Mystery Rays from Outer Space

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

June 17th, 2010

Dendritic cells that don’t prime

Langerhans cells in the skin
Dendritic cells in the skin (Langerhans cells) form a dense network of “sentinels” that act as first line of defense of the immune system.1

There’s a lot of interest in using dendritic cells as vaccines these days.  A paper in PLoS One2 offers a cautionary note.

Dendritic cells (DC) are the main cell type that drive T cells from their normal naive state to an active state.  In the naive state, a T cell can recognize its target, but doesn’t do anything about it; in the active state, the T cell does something, ranging from spreading inflammation to killing infected cells, and so on.  The DC is needed to bridge these states.  DC do many things, but at the simplest level they connect  the presence of an antigen (a T cell target, in this case) with the presence of something dangerous or abnormal — a pathogen, or tissue damage.

There are some conditions where we’d like an immune response, where DC don’t detect one or the other of their components (i.e. antigen or danger).  For example, there may be a situation that we know is dangerous, but where there’s  little evidence of “danger” for the DC.  A vaccine, for example, doesn’t want to deliver a huge amount of tissue damage, but we’d still like to get a strong response to an antigen.  For a natural situation, cancers are often ignored by the immune system even though there may be lots of cancer antigens, and one reason (of many) for this ignorance is that the DC may not perceive a lot of danger in the context of the cancer.

So why not take the DC out of the system, alarm them with some danger information in the test tube, load them up with antigen, and then return them to the body? That’s called a dendritic cell vaccine, and there’s fairly intense interest in the approach.

There’s been some success using this approach, but perhaps less than you’d expect from the biology as we understand it.

Several clinical trials conducted over the past decade have demonstrated that DC vaccines can prime and boost antigen-specific CD8+ T cells in humans. However, their clinical efficacy remains to be definitively demonstrated [6], [19], [20], [21]. The lack of success has been variously attributed to several factors: administration of relatively low cell numbers of DCs, suboptimal route of administration, improper antigen dose, poor choice of antigenic targets, unsuitable maturation state of DCs, and inappropriate frequency of injections. However, understanding exactly which of these concerns represent true problems may be difficult because little is known regarding the fate and function of ex vivo generated DCs after they have been injected 2

Dendritic cell

Yewdall et al asked what happens to DC after they’re given this course and returned to the patient (mice, in this case).  Their surprising conclusion is that the DC don’t work to prime T cells directly.  Instead, they have to hand off their antigens to other cells in the body that have never left:

Contrary to previous assumptions, we show that DC vaccines have an insignificant role in directly priming CD8+ T cells, but instead function primarily as vehicles for transferring antigens to endogenous antigen presenting cells, which are responsible for the subsequent activation of T cells. … This reliance on endogenous immune cells may explain the limited success of current DC vaccines to treat cancer and offers new insight into how these therapies can be improved. Future approaches should focus on creating DC vaccines that are more effective at directly priming T cells, or abrogating the tumor induced suppression of endogenous DCs. 2

As always in science, a single paper needs to be confirmed by others, so we won’t get too distressed until we see if other groups replicate this, and if it’s a universal truth or something specific to the particular system these authors were looking at.  (And, of course, this doesn’t trump actual evidence of efficacy for DC vaccines.) My own suspicion is that the work is accurate but limited, and there’s something about this particular system which prevented the transferred DC from being good primers; but as I say, I’d like to see some followup from another group.

  1. Tolerogenic dendritic cells and regulatory T cells: A two-way relationship. (2007) Karsten Mahnke, Theron S. Johnson, Sabine Ring and Alexander H. Enk. J of Derm Sci 46:159-167 doi:10.1016/j.jdermsci.2007.03.002 []
  2. Yewdall, A., Drutman, S., Jinwala, F., Bahjat, K., & Bhardwaj, N. (2010). CD8+ T Cell Priming by Dendritic Cell Vaccines Requires Antigen Transfer to Endogenous Antigen Presenting Cells PLoS ONE, 5 (6) DOI: 10.1371/journal.pone.0011144[][][]
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 22nd, 2010

Modeling disease and epidemics

Blyuss & Kyrychko, Fig. 5
Fig. 5.  Boundary of the Hopf bifurcation of the endemic steady state … 1

I don’t pretend to be a mathematician or to understand the more complex disease models that are out there, but I do think modeling is an essential way of understanding how to effectively deal with diseases.  A recent paper1 looks at epidemic diseases and seems to reach some interesting conclusions (though I will cheerfully admit that I don’t even remotely understand this paper, which is heavily mathematical).

The authors have built on models of infectious disease that incorporate immunity to the disease, and incorporated the assumption that immunity to the disease can wane over time, as opposed2 to the simpler, but less realistic, assumption that the immunity is either on or off.  I don’t think they are the first to do this, and I don’t understand any of the details of how their techniques differ from other models,3 but what I think they’re saying is that temporary immunity can lead to disruption of a simple, constant level of infection, and can actually drive periodic epidemics:

[W]hen the temporary immunity period is within a certain range, there will be periodic outbreaks of epidemic, and the disease will not be eradicated from the population. … The main feature is that temporary immunity leads to a possible destabilization of endemic steady state, and an interesting open question is what effects would vaccination have on the dynamics of an epidemic in such situation. 1

(My emphasis)  If I’m understanding this correctly, it leads to the possibility that where vaccines lead to relatively short-term immunity compared to the natural infection,4 it’s conceivable that vaccination could actually shift the disease from a steady state to an epidemic mode.  Obviously, if this can happen, it would be nice to be able to predict it.

Offhand, I can’t think of any examples where this might have happened in real life.  The most notorious epidemics, like influenza and norovirus, both tend to have fairly short-term immunity to start with. Something like Marek’s Disease of chickens would be an interesting case study, but the logistics of the poultry agribusiness is going to have a bigger impact than the vaccine (I would think).  The chicken-pox vaccine is the best example I can think of for a vaccine with relatively short-term immunity where the disease was endemic before the vaccine, and we’re not seeing any sign, that I know of, that chicken-pox is entering an epidemic situation.

The more relevant situation, I think, is for the natural epidemics.  As I say, both influenza and norovirus are well known for short-term immunity from natural infection, so maybe this is a factor there.  On the other hand, measles, which is spectacularly epidemic, has pretty long-term immunity from both the vaccine and the natural disease, and I don’t see any sign that the vaccine changed the personality of measles epidemics qualitatively (though of course, quantitatively the epidemics are much smaller now).

On the other other hand, of course it’s entirely possible that I completely misunderstand this paper, so if someone has a better grasp than I do please feel free to correct me.

  1. Blyuss, K., & Kyrychko, Y. (2009). Stability and Bifurcations in an Epidemic Model with Varying Immunity Period Bulletin of Mathematical Biology, 72 (2), 490-505 DOI: 10.1007/s11538-009-9458-y[][][]
  2. I think[]
  3. “For numerical bifurcation analysis of system with weak and strong kernels, we use a Matlab package traceDDE, which is based on pseudo-spectral differentiation and allows one to find characteristic roots and stability charts for linear autonomous systems of delay differential equations … “[]
  4. This is true for some vaccines, though not all[]
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.[]

April 15th, 2010

Living in the future: Mouse TcR clones

T cell receptor (top) interacting with MHC

It would be nice if I could claim that advances in biology are driven by pure intellectual processes, by hermits on mountaintops achieving new theories through mediation and  deep, pure thoughts. Of course, that’s not the case.  I think its fair to say that many, if not most, of the advances in immunology and virology are driven by new technology. Every so often, some lab comes up with some new way of looking at cells (say, multicolor intracellular staining and flow cytometry) or measuring something about the cells (MHC tetramers, maybe), and we go back to look again at the problems we’ve been struggling, using this new approach, and sometimes the new approach cracks open the problem (usually revealing new and even more interesting problems inside, but that’s why we do this, right?)

(I’m not trying to say that all the advances in the field are technique-driven. Charlie Janeway’s “Dirty Little Secrets” essay didn’t rely on new techniques, and neither did the concept of cross-priming, or lots of others. I’m just saying that new techniques do have a huge influence.)

A particularly cool new technique was just described by Hidde Ploegh, in association with Rudi Jaenisch. 1 Basically, it’s a new way of making TcR-transgenic mice.  TcR transgenics have been around for a long time2 and have led to a quite a few advances in immunology  — they’re now just another tool that’s used in lots of basic research.

thymocytes in the hthymus
Thymocytes developing in the thymus

But making a TcR transgenic mouse is a fair bit of work.  You need to find the T cell you’re interested in, clone out both TcR chains, clone them into the right transgenic vector and transfer them into a stem cell, then make a mouse from that and usually backcross it to a RAG knockout for a dozen generations before you can actually use it. And then you can ask whatever question you had, a couple of years ago when you started all this.

(If that didn’t mean much to you: The TcR is the T cell receptor. It’s what makes T cells specific. Each T cell as it comes out of the thymus has its own, distinct receptor.  It’s distinct at the protein level, and the reason its distinct is that the genome of the T cell is also unique.  The genome of T cells gets sliced and diced and glued back together in a unique way.  If you want to get a duplicate of that receptor you grab that DNA, for both halves of the receptor [that is, the alpha and the beta chains] and plunk it back into other t cells, and hen those t cells will all recognize the same thing.  This is wildly oversimplified, of course, but it’s close enough.)

Ploegh’s group figured a way around most of this, just by cloning a mouse straight from the T cell.

(By the way, saying “just cloning a mouse” — I know we’re living in the future right now. 3  It reminds me of when I was in Worcester, in the early 2000s when Advanced Cell Technology was cloning cattle, seeing a group of protesters at the corner of my street holding signs protesting cloning.  “Ban cloning! No to flying cars! Martians go home!”)

T cell receptor
T cell receptor

Anyway, here in the future, we, or at least Jaenisch, have reached a point where they can quite routinely clone mice from somatic cells; that is, from skin cells or from, say, T cells.  So that’s what they did. They took a T cell that recognized the specific antigen they were interested in,4 and used that to clone out a mouse.  Since the T cell had already undergone its genome rearrangement (and since that can only happen once), all the cells in the new, cloned, mouse ended up with the properly rearranged DNA.  That means the TcR in these mice is fixed, so all the T cells in these mice will recognize the antigen you want, instead of several trillion different antigens.

Essentially these are TcR transgenics, only faster and better.  Better, because, for example, there’s no endogenous TcR to eliminate, so no back-crossing to RAG mice — though they did have to do some back-crossing — and the TcR gene is in the right place under all the right regulation and so on.  They also point out that the standard procedures for making TcR has to start with activated T cells that have been through repeated rounds of stimulation, whereas this approach lets you start with naive cells.  I’m not quite sure this is a huge factor, but I won’t argue the point.

It’s one of those things that seems fairly obvious once it’s done, but (at least to me) was not at all obvious until they actually did it.  I have a feeling that it’s probably not quite as easy as they made it sound, but is still doable by most labs if they really want to do it; so I think it’s something we’re going to see quite a bit of in the next few years. It should help move the mouse field into testing more relevant and accurate systems.

The time-consuming generation of transgenic mouse models has led to the widespread use of a limited number of surrogate antigens, such as ovalbumin (recognized by the OT-I and OT-II transgenic mice) to study the immunobiology of infectious disease. Pathogens engineered to produce fragments of ovalbumin, and the immune reaction against it, are unlikely to capture all essential aspects of the physiological response. 1

  1. Kirak O, Frickel EM, Grotenbreg GM, Suh H, Jaenisch R, & Ploegh HL (2010). Transnuclear mice with predefined T cell receptor specificities against Toxoplasma gondii obtained via SCNT. Science (New York, N.Y.), 328 (5975), 243-8 PMID: 20378817[][]
  2. I think the first one was von Boehmer’s, in 1988:
    Kisielow P, Blüthmann H, Staerz UD, Steinmetz M, & von Boehmer H (1988). Tolerance in T-cell-receptor transgenic mice involves deletion of nonmature CD4+8+ thymocytes. Nature, 333 (6175), 742-6 PMID: 3260350[]
  3. Also, living in the future-wise, I wrote this a first draft of this on my iPad while sitting at my son’s soccer practice. (The iPad turns out to be fine for typing, but not so much for WordPress input — links and images are a problem.)  []
  4. This is the new part — previous clones have been made from lymphocytes, but the target was unknown.[]
April 10th, 2010

Immune databases and hypotheses

The folks associated with the IEDB (Immune Epitope Database) have published a very nice and useful guide to all the serious contenders in the immune database field.  1 If you have a particular need, this is an excellent starting point for choosing the appropriate starting point.  (It’s an open access article, too.)

They’ve obviously looked in a lot more depth than I have, but they make a few comments that my more limited assessment strongly supports:

… our survey highlights clear shortcomings in the predictive tools available. Namely, MHC class II and B cell epitope predictive tools merit improvement, both in terms of predictive performance and, for MHC class II, in terms of coverage of species and alleles currently available. 1

They comment that most (80%) of citations of the databases are attributable to “practical applications”, which I take to mean direct use of the prediction tools (identification of epitopes in new flu strains, for example), construction of new tools (e.g. better prediction of epitopes), and maybe papers that review the databases (which is rather circular, I think).

Hearn et al 2009 FIgure 8
FIGURE 8. Aminopeptidases influence amino acid frequency N-terminal of naturally presented MHC I epitopes. Regions N-terminal to naturally processed MHC I epitopes, or selected randomly from protein pre- cursors, were identified as described in Materials and Methods. A, Probability of divergence occuring randomly (Chi2 test) vs position relative to epitope start site. B, Observed amino acid frequencies at position 1 (P1) relative to epitope start vs no divergence from back- ground (45-degree line). The amino acids that diverge +/-2 SDs from background frequency are indicated.2

The other 20% of citations are, I guess, using the databases to generate and test hypotheses.   This seems high, to me.  I don’t think I’ve seen very much basic science in immunology that builds on this sort of resource.  I think we’re reaching the point where these databases are usable to test and develop new hypotheses, though, and I hope to see more of this in the near future.

One example is our recent paper,2  where I used the IEDB to ask what influence ER aminopeptidases have on MHC class I epitopes (see the Figure to the left). (If you care, we concluded that aminopeptidases were probably most important for trimming N-terminal extensions of up to three residues, and that there was a global preference for a half-dozen amino acids and a bias against valine and, of course, proline — proline is resistant to aminopeptidase trimming in general, so that finding supported the approach.)

We weren’t the first to use this general approach (Schatz et al3 came up with the same idea independently and published before we did) but we used the IEDB, instead of the SYFPEITHI database, and were able to identify many more epitopes.   (My last run at the database coincided with the database being revised and half the search tools I needed stopped working, which was annoying, but the manager [Randi Vita] was very helpful and we managed to grind through the queries, albeit in slow motion compared to earlier runs.)

  1. Salimi, N., Fleri, W., Peters, B., & Sette, A. (2010). Design and utilization of epitope-based databases and predictive tools Immunogenetics, 62 (4), 185-196 DOI: 10.1007/s00251-010-0435-2[][]
  2. Hearn, A., York, I., & Rock, K. (2009). The Specificity of Trimming of MHC Class I-Presented Peptides in the Endoplasmic Reticulum The Journal of Immunology, 183 (9), 5526-5536 DOI: 10.4049/jimmunol.0803663[][]
  3. Schatz MM, Peters B, Akkad N, Ullrich N, Martinez AN, Carroll O, Bulik S, Rammensee HG, van Endert P, Holzhütter HG, Tenzer S, & Schild H (2008). Characterizing the N-terminal processing motif of MHC class I ligands. Journal of immunology (Baltimore, Md. : 1950), 180 (5), 3210-7 PMID: 18292545[]
April 9th, 2010

Immune evasion versus superinfection

HCMV from J Virol
Human cytomegalovirus-infected cell

A number of viruses, especially herpesviruses, block the MHC class I antigen presentation system. It’s been widely assumed that this is for the obvious reason and that it allows the virus to avoid T cell recognition and elimination. But there’s been an awkward lack of experimental support for that assumption, to the point that I’ve begun to question it (and, more productively, to develop experimental systems with which to test it).   (See the list of posts below for some of my earlier comment on the subject.)

Now, at last, Klaus Fruh offers actual evidence that this assumption may be correct. 1

This deserves a long post, which it’s not going to get today.2 Briefly, Klaus’s group used a herpesvirus of monkeys (rhesus cytomegalovirus; rhCMV) to test this. This is closely related to the human herpesvirus human cytomegalovirus, which is a ubiquitous virus; the vast majority of humans have it, are infected with it as toddlers, remain infected with it throughout their lives, and don’t suffer any problems with it. It’s a rare cause of a mono-like disease, and it’s a concern in immune-suppressed people (especially transplant recipients), but mainly it seems to be a pretty innocuous hitchhiker.

Previous posts on MHC class I immune evasion

Immune evasion does work
Herpesvirus immune evasion: An emerging theme?

Immune evasion: Who needs it?

Viral T cell evasion in vivo: The vanishing evidence

Immune evasion: What is it good for?

The CMV family of herpesviruses carry a particularly impressive arsenal of anti-MHC class I immune evasion genes. (MHC class I is the target that antiviral T cells, also known as cytotoxic T lymphocytes or CTL, recognize. There’s an outline of the process that permits that recognition here.) Whereas herpesviruses like herpes simplex, or chicken-pox virus, and so on, seem to use only one gene to block MHC class I, CMVs seem to use three or four. This would suggest that this sort of immune evasion is really important for these viruses, but when Ann Hill actually tested that notion in mice3 removing these immune evasion genes had only a very small impact.

Fruh’s group has now done something similar using his rhesus model, and looked at an unusual characteristic of CMVs: They are able to repeatedly superinfect the same host. That is, someone4 can be infected with CMV, can have an apparently effective immune response to CMV, and yet can be infected by a new CMV virus. This is pretty unusual, of course. You’d expect that there would be a vaccine-type effect, in which the natural infection would drive a protective immune response. As far as I know, you don’t often see this sort of superinfection even with other herpesviruses, which is why, for example, the chicken-pox vaccine works.

Virus-Cell Interaction; Joerg Schroeer; Art of Science
“Human cytomegalovirus infected human endothelial cells”
by Joerg Schroeer

They made a pretty drastic mutant of the RhCMV to eliminate all four of the MHC class I immune evasion genes (taking out another half-dozen genes as collateral damage, but they checked that these weren’t confounding the story). This mutant virus, in spite of having completely lost its ability to block MHC class I, was perfectly able to infect monkeys and to set up a long-term infection — just like Ann Hill’s findings with mouse CMV. What the mutant virus was not able to do was superinfection.

Together, our results suggested that RhCMV was unable to superinfect in the absence of the homologs of US2, US3, US6, and US11 because the virus was no longer able to avoid elimination by CTL. 5

But when the pre-infected monkeys had their CTL temporarily eliminated, then the mutant viruses were able to superinfect. What’s more, after the virus got in and set up its new infection, CTL couldn’t clear them, even though the viruses still had no ability to evade MHC class I:

Our data imply that T cell evasion is not required for establishment of primary CMV infection or once the sites of persistence (e.g., kidney and salivary gland epithelial cells) have been occupied, but rather it is essential to enable CMV to reach these sites of persistence from the peripheral site of inoculation in the CMV-immune host. 5

This is really cool stuff. It offers an explanation for why Hill’s group didn’t see an effect for MHC class I immune evasion in their mouse CMV model — they didn’t specifically look at superinfection, though they looked at many other aspects of infection. (Does mouse CMV superinfect as robustly as human?)  It also offers an explanation for why experimental CMV vaccines have been ineffective — the immune evasion functions allow the virus to temporarily evade the immune response.

As I say, I don’t think superinfection is so common in other families of herpesviruses, so this may not be a universal explanation for MHC class I immune evasion by herpesviruses; but then, it’s been the CMV system that’s been most puzzling, anyway, so we may not need to go so far to look for answers after all.

  1. 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[]
  2. I’m still chilling with my kids on their spring break, not to mention a dozen other distractions[]
  3. Gold, M. C., Munks, M. W., Wagner, M., McMahon, C. W., Kelly, A., Kavanagh, D. G., Slifka, M. K., Koszinowski, U. H., Raulet, D. H., and Hill, A. B. (2004). Murine cytomegalovirus interference with antigen presentation has little effect on the size or the effector memory phenotype of the CD8 T cell response. J Immunol 172, 6944-6953.

    Pinto, A. K., and Hill, A. B. (2005). Viral interference with antigen presentation to CD8+ T cells: lessons from cytomegalovirus. Viral Immunol 18, 434-444.

    Lu, X., Pinto, A. K., Kelly, A. M., Cho, K. S., and Hill, A. B. (2006). Murine cytomegalovirus interference with antigen presentation contributes to the inability of CD8 T cells to control virus in the salivary gland. J Virol 80, 4200-4202.[]

  4. Or some monkey[]
  5. 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[][]
March 24th, 2010

DAMPs and PAMPs: The enemy within

The Enemy WithinThe immune system is, by its nature, destructive. Its function is to eliminate problems. Because it’s so destructive, there are many layers of control that constantly check and limit the response. Equally, there are controls that try to ensure that the response doesn’t start until it’s needed.

How does the immune response know when it’s needed? It has to eliminate problems, which means it needs to detect problems. So, what’s a problem?

In general, the immune system perceives two conditions as “problems”. One is when microbes are detected, and another is when damage is detected. These conditions are both detected through specific sets of receptors, and both lead to similar cascades of events that culminate in the response we think of as classically “immune” – first an innate immune response, and then if appropriate, an adaptive immune response that’s triggered by the initial innate response.

I’ve talked before about these two forms of problem detection. To summarize and grossly oversimplify some of the history: Charlie Janeway predicted the first form, which we now call “Pathogen-associated molecular pattern” (PAMP) detection; Polly Matzinger predicted the latter form, which we can call “Danger-associated molecular pattern” (DAMP) detection. PAMPs include things that are unique to bacteria or viruses — cell wall components that are present in bacteria, but not in vertebrate cells, for example; or double-stranded RNA, which is found in lots of viruses but would be unusual in our own cells. “Danger” signals, on the other hand, are indications of cell death — internal components of a cell, for example, that have leaked out as the cell dies. 1 For a while, it looked as if PAMPs were the major signal leading to innate and then adaptive immunity, but more recently it’s become clear that DAMPs are also very important.

One example of DAMP recognition would be tumor recognition. We know that tumors are recognized by the immune system — by T cells and B cells, which are adaptive immunity. We know that adaptive immunity is very inefficient without an innate response to set up the proper conditions. We also know that tumors aren’t pathogens as such, and so you wouldn’t expect them to trigger PAMP receptors. So what’s triggering the immune response to the tumor? The answer seems to be DAMPs. As tumor cells die, which they tend to do much more exuberantly than normal cells, they release internal components that the immune response registers as evidence of danger. 2 It’s even been proposed that the massive tumor cell death caused by chemotherapy is the real reason chemo works: The cell death is detected by the immune system as evidence of massive danger, and it’s the resulting immune response that actually eliminates the tumor, not the chemo per se.


So, historically, DAMPs are DAMPs and PAMPs are PAMPs, and never the twain shall meet. After all, internal cell components are quite different from microbes, right?

Well, except for the internal cell components that actually are microbes. Mitochondria, of course, are actually exceedingly symbiotic bacteria that live inside our cells, right? And it turns out that, yes, some DAMPs actually are PAMPs, because some of the danger responses are actually triggered by mitochondrial components that are really bacterial in origin. A lovely paper from Carl Hauser’s lab3 shows that mitochondrial components, released from cells after damage, trigger innate immune responses through pathways that are more traditionally associated with pathogen-specific patterns.4

As I say, immune responses can be very destructive, and Hauser’s interest in this arises from the destructive aspect.  Trauma that produces lots of tissue damage can lead to severe inflammation that looks a lot like sepsis, even though there are no bacteria involved, so he has been looking for triggers for this sterile systemic inflammatory response syndrome (SIRS):

Cellular disruption by trauma releases mitochondrial DAMPs with evolutionarily conserved similarities to bacterial PAMPs into the circulation. These signal through innate immune pathways identical to those activated in sepsis to create a sepsis-like state. The release of such mitochondrial ‘enemies within’ by cellular injury is a key link between trauma, inflammation and SIRS.3

I found it particularly interesting  that one of the mitochondrial DAMPs is formylated peptides. Formylation of peptides is typical of bacteria, not eukaryotes, so it’s a good way of detecting pathogens. Indeed, there are receptors for formyl peptides on neutrophils (FPR1), among other cells, and the mitochondrial DAMPs (including the formyl peptides) cause neutrophils to migrate toward the source (chemotaxis) — see the movie below:

Neutrophils migrate toward a pipette tip that is releasing mitochondrial DAMPs3

(Compare to this other movie I posted a while ago, which shows neutrophils in a mouse’s ear, being attracted to areas of tissue damage.)

H-2M3 structure
H-2M3 crystal structure5

In fact, formylated peptides have been long known to be a PAMP, but not just via the FPR1; they’re also presented by a mouse non-classical MHC class I molecule, H-2M3.  (I didn’t include a picture of H-2M3 in my Field Guide to the MHC earlier, so here’s a picture to the left. 5 Heavy chain in grey, beta2-microglobulin6 in red, peptide in green with the formylated end of the peptide — see how neatly it tucks into the peptide-binding groove there? in magenta.) And again, H-2M3 presents formylated peptides, not just from bacterial pathogens, but also from mitochondria. 7

Most people probably don’t think of MHC-family molecules as innate immune detectors, but many of the non-classical MHC molecules are true PAMP receptors (pattern recognition receptors, PRRs). It’s even been argued — based on H-2M3 itself, in fact — that this broad pattern-recognition ability is the original function of MHC molecules, and the role of MHC molecules in adaptive immunity is the latecomer:

F. M. Burnet asserted that it was their polymorphism that made MHC genes biologically significant. Certainly this is true for I-a8 function, but modern PRR-like I-b molecules9 suggest an alternate model for MHC origins. … Because most genes are monomorphic or minimally oligomorphic, and most class I-like genes not linked to the MHC are monomorphic, parsimony suggests the ancestral MHC locus was also monomorphic. This primitive MHC molecule, functioning as a PRR, would have been preadapted for the evolution of polymorphic class I-a molecules in the evolving adaptive immune system. 10

  1. In apoptosis, the programmed cell death that’s a normal part of tissue growth, internal cell components are carefully packaged up in such as way as to prevent this kind of response.[]
  2. Which, of course, it is.[]
  3. Zhang, Q., Raoof, M., Chen, Y., Sumi, Y., Sursal, T., Junger, W., Brohi, K., Itagaki, K., & Hauser, C. (2010). Circulating mitochondrial DAMPs cause inflammatory responses to injury Nature, 464 (7285), 104-107 DOI: 10.1038/nature08780[][][]
  4. Thanks to Alex Ling, who wrote to me suggesting I talk about this paper.  I’d filed it in with the other 512 papers that I want to talk about here, some time, but her email made me take another look and appreciate how neat the work is.[]
  5. Wang, C. R., Castano, A. R., Peterson, P. A., Slaughter, C., Lindahl, K. F., and Deisenhofer, J. (1995). Nonclassical binding of formylated peptide in crystal structure of the MHC class Ib molecule H2-M3. Cell 82, 655-664.[][]
  6. Why doesn’t the beta symbol ? stick? No matter how often, or how, I enter the code, it changes to a ? in the published post.[]
  7. Loveland, B., Wang, C. R., Yonekawa, H., Hermel, E., and Lindahl, K. F. (1990). Maternally transmitted histocompatibility antigen of mice: a hydrophobic peptide of a mitochondrially encoded protein. Cell 60, 971-980.
    Shawar, S. M., Vyas, J. M., Rodgers, J. R., Cook, R. G., and Rich, R. R. (1991). Specialized functions of major histocompatibility complex class I molecules. II. Hmt binds N-formylated peptides of mitochondrial and prokaryotic origin. J. Exp. Med. 174, 941-944.[]
  8. I-a are the classical MHC class I molecules that present peptides to cytotoxic T lymphocytes[]
  9. And, logically enough then, I-b are the non-classical MHC molecules that often present fairly conserved molecules.[]
  10. Doyle, C. K., Davis, B. K., Cook, R. G., Rich, R. R., and Rodgers, J. R. (2003). Hyperconservation of the N-formyl peptide binding site of M3: evidence that M3 is an old eutherian molecule with conserved recognition of a pathogen-associated molecular pattern. J. Immunol. 171, 836-844.[]
February 10th, 2010

A scarifying story

God of Smallpox
Sopona, the Yoruba god of smallpox

A while ago I listed a number of reasons why smallpox was eradicated, whereas other diseases haven’t been (yet). One of the reasons was that the vaccine against smallpox1 is so effective. Vaccinia immunization induces immunity for an extraordinarily long time — memory immune responses have been shown for up to 60 years after vaccination.

So why is vaccinia such an effective vaccine? Part of it is that vaccinia is a live virus: It replicates after you’re inoculated (so there’s lots of antigen there), and it stimulates the innate immune response (which is geared toward detection of live viruses, among other things). (The yellow fever vaccine is another live virus vaccine that’s also famous for inducing long-term immunity.) Vaccinia is also a large virus that has a lot of antigens available, so that there are lots of different modes of immunity triggered. That is, both B cell (antibody-based) immunity, and broad T cell-based immunity, are likely to be present and to have lots of different targets.

A recent paper2 suggests that the route of vaccination is also important. Unlike most vaccines, which are given by intramuscular (e.g. influenza vaccine) or subcutaneous (e.g. yellow fever) injection, or orally (the live polio vaccine), vaccinia is delivered by scarification — scraping the most superficial layers of the skin. I don’t think this was the result of deliberate comparisons — scarification was the traditional method, and it was easy and convenient. Before 1967:

A scratch about 5 mm long was made in the skin with a needle, a lancet or a small knife and the vaccine suspension was rubbed into the site. A single cut or cross cuts were made, in 1 , 2 or 4 different sites. This was essentially the same method as had been used for variolation in Europe during the latter part of the 18th century. ((Smallpox and its Eradication (Chapter 7). F. Fenner, D. A. Henderson, I. Arita, Z. JeZek, I. D. Ladnyi. World Health Organization, Geneva, 1988))

Later, a bifurcated needle was used:

Experiments soon showed that the multiple puncture method, in which the bifurcated needle was held at right angles to the skin, which was then punctured several times with the prongs, was very efficient and very easy even for an illiterate vaccinator to learn. It became the standard method of vaccination throughout the world. 3

Scarification was a simple and convenient way to deliver the vaccine.  It turns out that scarification isn’t just a convenience, it’s the most effective way to get immunity:

VACV immunization via s.s. [skin scarification], but not conventional injection routes, is essential for the generation of superior T cell–mediated immune responses that provide complete protection against subsequent challenges.2

Langerhans cells J Dermatol Sci
Langerhans cells in the skin4

This includes protection against respiratory-spread disease, not just skin infection. My first thought was that this is probably simply because the vaccinia virus replicates better in the skin than by intramuscular injection, but the improved immunogenicity is also seen with a non-replicating version of vaccinia, “MVA”. 5

My next thought is that Langerhans cells are probably part of the reason. Langerhans cells (see the figure to the right) are a subset of dendritic cells, probably extremely good at triggering immunity, that form a dense network under the skin, and probably act very efficiently at filtering skin-delivered antigen and delivering it to the immune system.

Also, the fact that the skin is damaged in the process evokes Polly Matzinger’s “danger” concept of immune stimulation.

At any rate, something, even if we don’t know exactly what, about scarification leads to better immunity, at least for vaccinia virus. That’s useful to know. Having said that, I’m not quite sure why this paper appeared in Nature Medicine, a very high-impact journal — the mechanism wasn’t shown at all clearly, and this isn’t the first time that the general observation has been made:

This study strongly indicated that, although less reactogenic, vaccinia vaccine administered im [intramuscularly] at a dose of 105 pfu fails to induce an immune response comparable to that elicited by standard scarification. 6

Even more broadly, the skin inoculation concept has been shown to lead to high immunogenicity in other systems; for example, it was shown a couple of years ago that yellow fever vaccine is more immunogenic when delivered intradermally than when given by its conventional subcutaneous route:

Intradermal administration of one fifth of the amount of yellow fever vaccine administered subcutaneously results in protective seroimmunity in all volunteers. 7

BIfurcated needle
Bifurcated needle used for smallpox vaccination

(I do have to add that apparently scarification — which is much easier than intradermal injection — does not work for yellow fever, based on some experiments in the 1950s.8 I haven’t read those papers myself, though. I’d be interested to see if the bifurcated needles used in the late 1960s and on for vaccinia might be more effective for the yellow fever vaccine.)

Anyway, seeing this in at least two instances9 makes it seem possible that it’s a general effect. If skin administration enhances immunogenicity, perhaps this is a way of extending limited vaccine stocks in an emergency.

  1. That is, vaccinia virus[]
  2. Liu, L., Zhong, Q., Tian, T., Dubin, K., Athale, S., & Kupper, T. (2010). Epidermal injury and infection during poxvirus immunization is crucial for the generation of highly protective T cell–mediated immunity Nature Medicine, 16 (2), 224-227 DOI: 10.1038/nm.2078[][]
  3. Smallpox and its Eradication (Chapter 11).  F. Fenner, D. A. Henderson, I. Arita, Z. JeZek, I. D. Ladnyi.  World Health Organization, Geneva, 1988[]
  4. MAHNKE, K., JOHNSON, T., RING, S., & ENK, A. (2007). Tolerogenic dendritic cells and regulatory T cells: A two-way relationship Journal of Dermatological Science, 46 (3), 159-167 DOI: 10.1016/j.jdermsci.2007.03.002[]
  5. At any rate, it’s claimed to be non-replicating, but I don’t remember seeing it formally shown that MVA doesn’t replicate, even temporarily, in the skin. Anyone know if this has been tested?[]
  6. Immunologic Responses to Vaccinia Vaccines Administered by Different Parenteral Routes Author(s): David J. McClain, Shannon Harrison, Curtis L. Yeager, John Cruz, Francis A. Ennis, Paul Gibbs, Michael S. Wright, Peter L. Summers, James D. Arthur, Jess A. Graham Source: The Journal of Infectious Diseases, Vol. 175, No. 4 (Apr., 1997), pp. 756-763[]
  7. Roukens, A., Vossen, A., Bredenbeek, P., van Dissel, J., & Visser, L. (2008). Intradermally Administered Yellow Fever Vaccine at Reduced Dose Induces a Protective Immune Response: A Randomized Controlled Non-Inferiority Trial PLoS ONE, 3 (4) DOI: 10.1371/journal.pone.0001993[]
  8. Ann Trop Med Parasitol. 1953 Dec;47(4):381-93.  Vaccination by scarification with 17D yellow fever vaccine prepared at Yaba, Lagos, Nigeria.CANNON DA, DEWHURST F.

    Am J Hyg. 1952 Jan;55(1):140-53.  A preliminary evaluation of the immunizing power of chick-embryo 17 D yellow fever vaccine inoculated by scarification.DICK GW.[]

  9. And I’m pretty sure I’ve seen at least one other example, but I’m blanking on the details[]
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.[]