Abstract : In infectious mononucleosis (IM), anti-EBNA-1 antibodies are produced which cross-react with multiple normal human proteins. The cross-reactions can be inhibited with synthetic peptides representing the glycine/alanine repeat in EBNA-1, which implies that the cross-reactivity is due to anti-gly/ala antibodies that cross-react with host proteins containing configurations like those in the EBNA-1 repeat. Here we report the isolation of five gene fragments from a Raji B lymphocyte cDNA library encoding peptides reactive with autoantibodies in an IM serum. One of these, p542, encodes a glycine rich 28-mer which constitutes its cross-reactive epitope, as shown elsewhere. By Northern blots, p542 was identifiable in three B lymphocyte lines, a T cell line, and an epithelial cell line. In a search of the GenBank for proteins with sequence similarity to p542, we found a high degree of identity with the mid- and 3' terminal regions of the recently published mouse gene, Raly, which encodes a protein with the structure of a heterogeneous nuclear ribonuclear protein (hnRNP). We confirmed by anchored RT-PCR our presumption that the 5' sequences of p542 also have a high degree of identity with Raly, including presence of RNA binding motifs characteristic of hnRNPs. There was also sequence homology with human hnRNP C2. From these observations and our previous studies, we conclude that the autoantigen for one of the cross-reactive autoantibodies generated during immune responses to the Epstein Barr virus, anti-p542, is probably an hnRNP.

Abstract : A variety of gene delivery technologies can be used to express antigens within somatic tissues, resulting in systemic humoral and cellular immune responses. This observation has led to the development of polynucleotide vaccine preparations for stimulation of systemic immunity. Mucosal immune responses are functionally distinct from systemic immune responses, and are stimulated by antigen presentation within specialised mucosal-associated inductor tissues. We hypothesize that mucosal genetic vaccine will require gene transfer methods which target mucosal-associated inductor tissues such as the oropharyngeal Waldeyer's ring or intestinal Peyer's patches. We have tested this hypothesis by expressing a test antigen using a replication-defective recombinant Semliki Forest Virus (SFV) preparation. Mice treated with recombinant SFV via an intravascular or intratracheal route generated systemic immune responses against the test antigen. In contrast, intranasal inoculation resulted in the production of IgA within pulmonary fluids, one hallmark of a mucosal immune response. These results indicate that transfection of mucosal effector tissues may not be sufficient for the generation of a universal mucosal immune response. Furthermore, the results predict that techniques which target transfection or transduction to mucosal inductor tissues will enable the development of a new class of polynucleotide vaccines which exploit current concepts in mucosal immunology.

Harvey B. "Dusty" Rhodes ( Feb 15, 1918 - Aug 4, 2014 )

Harvey "Dusty" Rhodes, the founding president of Columbia Community College, passed away peacefully at his home on Phoenix Lake in Sonora, California, after a long and accomplished life. He was 96.

Born to Irene (Canale) and Harry A. Rhodes, he grew up on a family ranch near Tracy, California, and lived with his grandparents during the week in nearby Banta, where he attended elementary school. He graduated from Tracy High School in 1935 and San Jose State in 1940, where he was captain of the baseball team, chairman of social affairs, and earned a degree in biological science. He later received a master's degree from the University of Southern California and a doctorate from the University of California at Berkeley.

Rhodes joined the U.S. Navy in December 1941, days before the attack on Pearl Harbor, and served his country in the South Pacific during World War II. He remained in the Naval Reserves until 1970, during which time he rose to the rank of commander and helped form the Central Valley Naval Reserve unit.

After returning to California following the war, Rhodes began a successful career in education. He worked more than a decade at Modesto High School as a teacher, coach, dean, and vice principal. He later joined the faculty of Modesto Junior College as director of continuing education, and then moved to Tuolumne County as the founding president of Columbia College near Sonora. He retired from his position at the college in 1979.

Upon retirement, Rhodes remained an active member of the community in numerous organizations, including the Sonora Lions Club, Sons in Retirement, Tuolumne County Historical Society, Association of California School Administrators, and Yosemite Natural History Association. He loved the San Francisco Giants, world travel, food and wine, and spending time with his family.

He was preceded in death by his parents, his oldest son Gary H. Rhodes, and grandson John Rhodes. He is survived by his beloved wife Patricia Hertert Rhodes, daughter Sandra Rhodes Conti (Richard), son Gregory Rhodes (Sally), six grandchildren, and seven great grandchildren.

All Answers

• 7th Aug, 2014 : Harm Geerligs ( Zoetis )

  • • In my opinion that will be the future. We will need to be able to respond directly against threats like this. We need to improve our technologies to make this possible.

• 7th Aug, 2014 : [Dr. Anne Searls De Groot (born 1956)] ( [EpiVax] )

  • • I was thinking that combining a fully recombinant B cell target with T helper epitopes would be ideal.

• 7th Aug, 2014 : Stephen Gracon ( Consultant )

  • • It is possible with the novel nanoemulsion technology we are developing which can use the nanoemulsion to completely inactivate and disrupt live pathogens, preserving key epitopes and create an effective vaccine. The vaccine can be given intranasally or intramuscularly. An important consideration is whether the live pathogen contains enough of the key epitopes to elicit a protective immune response. This approach is simple, rapid, cost effective and can be accomplished with recombinant proteins as well as live pathogens.

• 8th Aug, 2014 : Timothy Mark Doherty ( GlaxoSmithKline )

  • • It's not going to be easy.

    • I was involved in an EU-wide project (FASTVAC) over the last few years looking at exactly this question: how do we produce vaccines faster in response to new biological threats?

    • There are two aspects - first you need to actually identify the pathogen, and generate a response that is protective. In the case of a new pathogen, that might not actually be very obvious - look at how long it took to work out what a protective vaccine against HIV might look like. Anyone who thinks that all you need to do is identify a few immunodominant antigens and inject them is pretty much doomed to failure: vaccine development history is littered with immunogenic antigens that offer no protection at all, when used as vaccines and a few which actually make the disease worse.

    • That leads to the second aspect - regulation, release and safety. Vaccines are different from almost all pharmaceuticals in that they are potentially given to millions or even hundreds of millions of perfectly healthy people. As a result, you cannot ignore rare side effects: even a side effect in the "very rare" category could lead to thousands of cases if a vaccine is broadly used. That's not going to be acceptable even in an epidemic situation.

    • So any new vaccine is going to have to go through clinical trials, both for efficacy and for safety. Typically this can take 10 years or more from first application, and clearly that's no good for emerging threats. It can be done faster though: we routinely make new influenza vaccines every year, and researchers were able o produce a protype SARS vaccine ready for trials in about 6 months. In both cases, though that is possible because we already know a lot about the pathogen in question (or related pathogens) and immunity to these types of virus, and because there was already a process in place for making that type of vaccine.

    • So while I have great respect for Annie and the Epivax team, I don't buy the idea that we can (yet) create vaccines on demand. So far, computational identification has not proven to be a success in terms of rapid vaccine design, simply because the vast majority of targets identified are not protective. We've been playing with epitope identification for vaccines since the 1980's (it's what I did my PhD on, in fact!) and so far, we don't have a single success to point to. It does speed the process of identifying potential targets, but those targets then have to be tested in vivo in the old-fashioned way.

    • The Bexsero vaccine is a good example of this. Computational analysis identified 570 potential antigens which should be immunogenic - these were cloned and tested. 28 of them showed some degree of protective ability. That's a 5% hit rate, which is actually pretty good. Of those 28, only 7 showed actual vaccine potential - for a final hit rate of 1% and that's fairly typical, based on what's been published and my own experience. But to work that out, and then demonstrate safety and efficacy took 12 years. Identifying targets was by far the fastest and easiest part of that process. Computational analysis can speed that first step, but it doesn't remove the need for the subsequent steps, which is actually what takes the most time.

    • So if we genuinely want to speed up vaccine development, we need:

      • • 1) better understanding of biological processes, so we can predict what's protective, not just what's seen by the immune system. This is going to be a major challenge for computational biology in the years ahead

        • 2) better processes for approval (and preferrably pre-approval) of vaccine production, so that we can avoid having to plod step-by-step through the process in an emergency.

• 11th Aug, 2014 : Timothy Mark Doherty ( GlaxoSmithKline )

  • • There are two problems with vaccine development that work against vaccines for rare diseases. The first problem is that without demand, you have no market, so anyone making a vaccine for (for example) Ebola, has to be prepared to spend tens, if not hundreds of millions of dollars, with no prospect of ever getting that money back.

    • Governments can do that, but commercial firms have a hard time justifying it. I don't think it's a coincidence that most of our current vaccines were first developed by governments and then later commercialised by companies. I don't see anything wrong with that - in fact, I think it's an ideal model, where governments do basic research (which they traditionally have been better at than the private sector) and then companies handle marketing and distribution (which traditonally they have been better at than the public sector). GSK (the company I work for) has an Ebola vaccine in the pipeline, which was developed with theNIH in exatly this fashion. There's a good case for governments to cover the cost of developing new vaccines, because governments are the ones who have to pay the costs of diseases anyway. Even if you ignore the lives lost, it's estimated that current Ebola outbreak will cost 100 million USD to control - and that's even before you take the indirect cost on trade and business caused by border closings and quarantine.

    • That's easily enough money to pay for vaccine development.

    • But there is a second problem with vaccines for rare or epidemic diseases. How do you test them for efficacy? We have a deecent anmal model for Ebola, but we have no good correlate of immunity in humans, so the only way to test an Ebola vaccine right now is to wait for an outbreak (which might take decades) and then test the vaccine on a relatively large scale, in the middle of a public health emergency. That's a very risky approach, and extremely difficult and expensive to do. It also runs directly against our current development paradigm, where safety is front and centre.

    • It's not impossible, but it reflects my comment above: our greatest challenges for many diseases are not technical, but regulatory. How do you test vaccines for rare, new or epidemic diseases in a way that protects public health and safety, preserves confidence that the vaccine itself is safe, and also allows the information needed for product registration to be collected?

    • It's not an easy question to answer, and companies need to work with public health authorities to develop solutions for this. Unfortunately, it's a hard sell, because there is significant risk here and no-one really wants to cover that risk.

• 11th Aug, 2014 : [Dr. Anne Searls De Groot (born 1956)] ( [ EpiVax ] )

  • • All good points, and I agree that Regulatory is the biggest hurdle. That's what we're focusing on now - are there 'platforms' (similar to the flu situation) that can be used to speed approval? Can we identify a platform that is approved, to which we hitch the novel vaccine antigens or epitopes? Other groups have developed such platforms. See Mark Poznansky's work with HSP-biotin-avidin a.k.a. "ASAP" for example: (see Mark's link on the mesothelin vaccine).

    • In silico methods for predicting which antigens are likely to be immunogenic took a giant leap forward with JanusMatrix - our tool that helps define which antigens are less cross-conserved with the human genome. (see: JanusMatrix link). Turns out that pathogens try to evade immune response by inserting 'self'-like T cell epitopes - that may be one of the as-yet-undiscovered stumbling blocks in computational vaccinology. We validated this hypothesis several ways - see Viral Camouflage link) for example, other papers (showing H7N9 has Treg epitopes, for example) are hopefully coming out soon.

    • Lastly, the Bexero vaccine was developed using bioinformatics, not computational vaccinology. May seem like semantics, but the methods used still went through mice, and we do not believe that mice give a great signal for immunogenicity.

    • Thank you Tim for your kind words about EpiVax. Much appreciated.

    • Article The two-faced T cell epitope

    • [ and http://bit.ly/Viral_Camouflage ]

    • http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3943805/

    • Article Low immunogenicity predicted for emerging avian-origin H7N9

    • Article Cross-conservation of T-cell epitopes Now even more relevant...

• 23rd Oct, 2014 : Brian Hanley

  • • Sure. We can do this. It's called DNA vaccination. Just express major viral protein sequences in a plasmid and inject. There's no need to play around identifying epitopes and all the rest of that. The immune system does it. It's worked every time in animals. It's very fast. In theory, 1,000 doses can be produced in 10-15 days if you have the virus sequence. But DNA vaccine doesn't produce high antibody titers, which are the surrogate endpoint used for humans, despite the fact that antibodies cannot not cure viral diseases. T-cells and NK cells do that. Cellular immunity defeats viruses. Antibodies just help, and can prevent reinfection.

• 27th Oct, 2014 : Timothy Mark Doherty ( GlaxoSmithKline )

  • • @ Brian. Unfortunately, it's not so simple. I've worked with DNA vaccination for about 20 years now, and while - in theory - it's simple and fast, in practice, it's proven to be no different from other forms of vaccination. Far from working every time, the vast majority of DNA vaccines have failed to work at all. It's why after more than 20 years work, we still don't have a single working DNA vaccine for human diseases. It's also why in my previous job, after testing multiple DNA vaccine constructs, we went with an adjuvanted protein for human clinical trials. The same antigens delivered as DNA gave weak or insignificant protection versus solid, reproducible protection for the protein - in both cases, tested across multiple species.

    • The appeal of DNA vaccination is that nucleic acids are relatively cheap and easy to synthesise and purify in bulk. In addition, it was hoped that foreign DNA (especially bacterial/viral DNA) would be recognised and stimulate a response via PAMP signalling. In effect, it was hoped that DNA vaccines would be self-adjuvanting. This has proven not to be the case in humans and in nonhuman primates. Today, a lot of companies working with DNA vaccines are using adjuvants or delivery systems to address these weaknesses - just as we do with recombinnat protein vaccines.

    • When it comes to recognition, the DNA vaccines need to be able induce production of a protein that can then trigger an adaptive immune response: DNA by itself may be able to trigger a weak local innate response, but without a defined antigenic target, you won't get good T cell responses, and thus no immune memory.

    • A segment of DNA injected into a host has to pass through several gates to be able to induce a good T cell response. First the plasmid has to be talken up efficiently, without being degraded. Many constructs fail this test, which is why so many DNA vaccines induce weak responses or no responses. Secondly, once being taken up, it has to induce translation in the host - and again, many DNA constructs don't - or least do so poorly. Last of all, the product produced - if there is one - needs to match the protein expressed by the pathogen during invasion/disease - and it needs to be appropriately processed and presented. Otherwise the immune response will likely be irrelevant. And for T cell responses, context is critical: the epitopes presented need to match those recognised in the context of infection. For this, sophisticated analysis of the product, epitope structure and immunodominance needs to be in place: small changes in the DNA construct can have major changes in how the final product folds and is processed and presented. After all translation in host cells occurs in a very disfferent context to translation in the pathogen - or even in a virally-infected host cell. We had hoped in the past that we could let the body sort this out itself - but that hope hasn't panned out.

    • Personally, I think DNA vaccination has potential that still needs to be explored, but the failure of DNA vaccines has been so widespread over the last couple of decades, that it's given rise to a joke among vaccine developers that DNA vaccination is the technology of the future - and always will be.

• 27th Oct, 2014 : Brian Hanley

  • • 1. Failure. Are you familiar with the literature? Perhaps you should familiarize yourself. These papers aren't the only ones around.

      • • http://www.sciencedirect.com/science/article/pii/S0042682202000363 - DNA vaccination of infants in the presence of maternal antibody: a measles model in the primate (2003) Mary Premenko-Lanier, Paul A Rota, Gary Rhodes, David Verhoeven, Dan H Barouch, Nicholas W Lerche, Norman L Letvin, William J Bellini, Michael B McChesney

        • http://jid.oxfordjournals.org/content/189/11/2064.full.pdf - Protection against Challenge with Measles Virus (MV) in Infant Macaques by an MV DNA Vaccine Administered in the Presence of Neutralizing Antibody (2004) Mary Premenko-Lanier, Paul A. Rota, Gary H. Rhodes, William J. Bellini, and Michael B. McChesney

        • http://jid.oxfordjournals.org/content/203/1/95.full - Multivalent Smallpox DNA Vaccine Delivered by Intradermal Electroporation Drives Protective Immunity in Nonhuman Primates Against Lethal Monkeypox Challenge (2011) Lauren A. Hirao, Ruxandra Draghia-Akli, Jonathan T. Prigge, Maria Yang, Abhishek Satishchandran, Ling Wu, Erika Hammarlund, Amir S. Khan, Tahar Babas, Lowrey Rhodes, Peter Silvera, Mark Slifka, Niranjan Y. Sardesai, and David B. Weiner

    • 2. That there is no human vaccine yet has absolutely nothing to do with whether DNA vaccines provide disease protection or not.

      • • Human vaccines require meeting the surrogate endpoint of antibody titers. It also requires a company to carry it forward at a time when vaccines are under all kinds of ridiculous pressure from anti-vaccine activists. Gary Rhodes' vaccine against measles works, no question about it. He created it to fight measles in Africa where vaccine efficacy averages around 50%. Read those papers and tell me that vaccine doesn't work. In fact, under the animal rule it should be allowed. But it isn't because of the surrogate endpoint of antibody titers.

        • The other reason why there isn't a DNA vaccine is that most of the useful work has been done in academia. Gary's stuff was not patented. Without patents companies won't bother with it. Why UCD didn't patent his DNA vaccine is anyone's guess. Mine is that the Tech Transfer office didn't score it high enough for ROI potential. Plus, patents in that area are uniquely difficult to obtain.

        • Commercial pharma requires that to produce a vaccine they have patents on the technology. That's just SOP for pharma. And as I alluded to, because of the Singh decision, and the fact that DNA vaccines are considered a type of biologic, it's quite difficult to get a patent in the area.

    • 3. Cellular assay is adaptive immune response. So is any antibody response, even if it is low.

    • 4. Unless you are making constructs with selected epitopes of a viral protein, what are you talking about?

      • • The proteins produced by virus RNA translation in a mammalian cell fold the same as the same mRNA sequence translated in a mammalian cell. That's the environment. Or are you suggesting that shepherd proteins from a virus (none I'm aware of) are dense enough to change the folding of translated proteins out of ribosomes? There are differences in protein folding (and expression) in bacterial cells, but that's quite different from what you said. You seemed to say that there is something mysterious going on with expression of viruses in mammalian (and primate) cells that causes virus proteins to fold differently when they are translated from EXACTLY the same sequence. Please. Elucidate. I'm very interested in how that works.

    • 5. If you are referring to selected epitope expression attempts in DNA vaccination, all I can say is don't do that. If you try to express 8-20aa with DNA vaccination, it's as likely that you will produce an miRNA that silences expression as a working mRNA--unless you pad it out quite a bit. Even without a U6 promoter, it can do that. If you use a short peptide produced by fermentation, (again 8-20aa) it's a lousy technique for vaccination. Yes, that can be made to work in animals for producing epitope-targeted antibodies for research use--if you use extremely harsh adjuvants like Freunds. Try that in humans, and it's guaranteed to fail in the majority. Yes, I have crunched the data from such efforts. As a rule of thumb you will get about 1/3rd with good titers, 1/3rd with iffy titers, and 1/3rd with nothing noticeable. Forget targeted epitopes for human vaccines. Waste of time.

    • 6. Of course it's true not all constructs express as well as others.

      • • And commercial pharma will want to find a vector and a promoter they can protect as IP before they will touch a product. But I can tell you for a fact, that with the pNK vector Gary Rhodes used, every vaccine he made worked. It is based on the CMV promoter, and as everyone in the area knows, the CMV promoter is one of the gold standards for high expression.

        • Gary never did anything fancy like in-vivo electroporation himself, although others did who he participated with. He just used simple injection. Those monkey studies that protected against measles were simple injection. Take a look.

        • I won't belabor the literature on non-primate animals. There's plenty showing those work, from mouse to tursiops truncatus. But I presume you know that.

        • Gary Rhodes was a major mentor of mine. I worked on a related system in his lab before he died. Sadly, he passed away, Dec 26, 2013 of a stroke.

        • I would suggest surveying measles mortality and morbidity in Africa, and the problems of delivery. I would also suggest that you total up that impact between 2004 when that measles DNA vaccine in infant rhesus macaques worked and today (10 years) without it.

        • I think I have Gary's measles vaccine sequence around somewhere. I've used his pNK vectors. I'll find a way to get it to someone who wants to produce it.

• 28th Oct, 2014 : Timothy Mark Doherty ( GlaxoSmithKline )

  • • @Brian.

    • I'm sorry to hear that Gary has passed away: I didn't know that. I'd met him in the past to discuss heterologous vaccination strategies.

    • As for the vacccines themselves, if "failure" seems too harsh, you could substitute "not successful". Over the last 2 decades, vaccine development has been more productive than ever before. We have seen about two dozen new vaccines progress successfully through phase III - meningococcal meningitis, rotavirus AGVE, avian influenza, pneumococcal disease, cervical cancer, dengue, malaria, Borellia etc etc. Many of these have employed new technologies, so it's not that the lack of a proxy marker such as serology is an impossible stumbling block. Some of them (Lyme disease, Malaria, etc) are for vaccines with niche markets or for developing country diseases, so the lack of a blockbuster market does not stop companies developing vaccines. Ebola is a good example: even before the current outbreak, there were multiple vaccines already on the point of entering clinical trials - and this is never going to be a big money-maker.

    • So the lack of success for DNA vaccines is not down to external factors. Other vaccine technologies facing the same stumbling blocks have succeeded. It's all about the vaccines. There have been DNA vaccines that have reached clinical trials - HIV, cancer, Hepatitis B, etc (see PMID: 21198665 for a review, for example). None of them have passed the acid test though: do they work, under practical conditions? [Dr. Margaret Ann Liu (born 1956)] has been a champion of DNA vaccination for decades now, and she calls the results "disappointing". Rule #1 about vaccine development is that generating an immune response does not indicate efficacy. That's actually rules #2 though #5, as well. Unless you have a clinically valid proxy marker, the only way to calculate efficacy is to show efficacy.

    • Now - that doesn't mean that DNA vaccines can never work: just that getting them to work is no easier than it is with other vaccine technologies. There's no magic bullet, and just getting your genes into the host, by itself means *nothing*. Your own cites indicate exactly what I was referring to when I pointed out that the early hopes that DNA vaccines would be an easy way forward haven't panned out. The MV-DNA vaccine generates an immune response - but by itself, that response protects only around half the challenged macaques (versus nearly all, using the same model but the licensed vaccine). The most recent publications show that you can get similar levels of protection to the licensed product with MV-DNA using IL-2 as an adjuvant ... but of course this eliminates the potential advantages of production cost and not needing a cold chain. It's really nice work, but as it stands, it's not a realistic product for developing countries.

    • Our own experience mirrors that: in my last job, we tested multiple DNA vaccines (for mycobacterial diseases, for Chlamydia and for Streptococcus) - some of them using the CMV promoter, which is a fairly standard tool. So this is not exactly new territory for me. The best of these constructs generated measureable CD8 and even CD4 responses (and low, but detectable antibody levels as well, for what that's worth). They were even protective against bacterial dissemination and growth on challenge. But - and here's the rub - the level of protection obtained was very low and variable, compared to virally vectored vaccines or adjuvanted protein vaccines, in direct head to head studies, using multiple different models (some of that work for the TB vaccine is presented in PMID:15687025, you can find the rest - and there's a lot - in associated publications). When we had to decide where to place our bets - and a lot of research money - for clinical trials, it was a no-brainer. We chose the technologies that delivered high, consistent protection across multiple species and multiple challenge models. Even that is no guarantee that these vaccines will work long-term in humans: it's simply our best shot.

• 28th Oct, 2014 : Brian Hanley

  • • No, you are both misstating protective versus sterilizing immunity, and misstating the results of the studies. The MV-DNA vaccine protected all the monkeys from any serious disease, including infants, and no adult showed disease symptoms. Half the infants who were vaccinated during the period of maternal antibody showed minor symptoms. No other vaccine type can produce protective immunity in infants during maternal antibody. The IL-2 (or IL-15) adjuvant was only to raise Ab titers. (Similarly, DNA vaccine for Ebola protected all monkeys from challenge.)

    • What the DNA vaccine did not do is generate sterilizing immunity. But that is true of yearly flu vaccines as well, and appears to be true for live measles vaccine to some extent. Probably at least 1/3rd or so of those who get yearly flu shots develop a light case of the flu when exposed, because the epidemiology says so. Flu vaccines don't generate the degree of herd immunity that they should. Those vaccines get approval, because of the surrogate endpoints accepted.

    • The question also is, what is the problem you are trying to solve? Are you trying to protect people (such as West Africans) from death and permanent disability? Or are you trying to eradicate the disease operating in the developed world? In Africa, where lack of cold chain puts vaccination efficacy in the 50% range, DNA vaccines make excellent sense. They are pretty heat stable when lyophilized, they protect people from serious disease, usually eliminate any noticeable symptoms (from viral illness) they work on infants during or shortly after birth (which is the one time when African health care might see the child fairly reliably), and they are safe to inject into anyone, HIV infected or not.

    • For addressing most viral disease, DNA vaccines are excellent, and particularly good in the parts of the world where vaccination doesn't work very well.

28th Oct, 2014 : Brian Hanley

• • • The other examples, Tim, are, first of all, not good targets for DNA vaccination, and many of them are diseases where vaccination of any kind doesn't work reliably, or cannot work.

    • I don't think that any bacterial disease is a good target for DNA vaccination. The reason is simple. The cellular systems for making the vaccination targets from DNA are different in bacteria and mammalian cells. The DNA provided to a mammalian cell is not going to make the antigens of bacterial pathogens as well. There will be differences in glycosolation, sometimes differences in folding. (One finds the reverse happens also, when producing proteins by fermentation in E coli.)

    • TB vaccines are all lousy. BCG is only used because it's somewhat effective. When I looked into TB vaccines last, the most promising work was from way back, a guinea-pig study that used cord factor. (Prompted by Beaman.) There is no particular reason to think a DNA vaccine would work on TB very well in humans, doubtful it would work as well as BCG which isn't very good.

    • HIV vaccines don't work for a host of reasons. No vaccine works on HIV. So why would a DNA vaccine be expected to work? HIV can infect cells by membrane-to-membrane contact. In fact, based on the experience of lab techs growing HIV in cell culture, that is the most effective infection method there is. (Which means that all the protocols for HIV vaccine testing in monkeys are wrong, but nobody cares. The grant money rolls in, and those protocols haven't changed.) HIV vaccines keep being worked on, and most of that work is known to be worthless by those who work on it, but grants keep coming. (The exception may be synthetic vaccination, which could provide some protection.)

    • So my question would be, why pursue DNA vaccination for chlamydia, streptococcus, or TB? In an academic grant environment, perhaps one could justify that. But I can't think of one for a straight DNA vaccine. Saying that DNA vaccination tilts toward Th1 is not good enough. The target of a DNA vaccine should be reliably eradicated by Th1 response, and the antigens should be produced in mammalian cells in the normal course of the disease. (If they aren't one needs to show how the antigen is substantially identical.) Mouse model is not a good one for humans with DNA vaccination. Monkey model is, unfortunately, needed.

    • A Hep B DNA vaccine is not one that I would expect to work very well. The reason is that Hep B is not always eradicated by cellular immunity in the normal course of disease. The reason for that failure is unclear. The current vaccine has a 5%-15% failure/non-response rate. (Though some of those are infected.) I could see DNA vaccine used together with a classic vaccine perhaps, and it may do no worse, but I wouldn't expect DNA vaccine to do significantly better. To prevent Hep B in everyone, you need high antibody titers so that the infectious dose of virus can be destroyed before it infects. For the majority the vaccine will probably work through cellular immunity, but it is unlikely for all. Thinking it over, I suppose it is worth checking a DNA vaccine, it would need a large population. It might be marginally better, or improve vaccine response as a component of a prime-boost.

    • That Liu review mostly agrees with my views. There are animal vaccines available, and part of the reason for this is that animals respond better. For instance there was success with a DNA vaccine against snake venom in animals. http://www.ncbi.nlm.nih.gov/pubmed/10931154 That is a result that is not going to happen in humans. I am, however, less sanguine than she is about DNA vaccination effectiveness for cancer. We shall see.

    • The place to use straight DNA vaccines is on classical virus diseases. (By classical, I mean, under normal course of the disease, the host is infected by the virus and they either get well or die, and the immune system eradicates or permanently controls the virus.)

    • DNA vaccines are particularly useful for very rapid response to a novel classical virus, and for vaccination in regions where cold chain isn't practical. For vaccination in the presence of antibody (for infants) it's the best and safest method.

• 29th Oct, 2014 : Timothy Mark Doherty ( GlaxoSmithKline )

  • • @Brian.

    • I think you are making a lot of very questionable assumptions and simplifying the immunology far too much: more than the science can actually bear, in fact. For example, you mention that DNA vaccination would be good for classical viral infections ... but then dismiss DNA vaccination against Hep B, which in fact fits just fine into the paradigm of a classical viral disease, as you defined it.

    • In truth, it's never a good idea to divide immune responses into black/white antibody or CMI. Without good T cell memory, you are unlikely to get decent or sustained antibody responses, and of course very few infections or vaccines can demonstrate a cellular response without at least some antibody response. It's pretty much never either/or, but both - the difference being one of degree.

    • Hepatitis B vaccination is a good example. Although the current vaccines generate a significant B cell response, they also generate a sustained antigen-specific T cell response - indeed, chronic Hepatitis B disease appears to be correlated not with antibody levels, but with T cell dysfunction: the inability to eradicate the infected cells. Most infected individual eradicate the infected cells just fine.The T cell memory response induced by hepatitis vaccination explains why individuals who become antibody-negative or -low in the years after vaccination retain such a very high degree of protection, not just for years, but for decades. In fact, the lack of direct correlation between antibody levels and protection is why the practice of testing serology and revaccinating if antibody levels are low is no longer generally recommended. In other words, serology is a proxy, not a correlate.

    • So to answer your question "So my question would be, if anyone working on those projects you worked on had a clue about DNA vaccination, why would they even consider it for chlamydia, streptococcus, or TB?" the answer is simple: because they hoped it would work. And - contrary to your expectations - it did work: suggesting that in fact, they did know what they were doing. A quick Pubmed search turns up several hundred articles on DNA vaccination against TB with multiple demonstrations of efficacy in animal models (see, for example, PMID: 21704108). The reason that these haven't progressed further than proof of principle, is that - as in the paper cited above - the level of protection with DNA vaccination has consistently been at or slightly lower than the level conferred by BCG (the current vaccine). The best virally-vectored and recombinant protein vaccines attain levels of protection significantly higher than that - usually about double the best effects obtainable with DNA vaccination. That's why they are now in phase IIb and candidate DNA vaccines are not.

    • To short-circuit the argument that it didn't work because it's the wrong pathogen, it's worth noting that exactly the same is true in the measles model that Gary Rhodes and colleagues published: the unadjuvanted DNA vaccine afforded the primates about half the protection obtainable with the current vaccine candidate in the same model.

    • And that's why if you do a Pubmed search on DNA vaccination for pretty much any disease (DNA vaccination for TB, for example turns up over 400 articles), you'll note a very clear pattern. The early papers started from similar premises to your current position: that you would get usable protection by just whacking the genes into the host and letting nature take its course. That's been tried, and failure has been pretty much universal. You can sometimes get protection, but not at levels superior to - or even equivalent to - competing technologies. The focus now has shifted to DNA vaccination in combination with other technologies - codon optimisation, adjuvants, heterologous vaccination strategies, etc. It's still too early to say for sure, but it looks like that's the way forward for DNA vaccines: at any rate the results - including the ones you linked to yourself - look more promising than DNA vaccination alone. And really, that's the same path that other vaccine technologies have followed: we would not have gotten very far with recombinant protein vaccines, if we had stuck with delivering unmodified, unadjuvanted proteins, for example.

    • Now, of course, it's possible that the thousands of experts working in the field are idiots, that they have learned nothing at all from the last 20 years of work, and that they should listen to you instead. I'd just gently suggest you reflect on what the odds are that that's the case.