Mystery Rays from Outer Space

Meddling with things mankind is not meant to understand. Also, pictures of my kids

August 18th, 2008

Quality and quantity once again, with vaccination

Budding HIVIt’s rapidly becoming accepted, if not quite dogma, that T cell quality (rather than, or as well as, quantity) is a critical factor in controlling HIV infection. (I’ve talked about T cell quality several times previously. What it means, simplified, is that antiviral cytotoxic T cells can have a range of different functions, and those CTL with multiple functions seem to do better at controlling HIV than those with only one or a handful of functions.) As a result, there’s a lot of interest in developing vaccines that induce multi-functional CTL, in the hope that those vaccines will better control the virus itself. A recent paper1 from Norm Letvin’s lab, though, supports the concept but doesn’t offer a lot of encouragement for the vaccine strategy.

Letvin’s group vaccinated monkeys against immunodeficiency virus using several different vaccine strategies, and evaluated the quality of the antiviral CTL elicited by those vaccines. As we have now come to expect, there were big differences in both the quantity and the quality of T cells with the various approaches. No surprises so far.

Next, they challenged the vaccinated monkeys with virus. Again, as expected, those monkeys who controlled the virus best, had the largest and most multifunctional CTL response to the challenge (“both the magnitudes and functional profiles of the virus-specific CD8+ T cells generated by vaccination were associated with control of viral replication following SHIV-89.6P challenge“).

The unexpected part, though, was that the vaccine response didn’t tell you anything about the challenge response. That is, even though some vaccines gave lots of multifunctional T cells and others gave relatively little, that did not correlate with the eventual response after challenge; “Although the different vaccination regimens generated qualitatively different virus-specific T-cell populations, those differences were lost following the virus challenge.” Letvin’s group concluded that the similar levels of virus after challenge overrode the vaccine pattern.

The good news — kind of — is that any of the vaccines seemed to work relatively well. After challenge, “the profile of cytokine production by the virus-specific T lymphocytes in the control monkeys was heavily biased toward cells that produce only IFN-?, while the virus-specific T lymphocytes of all of the experimentally vaccinated monkeys following challenge were uniformly polyfunctional.” That is, even though the vaccines didn’t ultimately differ from each other, vaccination did lead to a different, and probably better quality, CTL response than in unvaccinated monkeys.

This might suggest that even testing CTL quality as well as quantity after vaccination may not be very predictive. However, it’s also possible that the monkey model is once again being deceptive. For example, if their suggestion that the challenge dose re-set the CTL quality is correct, this might be highly sensitive to both the number of infecting viruses and, even more, to the precise kinetics of early viral replication; and there are a myriad of other differences as well. The bottom line, though, is a reminder that we really don’t understand antiviral immunity very well in any system, let alone the baroque interactions between HIV and the immune system.

  1. Sun, Y., Santra, S., Schmitz, J.E., Roederer, M., Letvin, N.L. (2008). Magnitude and Quality of Vaccine-Elicited T-Cell Responses in the Control of Immunodeficiency Virus Replication in Rhesus Monkeys. Journal of Virology, 82(17), 8812-8819. DOI: 10.1128/JVI.00204-08[]
July 21st, 2008

On T cell quality

Our results suggest that some CD8 T cells induced by vaccination are more efficient than others at responding to a viral challenge. These findings have implications for future AIDS virus vaccine studies, which should consider the “fitness” of vaccine-induced T cells in order to generate robust responses in the face of virus exposure.

Limited Maintenance of Vaccine-Induced Simian Immunodeficiency Virus-Specific CD8 T-Cell Receptor Clonotypes after Virus Challenge.   Miranda Z. Smith, Tedi E. Asher, Vanessa Venturi, Miles P. Davenport, Daniel C. Douek, David A. Price, and Stephen J. Kent. Journal of Virology, August 2008, p. 7357-7368, Vol. 82, No. 15 doi:10.1128/JVI.00607-08

For more information see:

July 20th, 2008

Quantity vs quality again

PlasmodiumAll you want in a vaccine is that (1) it doesn’t do any harm, and (2) it prevents disease. When you’re running initial tests on a potential vaccine, though, you often can’t actually include (2) in the tests — especially for a human vaccine– because it’s rarely acceptable to infect your volunteers with, say, HIV. Instead, you identify surrogate measures like level of antibody, or number of T cells, and you judge your vaccine on those surrogate measures at first. If your vaccine doesn’t induce a lot of antibody, say, cytotoxic T lymphocytes (CTL), or whatever it is that you’re measuring, then back to the drawing board.

The problem with that approach, of course, is that we still don’t understand the immune system all that well, and so the surrogate measures that get used may not be ideal. We’ve been seeing this with a number of HIV vaccines, which in preliminary tests induced great surrogate measures but ended up not protecting against disease. This (among other things) has led to a recent focus of quality of immune responses as well as quantity. A recent paper emphasizes this, coming at the issue from a very different direction.

Malaria vaccines haved been a huge challenge to develop. Getting strong immune responses against protective antigens has been pretty difficult, and then moving these into clinical settings has usually shown rather underwhelming efficacy. One of the approaches that was tested, as a way of safely inducing immune responses, was genetic immunization (or DNA vaccines). In this approach, rather than a protein antigen, you inject your patients with DNA encoding the antigen of interest. The DNA gets taken up by cells, expresses your antigen of interest, and hopefully that antigen then induces an immune response.

As I understand it, this approach has worked pretty well in mice, but not so well in humans. Immune responses in humans vaccinated with DNA have usually been very low; so low that (based on these surrogate measures) the vaccines were abandoned and people weren’t challenged with the pathogen.

… it became clear that, for reasons that remain poorly understood, the same high immunogenicity could not be reproduced in humans.. Genetic immunisation of volunteers could induce at best CTL cells but low, or absent, CD4 T-cell and antibody responses. The immunogenicity was so low that, although reported, several clinical trials were considered unsuitable for publication. In many of these trials, challenge by the infectious pathogens was either not possible or decided against in view of the limited responses induced.1

Plasmodium & red blood cellsPierre Druilhe, at the Pasteur institute, decided to test the approach more directly, with a challenge experiment.1 They vaccinated some chimps with a DNA vaccine against a malaria antigen, an approach that had previously led to very weak immune responses in humans. Here again, the immune responses were not very exciting: there was no humoral (antibody) response, though there were detectable MHC class I and MHC class II-restricted T cell responses. But the effect on malaria infection was dramatic; there was 
sterilizing immunity, no detectable parasites, in 3 of the 4 chimps.

The authors comment “these findings suggest that the relative scarcity of an immune response should not necessarily exclude the assessment of the protective efficacy of a vaccine candidate when ethically feasible,” which is a reasonable suggestion, though I think their data don’t really show that this was a “scarce” immune response. Did they really see a low response when you consider T cells, or is it actually a strong response that is strictly biased to the cell-mediated immune response? They didn’t include some information that I’d consider critical — how the T cell responses here compared to a more conventional vaccine approach. They cite an earlier paper2 showing some protection against schistosomiasis in cattle after a DNA vaccine in spite of undetectable antibody responses, to argue that DNA vaccine protecting in spite of a low immune response may be a general effect; but since the schistosomiasis study didn’t even look at T cell responses as far as I can see, it doesn’t answer that question either.

Overall, I think this is not a very strong paper. It’s just a handful of animals (and some of their stats are highly questionable; I don’t think you can challenge 4 animals twice and call that a cumulative N=8), and they don’t show me how the T cell responses compare to other approaches, so I don’t know if it’s even a “weak” immune response. Still, it’s an interesting observation, and emphasizes that it’s important to understand the quality of an immune response as well as quantity.

  1. Daubersies P, Ollomo B, Sauzet J-P, Brahimi K, Perlaza B-L, et al. (2008) Genetic Immunisation by Liver Stage Antigen 3 Protects Chimpanzees against Malaria despite Low Immune Responses. PLoS ONE 3(7): e2659. doi:10.1371/journal.pone.0002659[][]
  2. Field testing of Schistosoma japonicum DNA vaccines in cattle in China. Shi F, Zhang Y, Lin J, Zuo X, Shen W, Cai Y, Ye P, Bickle QD, Taylor MG. Vaccine. 2002 Nov 1;20(31-32):3629-31. []
July 3rd, 2008

Quality vs. quantity in cancer vaccination

XVivo: Cancer cell attackAlthough 750-odd tumor antigens may seem like quite a few potential vaccine targets, it’s really not so much when you’re dealing with billions of individual tumors; and so when designing a tumor vaccine, you may have to make some compromises. The peptide may bind to the MHC class I with low affinity, for example, making it relaitvely non-immunogenic. Several groups who are working on tumor vaccines have tried to work around this problem by optimizing tumor antigens in various way, hoping to boost the immunogenicity while retaining the specificity of the peptide. This has often seemed to work quite nicely, cranking up immune responses significantly while keeping the response focused on the tumor. Nevertheless, a recent study1 suggests that this may not be a good idea.

Melanomes (Wellcome)It has been suggested2 that cancer antigens tend to be poor MHC binders. That would mean that the peptide falls out of the MHC complex relatively rapidly and becomes invisible to T cells, so that to keep a certain level of target on the cell surface, you’d need to start with much more; in other words, the peptide would be less immunogenic. (This has been explicitly shown for a number of tumor epitopes, but as far as I know has not been globally demonstrated. It occurs to me that the Immune Epitope Database [IEDB]   may have enough information to at least make a start at that analysis; maybe I’ll take a run at it, in whatever of my  free time isn’t taken up by playing baseball with my fanatic son.)

If the natural peptide doesn’t bind stably to MHC, perhaps an analog peptide — a peptide with a slightly different amino acid sequence — can be made, with the same T cell recognition properties, that does bind well; and this analog could be used for immunization. That’s just what has been done in a number of clinical trials,3 and the results have actually looked good; for example, mice immunized with an analog peptide of a melanoma tumor antigen generated far more T cells than with the natural antigen. 4

But bigger is not always better, and now Speiser et al. have examined the effects of an analog peptide qualitatively as well as quantitatively. Again using a melanoma antigen, they compared  the immune response to a natural and an analog peptide vaccination. As with other studies, the analog peptide induced more T cells; about twice as many. But the quality5 of the T cells induced by the natural peptides was much better, to the point that the less abundant natural response, was more effective in its anti-tumor function than the more abundant response induced by the analog peptides.

At first, it seems paradoxical that the “less immunogenic” natural peptide induced more strongly functional T cells. … CD8 T cells must be able to recognize low amounts of viral peptide antigen for protection. More recently, in vivo experiments in mice showed that the peptide concentration used for DC labeling and priming inversely correlated with the avidity of TCRs of memory cells. Thus, one may conclude that vaccination should be done with low peptide doses and/or peptides with low HLA binding stability (provided that one can still elicit a reasonably strong T cell response).

(My emphasis.)  This is actually strikingly reminiscent of some of the recent work on viral — especially HIV — immune responses, where T cell quality (induction of “multifunctional” T cells) seems to be more important than maxing out the number of T cells. 6.  I guess it’s not surprising that the anti-cancer and anti-viral responses are similar in this, as they are in many other ways.

  1. Speiser, D.E., Baumgaertner, P., Voelter, V., Devevre, E., Barbey, C., Rufer, N., Romero, P. (2008). Unmodified self antigen triggers human CD8 T cells with stronger tumor reactivity than altered antigen. Proceedings of the National Academy of Sciences, 105(10), 3849-3854. DOI: 10.1073/pnas.0800080105[]
  2. For example: Poor immunogenicity of a self/tumor antigen derives from peptide-MHC-I instability and is independent of tolerance. Zhiya Yu, Marc R. Theoret, Christopher E. Touloukian, Deborah R. Surman, Scott C. Garman, Lionel Feigenbaum, Tiffany K. Baxter, Brian M. Baker, and Nicholas P. Restifo. J Clin Invest. 2004 August 16; 114(4): 551-559. doi: 10.1172/JCI200421695. []
  3. Speiser et al. cite a half dozen instances; I won’t parrot them[]
  4. Parkhurst et al. (1996) Improved induction of melanoma-reactive CTL with peptides from the melanoma antigen gp100 modified HLA-A*0201-binding residues. J Immunol 157:2539-2548. []
  5. Quality in this case means functional ability to deal with the cancer; it includes things like activatability, amount of cytokine production, and amount of lytic proteins produced[]
  6. For example: Induction of multifunctional human immunodeficiency virus type 1 (HIV-1)-specific T cells capable of proliferation in healthy subjects by using a prime-boost regimen of DNA- and modified vaccinia virus Ankara-vectored vaccines expressing HIV-1 Gag coupled to CD8+ T-cell epitopes. Goonetilleke N, Moore S, Dally L, Winstone N, Cebere I, Mahmoud A, Pinheiro S, Gillespie G, Brown D, Loach V, Roberts J, Guimaraes-Walker A, Hayes P, Loughran K, Smith C, De Bont J, Verlinde C, Vooijs D, Schmidt C, Boaz M, Gilmour J, Fast P, Dorrell L, Hanke T, McMichael AJ. J Virol. 2006 May;80(10):4717-28. and references therein.[]
June 19th, 2008

Adjuvants: Quality as well as quantity

Jenner vaccination bookVaccination is one of the (if not the most) important medical advances in history. The problem today is that most of the easy diseases already have vaccines available, and now we’re trying to develop vaccines against the hard ones. Fortunately, I think we’re entering a new golden age of vaccine development, as we begin to understand why immunization works at the molecular level, to the point where we may soon be able to deliberately tweak them for optimal efficacy.

Back in the dark ages, when I was first working with vaccines,1 adjuvants were a witches’ brew of newts’ eyes and frogspawn, and the ones that worked, just sort of … worked. No one really knew why. But around the time I backed away from vaccines, (partly because of this empirical adjuvant stuff) new theoretical frameworks were being developed that began to explain how and why adjuvants work, and now — some 20 years later — we are at the point where theory is moving solidly toward practice.

I’ve commented several times on the issue of immunodominance. T cell responses to antigens aren’t smoothly distributed over all the possible targets in the antigen; instead, a handful of targets get the lion’s share of the T cell response. Sometimes this is a good thing (for example, it’s a way of getting a screaming hot response to a target, instead of having a bunch of wimpy little responses); sometimes it’s bad (if it’s a moving target, as with rapidly-mutating viruses such as HIV, then your screaming hot response may be to a target that no longer exists, whereas having a bunch of targets at least nearly guarantees that you’ve got something to shoot at.)

In spite of its importance, though, the underlying mechanisms that drive immunodominance aren’t well understood. For example, one possible explanation is that the T cell that ends up becoming dominant, started out as the most abundant clone originally. A paper last year2 (I talked about it here) supported that possibility, but a more recent study3 that I talked about earlier this week suggested that while clonal abundance is one factor, there must be other, equally important, influences on the response.

That fits with another paper that came out in May,4 looking at the effects of different adjuvants on the immune response. Of course this has been done many times in a quantitative way — which adjuvant gives the biggest response? — but Malherbe et al. asked the question qualitatively: What exactly happens to the T cell response? That is: We know that different adjuvants can cause higher or lower responses to an antigen; but are the different responses made up of the same CTL5, or do different adjuvants crank up different sets? Can we drive a T cell response that is qualitatively, as well as quantitatively, better?

Smallpox vaccine vialI, for one, (and I think most of the field) would have said “No”; no matter what your adjuvant is, the response would be qualitatively the same. Why would one particular CTL precursor clone be stimulated better or worse by a particular adjuvant? That’s the answer that would be predicted from the first study, that suggested that immunodominance is determined mainly by the precursor frequency: You can’t really affect the precursor frequency (that’s set during thymic development), so no matter what you do with your antigen you should get the same relative response (even though the total response may be higher or lower, it would contain the same proportion of T cell clones).

In fact, that’s not what happens. Malherbe et al. compared five different adjuvants, mixed with the same antigen. The adjuvants are known to act through different mechanisms. (That is, while they all act by stimulating innate immune recognition molecules, they stimulate different innate receptors — different TLR molecules, or [as we now know6 ] pattern recognition receptors that are different from TLRs altogether.) Then they assessed the subsequent immune response by comparing the immunodominance hierarchies that came out of the immunization. The different adjuvants drove expansion of different T cell clones, so that the response was qualitatively different.

In particular, adjuvants drove expansion of higher-affinity clones:

…adjuvants regulate clonal composition by using a mechanism that alters initial TCR-based selection thresholds and that relies most heavily on blocking the propagation of antigen-specific clonotypes expressing low-affinity TCR. … Thus, adjuvant formulation can modify the TCR-based selection threshold that regulates Th cell clonal composition in response to protein vaccination.

How adjuvants do this remains unknown. It wasn’t related to the antigen dose (which has previously been shown to affect the TcR affinity). Possibilities include differential dendritic cell maturation, altering local antigen contentration (the “depot” effect that has been the classic explanation for alum’s mechanism of action — though that explanation is at least partly rendered obsolete by the recent paper7 from Richard Flavell’s group), and direct stimulation of T cell clones — but who knows.

Assuming this holds up for different antigens (they’ve only looked at one, so far) the key thing, in clinical terms, is that it’s possible to alter immunodominance without changing the antigen. We need to understand how this works, because it may be a much simpler way of improving immune responses than altering the antigen itself.

  1. It looks as if I may be doing so again; our proposal for a Vaccine Center here has been funded, at least for a few years; although I’m only a small part of the group[]
  2. Naive CD4(+) T Cell Frequency Varies for Different Epitopes and Predicts Repertoire Diversity and Response Magnitude. Moon JJ, Chu HH, Pepper M, McSorley SJ, Jameson SC, Kedl RM, Jenkins MK. Immunity. 2007 Aug;27(2):203-13.[]
  3. Obar, J., Khanna, K., LeFrancois, L. (2008). Endogenous Naive CD8+ T Cell Precursor Frequency Regulates Primary and Memory Responses to Infection. Immunity, 28(6), 859-869. DOI: 10.1016/j.immuni.2008.04.010[]
  4. Malherbe, L., Mark, L., Fazilleau, N., McHeyzer-Williams, L., McHeyzer-Williams, M. (2008). Vaccine Adjuvants Alter TCR-Based Selection Thresholds. Immunity, 28(5), 698-709. DOI: 10.1016/j.immuni.2008.03.014

    Commentary at:
    Immunity 28:602-604 (16 May 2008) doi:10.1016/j.immuni.2008.04.008
    Preview: Taking a Toll Road to Better Vaccines
    Sharon Celeste Morley and Paul M. Allen[]

  5. CTL: Cytotoxic T lymphocytes[]
  6. Eisenbarth, S.C., Colegio, O.R., O’Connor, W., Sutterwala, F.S., Flavell, R.A. (2008). Crucial role for the Nalp3 inflammasome in the immunostimulatory properties of aluminium adjuvants. Nature DOI: 10.1038/nature06939[]
  7. Eisenbarth, S.C., Colegio, O.R., O’Connor, W., Sutterwala, F.S., Flavell, R.A. (2008). Crucial role for the Nalp3 inflammasome in the immunostimulatory properties of aluminium adjuvants. Nature. DOI: 10.1038/nature06939[]
May 4th, 2010

Does immune evasion allow rapid HIV progression?

How not to be seenI was getting a little concerned and distressed by the lack of evidence for any function of viral MHC class I immune evasion. It’s kind of a relief to see articles demonstrating function coming out.

MHC class I is the target for cytotoxic T lymphocytes (CTL), which are generally believed to be pretty important in controlling viral infection. So when some viruses were shown to block MHC class I in cultured cells, it seemed pretty obvious that this would be a big benefit for the virus. You’d expect these viruses to be exceptionally resistant to CTL, for example.

But when people actually looked in animals (as opposed to in tissue culture), the ability to block MHC class I didn’t seem to do all that much. I’ve summarized some of those experiments here and here. For example, the MHC class I immune evasion genes in adenoviruses and in mouse cytomegalovirus (MCMV) didn’t show much effect on the actual infection at all.1 Mouse herpesvirus 68 (MHV68) had shown an effect, but not at the time point that you might expect — not early after infection, when CTL are kicking in and clearing virus, but rather later on, during the latent phase.2

We all believed there must be a function, because viruses don’t hang on to genes for millions of years unless those genes are important,3 but I was starting to wonder if perhaps we were looking in the wrong places — whether any immune effects might be spillover from some other function, say. But, as I say, we’re starting to get confirmation that these things really are doing more or less what we’d expected all along.

A little while ago, Klaus Fruh and Louise Pickert showed a significant effect of MHC class I immune evasion in rhesus cytomegalovirus: without that ability new viruses couldn’t superinfect hosts that already carry the virus. 4 (I talked about it here.) It’s quite possible — though of course not certain until it’s actually tested — that this is also true for human cytomegaloviruses (which are very closely related to the rhesus version) and for mouse CMV (which are less closely related but in the same family). So now we have functional data for MHC class I immune evasion for representatives of two broad groups of viruses, the betaherpesviruses (the cytomegaloviruses) and the gammaherpesviruses (the MHV68 story).

Now there’s another paper5 showing a function for the MHC class I immune evasion ability of HIV (actually for SIV, but again it’s probably true for the closely-related HIV).

HIV has a gene, nef, that can block MHC class I expression. This has been shown in cultured cells, but understanding its relevance in actual infections has been difficult:

Although these data suggest that Nef-mediated immune evasion could play an important role in AIDS pathogenesis, there has been little direct evidence linking disease progression with MHC-I downregulation in vivo. 5

Obviously you can’t make a nef-less HIV and just throw it into people to see what happens. Even doing the experiment in monkeys with SIV is complicated by the fact that nef is very polyfunctional — as well as downregulating MHC class I, it also targets a number of other molecules.

But you can take advantage of natural variation, both in the virus and the host.  Nef isn’t equally effective on all MHC class I types, for one thing. As well, nef can develop mutations within the host.  It turns out that rapid disease progression correlates with the extent of MHC class I downregulation, whereas effects on other genes affected by nef (CD3 and CD4) didn’t correlate:

The extent of MHC-I downregulation on SIV-infected cells varied among animals …  the level of MHC-I downregulation on SIV-infected cells was significantly greater in the rapid progressor animals than in normal progressors.  … high levels of MHC-I downregulation on SIV-infected cells are associated with uncontrolled virus replication and a lack of strong SIV-specific immune responses.5

This is strictly a correlation study, so we can’t confidently say that MHC downregulation causes disease progression. Still, it’s an interesting finding, and perhaps one that can be followed up in human studies.

  1. Gold MC, Munks MW, Wagner M, McMahon CW, Kelly A, Kavanagh DG, Slifka MK, Koszinowski UH, Raulet DH, & Hill AB (2004). Murine cytomegalovirus interference with antigen presentation has little effect on the size or the effector memory phenotype of the CD8 T cell response. Journal of immunology (Baltimore, Md. : 1950), 172 (11), 6944-53 PMID: 15153514
    Only slightly qualified by
    Lu, X., Pinto, A., Kelly, A., Cho, K., & Hill, A. (2006). Murine Cytomegalovirus Interference with Antigen Presentation Contributes to the Inability of CD8 T Cells To Control Virus in the Salivary Gland Journal of Virology, 80 (8), 4200-4202 DOI: 10.1128/JVI.80.8.4200-4202.2006[]
  2. Stevenson, P., May, J., Smith, X., Marques, S., Adler, H., Koszinowski, U., Simas, J., & Efstathiou, S. (2002). K3-mediated evasion of CD8+ T cells aids amplification of a latent ?-herpesvirus Nature Immunology DOI: 10.1038/ni818[]
  3. I will admit there’s a certain circular quality to this argument.  “The gene must be important, because viruses don’t carry unimportant genes.  We know that, because this gene that they’ve hung on to must be important.”[]
  4. Hansen, S., Powers, C., Richards, R., Ventura, A., Ford, J., Siess, D., Axthelm, M., Nelson, J., Jarvis, M., Picker, L., & Fruh, K. (2010). Evasion of CD8+ T Cells Is Critical for Superinfection by Cytomegalovirus Science, 328 (5974), 102-106 DOI: 10.1126/science.1185350[]
  5. Friedrich, T., Piaskowski, S., Leon, E., Furlott, J., Maness, N., Weisgrau, K., Mac Nair, C., Weiler, A., Loffredo, J., Reynolds, M., Williams, K., Klimentidis, Y., Wilson, N., Allison, D., & Rakasz, E. (2010). High Viremia Is Associated with High Levels of In Vivo Major Histocompatibility Complex Class I Downregulation in Rhesus Macaques Infected with Simian Immunodeficiency Virus SIVmac239 Journal of Virology, 84 (10), 5443-5447 DOI: 10.1128/JVI.02452-09[][][]
April 20th, 2010

Rotavirus vaccine and herd immunity

Rotaviruses are one of the most common causes of gastroenteritis in children.  A new rotavirus vaccine was introduced a few years ago; what impact has it had on disease?

This study confirms on a national scale that the 2008 rotavirus season among children aged <5 years was dramatically reduced compared to pre-RV5 seasons.  …  Based on the observed decrease during the 2008 season, we estimated that ~55,000 acute gastroenteritis hospitalizations were prevented during the 2008 rotavirus season in the United States. A decrease of this magnitude would translate into the elimination of 1 in every 20 hospitalizations among US children aged <5 years.1

(My emphasis)

Here’s what that looks like:

Rotavirus vaccine vs. gastroenteritis

Monthly acute gastroenteritis and rotavirus-confirmed hospitalization rates.  The rotavirus vaccine was introduced in 2006; in 2007 about 3% of children were completely vaccinated; in 2008 about 33% were vaccinated 1

Interestingly, the reduction in gastroenteritis wasn’t only in vaccinated children:

In 2008, acute gastroenteritis hospitalization rates decreased for all children aged <5 years, including those who were either too young or too old to be eligible for RV5 vaccination. …These findings … raise the possibility that vaccination of a proportion of the population could be conferring indirect benefits (ie, herd immunity) to nonvaccinated individuals through reduced viral transmission in the community1

(My emphasis, again)

Assuming this continues to hold up (and similar studies2 have found similar large reductions) it’s a striking example of herd immunity.

(Added later: The vaccine this paper looked at was RotaTeq.  This is not the vaccine that was recently found to be contaminated with porcine circovirus genomic fragments; that was the other rotavirus vaccine, Rotarix.)3

(Second update: RotaTeq apparently also is contaminated with porcine circovirus genomic fragments.)

  1. Curns, A., Steiner, C., Barrett, M., Hunter, K., Wilson, E., & Parashar, U. (2010). Reduction in Acute Gastroenteritis Hospitalizations among US Children After Introduction of Rotavirus Vaccine: Analysis of Hospital Discharge Data from 18 US States The Journal of Infectious Diseases DOI: 10.1086/652403[][][]
  2. For references see
    Weinberg, G., & Szilagyi, P. (2010). Vaccine Epidemiology: Efficacy, Effectiveness, and the Translational Research Roadmap The Journal of Infectious Diseases DOI: 10.1086/652404[]
  3. I haven’t talked about the Rotarix withdrawal because I think it’s been widely and very well covered on other blogs.  (I have 536 papers in my list of things I want to talk about here some time, so I usually don’t bother blogging about findings other places cover in detail.)  Vincent Racaniello at the Virology Blog has his usual high-quality commentary on it here.  He also made an important point on his podcast, This Week In Virology (either number 75 or number 77, I don’t remember which), which I don’t see explicitly on the post: The circovirus-containing vaccine went through all the safety trials, and no problems were seen.

    Obviously circovirus genomes aren’t supposed to be in the vaccine and they’ve got to go.  But (1) we don’t know if the genomes are infectious, or just fragments; (2) there’s no evidence, in spite of centuries of exposure to porcine circovirus, that it has any effects in humans; (3) the vaccines were shown to be safe, at least in the short term.

    On a larger scale, we’re entering a new era of analysis.  I suspect more of this sort of contamination will turn up as the sensitivity of our screening techniques improve, much like chemical detection: As we improve chemical detection to the parts-per-billion and parts-per-trillion level there needs to be better understanding of safety levels. Is this true for biologics? There are good arguments that there may be no safe level for some biologics, and any detection should lead to withdrawal, but on the other hand there clearly is a safe level for other biologics.  Human poop is loaded with vast amounts of viruses of peppers, for example; now that we know that should we regulate pepper mottle virus?

    I don’t have answers, which is why I relegate this to a footnote, albeit a long a rambling footnote.[]

March 18th, 2010

Measles week, part IV: Some of the answers

Workers punish a god of measles
Various workers affected by measles punish a god of measles, while a doctor and drugstore keeper try to protect the god from them. (1862

Well, here we are already at Part IV of Measles Week.  Doesn’t time fly? Remember how young we all were, back at Part I, when I raised the question I’m trying to answer today? And those merry, innocent days of Part II (The origin of measles)? And then Part III (The probably-wrong explanations) — doesn’t it seem just like yesterday?

Today, Part IV is all about the explanations for the spectacular drop in measles case-fatality rates (between 40 and 150 times lower death rate per case of measles) in the first half of the 20th century (chart).  No one of these explanations alone seems to be completely adequate to explain the spectacular decline in measles deaths, but perhaps — probably — the combination of all of them put together, perhaps with contributions from some of the Part III explanations, account for the drop.

Explanations that (might be) right:

  • Better treatment of measles, especially antibiotics. Measles is a viral disease and so not treatable by antibiotics, but it’s the secondary infections that kill; and those could be controlled by antibiotics.
  • Reduction in crowding. There’s evidence that overcrowding, per se, can make severe measles disease more common. Improved living conditions might have helped with this.
  • Demographic changes. This is a little vague, but I have a couple of specific aspects in mind.
  • Nutrition. This is the most popular, and probably most important, explanation. But (to me, anyway) it doesn’t seem to be enough, all by itself.
  • Vitamin A. As a subset of nutrition, and also as a treatment on its own.

Let’s leave nutrition to the last and quickly run through the other explanations first.

First: Better medical treatment of measles patients. As I say, antibiotics probably put a big dent into the toll from secondary bacterial infections.  There were also advances in things like oxygen treatment during pneumonia and so on. Probably important factors, but the problem with this explanation is that by the time antibiotics became available, the trend to reduced measles mortality was already well under way.  You don’t see sudden drops in mortality associated with these things kicking in, just a continuation of the ongoing decline.

Death rates in the 20th century & antibiotics
Antibiotics and 20th-century mortality rates 1

Compare to the chart of overall mortality rates in the 20th century1 (to the left; this is the inset from the larger chart here). It shows two curves being fit to the data — one in the first 30-odd years of the 20th century, one in the second half of the 20th century.  From 1938 to 1953, between those smooth curves, there’s an especially dramatic drop in mortality rates.  That abrupt drop corresponds to the introduction of antibiotics. You don’t see that abrupt drop with measles death rates.

• Reduction in crowding.  This seems like a simple thing, but it’s been proposed (first, I believe, by Aaby and Coovadia,2 in 1985) to be a major influence on measles mortality.

Their observations suggested that severe measles cases are most closely associated, not with malnutrition as you might expect, but with overcrowding. (Obviously, the two are both tightly linked to poverty in general, so pulling them apart is a little tricky.) They argue that you’re more likely to get a large dose of measles virus if you’re crowded together with a measles patient, and that getting more virus at the outset correlates with having more severe disease:

It was found that severe measles was not associated with PEM [protein-energy-malnutrition] but frequently accompanied overcrowding in Guinea-Bissau. Secondary cases fared worse than index patients. … The hypothesis which fits most of the observed facts postulates that the transmission of a large inoculum of virus particles to susceptible children is an important cause of severe disease2

(My emphasis) So, as overall wealth improved in the early 20th century and quality of life became better, children became less crowded, less likely to receive massive doses of virus, and were better able to control the lower doses they did get.

This makes sense, but I don’t think there’s enough of an effect to account for the drop in mortality — again, we need to explain a hundred-fold reduction in the case-fatality rate.  This is probably one significant factor, but not enough to account for everything.

• Reduction in crowding is a part of the next category, Demographic changes. This is much harder, for me anyway, to put together with hard data, but follow me here:

We  know that measles mortality rates are by far the highest in the youngest of its victims.  Children over, say, 5 years old or so are much less likely to die than are infants.3  So, any changes in society that would make measles more likely to infect older — even slightly older — children, would have a massive effect on mortality rates.  We see this even today, where small changes in age at infection lead to significant changes in survival. 4

Meanwhile, we see measles mortality rates beginning to drop just around the time of one of the biggest demographic changes in UK and US society — World War I.  What I don’t know, not being a historian, is just how WWI would affect measles epidemics. Were children mixed more, or less, as their fathers went off to war? Were children taken out of London and other cities, into rural areas, as they were in the second World War?  (We know that measles is an urban disease.)  And so on.  I don’t know enough about population movement and changes in this period to put the story together, but I’m personally convinced that this had a significant effect on measles mortality, and most likely because (somehow) it led to children being infected just a little bit later in their life.

(Edit: In the comments, Tsu Do Nimh [if that’s his real name] points out that 1915 was the time family planning and birth control started.  That’s another potential cause of a significant demographic change toward smaller families, which in turn could lead to exposure to measles at a later age.)

I don’t think this is the whole story, but it does seem to be one of the explanations that (in principle) does have enough power to cause a hundred-fold drop in measles case-fatality rates.

Measles quarentine

• Moving on to the last two categories, which are closely related.  It’s well known, now, that vitamin A deficiency greatly increases the risk of death after measles infection.  And in England, at the least, in the first third of the 20th century, vitamin A deficiency was common,5 especially in the poor (who were almost entirely at risk of measles-related death; measles was never a big risk to the wealthy).

So vitamin A supplementation presumably must have had a big impact on measles mortality, once it became widespread.

But: First of all, vitamin A supplementation didn’t become part of measles treatment until the early 1930s.6 By that time, the case-fatality rate had already started to drop pretty dramatically, and, as always, we don’t see any sudden drop in the death rate that’s associated with any one factor.

Second, the effect doesn’t seem to be great enough — vitamin A supplementation reduces measles mortality about 2 to 3-fold,7 which is great, but nowhere near enough to account for the hundred-fold reduction in death rates we see.

Measles week
Part I: Introduction
Part II: Emerging disease
Part III: Not the answers
Part IV: Some of the answers
Part V: What about the vaccine?

So, yet again: Part of the story, but far from the whole story.

• Finally: Nutrition. It’s very clear that malnourished measles patients do much, much worse than those who are well nourished.  It can be a huge effect, certainly enough to account for the differences in 1910 and 1950 death rates.   Patients in the developing world, today, may suffer case-fatality rates that are much more like 1910 London (10-30% death rates) than 1950 and present-day London (0.025% death rates). 8

But my question — and again, I’m no historian — is, how badly malnourished were children in the 1920s?  The biggest loss in survival comes from the most malnourished children, from children who are severely malnourished. Just “ordinary” levels of malnourishment “only” cause about a 2- to 5-fold difference in survival.9 Yet again, not enough to account for the 100-fold change in survival by 1950.

Were children in England and the US, in 1920, really comparable to severely malnourished third-world children today? Of course it was almost entirely the poor who died; measles even in the 1910s were known to spare the rich and kill the poor. But even so — Am I naive, or were the ordinary working poor in those days really malnourished to the border of famine?

So there are the general explanations for the increased measles survival from 1915 to 1955.  Each of those factors I can easily see causing a 5- or even 10-fold improvement in mortality, but none of them seems, to me, to be enough for the effect we see.  There’s some room for synergistic effects, multiplying survival rates rather than additive (better-nourished patients who are less crowded and therefore receive lower doses of virus, getting better treatment after they do get sick) — but equally, there’s a lot of overlap (vitamin A deficiency isn’t completely separate from overall malnourishment).

(This is why I’d really, really like to see if modern measles virus and 1910 measles virus actually were similar at the genome level, or if there might be some change in the virus after all.)

As I said earlier, I’m not an expert on any aspect of this, and I welcome any corrections.  (But, again, comments that are your opinion aren’t going to be much help; data and references, please.)

  1. Armstrong, G. (1999). Trends in Infectious Disease Mortality in the United States During the 20th Century JAMA: The Journal of the American Medical Association, 281 (1), 61-66 DOI: 10.1001/jama.281.1.61[][]
  2. Aaby, P. (1985). Severe measles: A reappraisal of the role of nutrition, overcrowding and virus dose Medical Hypotheses, 18 (2), 93-112 DOI: 10.1016/0306-9877(85)90042-8[][]
  3. Wolfson LJ, Grais RF, Luquero FJ, Birmingham ME, Strebel PM (2009) Estimates of measles case fatality ratios: a comprehensive review of community-based studies. Int J Epidemiol 38:192–205.[]
  4. Marufu T, Siziya S (1998) Secular changes in rates of respiratory complications and diarrhoea among measles cases. J Trop Pediatr 44:347–350.[]
  5. Semba RD (2003) On Joseph Bramhall Ellison’s discovery that vitamin A reduces measles mortality. Nutrition 19:390–394.[]
  6. JB. E (1932) Intensive vitamin therapy in measles. BMJ 2:708.
    Semba RD (2003) On Joseph Bramhall Ellison’s discovery that vitamin A reduces measles mortality. Nutrition 19:390–394.[]
  7. Madhulika Kabra SK, Talati A (1994) Vitamin A supplementation in post-measles complications. J Trop Pediatr 40:305–307.

    D’Souza RM, D’Souza R (2002) Vitamin A for the treatment of children with measles–a systematic review. J Trop Pediatr 48:323–327.

    Tielsch JM, Rahmathullah L, Thulasiraj RD, Katz J, Coles C, Sheeladevi S, John R, Prakash K (2007) Newborn vitamin A dosing reduces the case fatality but not incidence of common childhood morbidities in South India. J Nutr 137:2470–2474.

    Mishra A, Mishra S, Jain P, Bhadoriya RS, Mishra R, Lahariya C (2008) Measles related complications and the role of vitamin A supplementation. Indian J Pediatr 75:887–890.[]

  8. Alwar AJ (1992) The effect of protein energy malnutrition on morbidity and mortality due to measles at Kenyatta National Hospital, Nairobi (Kenya). East Afr Med J 69:415–418.

    Latham MC (1975) Nutrition and infection in national development. Science 188:561–565.

    Morley D (1983) Severe measles: some unanswered questions. Rev Infect Dis 5:460–462.

    Kaler SG (2008) Diseases of poverty with high mortality in infants and children: malaria, measles, lower respiratory infections, and diarrheal illnesses. Ann N Y Acad Sci 1136:28–31.[]

  9. Alwar AJ (1992) The effect of protein energy malnutrition on morbidity and mortality due to measles at Kenyatta National Hospital, Nairobi (Kenya). East Afr Med J 69:415–418.[]
March 15th, 2010

Measles week, part I: Introduction

Zhong Kui punishing two gods of measles.
Zhong Kui, a Chinese god, punishing two gods of measles (1862)

I’ve talked before about measles incidence and the effect of vaccination.  Now I’m going to spend this whole week talking about measles deaths, because I ended up with more than I could cover in one or two posts.  So this is Part I of a five-parter.

A group of diseases which … even now are considered to be unavoidable are scarlet fever, measles, and whooping cough. … According to the statistics collected in the census of 1900, these three diseases were responsible for upward of thirty thousand deaths in the course of a year.”

–“The Conservation of the Child”, by Earl Mayo.  in The Outlook. A Weekly Newspaper. Volume XCVII.  January-April, 1911 (pp. 893-903)

That was the situation in 1911 and in the early 20th century generally, and for centuries before that.  Almost every child caught measles, and a lot of them died.  Measles wasn’t quite as lethal as smallpox, but it wasn’t too far behind:

Measles should no longer be considered a “minor” infection. It is a major illness causing a considerable mortality and a much greater morbidity among young children affected by it. 1

(By the way, as well as citing my direct quotes in footnotes as usual, I’ve collected the 40-odd references I read while trying to figure this story out and put them up here.)

But, starting somewhere around 1915, that began to change.  Very gradually (so gradually that it almost escaped attention) measles stopped being a fatal disease.  In 1945, William Butler said:

In three-score years or so, during which the population of England and Wales has nearly doubled, the gross annual contribution of deaths from measles has fallen to about one-twelfth of the mean figure at which during several quinquennia it stood in the eighties and nineties of the last century.  Nor is there reason to believe–on the contrary–that measles is now less prevalent than it was. It is still true that nearly everyone at one time or another has measles. 2

And the trend didn’t stop there.  In 1945, about 163 out of every 100,000 measles cases died.  In 1955, just 25 of 100,000 died, and it’s hovered around there since.

In other words, a person who caught measles in 1900 was between 40 and 150 times more likely to die than someone who caught the virus in 1955.  You can play with the numbers in various ways, but no matter what you do there has been an absolutely, spectacularly, incredible drop in measles case-fatality rates.

Below are a couple of charts to illustrate this.  The UK (number of measles deaths in England and Wales) is on the left, the US (measles deaths per 100,000 population) on the right (click for larger versions).  The dashed blue lines are the actual numbers.  Because measles is a very, very epidemic disease, the numbers change hugely every year, so I’ve applied a crude smoothing to the raw numbers (the green solid lines) to make the trends easier to see.  My US numbers only go up to 1940, but here’s a chart through the 1960s, if you like – there’s no surprises in it, it’s pretty much like the UK.

US Measles deaths - 20th century US Measles deaths - 20th century
Measles deaths in the UK, 1900 – 1965 Measles deaths in the US, 1900 – 1940

Important note: In England, measles was not a notifiable disease between 1919 and 1939 (and I believe the rules for notification changed for a few years before 19193 as well), and the effect of this is easily seen — the abrupt drop in reported deaths just before 1920, and the flatter line for a few years afterward, is almost certainly not real (I’ve boxed that non-notifiable period off in red.)  But the overall trend is still easy to see even so.

This had nothing to do with the measles vaccine, because this survival increase happened entirely before the vaccine was available in 1963.  There was essentially no change in the number of measles cases over this period (adjusted for population, of course), it’s just that once you caught measles you weren’t as likely to die.  And case-fatality rates didn’t change significantly after the vaccine was introduced.  The death rate per case in 1955 (pre-vaccine) is pretty much what we see today in first-world measles outbreaks.

The vaccine did spectacularly reduce the number of cases, of course, and therefore did reduce the total number of deaths.  Also, equally obviously, vaccines aren’t only given to prevent deaths.  Even if measles doesn’t actually kill your child, she’ll still, quite possibly, be pretty sick; there’s a pretty good chance she’ll be hospitalized; and a significant number of survivors have some form of medium- or long-term complications.

Was measles unusual? Overall mortality, and especially childhood mortality due to disease, did drop over this period, and quite dramatically so:

The infant mortality rate has shown an exponential decline during the 20th century.  … For children older than 1 year of age, the overall decline in mortality during the 20th century has been spectacular. … Between 1900 and 1998, the percentage of child deaths attributable to infectious diseases declined from 61.6% to 2%.  4

Here’s the famous chart of 20th-century mortality.5  (Note the brief, huge spike in 1918, due to the 1918 pandemic influenza!)

20th century mortality rates
Mortality rates in the US through the 20th century

So yeah, in general mortality rates did improve greatly since 1900, flattening out in the 1950s, just the same pattern as with measles deaths.  But check the scale, and compare to the UK (especially) chart above: Measles outpaced this overall improvement, and by a huge amount.  Overall, during this period childhood mortality rates improved perhaps 8-10-fold — clearly a tremendous improvement, but still, at best a quarter of the improvement in measles survival. (And measles was a late starter, too — overall mortality had been dropping for at least 15 years before measles case-fatalities started to go down.)

So what happened between 1915 or so, when measles death rates began their decades-long drop, and 1955, when the drop stopped?  That’s the subject of this entire week’s worth of posts, but to give you a peek at the answer I came up with: It beats the hell out of me. There really isn’t a single, simple explanation for this, as far as I can find.

(I’m not a historian, a medical doctor, or a measles researcher, and I’m more than happy to be corrected.  Anyone who has actual information on this, please let me know.  If you have an opinion, no offense, but I’m not interested unless you have data to back it up.)

The problem is that the usual answers are either too vague to be useful (what exactly does “quality of living” mean, medically?) or inadequate (improved nutrition is certainly important, but as far as I can see probably only improves survival maybe 5-fold, not 100-fold).  Specific advances (antibiotics, etc) undoubtedly helped, but you don’t see abrupt short-term drops in mortality, as you’d expect if any single advance was a major factor; rather, you see a constant, gradual, improvement.

I’m left with the unsatisfying conclusion  that either a conglomeration of many factors may have acted together (the most likely situation, and that’s what the real world is often like — no simple answers), or that there’s some specific factor that I haven’t found out about.  I’ll talk about specific causes later this week.

Here’s my plan for Measles Week:

  1. Monday: Explanation of the question, and evidence for it being a real question. Done!
  2. Tuesday: History of measles virus
    • Origins and impact
  3. Wednesday: Answers that are (probably) wrong
    • Changes in surveillance or notification
    • Sanitation
    • Change in the virus
    • Antiserum treatment
  4. Thursday: Answers that (might be) right
    • Nutrition
    • Vitamin A
    • Less overcrowding
    • Antibiotics and other treatments
    • Demographic changes
  5. Friday: What would measles be like today, without the vaccine?
    • Mortality and complication rates in modern 1st-world epidemics

Put on your party hats, blow up a balloon, pull up a chair, and stick around.

  1. Prevention of Measles in a Children’s Hospital. W. E. Crosbie. Br Med J 1938;1:1003-1004[]
  2. The Fatality Rate of Measles: A Study of its Trend in Time William Butler Journal of the Royal Statistical Society, Vol. 108, No. 3/4 (1945), pp. 259-285[]
  3. Butler W (1945) The Fatality Rate of Measles: A Study of its Trend in Time. Journal of the Royal Statistical Society 108:259–285.[]
  4. Guyer B, Freedman MA, Strobino DM, Sondik EJ (2000) Annual summary of vital statistics: trends in the health of Americans during the 20th century. Pediatrics 106:1307–1317.[]
  5. Armstrong, G. (1999). Trends in Infectious Disease Mortality in the United States During the 20th Century JAMA: The Journal of the American Medical Association, 281 (1), 61-66 DOI: 10.1001/jama.281.1.61[]
January 22nd, 2010

A flood of DRiPs

"Untitled (Green Silver)” - Jackson Pollock
“Untitled (Green Silver)” – Jackson Pollock

In the past few weeks not only did I post a short update on the DRiPs hypothesis here, but coincidentally a bunch of papers on DRiPs have also been published. I’ll probably cover some of these in more detail at some point, but here are some of the recent papers and my brief comments.

Just as a reminder: the DRiPs (“Defective ribosomal products”) hypothesis proposes that most of the peptides presented to cytotoxic T lymphocytes don’t come from the actual proteins that we normally measure — rather, the immunologically relevant peptides come from deformed and defective proteins that are mis-read and misfolded during their translation. (More explanation of DRiPs here and here; more explanation of how T cells recognize cells and where peptides come in, here.)

Jon Yewdell’s insight,1 which is still somewhat controversial, was that defective proteins may actually be very common. Instead of being rare and abnormal events, he argued, protein production is a highly error-prone business, and a large fraction of newly synthesized proteins are broken. These defective products are very rapidly recycled into peptides and amino acids, and because of this rapid recycling they are the major source of peptides for T cell recognition.

On his original publication I had no problem with the underlying concept, but wasn’t overwhelmed by the data, and felt that there were too many counterexamples; since then he, and others, have put forward more and more examples, and I think it’s also fair to say that Jon has softened a little on the original hypothesis.2 I’m more or less convinced that DRiPs are one important source of peptides, though I remain dubious that they are the only, or (and here I get very uncertain) even the major source.

Anyway, in the past few weeks, we’ve seen these papers:

  • The Synthesis of Truncated Polypeptides for Immune Surveillance and Viral Evasion3

This is from Nilabh Shastri, and it’s not a big conceptual departure from some of his previous work. He’s argued for quite a while that aberrant proteins are major sources of T cell targets (see my posts here and here, for examples). Here he extends the argument to the EBNA1 protein from Epstein-Barr virus. This is a remarkably interesting protein for many reasons, one of which is that there’s reason to believe that DRiPs must be the only real source of T cell targets from EBNA1. Here, Shastri shows that in fact DRiPs (in the forms of truncated synthesis products) are in fact targets for T cells (“Thus, translation of viral mRNAs as truncated polypeptides is important for determining the antigenicity of virus proteins“). (I don’t know if it’s fair to generalize to all viral mRNAs from this very unusual protein, though.)  Very intriguingly, he also shows that DRiPs seem to be specifically blocked by EBNA1 mRNA!

Regulating production of DRiPs at the level of mRNA translation may serve as an immune evasion strategy for latent viruses. …  It is tempting to speculate that episome maintenance proteins, found in herpesviruses of various species, might have evolved to inhibit pMHC I presentation by interfering with production of DRiPs.

Is this a new viral immune evasion mechanism? And if so, how widespread is it? I know Nilabh (or someone from his lab) reads this blog occasionally, and I’d be interested in hearing their ideas on this — is it pure speculation, or do they have reason to extend the observation?

  • Viral adaptation to immune selection pressure by HLA class I–restricted CTL responses targeting epitopes in HIV frameshift sequences4
HIV-1 frameshift inducing element
HIV-1 frameshift inducing element

These authors looked at proteins produced by reading frame shifts from HIV.  Although HIV does a lot of frame-shifting “deliberately”, here we’re looking at frame-shifts that are (probably) not “real”.  That is, while it’s possible that some of these proteins have a biological function, for the most part they’re probably nonsense proteins, the product of incorrect selection of reading frames by the ribosome, and therefore you’d expect them to be recognized as improper proteins by the quality-control system and rapidly destroyed. In that sense they fit into the “DRiPs” concept. This fits neatly with Shastri’s previous work on frame-shifting, as well as providing modest support of the DRiPs concept.

The interesting thing here is that this paper offers evidence for large-scale immunological importance of peptides from frame-shifted proteins.  Shastri has previously shown convincing evidence that peptides derived from frame-shifted proteins can be recognized by T cells, but I always wondered if that was just a test-tube novelty. In this paper, though, Berger et al. argue that these frame-shifted potential targets show evidence of evolutionary selection, suggesting that they are recognized often enough to be a significant factor in the viral life-cycle.

  • CD8 T cell response and evolutionary pressure to HIV-1 cryptic epitopes derived from antisense transcription. 5

And this is a very similar paper, showing the same thing for antisense-derived peptides. Like the frame-shifted proteins discussed above, these antisense proteins would probably be nonsense and rapidly degraded — defective ribosomal products, in other words — and again, there’s some evidence that these are under immunological selection, suggesting that this recognition is a real-world phenomenon.

These findings indicate that the HIV-1 genome might encode and deploy a large potential repertoire of unconventional epitopes to enhance vaccine-induced antiviral immunity.5

  • The antiviral factor APOBEC3G improves CTL recognition of cultured HIV-infected T cells. 6

This is a particularly cool paper.7 We know that APOBEC3G — a host protein that evolved, apparently, to provide protection against infection with retroviruses such as HIV — acts by driving hypermutation of infecting retroviral genomes. HIV resists this effect through its protein vif, which in turn drives rapid degradation of several APOBECs.

But in spite of this vif-mediated protection, it’s probably true that APOBECs still have some effect on HIV, especially very early in an infection before vif can take them out; so there’s a background of mutation in HIV driven by APOBECs. This paper shows that APOBEC-driven mutation improves T cell recognition of HIV-infected cells, and the effect is probably because the mutations force HIV to make even more defective proteins, so that there are more T cell targets. This was done in rather an artificial system (mainly by either eliminating vif altogether, or by cranking up the levels of APOBEC3G artificially), so it’s not clear how important it would be in a natural infection.

I also wonder if this argues against the notion that DRiPs are normally a big factor, because if so the background of DRiP-derived peptides should be quite high and increasing it might not be a big factor; but that’s a quantitative issue that’s hard to deal with. Still, an interesting take on antiviral effects.

  • Defective Ribosomal Products Are the Major Source of Antigenic Peptides Endogenously Generated from Influenza A Virus Neuraminidase 8
"Drips" (Inger Taylor)
“Drips” (Inger Taylor)

This is the paper that most explicitly tests DRiPs, which is not surprising, since it comes from Jon Yewdell himself.9 The paper starts with quite a fair summary of the hypothesis’s status, including some of the problems with previous experiments:

In all of these studies, we used recombinant vaccinia viruses (VVs) to express SIINFEKL-containing source Ags. It is possible that we grossly overestimated the contribution of DRiPs to Ag processing in these studies due to the use of VV to express non-VV genes. We recently showed that differences in viral translation mechanisms can greatly increase the fraction of DRiPs; expression of influenza A virus (IAV) nuclear protein by an Alphavirus vector resulted in the defective translation of >50% of nuclear protein recovered from cells. VV expression is known to modify the Ag processing pathway of some inserted viral gene products compared with their natural infection context. Further, the fusion of multiple genes to create chimeric proteins can greatly decrease the fidelity of protein synthesis or protein folding …8

In an attempt to get around some of these problems, they tried to come up with a more natural system.  What they built is more natural, but still is fairly artificial (as they acknowledge); still, their findings did add more support to the basic idea. (As a sign that Jon has softened his position some in the past decade, their comment “Although DRiPs are clearly a major source of antigenic peptides, it is important to recognize that peptides are also generated from natural protein turnover” is one that I think all but the most hardened anti-DRiPers would agree with; it’s coming down to a question of quantitation, of what “major” actually means, rather than absolutes.)

I still suspect that there are cases where DRiPs are critical, and cases where they’re not particularly important, and I don’t have a good sense for how many instances of each there are. My gut feeling is about half and half, but it’s not something I’d defend with my life.

  1. Yewdell, J. W., Aton, L. C., and Benink, J. R. (1996). Defective ribosomal products (DRiPs): A major source of antigenic peptides for MHC class I molecules? J. Immunol. 157, 1823-1826[]
  2. Which has made it a bit of a moving target when it comes to disproving it, unfortunately[]
  3. Cardinaud, S., Starck, S., Chandra, P., & Shastri, N. (2010). The Synthesis of Truncated Polypeptides for Immune Surveillance and Viral Evasion PLoS ONE, 5 (1) DOI: 10.1371/journal.pone.0008692[]
  4. Berger, C., Carlson, J., Brumme, C., Hartman, K., Brumme, Z., Henry, L., Rosato, P., Piechocka-Trocha, A., Brockman, M., Harrigan, P., Heckerman, D., Kaufmann, D., & Brander, C. (2010). Viral adaptation to immune selection pressure by HLA class I-restricted CTL responses targeting epitopes in HIV frameshift sequences Journal of Experimental Medicine, 207 (1), 61-75 DOI: 10.1084/jem.20091808[]
  5. Bansal, A., Carlson, J., Yan, J., Akinsiku, O., Schaefer, M., Sabbaj, S., Bet, A., Levy, D., Heath, S., Tang, J., Kaslow, R., Walker, B., Ndung’u, T., Goulder, P., Heckerman, D., Hunter, E., & Goepfert, P. (2010). CD8 T cell response and evolutionary pressure to HIV-1 cryptic epitopes derived from antisense transcription Journal of Experimental Medicine, 207 (1), 51-59 DOI: 10.1084/jem.20092060[][]
  6. Casartelli, N., Guivel-Benhassine, F., Bouziat, R., Brandler, S., Schwartz, O., & Moris, A. (2009). The antiviral factor APOBEC3G improves CTL recognition of cultured HIV-infected T cells Journal of Experimental Medicine, 207 (1), 39-49 DOI: 10.1084/jem.20091933[]
  7. I’m presenting this one on Friday in the Immunology Journal Club I run here.[]
  8. Dolan, B., Li, L., Takeda, K., Bennink, J., & Yewdell, J. (2009). Defective Ribosomal Products Are the Major Source of Antigenic Peptides Endogenously Generated from Influenza A Virus Neuraminidase The Journal of Immunology, 184 (3), 1419-1424 DOI: 10.4049/jimmunol.0901907[][]
  9. Interestingly, it looks as if Jon has turned his attention back to influenza viruses in the past year — he cut his teeth on influenza, quite a number of years back, but it hasn’t been his main focus for a while. I guess H1N1 gave him the excuse he needed to move back that way.[]