Mystery Rays from Outer Space

HapMap 3, officially announced in today’s issue of Nature,¹ is an “integrated data set of common and rare alleles” in human populations, built from “1.6 million common single nucleotide polymorphisms (SNPs) in 1,184 reference individuals from 11 global populations“. 

As well as being a resource for genome-wide studies, there are a number of things that can be done with the data directly. One of those is to help identify regions that are under positive natural selection. The authors found a number of them, including several immune-related genes in the Kenyan population.

A little sadly for me, none of these genes are ones I’m particularly familiar with. The three that are listed are:

• CD226.  This is an activating NK cell receptor. An allelic variant in CD226 has been linked to a number of autoimmune diseases,² so it wouldn’t be surprising to learn that it’s under some form of selection.  I didn’t check the actual SNP that was shown to be selected, to see if it’s the same one that’s linked to autoimmunity.

• ITGAE.  This is an integrin³ that’s apparently involved in lymphocyte trafficking.  Allelic variants in ITGAE have been linked to a number of diseases including sarcoidosis⁴ and ischemic stroke.⁵

• DPP7 is dipeptidyl-peptidase 7.  Although I’ve had a strong interest in peptidases for a while⁶ because of their influence on MHC class I antigen presentation, DPP7 seems to have an unrelated role, that of preventing apoptosis of resting lymphocytes. I don’t know of any links between DPP7 and disease, but obviously altering lymphocyte survival could impact lots of things. 

I’m sure that any more immune-related genes are under strong selection — we know that MHC genes are very strongly and rapidly selected, for example — but they don’t necessarily send up flags in this sort of analysis. 

1. The International HapMap 3 Consortium (2010). Integrating common and rare genetic variation in diverse human populations Nature, 47, 52-58 DOI: 10.1038/nature09298[↩]
2. Douroudis K, Kingo K, Silm H, Reimann E, Traks T, Vasar E, & Kõks S (2010). The CD226 Gly307Ser gene polymorphism is associated with severity of psoriasis. Journal of dermatological science, 58 (2), 160-1 PMID: 20399620

   Maiti AK, Kim-Howard X, Viswanathan P, Guillén L, Qian X, Rojas-Villarraga A, Sun C, Cañas C, Tobón GJ, Matsuda K, Shen N, Cherñavsky AC, Anaya JM, & Nath SK (2010). Non-synonymous variant (Gly307Ser) in CD226 is associated with susceptibility to multiple autoimmune diseases. Rheumatology (Oxford, England), 49 (7), 1239-44 PMID: 20338887[↩]

3. Intergrins are cell-surface molecules often involved in cell-cell interactions[↩]
4. Heron M, Grutters JC, Van Moorsel CH, Ruven HJ, Kazemier KM, Claessen AM, & Van den Bosch JM (2009). Effect of variation in ITGAE on risk of sarcoidosis, CD103 expression, and chest radiography. Clinical immunology (Orlando, Fla.), 133 (1), 117-25 PMID: 19604725[↩]
5. Luke MM, O’Meara ES, Rowland CM, Shiffman D, Bare LA, Arellano AR, Longstreth WT Jr, Lumley T, Rice K, Tracy RP, Devlin JJ, & Psaty BM (2009). Gene variants associated with ischemic stroke: the cardiovascular health study. Stroke; a journal of cerebral circulation, 40 (2), 363-8 PMID: 19023099[↩]
6. Pubmed link to my peptidase papers[↩]

From A History of British Fish (William Yarrell, 1835)

I’ve talked about lamprey immune systems several times (here, here, and here). I find them fascinating because it shows both how our own immune system developed, and also shows alternate routes that can lead to a pretty good, but very different, immune system.

Quick background: In order of evolutionary appearance you have sea urchins, lampreys, sharks, reptiles, mammals. (Note that this is not true, it’s no more than a sloppy shorthand for common ancestry, but it’s a handy shorthand for this purpose.  See a phylogenetic tree here.) Mammals have a form of adaptive immune system that includes T lymphocytes and antibodies, and at first glance this whole complex system arose, almost fully-formed, in sharks.¹

This has always amazed me, because an adaptive immune system doesn’t work in isolation; the pieces don’t work alone. You need all kinds of moving parts — all the complex molecular pieces that chop and snip DNA to form T cell receptors and antibodies, all the multiple parts of a thymus that screen T cells for functional and safe receptors, the MHC molecules that the receptors see and all the pieces that snip and shuffle around peptides for that system, the spleen and lymph nodes that let lymphocytes interact with other cells, — and it seemed that all these pieces abruptly appeared and put themselves together, like a fine watch, in one evolutionary blink.

When I first learned about this, some 15 or 20 years ago, I told myself that this was an illusion, that once more species were looked at we’d see the history of these moving parts in other common ancestors. Of course, this is exactly what’s happened since then. We see accidental, random parts in sea urchin genomes (I talk about that here) and we see other bits and pieces arising in lampreys and hagfish (in the links at the top).

So in reality, the adaptive immune system didn’t arise all that suddenly after all; the pieces gradually were added over a hundred million years or more, sometimes purely by chance, sometimes for other purposes altogether, and sometimes as components of a prototypic immune system that acted as a foundation for the whole shark thing.²

So that’s the first part of the background: In lampreys, which diverged from the mammalian lineage maybe 450 million years ago, we see many of the pieces of a mammalian adaptive immune system. There are cells that look a lot like lymphocytes, there is something that looks like a spleen. But, as I say, there are none of the familiar pieces that we think of as an adaptive immune system. Lampreys flatly do not have our adaptive immune system:

Nevertheless, the cardinal elements of adaptive immunity, namely Ig, TCR, RAG1 and 2, and MHC class I and II, were conspicuously absent.³

Lamprey variable receptor with bound antigen4

But step back a little, and look a little deeper, and we see some familiar parts. Lampreys do, in fact, have variable receptors, just like T cell receptors and antibodies, and those receptors are made by chopping and shuffling genome DNA, just like TcR and antibodies, and are expressed in their lymphocyte-like cells, and some are secreted (like antibodies and B cells) and some are cell-associated (like T cell receptors).

And here’s the other amazing thing: At the molecular level, the lamprey receptors are completely unlike T and B cell receptors. The lamprey lineage came up with a completely different system that allows them to do pretty much the same thing as the shark lineage. Their receptors are different kinds of molecules, and the system that shuffles the genomic DNA is different. ⁵ Yet, the functional end product is the same — a system that has immunological memory. An adaptive immune response, that’s quite alien to our own, but that works pretty damn well.

Although the Ig-based and VLR-based adaptive immune systems in jawed and jawless vertebrates use different genes and assembly mechanisms, both systems generate diverse repertoires of anticipatory receptors capable of recognizing almost any Ag through the combinatorial assembly of large arrays of partial gene segments. The development of clonally diverse lymphocytes allows for Ag-specific responses and memory, which are lacking in innate immunity.³

There is still a lot we don’t know about lamprey immunity (how does it present self-reative receptors, with no thymus?) but what we do know is just so amazing, I’m completely fascinated by it. It beautifully illustrates two of the basic features of evolution — building on previous structures, whether related or not; and alternate solutions to the same problem. Herrrin and Cooper have a short and dense, but very interesting, review, ³ that prompted this particular post.

1. That is, in the common ancestor of sharks and mammals, to use a slightly less-sloppy terminology.[↩]
2. And of course, the system has continued to evolve. The mammalian system is remarkably similar to the shark in broad strokes, but it’s also very different in many ways.[↩]
3. Herrin, B., & Cooper, M. (2010). Alternative Adaptive Immunity in Jawless Vertebrates The Journal of Immunology, 185 (3), 1367-1374 DOI: 10.4049/jimmunol.0903128[↩][↩][↩]
4. B. W. Han, B. R. Herrin, M. D. Cooper, I. A. Wilson (2008). Antigen Recognition by Variable Lymphocyte ReceptorsScience, 321 (5897), 1834-1837 DOI: 10.1126/science.1162484[↩]
5. Though there are some common pieces that hint at a common ancestor of the two systems, maybe.[↩]

TcR interacting with artificial membrane1

Why does autoimmune disease (sometimes) follow viral infection?²

It’s a pretty well-known phenomenon, but a definite answer isn’t yet known — and of course there may not be a single answer, there may be multiple causes. We know that many autoimmune diseases seem to be triggered by some sort of infection or inflammation. A classic example is Guillan-Barre syndrome, which is a little more common (though still very rare) in people who have received certain influenza vaccines, but there are plenty of other examples.³ It’s not believed that the infection actually “causes” the disease, but rather that someone who already has a genetic predisposition to the autoimmune disease needs to have some kind of environmental trigger to have the disease actually kick in; and, very rarely, a viral or other infection will provide that trigger.

(The genetic predisposition is clear because, among other points, identical twins are much more likely to both get autoimmune disease than are fraternal twins; whereas the need for an environmental trigger is clear because even if your identical twin gets an autoimmune disease, you’re usually less than 50% likely to get it yourself. Note that I’m lumping together hundreds of different diseases into the “autoimmune” package, and the specific odds and so on differ for each one.)

OK, so if you have a genetic predisposition to autoimmunity — and let’s get more specific, the paper I’m looking at deals with multiple sclerosis (MS) — there’s a small chance that a viral infection will trigger that disease. One of the most popular models for this is “molecular mimicry”. Simplified: This is the notion that a viral protein looks, to a T cell, a little bit like a self protein. The viral protein appears in the context of infection, with its concomitant inflammation and tissue damage and so on, and the T cell is activated to it. The T cell wouldn’t be activated by the self protein because it hasn’t been seen in the context of inflammation before, but once over the activation hurdle the T cell is now able to attack the self protein, and this is autoimmunity.

T cell receptor (top) interacting with MHC

Molecular mimicry is an attractive model, but there’s not a lot of direct evidence for it.  Another possibility has been proposed for a while: Dual TcRs. Normally, T cells can only recognize a single target. This is by “design”;⁴ if the T cell can see two targets, it could get activated by one, and then attack the other, even if the second target was never present during inflammation. This sort of dual target recognition is obviously dangerous, and there are safeguards that mostly prevent it; but some T cells do sneak through with at least the theoretical potential for dual recognition. So what could happen here is that one TcR could be directed against the pathogen, and activate the T cell; then the other TcR, recognizing self, could run amok because it’s now on an activated T cell.

T cells with dual specificity do exist, at a fairly significant frequency (1-8%; at least one source claims as high as 33%, which seems much too high to me), but whether they actually do anything in autoimmunity is up in the air. This idea has been around for a while, but I don’t think there’s been much evidence for it happening naturally. In at least one case, where it was tested in an artificial system, dual TcRs did not seem to be responsible for an automimmune disease. ⁵

The most recent paper offers evidence that (in quite an artificial system) dual-specificity T cells are responsible for multiple sclerosis: ⁶

Our results demonstrate the importance of dual TCR–expressing T cells in autoimmunity and suggest a mechanism by which a ubiquitous viral infection could trigger autoimmunity in a subset of infected people, as suggested by the etiology of multiple sclerosis.

It’s an interesting and solid paper as far as it goes, but we’re left with the issue of this being a highly artificial system — mice with manipulated TcRs and manipulated autoimmune disease. Is this a real issue in natural autoimmunity and natural infections? This paper doesn’t really address that, but it does support the notion that it’s something to look more closely at.  (And again, different autoimmune diseases, or even different people with the same disease, may have altogether different triggers.  Maybe some people have molecular mimicry as the trigger while others have dual TcRs and other have who knows what.)

1. By Raghuveer Parthasarath, then in the Groves lab[↩]
2. Also, why are so many of my keyboard keys sticking together? An altogether easier question quickly answered by pointing to my kids “helping” me with my work while holding popsicles[↩]
3. For a review:
   Fujinami, R. (2001). Can Virus Infections Trigger Autoimmune Disease? Journal of Autoimmunity, 16 (3), 229-234 DOI: 10.1006/jaut.2000.0484[↩]
4. I.e. evolution.[↩]
5. McGargill MA, Mayerova D, Stefanski HE, Koehn B, Parke EA, Jameson SC, Panoskaltsis-Mortari A, & Hogquist KA (2002). A spontaneous CD8 T cell-dependent autoimmune disease to an antigen expressed under the human keratin 14 promoter. Journal of immunology (Baltimore, Md. : 1950), 169 (4), 2141-7 PMID: 12165543 [↩]
6. Ji, Q., Perchellet, A., & Goverman, J. (2010). Viral infection triggers central nervous system autoimmunity via activation of CD8+ T cells expressing dual TCRs Nature Immunology, 11 (7), 628-634 DOI: 10.1038/ni.1888[↩]

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

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[↩][↩][↩]

I 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[↩][↩][↩]

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[↩]

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:

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.[↩]

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 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

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.[↩]

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).

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[↩]

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.

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.

“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[↩][↩]