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How You Make an Adenovirus Vaccine

The other day I had a look at the process used to make the mRNA vaccines, so I thought it would be a good idea to do the same for the adenovirus vector ones, such as J&J, Oxford/AstraZeneca, CanSino, Gamaleya et al. It’s a different system, with its own advantages and disadvantages, and that’s the broad story of scale-up manufacturing all the way: tradeoffs at every turn. It’s always tough to break out of the constraints of the Engineer’s Triangle: “Fast, Cheap, Good: Pick Any Two”. That is, if it’s good and cheap, it’s unlikely to be fast, and if it’s fast and good it’s unlikely to be cheap. And of course, if it’s fast and cheap, it’s unlikely to be any good!

Update: for further reading, reviews on this whole topic can be found here, here, here, here, and here.

Intro: Adenoviruses and Infection

Adenoviruses are extremely common double-stranded DNA-containing pathogens, and it seems like a sure bet that every single person reading that has been infected with several of them over the years. They tend to cause mild respiratory symptoms and sometimes show up as ear infections or conjunctivitis. There are no antiviral drugs that target them, and no adenovirus-targeting vaccines that are available to the general public (although there have been anti-adenovirus vaccination programs in the military for a couple of subtypes). Subtypes, indeed: there are at least 88 of them that infect humans (more since that paper was published!), divided into several related groups. Among the more common ones is adenovirus 5 (Ad5), and in some regions of the world you find 80 to 90% of the population is already seropositive to it (the US figures seem to be close to 40%).

The adenoviruses have long been used as tools in molecular biology, since they have plenty of room to carry a modified DNA payload, show no tendencies to integrate DNA into the host genomes, and can infect both dividing and nondividing cells. But as those figures just cited show, a downside of using them as human therapies is that many people may well already have antibodies to your viral vector tool right at the start, which will surely knock down its effectiveness. For this reason, there has been a long-running search for rare and unusual Ad forms to use as platforms, which explains why you see J&J and Gamaleya using Ad26, Oxford/AZ using a virus from chimpanzees that’s not in the human population, ReiThera using a gorilla adenovirus, etc. And it’s also why people wonder about CanSino’s efficacy in general, since they stayed with Ad5.

No matter what variety you use, though, you also have to wonder about what happens if you administer a second shot of the same vaccine: how much makes it through? The current wave of trials, I have to say, is going to provide more real-world data to answer this question than we could ever have imagined having so quickly. That’s closely related to an even larger question: once you’ve had a vaccine (or even a gene therapy?) with a particular adenovirus vector, what happens if you want to get another vaccine for a different disease that uses the same vector? Are you going to cross off large numbers of people from being dosed in Ad5 space, Ad26 space, and so on? Another unanswered question, as of yet.

No matter what the adenovirus types, the end result of such a vaccination is rather similar to what happens with an mRNA vaccine like Moderna or Pfizer/BioNTech. The adenovirus goes in and does its normal infection route; all that machinery is intact.. But in this case, the DNA payload that’s delivered into your cells is not a big set of instructions for making more adenoviruses, it’s a much shorter sequence that codes for the coronavirus spike protein instead. So the modified DNA gets transcribed to messenger RNA in your cells (and that’s the exact step that the mRNA vaccines jump in at if you take them), and this mRNA is taken up by ribosomes and translated into the Spike protein itself. And production of that foreign protein sets off your immune system, which to be sure has already been ringing alarm bells because it is sensing that a foreign virus is attacking. That’s why you don’t need an adjuvant added to either the viral vector vaccines or the mRNA ones; they set off the various Intruding Virus Detected machinery very well on their own, whereas just injecting you with the Spike protein itself skips past some of those warning systems. Those are generally set up around sensing foreign DNA and RNA, and jumping past them with an injected protein leads to a less vigorous response (thus the need for an adjuvant in the mix).

Making an Adenovirus Vector

So let’s move on to how you make such vaccines. You’ll need to produce a large mass of infectious viral particles, each with the modified stripped-down DNA that you want to target to a patient’s cells. You especially want to take out the part of the adenovirus genome called E1; removing that makes it impossible for the virus to replicate. If you need more room for your own payload, you can delete the E3 region as well. This stuff has all been explored some years back; there are number of regions in the viral genome that have been shown to be suitable for splicing in your own sequences.

But getting this done and into a system that will make a pile of new virus requires some genomic dance steps. The most common way to do this, in broad outlines, is to make a bunch of (linear) adenovirus DNA, with your own modifications, and then get that into a big reactor full of human cells. And (this is key) these human cells have already been engineered to make the proteins that the viral E1 region makes, the ones the virus needs to replicate. This complementation trick allows the modified adenovirus to replicate away in the human cells and give you a much-increased yield of new infectious virus particles, but ensures that these viruses themselves are still unable to replicate. The E1 proteins they’d need are not coded for in their own genomes (you took that part out), but were just present for them inside the human cells. And when injected into a patient, they will most definitely not be encountering any other human cells that are cranking out viral E1 proteins for them.

So the first step in this process is to engineer the viral DNA that you need and make a lot of it. This step has often been done in bacteria because bacterial DNA is relatively straightforward to handle and to get replicated. There are actually commercial systems you can buy to do this on a laboratory scale – that is, you can purchase plasmids (the circular DNA molecules used by bacteria) that already have the A5 adenovirus genome in them, with the E1 and E3 regions already removed, and with the sequences set up for easy insertion of whatever DNA you want. Another way to do that is with a variety of plasmid called a bacterial artificial chromosome (BAC), and you can buy those with the features you need for modification. But you’ll recall that J&J (and Gamaleya) are both using Ad26, while Oxford/AZ are using a chimpanzee adenovirus, so the commercial Ad5 reagents won’t be of any use – the teams involved have been working up their own tools for the job. Earlier on, CanSino reported using the commercial AdMax system from Microbix (Toronto) for their plasmid work. In any case, though, you’re forcing the bacteria (often good old E. coli) to make copies of these plasmids, as many as it can stand. You then lyse (break open) the bacteria, isolate your DNA, and then break open the circular plasmids to get a linear DNA molecule. Some of the BACs can be engineered so that they do that to themselves, saving you a step. You need the linear DNA because it turns out that in that form It can directly infect human cells (with the help of some additives in the cell culture to get it through the cell membranes more efficiently). Update: you need the linear DNA form for adenovirus replication/packaging once it’s in the cell. The earlier Oxford papers reference this book chapter for their methods. As for J&J, this patent would appear to have some of the details of their system. Updated: you can get adenovirus DNA in as the circular plasmid form, too

From this paper and this one, it appears that the Oxford/AZ team is using a BAC, engineered from combination of two different plasmids through “recombineering” to bring in their sequence for the Spike protein into what used to be the E1 region of the adenovirus sequence. (I’m skipping the details of that process to save time, space, and patience). Meanwhile, you can read about what appears to be the J&J plasmid system here and here (that last one detailing another adenovirus subtype, but apparently with similar techniques used for Ad26).

Now it’s time to get those linear DNA molecules into human cells. Here we get into some controversy, depending on your beliefs. It looks like Oxford/AZ is using a complementation-engineered version of HEK293 cells for this process, as are Gamaleya and CanSino, while J&J is using a line called PER.C6. These two have both been around for a while. The HEK initials stand for “human embryonic kidney”, and it was indeed first isolated from aborted fetal tissue in the early 1970s at Leiden University. PER.C6 as a complementation strain goes back to 1998, but the origin of the cell line is back in 1985 in Leiden as well, also from aborted tissue, with the “ER” part standing for “embryonic retinoblasts”. As you can well imagine, people with strong anti-abortion beliefs are not enthusiastic about taking vaccines that touch on this area in any way for their production, while other with different beliefs are not bothered at all. No matter what, though, it seems crucial for the linear DNA to be transfected into some sort of human cell complementation line; that’s the only way you’re going to get amplified yields of the final viral particles used in the vaccine.

As you read about vector vaccine production, you’ll sometimes see the phrases “virus seed stock” and “host cell bank”. You’ll see below that there are manufacturing sites all over the world for these vaccines, and the last thing you need is for everyone to be out there going it alone. Batches of the plasmids, the linear DNA, the complentary cells, and the final adenovirus are all going to be stored for future reference and/or distribution, and exhaustively characterized. You definitely want to keep a close eye on the batches of these things to make sure that you’re dealing with the same stuff at all the production sites.

Human cell culture – any cell culture – is simultaneously a scientific process and an art form. Ask anyone, literally anyone who’s done it, and if you can find someone who’s worked on it at an industrial scale, they’ll confirm that all the more vigorously. This is (or can be) the weak point of the entire viral-vector production process. When everything is working, this method for infecting living cells and turning them into virus factories is hard to beat. But it doesn’t aways work the way it’s supposed to. It appears that AstraZeneca has been having problems because one of their largest production facilities has been experiencing problems with low yields of virus, even though everything should be the same (same viral DNA, same cell line, etc.)

To give you an idea, HEK293 cells themselves come in varieties that grow on the surfaces of a culture vessel (adherent, HEK293A) or grow floating around in suspension (HKE293S). You may well want the latter for serious scaleup (not least because you’re growing in three dimensions instead of two), but it can be done either way. Adherent cells grow until they touch and form a confluent layer on their surface, and some lines are OK when that happens and some aren’t (or gradually become less happy about it). Suspension cell lines divide and make a thicker, more concentrated suspension, and all of them react somewhat differently to that process, too. You have to think about what media all these things are growing in and what nutrients to provide (and in what concentration), the buildup of waste products (and debris from dead cells), the washing of adherent lines with fresh media and the stirring rates and techniques for suspension ones. . .oh, it’s glorious.

For example, when using engineered cells to make modified human proteins (an extremely common task in both academic and industrial molecular biology), I have been on a project where the yield of protein changed dramatically using the same damn cells grown in cylindrical “roller bottles” which were stirred (as the name implies) by slow rotation (rather like a convenience store hot dog machine), versus being grown in “shaker bags”, a more free-form affair that was sloshed around slowly by rotary oscillation. Why did the cells care? You tell me – but under one set of conditions they made a lot more protein than the other. Why is one of AZ’s plants making less virus than it should? Who knows?

Purification and Packaging

Isolation of the viral particles is likely pretty similar for all of these vaccines. I’ve been unable (no great surprise) to find detailed production information for any of the current vaccines, but this was likely one of the less stressful parts of the process optimization, given all the work that has already been put into adenoviruses over the years. You’ll lyse the cells in the cultures and start with some rough filtration to pass the viral particles and retain the cellular debris. From this AstraZeneca page, it looks like they’re using a series of filtration steps, followed by membrane chromatography (likely some sort of ion-exchange technique, in this case, based on the charged residues of the viral surface proteins), followed by an ultrafiltration step. You can bet that the organizations involved already had a pretty clear idea of what steps they’d be taking, although all of this stuff needs some tweaking for optimization and also validation at every step. The regulatory agencies involved will have seen these details, but I don’t think we’re going to.

And then you have to formulate the viral particles, which is a much less fraught process than it is with the mRNA vaccines. The other ingredients for the vaccine itself are going to be pretty innocuous stuff, no weird lipids as needed for the lipid nanoparticles. Here’s the list for the Gamaleya vaccine (see the first page of text); there’s nothing on it that looks to be any sort of supply problem. Now it’s time for fill-and-finish, which has been a common step for everyone, rounding up enough production-line capacity for filling and capping sterile vials.

I see that the earliest batches of the Oxford/AZ vaccine were produced at Oxford itself, and later on were manufactured and packaged by a company called Advent (in Pomezia, Italy) and by COBRA Biologics (in Keele, UK) with vial-filling by Symbiosis (in Sterling, UK). They’re working with the large contract firm Catalent in both the US (Harmans, MD) for production and Europe (Anagni, Italy) for fill-and-finish. There is production in the Netherlands (Halix) and Belgium (Novasep, in Seneffe). The last one is apparently the site with the yield problems. It’s also being packaged in Dessau, Germany by IDT Biologika. Russian manufacturer R-Pharm has a plant in Germany that’s in production for export back into the CIS countries (they’re also producing the Gamaleya vaccine). Insud in Spain is involved as well, as is a new plant of theirs in Argentina. AZ also has a big production deal with India’s Serum Institute, and WuXi is involved in China and at a plant in Wuppertal, Germany. And I’m sure I’ve missed some deals.

J&J, for their part, has a lot of capacity in the Netherlands (such as in Leiden), and they have signed deals with Emergent to produce the vaccine in Baltimore (who are also working with AstraZeneca, and indeed with Novavax, producing their protein vaccine at a separate Maryland plant). They’re also working with Catalent (at their Bloomington, Indiana plant and also at the Anagni site in Italy), Reig Jofre in Barcelona, Aspen Pharmacare (in Port Elizabeth, South Africa), Biological E in India (who just bought another facility in Himachal Pradesh), and with PCI Pharma for cold storage and shipping. No doubt there are more deals out there, too.

So there you have it, in outline form anyway. Any one of these steps can be zoomed in on to reveal a forest of further details, but that should give you an idea of what’s happening (and in many cases may provide even more than you ever wanted to know!) As you can see, it’s a fundamentally different process than the mRNA vaccines, with its own features (good and bad). That may well become important if we have to retool the existing vaccine candidates for new variants, but that’s a post for another day!

58 comments on “How You Make an Adenovirus Vaccine”

  1. c says:

    Hey Derek, any interest in commenting on “RadVac” and its synthesis/use by non-experts?

    Maybe not worth your time, but would be interesting.

    1. Lapsed Chemist says:

      I would recommend watching this documentary to get a sense of the ethical issues
      this raises ( ). Charlatans abound in this space.

    2. biff_ditt says:

      I second this. I would very much like to see your take on Radvac

      1. Lapsed Chemist says:

        I do not want to speak for Derek, but his views would be quite obvious if you read these groups of posts.

        I learned the phrase – “Homo homini lupus” – A man is a wolf to another man form one of these blogs 🙂

    3. gippgig says:

      I have no idea how well this would (or could) work but I think it is very important to determine. IF it works this could be an ideal way of dealing with those pesky variants. Any plans anywhere to test for mucosal immunity in those who’ve used RadVac? Somebody do it!
      To totally change the subject, is there any evidence of what would happen if someone was vaccinated at the same time they got infected by the Ad (or a very similar subtype) the vaccine used? The wild Ad would provide the missing E1 (& E3 if needed) genes allowing the vaccine to replicate (unless there’s some sort of fail-safe I don’t know about). It seems most likely that this would interfere with the wild Ad replication (see defective interfering viruses) and boost the immune response to the vaccine, which would be a win-win, but with the immune system you never know…

  2. Quin says:

    There is hesitance to talk about vaccine safety, since we want to assure people that the vaccines are safe. But these vaccines are not FDA-approved and are under EUA only.

    But is there some reason to think that there might be less risk of longterm adverse effects for vaccines using an Ad platform rather than an mRNA platform (the mRNA platform being entirely new).

    Any info is appreciated.

    1. Julie Crudele says:

      The mRNA vaccine platform is not entirely new. It’s been under development for decades and in humans since 2013 with aero safety issues. Given mRNA’s inherent transience, there’s no reason to think that there would be long-term issues, and there’s been no evidence of any such issues over the past 8 years.

      1. Doug H MD says:

        can you point to studies showing hat 8 year safety record? i could not find any…

        1. Alberto J. Villena says:

          There are not 8 years follow up safety tracks for mRNA vaccines, but some clinical trials were done in 2017, and until now no adverse effects have been reported:
          Alberer M, Gnad-Vogt U, Hong HS, Mehr KT, Backert L, Finak G, Gottardo R, Bica MA, Garofano A, Koch SD, Fotin-Mleczek M, Hoerr I, Clemens R, von Sonnenburg F. Safety and immunogenicity of a mRNA rabies vaccine in healthy adults: an open-label, non-randomised, prospective, first-in-human phase 1 clinical trial. Lancet. 2017 Sep 23;390(10101):1511-1520. doi: 10.1016/S0140-6736(17)31665-3. Epub 2017 Jul 25. PMID: 28754494.

      2. Doug H MD says:

        can you point to some studies confirming htat 8 year track record of safety?

  3. sgcox says:

    I want my vaccine shaken, not stirred

    1. Ogamol says:

      Spin for the Win! 😉

  4. Julie Crudele says:

    “You need the linear DNA because it turns out that in that form it can directly infect human cells (with the help of some additives in the cell culture to get it through the cell membranes more efficiently).”

    No, you need linear DNA because Ad requires a piece of linear DNA to package it’s genome. They use lipofectaminein that book chapter to get the DNA into the cells. Lipofectamine can be used to get plasmids into cells as well.

    1. Harvey 6'3.5" says:

      Julie Crudele is essentially right. Adenovirus normally has a linear genome with a protein called pTP (precursor to terminal protein) at each termini. As Derek said, you excise the E1 viral region (which has the genes that suppress the cell response) but you leave E2, which has the pTP and Adenovirus polymerase genes. Interestingly, pTP may be more important than polymerase because they share some overlapping sequence and pTP is conserved at the expense of the polymerase sequence.

  5. Melon says:

    Are adenovirus vaccines less effective when used in winter? Presumably the background level of adenovirus antibodies is much higher in winter when people have adenovirus related colds. I’d assume that even if cross-reactivity of antibodies against different adenoviruses is low, cross-reactivity will become relevant once the titre of anti adenovirus antibodies rises following an infection.

    1. AVS-600 says:

      Most colds are caused by rhinoviruses and coronaviruses. Adenoviruses are a small subsegment of cold-causing viruses, so it’s likely the titers aren’t hugely different seasonally overall (i.e. they’re fairly close to zero in both seasons, even if the *relative* titer might be higher in winter). The titers for antibodies that actually cross-react with the vaccine vector adenoviruses should be smaller still.

      On the other hand, immunology is complicated, so who really knows before it’s tested in the field?

    1. George Dorbell says:

      …Half time score. Wrinklies coming on as subs in second half. Just have to see how game plays out.

      All a real world, grand scale Phase III trial, which pretty much bound to be from the outset. Participating myself tomorrow here in Limeyland – invited to local surgery for first jab. Pretty sure will be OXAZ. Wrinkly got nowt much to lose from giving it a go.

      From the above link…

      “Yet the South African trial… found such a low efficacy against mild and moderate disease, under 25%, that it would not meet minimal international standards for emergency use. But scientists are hopeful it might still prevent severe disease and death—arguably the most important job for any COVID-19 vaccine.”

      1. colintd says:

        The confidence bars on the data look rather wide, which is not surprising given the very small sample size. Similarly it isn’t clear how much protection the AZ vaccine gives against serious disease with the SA variant. I think I’ll hold off judgement either way, but suspect/hope it will still prevent serious illness.

      2. Some idiot says:

        Agree, particularly about the prevention of serious illness and death.

        Saw a headline in the last 24 hours about two residents of a German nursing home, who had already received both vaccinations (one of the mRNA vaccines), had contracted COVID19. Read the article, and further down in the article it pointed out that they had tested positive for COVID19, but were not showing any symptoms. Which, all in all, is the important thing…!

  6. AGMMGA says:

    For what it’s worth, the Catholic Church has officially decreed that, although the involvement of aborted fetal cells in vaccine manufacture is problematic, good catholics can get the vaccine, and should do so in order to avoid spreading Covid-19 and to protect others. So if you know any hesitant catholic, the URL of the official, Pope sanctioned proclamation (english; spanish also available) is linked in my nickname.

    1. John M says:

      Unfortunately the use of HEK293 cell lines in vaccine or drug production gets translated in social media to “the vaccine/drug contains aborted fetal tissue.” This claim has even been made against Pepsi because HEK293 cell lines were used by a company doing flavor consulting to Pepsi! I always feel the need to correct this misperception.

      1. This cell line was derived from one single fetus 50 years ago. It does not represent ongoing use of fetuses aborted over time.

      2. The reason why this fetus was available for research is lost to history. We don’t know whether it was an elective or medically necessary abortion, or even if the fetus was miscarried (“spontaneous abortion”).

      3. The HEK293 cells are used a cellular factories to produce a virus or protein, and given stringent purification of the viral or protein product there should be no element of the HEK293 cells in the final vaccine or drug. I.e. the vaccine or drug does not “contain fetal tissue”.

      1. Micha Elyi says:

        The ethical objection is to the use of elective-abortion sourced fetal tissue for any purpose. Any purpose. If the end product contains such tissue then it is doubly horrific.

  7. Patrick says:

    Somewhat along the lines of the aborted tissue discussion, do you know what media the cells are grown with?

    I heard a vegan saying they were uncomfortable about the use of horseshoe crab blood for LPS assays during manufacturing, but most cell culture I’ve been involved with has used animal serum of one form or another.

    Obviously it’s not something I’m particularly concerned about, but I’m sure it will come up as an issue at some point.

    1. johnnyboy says:

      Yes, by all means let’s look for additional potential ‘issues’, so that vegans can complain more ! Cuz whining vegans is really what we should worry about at the moment.

    2. Mammalian scale-up person says:

      We wean cell lines off FBS pronto – not because we’re all vegan but because it’s hideously expensive, requires a lot of expensive cold storage, and is a known source of zoonotic viruses contaminating our systems. Weaning cells down to defined media (glucose, vitamins and minerals, individual amino acids, synthetic shear protectant) is done at the same time we convert any adherent lines to suspension, and in suspension lines we mostly don’t use it anyway. Well, I don’t, I think there’s an antique cell line out there that still does, but it’s definitely not routinely done anymore.

    3. Roy v H says:

      JnJ uses serum free media, so don’t worry about it 🙂

    4. TabeaK says:

      In mammalian manufacturing processes serum use is increasingly uncommon. Too expensive, too much batch variability and a risk factor for zoonotic viruses. Most manufacturing processes aim at “animal free” medium compositions using recombinant protein supplements if needed.

      Now, having said that, early Discovery Research & Academic Research is a different kettle of fish, serum is still routinely used in that environment – so any new biologic drug is likely to have been worked up in this type of environment. Once you get to the manufacturing stage, however, it is usually not a factor in play anymore.

      And PS: Very personal opinion: Vegans need to get over themselves…

      1. Stroodle says:

        Remind me of how this pandemic started in the first place?

        But yes, academic use of a biproduct of the meat industry shouldn’t rate highly on anyone’s ethical agenda currently.

  8. Ilya Yasny says:

    Derek, thank you for an excellent review,

    It would be great to have more details on the validation, QA and QC part of the process. To my knowledge, this is something which is very hard to comply with within these aggressive timelines. ICH Q5E requires comparability studies which can get as far as to the clinical comparability, after almost any change in the manufacturing process. Not to mention scaling up or opening a new manufacturing site. This is apparently something that Sputnik still struggles with.

    1. Derek Lowe says:

      True, that’s a key part of the process and not always straightforward to get right. But QA is way out of my expertise, even by my standards! I’ll see what I can round up, though. . .

      1. Lapsed Chemist says:

        Agree – this is an area as challenging as manufacturing as demonstrating comparability of these species is difficult. For the AZ vaccine – one site (Novasep) appears to be struggling with the process, but others are manufacturing without issue. This is not atypical and demonstrates the complexity of the challenge. I’m sure scientists at both sites are grappling with the issue as we speak to improve consistency of performance and also that the material produced is comparable to other sites.

        I suspect that the groups leveraged a lot of work from earlier projects to get these assays ready in time. Given the compressed timelines, a pragmatic approach has been taken by the agencies which would be interesting to learn about.

        To show the importance of analytics – for the AZ vaccine – the quantification method for vg titre was changed mid study and has held up US release and caused a lot of additional work to rationalise ( )..

  9. A Nonny Mouse says:

    ……..vial-filling by Symbiosis (in Sterling, UK)…

    Not correct. This is done in the UK by Wockhardt in Wrexham (and also in Germany for the UK).

    1. Lapsed Chemist says:

      Symbiosis manufactured pre-commercial material (i.e. to support Phase 1/2 clinical studies)

  10. Dick says:

    There is an interesting description of how AI has and will speed vaccine “tweaking” in
    AlphaFold Proves That AI Can Crack Fundamental Scientific Problems. This is from the IEEE SPECTRUM, Feb 21 issue.

  11. Barry says:

    Do adenoviruses have tricks to get their DNA (and any payload we’ve attached) into the host nucleus for transcription? Or does transcription happen in the cytosol?

      1. Marko says:

        The CDC really does publish junk sometimes. From your link :

        “The genetic material delivered by the viral vector does not enter the cell nucleus and does not integrate into a person’s DNA.”

        This is half true and half pure crap. Of course the DNA-based viral vectors get into the nucleus. The sad thing is , lots of publications look to the CDC pages for guidance before they push out their articles to the public, now full of CDC misinformation.

        Who’s running that CDC dumpster now? Clean-up needed in Aisles 1 thru 97, STAT !

        1. Josh B says:

          I contacted the CDC and fortunately they corrected the article (the original can be viewed on wayback machine).

          1. Marko says:

            Nice work !

            My faith in the CDC is being restored, if only gradually.

  12. luysii says:

    Completely off topic but useful and even ethical. Here’s how I jumped to the head of the vaccine line and got vaccinated ahead of time.

  13. Rob McMillin says:

    First off, I really wanted to thank you for this blog. I have learned so much from it, and in the current environment, it’s indispensable reading. My question is this: Peter Hotez has repeatedly said he thinks mRNA vaccines will not scale well, yet you have the CEO of Sanofi making a very big bet in the opposite direction and in fact all but coming out and saying mRNA vaccines will be the way forward for new diseases. Thoughts?

    1. Mammalian scale-up person says:

      It is possible BUT there are several hurdles to overcome.

      1) Raw materials. You need recombinant *GMP quality* (as in, NOT lab quality) enzymes. These can certainly be made, the technology existed even when I was an undergrad back in the Clinton/Kurt Cobain era. Bacterial fermentation at smallish scale and relatively easy to do. Since they’re being used for industrial processing and not put into humans, you can do convenient things to improve the process like stick 6his tags on the end and tether them to IMAC resin to better control the reaction rate and set yourself up for continuous methods.

      2) Liposome/LNP formulation needs to be done by process engineers who actually know a little something about liposome manufacturing. Sanofi has these, or should have, or can afford to hire them and keep them.

      3) Different kinds of lipids and a wider variety need to be at least tried out, instead of this whole getting married to just one other startup’s lipid and then having a patent pissing contest with them. Never restrict yourself to a single source of anything, there is far too much risk that one source will go out of business and you’ll be screwed, unless you plan for vertical integration and make the lipids yourself at a small molecules site within your company. Which Sanofi also has the resources to do if they choose.

      4) The medicinal chemists need to choose targets appropriate for the PK of intracellular transient expression. Alnylam did an excellent job of thoroughly understanding the PK of their modality, and Sanofi should follow their example.

  14. Tony says:

    CSL in Melbourne, Australia is also delivering 1 million doses of Oxford/AZ per week from March.

    That said, I think CSL will switch their manufacturing to Novavax at some point.

  15. Mie says:

    I really love reading your article very nice and very interesting. And it was very informative.

    resin name badges

  16. georgios says:

    is it true that Adenovirus vaccines cannot be boosted/ re-dosed due to immune reponse

    1. sgcox says:

      No, it is not.
      Oxford/AZ and Sputnik reported higher titres after second job. That is why two doses are the default regime. Janssens should report soon results of single-dose and two doses trial soon. Will be interesting to compare

      1. Chris says:

        This is my question too, Clearly a second does and must work (as it is in the AZ regimen), but the immune system does produce anti-vector NAbs ( . Does this not impede the efficacy of boosters as some of booster is neutralised before it is able to infect a cell? Is the dose of the booster higher? Could there be additional benefits to using a different adenovirus vector for primary and booster shots?

  17. Peter says:

    >Russian manufacturer R-Pharm has a plant in Germany that’s in production for export back into the CIS countries (they’re also producing the Gamaleya vaccine).

    Apparently the link given in support of the statement above was misplaced, as the linked article contains no info on the German plant and moreover this article is related to the joint Gamaleya-AZ vaccine rather than the original Gamaleya one.

  18. fajensen says:

    …. because bacterial DNA is relatively straightforward to handle and to get replicated ….

    To think that only 20 years ago everyone would have understood that as a recognised expert in the field making an ironic insider joke to liven up a conference paper. We have come a very long way in a very short time!

  19. Arvin Solangon says:

    My main concern with the DNA vaccine as opposed to the mRNA one is the need for the DNA strand to be incorporated into the host cell’s nuclear genome in order for it to be transcribed into mRNA for subsequent translation. We all know the splicing of viral genomes into the cellular genome is one of the major mechanisms for inadvertently introducing mistakes/mutations that, when the cell’s mutation detection and repair mechanisms fail to fix, become permanent and possibly dangerous, which is a possibility that the mRNA vaccine entirely avoids and is, therefore, a major plus factor for it in my view. This isn’t something minor because it’s precisely the mechanism for a lot of cancers, for example. Articles either completely avoid delving into this detail or mention it briefly without explaining how something so unusual is made possible (like it’s something routine) like you or this physician* on YouTube did. I am not sure how wanting to avoid adding fuel to the fire of conspiracy theories, especially when it’s actually a legit concern during a time when resistance to new vaccines is particularly unwanted, plays into this decision consciously or subconsciously but if that is indeed the motivation then it is perfectly understandable. But it still doesn’t make the question go away anyway.

    So could you please explain how the Oxford/AstraZeneca and J&J vaccines are able to proceed with using the host cell’s machinery for reproduction of the viral spike protein without allowing the adenovirus vector to initially insert the DNA strand into the host’s own chromosomal DNA?


    1. Arvin Solangon says:

      So I’ve been able to answer my own question since then with these thanks to the rabbit holes of the internet. I just found this nugget out and realized yet again how much there is always something new to learn in molecular biology and how much we actually do not yet know and understand. So “episomal DNA” it is…interesting!

      “Furthermore, adenovirus vectors do not integrate into host genomes but stay as episomal DNA in the nucleus of host cells…”

      “The replication of HAdV genomes takes place within the host cell nucleus, which is a complex environment home to a vast repertoire of cellular processes. Accordingly, HAdV must co‐opt or antagonize many cellular pathways, while reorganizing the existing nuclear environment. These architectural changes include the formation of virus‐induced membraneless nuclear compartments that harbor viral genome replication, termed viral replication compartments (VRCs)…”

      1. Ramin P says:

        That’s helpful, but there’s no mechanism described! What prevents the adenovirus vector from integrating? And, how strong or fool-proof is that protection? I found this paper, analyzing the human genome from blood cells in over 8,000 people, identifying a small % of adenovirus DNA integrating into the samples! We have to be careful about saying ‘never’ in biology – we used to believe it was impossible for RNA to turn into DNA, until retroviruses were discovered and that mechanism was described. In theory, I think there is still a chance for DNA integrations to occur, especially when it’s been described with regular adenoviral infections.

  20. Pablo says:

    Expanding a bit on the list of vaccine production contracts, Oxford/AZ also has a contract of technology transfer with the Brazilian public institution Fiocruz/Bio-Manguinhos. The aim is to produce locally the doses for the Brazilian population.

  21. Gunnar says:

    How long does episomal DNA from the J&J vaccine remain active in the infected host cell nucleus? When does it stop being transcribed and degrade?

    1. Helena says:

      @Gunnar have you found an answer to your question? I truly wonder about this.

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