lipid membrane that was acquired from the cell wall of the cell that made the virion as it was shed. This is the membrane that needs to be fused with new cell’s membrane (bottom of frame) and embedded in which is the Spike (S) trimer.

    Click image for larger version  Name:	SARS cutaway and bi-layer.JPG Views:	3 Size:	217.4 KB ID:	905846
    The next image shows a single S protein with the Receptor binding domain (RBD) in purple and the N terminal domain (NTD) in turquoise with the C terminal domain, which is anchored in the viral membrane, right at the bottom. Three of these make the Spike homo-trimer but it is easier to see the key domains in a single protein.
    Click image for larger version  Name:	Single S.JPG Views:	3 Size:	107.1 KB ID:	905845

    The next image shows a trimer with one of it three S1/S2 furin cleavage sites marked.

    Click image for larger version  Name:	SARS S1-S2 cleavage site..JPG Views:	3 Size:	72.0 KB ID:	905847

    Having identified some of the key sites in fusion we can look at the sequence of events.

    The first is usually cleavage at the S1/S2 furin site. This is not found in most SARS like bat viruses and is not needed for infection but if it is not cleaved it makes a later step, which I will return to, more difficult. This can occur at any time after the viral capsid is made including before it leaves the cell that made it.

    The second step is the binding of the RBD to the ACE2 which causes a conformational change and allows the release of the S1 section of the Spike which can float away, if cleaved at step one, or if not it will float about tethered by the S1/S2 join and impede access of the host protease needed for step 3 cleavage.

    I have not shown a diagram for the step 3 cleavage site (S2') because it is buried deep inside the Spikes stalk to protect it from premature cleavage. This cleavage has to happen for infection but it is not until after the changes caused by step two that it becomes exposed. Once cleaved the protein undergoes a major conformational change that brings the NTD down to the cell membrane where it anchors itself. With the N terminal embedded in the host membrane and the C terminal in the virus the conformational change brings the two membranes together to cause a pore and cell entry.

    So to recap
    Step1 – Cleavage at S1/S2 – useful but not essential leading to minor conformational change effecting RBD.
    Step 2 – S RBD to ACE2 Binding – essential leading to conformational change releasing S1 cap and exposing the fusion peptide.
    Step 3 – S2 prime cleavage of the fusion peptide – essential as it allows anchoring of the NTD in the cell membrane so the attendant conformational change can first pull the virus and cell together and then surmount the energy barrier to fuse the host and viral lipid membranes.

    With an understanding of the process I hope this animation will make sense. I would watch it straight through and then again at one quarter speed to watch the concertinaing of the strands by the formation of alpha helixes from less structured strands. (I do not know how many Spike trimers are need to pull in concert to achieve this process but in flu it seems 3 HA trimmers are normally involved in fusion)

    Armed with the process the virus needs to happen we can look at the changes in the various circulating viruses and what effects they may have on the immune response or vaccines.

    Two more graphics, one showing the RBD to ACE2 interface with some of the key sites labelled the bottom figures shows the electrostatic surface charge in the bound and unbound forms and a table showing various monoclonal antibodies tested against engineered viruses expressing some of the main amino acid changes of interest.

    Click image for larger version  Name:	ACE2 RBD binding.JPG Views:	3 Size:	129.0 KB ID:	905849

    Click image for larger version  Name:	Variants.JPG Views:	3 Size:	315.5 KB ID:	905848

    The Class 1 & 2 mAbs bind to various areas within the RBD while Class 3 are neutralising but outside the RBD (at a guess most would be NTD as this is external in the pre-cleavage form but critical for attachment.) The circulating variants AA changes form the columns with the numbers in the boxes IC50 values in ng/ml so low numbers mean high binding affinity.

    I do not know what was used as the wild type (column 1 wt) but it does not seem to include S D614G (last column) although this is now dominant at that position (and AFAIK present in the vaccines), interestingly it is more susceptible to these mAbs than the wt in most cases as is R683G (column 2) but when R683G is combined E484K it is dramatically worse across much of the RBD. Unfortunately they have not included E484K alone for comparison. One other point to consider is the fact all of the main vaccine candidates, except Astrazeneca, have made a change to the sequence near the Step 3 cleavage site adding some Prolines which stiffens the joint but does not stop cleavage. The reason for the change relates to Step1 if that S1/S2 furin cleavage site is cleaved it alters the shape of the RBD, so antibodies may be made to both forms, but for the vaccine we want to maintain the pre-cleavage form so the immune system makes antibodies to that configuration which will give improved binding in a subsequent natural infection. The N-K changes at 439 & 440 both interfere with neutralising binding but outside the RBD. Each of these changes is being tested individually and, as can be seen with the R683G/E484K dual change, just knowing how they work alone does not necessarily predict how they behave in the more complex combinations found in nature.

    All of the above only covers the first step in viral infection, getting in the door, and needs to be performed again to get the nucleocapsid out of the endosome and into the cytoplasm. The exterior of the virion is covered in spike trimers and only a few will have been facing the cell surface which leaves plenty to repeat the process in getting through the endosome's lipid bi-layer. Up to this point Corona and Influenza virus both follow a very similar process but at this point the flu virus remains in the endosome which the cell acidifies as it turns into a lysosome flu uses its M2 ion pump to acidify its interior and destabalise the bonds that bind the M1 proteins which form its capsid.

    N.B. This is just my interpretation of my reading and may not be accurate in all aspects. If you think I am wrong on any particulars please post so I can research further and make corrections.
    As always I hope it helps.
    Last edited by JJackson; February 2, 2021, 05:50 PM.

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