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Pseudo vaccines?

by gravenewworld
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gravenewworld
#1
Jan27-12, 08:50 PM
P: 1,404
Is there a list of all known antigens for pathogens and the amino acid sequence for the proteins? If so, why doesn't someone just take that list, create the peptide in an automatic synthesizer, put the peptide into the syringe and activate the immune system by injecting it? Would it work? If you knew how many proteins you were synthesizing, you can then precisely control the dose given of the antigen.


How many peptides on average does an antigen need to be before it will be recognized? Of course our current technology won't allow for efficient synthetic synthesis of long peptides, but it did in the future, could we easily create pseudo vaccines from synthetic antigens?
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atyy
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Jan27-12, 09:07 PM
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http://people.ucalgary.ca/~ceri/cmmb...t/ANTIGEN.html
"2. Molecular Size: There is a correlation between size and immunogenicity. The best immunogens are in the range of 100,000 Da., while small molecules 5-10,000 Da are generally poor immunogens. Minimally they must be large enough to be processed."

http://en.wikipedia.org/wiki/Epitope_mapping
"Epitope mapping is the process of identifying the binding sites, or ‘epitopes’, of antibodies on their target antigens (which are proteins)."
Curious3141
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Jan27-12, 09:50 PM
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Quote Quote by gravenewworld View Post
Is there a list of all known antigens for pathogens and the amino acid sequence for the proteins? If so, why doesn't someone just take that list, create the peptide in an automatic synthesizer, put the peptide into the syringe and activate the immune system by injecting it? Would it work? If you knew how many proteins you were synthesizing, you can then precisely control the dose given of the antigen.


How many peptides on average does an antigen need to be before it will be recognized? Of course our current technology won't allow for efficient synthetic synthesis of long peptides, but it did in the future, could we easily create pseudo vaccines from synthetic antigens?
They're already doing this. Hepatitis B vaccine, for example is a recombinant subunit vaccine, where the viral surface antigen is expressed in genetically-modified yeast.

The issues with any subunit vaccine (which generally has fewer epitopes than native protein from a killed virus or bacterium) include reduced immunogenicity (which is often compensated for with adjuvants).

You might also be interested in naked DNA vaccines. Look it up.

Ygggdrasil
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Jan27-12, 11:50 PM
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Pseudo vaccines?

Quote Quote by gravenewworld View Post
Is there a list of all known antigens for pathogens and the amino acid sequence for the proteins? If so, why doesn't someone just take that list, create the peptide in an automatic synthesizer, put the peptide into the syringe and activate the immune system by injecting it? Would it work? If you knew how many proteins you were synthesizing, you can then precisely control the dose given of the antigen.


How many peptides on average does an antigen need to be before it will be recognized? Of course our current technology won't allow for efficient synthetic synthesis of long peptides, but it did in the future, could we easily create pseudo vaccines from synthetic antigens?
One problem, as you note, is that proteins are large and most cannot be made in an automatic synthesizer (which can only reliably string together polypeptides of up to ~100 amino acids, whereas many most protein consist of many hundreds of amino acids).

There are, of course, ways of producing proteins through recombinant DNA technology; you put the DNA for the protein of interest into a bacterium, yeast or other organism, and that organism produces the protein for you. However, most of the proteins that act as antigens are membrane proteins, which are difficult to produce in large quantities by these methods. Furthermore, many of viral antigens undergo chemical modifications (e.g. attachment of sugar molecules) that greatly affects how the immune system recognizes these proteins, but the recombinantly-expressed proteins generally lack these modifications or they are not modified in the correct way.

Ok, but surely antibodies aren't recognizing the whole protein. Can't we just take a small piece of the antigen, produce that in our automated synthesizer, and use that as a vaccine? Sure, but this approach does not always produce an effective immune response. Antibodies don't recognize the sequence of amino acids of a protein; they recognize the overall shape of the protein. It is very difficult to get a small peptide to adopt the correct shape in isolation. Some have tried to design scaffolding to hold the peptide in the correct shape, but often this generates antibodies that recognize the scaffolding as well as the peptide, so the resulting antibodies aren't effective against the antigen in the context of the pathogen.
gravenewworld
#5
Jan28-12, 12:06 AM
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Quote Quote by Curious3141 View Post
They're already doing this. Hepatitis B vaccine, for example is a recombinant subunit vaccine, where the viral surface antigen is expressed in genetically-modified yeast.

The issues with any subunit vaccine (which generally has fewer epitopes than native protein from a killed virus or bacterium) include reduced immunogenicity (which is often compensated for with adjuvants).

You might also be interested in naked DNA vaccines. Look it up.
This sounds like an incredible waste of time and effort. What do you have to do? Transfect a yeast cell with recombinant DNA and then purify the end product? You also have to deal with DNA in the first place. Why not just synthesize the protein directly? What I would like to know is, what is the current research out there on making synthetically derived short chain amino acids immunogenic? Can we improve it?


I simply don't understand why the US of freakin A doesn't treat protein synthesis like the Manhattan Project. Sure our technology these days can only synthesize peptide strands only about 100 amino acids long, but come on, people have been improving this technology since the 70s. How long until we can discover the proper organic synthesis reactions and conditions until we can create a peptide strands 1000 units long? People aren't going to give up until it happens. We need to start planning ahead of time.


What happens then when we can create peptide strands 1000 units long? 10,000 units long? All we will need is to input a computer program to synthesize our peptide strand and have it recognized by the immune system no? A protein on the surface of a bacterium can now be synthetically created and delivered with high precision, what could stop this from creating any vaccine that could treat almost any infectioous disease (as long as it isn't resistant?)?


If we could just make the antigen, no one would need "damaged" bacterial or viral cells anymore to create vaccines right?
Ygggdrasil
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Jan28-12, 12:18 AM
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An advantage of recombinant DNA is that once you make the gene encoding your protein, it is very easy to replicated the DNA and get a whole bunch of cells producing the protein for you. A few liters of yeast can produce protein much more quickly and cheaply than your automated synthesizer, even including the cost and effort of purifying the protein.

Another issue: let's say you can chemically synthesize a large polypeptide. It starts out in organic solvent after the synthesis. How do you get this large protein to fold correctly?
gravenewworld
#7
Jan28-12, 12:34 AM
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Quote Quote by Ygggdrasil View Post
An advantage of recombinant DNA is that once you make the gene encoding your protein, it is very easy to replicated the DNA and get a whole bunch of cells producing the protein for you. A few liters of yeast can produce protein much more quickly and cheaply than your automated synthesizer, even including the cost and effort of purifying the protein.

Another issue: let's say you can chemically synthesize a large polypeptide. It starts out in organic solvent after the synthesis. How do you get this large protein to fold correctly?

To your first point, sure you can get a yeast to produce tons of your protein, can you control for EXACTLY how much protein your yeast produces? Probably not? With automated programmable syntehsise you can control exactly how much protein is in your solution and I imagine immune system response is a function to how much dose you give. Let's say you have a yeast cell produce your protein. Is it easy to count 10,000 proteins out of what it produces? What if you need 100,000 proteins? We're talking about personalized medicine. Everyone's immune system is different, with an automated synthesis you could theoretically fine tune the concentrations you need to produce a response correct?

Second point. Good question, I was under the impression that the peptide sequence determines structure and folding. Could you create some sort of reactor to synthesize the peptide strand and have its end product "spit out" into a soup filled with features native to a cell environment such as chaperone proetins? Couldn't we just transfect a yeast with a chaperone protein DNA to produce the chaperones we needed?


I'm sure this sounds ridiculous, but I bet the atom bomb did too in the 1890s.
Ygggdrasil
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Jan28-12, 01:12 AM
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Quote Quote by gravenewworld View Post
To your first point, sure you can get a yeast to produce tons of your protein, can you control for EXACTLY how much protein your yeast produces? Probably not? With automated programmable syntehsise you can control exactly how much protein is in your solution and I imagine immune system response is a function to how much dose you give. Let's say you have a yeast cell produce your protein. Is it easy to count 10,000 proteins out of what it produces? What if you need 100,000 proteins? We're talking about personalized medicine. Everyone's immune system is different, with an automated synthesis you could theoretically fine tune the concentrations you need to produce a response correct?
If you have a large amount of purified protein it is trivial to measure out a certain quantity.

Could you create some sort of reactor to synthesize the peptide strand and have its end product "spit out" into a soup filled with features native to a cell environment such as chaperone proetins?
The ribosome is one such reactor... In fact, some recent studies suggest that features of the ribosome help to aid the folding of proteins to their native state. Although the primary sequence of proteins determines their structure and folding, in practice, it is pretty difficult to get large unfolded proteins to fold back into their native state.

I'm sure this sounds ridiculous, but I bet the atom bomb did too in the 1890s.
It's not completely ridiculous, but I'm just pointing out some potential problems with your idea. After all, if you can think of a solution to these problems, the idea won't be so ridiculous after all. Chemical synthesis makes it much more easy to add unnatural features to the protein you're synthesizing (such as unnatural amino acids, amino acids with the wrong chriality, sites for attachment of specific post-translational modifications, etc.), so it could definitely offer some advantages over recombinant expression.
bobze
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Jan28-12, 01:17 AM
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Quote Quote by gravenewworld View Post
Second point. Good question, I was under the impression that the peptide sequence determines structure and folding. Could you create some sort of reactor to synthesize the peptide strand and have its end product "spit out" into a soup filled with features native to a cell environment such as chaperone proetins? Couldn't we just transfect a yeast with a chaperone protein DNA to produce the chaperones we needed?


I'm sure this sounds ridiculous, but I bet the atom bomb did too in the 1890s.


No, much of the folding of a protein requires a specific cellular environment for folding to occur correctly (like in the ER).

There are other important reasons we could never do what you are asking (think autoimmunity or think why we don't have a recombinant vaccine to M-proteins in group A strep), but after a 12 hour study day I'm too tired to elaborate at the moment--I'll get back to you tomorrow.
Curious3141
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Jan28-12, 06:29 AM
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Quote Quote by gravenewworld View Post
This sounds like an incredible waste of time and effort.
Why? As things stand, it's cheaper, faster and greater-yielding to use a living expression platform.

What do you have to do? Transfect a yeast cell with recombinant DNA and then purify the end product? You also have to deal with DNA in the first place. Why not just synthesize the protein directly?
You can make anything sound as long-winded or as succinct as you like. For instance, instead of saying "synthesise protein directly", I could say first you have to find a solid support, like beads, which are specially treated so that they covalently bind peptides. Then we have to go through a long sequence of coupling, washing and deprotection to add amino-acids systematically to the growing chain. Each step has to be done with very high yield because, otherwise, the overall yield of the peptide-chain will be too low to be commercially viable. And then you have to cleave the covalent bonds linking the completed peptide to the beads.

And after all that, you still can't achieve very long peptide chains, and your protein may still fail to conform properly, like it would in-vivo.

So what's the point of going through all that?

You still haven't clarified your objection to using living expression systems. Is it safety? Because modern methods render the process very safe, so the point is moot.


What I would like to know is, what is the current research out there on making synthetically derived short chain amino acids immunogenic? Can we improve it?
Sure, with time, anything and everything will improve. It may still not supplant living expression systems because the latter may still prove cheaper, better, faster, and just as safe.


I simply don't understand why the US of freakin A doesn't treat protein synthesis like the Manhattan Project. Sure our technology these days can only synthesize peptide strands only about 100 amino acids long, but come on, people have been improving this technology since the 70s. How long until we can discover the proper organic synthesis reactions and conditions until we can create a peptide strands 1000 units long? People aren't going to give up until it happens. We need to start planning ahead of time.


What happens then when we can create peptide strands 1000 units long? 10,000 units long? All we will need is to input a computer program to synthesize our peptide strand and have it recognized by the immune system no? A protein on the surface of a bacterium can now be synthetically created and delivered with high precision, what could stop this from creating any vaccine that could treat almost any infectioous disease (as long as it isn't resistant?)?


If we could just make the antigen, no one would need "damaged" bacterial or viral cells anymore to create vaccines right?
Sure, if technology improves, we could have all that. We could also have warp-driven spacecrafts with replicators on board that can immediately synthesise the vaccine without going through any of the intermediate steps. All while you're relaxing on the holodeck playing the most realistic game of Halo ever while en route to site-see Alpha Centauri.

What's the point of speculating about a future neither of us can see?

Even if you can synthesise long peptides with great precision, what about production capacity? Commercially viable production may still not be feasible with automated synthesis. And even if the method is able to produce enough, it may cost way too much, so people continue to fall back on older methods.

As things stand, genetic manipulation is much further developed than protein manipulation. It just makes more sense to be able to transfect living, efficient, safe expression platforms with a gene or genes of interest, let them replicate, then let them do all the work.

There is a role for special protein synthetic methods. If custom peptides with unnatural amino acids are required, this is the only way to go. But for the purposes of common-garden vaccine production, this has not yet been required.
bobze
#11
Jan28-12, 02:00 PM
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Quote Quote by gravenewworld View Post
Is there a list of all known antigens for pathogens and the amino acid sequence for the proteins? If so, why doesn't someone just take that list, create the peptide in an automatic synthesizer, put the peptide into the syringe and activate the immune system by injecting it? Would it work? If you knew how many proteins you were synthesizing, you can then precisely control the dose given of the antigen.


How many peptides on average does an antigen need to be before it will be recognized? Of course our current technology won't allow for efficient synthetic synthesis of long peptides, but it did in the future, could we easily create pseudo vaccines from synthetic antigens?
There are, as I alluded to earlier some problems with this. First a very brief run through of antigen processing and presentation (cause I don't know how much immuno you've had).

Extracellular proteins get taken up by antigen presenting cells (APCs) and endocytized via endosomes. In these endosomes the protein is processed (broken down) into 13-18 long amino acid peptides. They are fused with vesicles containing major histocompatability complex (MHC) proteins--In this case, MHC II proteins (MHC I proteins mediate "internal surveillance, more on that later). The MHCII-antigen (16-18 aa's) then get presented to CD4 T cells (helper Ts or Th henceforth). There are "2 routes" your Th response can go from here. First they can take the Th1 approach, which is important for mediating cell-mediated immunity (for internal pathogens, like viruses for instance, CMI for short) or the Th2 response for humoral immunity (B cells, which make antibodies, HI for short). Which "path" gets chosen is a complex interaction of cytokines that modulate the response.

That now said we come to the first problem--Control. If we just "synthesized one long protein"--you'd have no control over how the pieces are "chopped up"--So certainly you'd generate some relevant antigen recognition, but you'd also generate a response to lots of "nonsense" antigen--Or antigens that don't necessarily exist as pathogenic antigens.

This rolls into the second and very big problem--Autoimmunity. While our bodies do a very good job at culling T cell lines that don't show self tolerance (searchable term), it isn't perfect. And from time to time and in individual to individual T cells sensitized to antigens against ourselves do unfortunately sneak through the selection process. This can happen for a variety of reasons, but the one most relevant to the discussion here is molecular mimicry. A common and well known example of this is with the highly immunogenic M proteins on the surface of streptococcus pyogenes (causative agent of strep throat). Post-streptococcal infection people can get rheumatic fever. The gist of which is--your body makes antibodies (Ab henceforth) against some of those M proteins, that also happen to have the right shape to bind to cardiac myocytes (we call this Ab cross-reaction). The problem then that occurs is the antibodies can be cytotoxic to the myocytes OR can cause Ab dependent cell cytotoxicity (ADCC)--mediated by other immune cells (like NK cells).

The reason then, no one is all that interested in making a M-protein vaccine (for which there are 100+ M proteins, last I had checked) against strep throat is because no one really wants to take the risk or bare the burden (lawsuits anyone???) of developing a vaccine with the black box warning of "MAY CAUSE SUBACUTE RHEUMATIC FEVER"--Though cardiologists might not mind the increase in business (okay, okay I kid, I kid--bad joke). And especially considering that S. pyogenes is highly susceptible to penicillins (dirt cheap), the risk isn't even close to the reward.

So in the case of your agglomerate peptide, who's processing we cannot control, you run a serious, serious risk of generating immunogenic peptides which will be cross-reactive with self-antigens and sensitizing your immune system to them. That last part being extremely bad for business.


Last quick problem with the idea involves those Th1 and Th2 responses I mentioned above. Some types of infections require a Th1 (CMI) response to clear them, while others require a Th2 (HI) response. I short-changed you above with the conventional "viruses=intracellular=Th1" above. Its actually more complicated than that. For instance TB (mycobacterium tuberculosis) is a bacteria and you might reason that the Th2 response is very important in mediating immunity. Makes perfectly logical sense, however nature doesn't like hard and fast rules. TB actually thrives intracellularly and requires a Th1 (CMI) response to clear. In deed, people who produce Th2 responses to mycobacterial infections don't tend to do so well--prognostically speaking.

Similarly, rotovirus--A leading cause diarrheal illness and morbidity/mortality the world over, is caused by a virus (a reovirus to be exact). You possibly would reason then that CMI and the Th1 response is important to preventing rotoviral infections--Again, nature and her disdain for our rules. Immunity to rotoviral infections is gained through sIgA (secretory immunoglobulin A), a humoral response--Th2 (for a variety of complex reasons outside the depth of this post). A vaccine which induced a CMI response would do little to help in providing roto-immunity.

The problem coming to shape here is that while a large, single protein sequence of "all antigens" could potentially sensitize people to said antigen, there isn't a guarantee that the adaptive immune response developed would produce the adequate response to establish immunity and memory.


-Bob
gravenewworld
#12
Jan29-12, 09:34 AM
P: 1,404
Thanks Bob for the excellent overview.


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