Mystery Rays from Outer Space

I don’t know why I read the ScienceDaily newsfeed, because it drives me crazy every single day.  I had naively thought that whoever massages the press releases they receive would have, maybe, a teeny tiny clue about what’s gone on in the field before, but they seem to have the historic awareness of tree squirrels.  Today’s gem:

It’s a paradox that has confounded evolutionary biologists since Charles Darwin published On the Origin of Species in 1859: Since parasites depend on their hosts for survival, why do they harm them? … The study, published in the early online edition of the journal Proceedings of the National Academy of Sciences, provides the first empirical evidence in a natural system of what’s called the “trade-off hypothesis.

It’s not a “paradox” at all, and while it may “baffle” the marketing department that wrote the press release ScienceDaily regurgitated, it certainly hasn’t baffled evolutionary biologists for a long time.  I’ve talked about this exact subject here:

… if there’s a link between increased transmission and increased virulence, then the balance will not favour the pathogen becoming benign.

Here:

I’ve previously talked about the common misconception that viruses evolve toward benignity. This is usually phrased something like, “Natural selection favours viruses with low pathogenicity/virulence (so they don’t eradicate their hosts)“, or “Viral pathogenesis is an abnormal situation of no value to the virus“. This claim is clearly wrong — “clearly” both through common sense, and through observation.

And here:

I’ve observed before that the common belief that viruses evolve toward avirulence is not particularly true. It’s more accurate to say that viruses evolve toward improved transmission. Some viruses are better transmitted if they let their host survive longer, but other viruses have to be virulent in order to spread. The former may evolve toward reduced (though not necessarily loss of) virulence, but the latter would “want” to maintain stable virulence.

We walk a fine line between death due to immune deficiency, smothered under the weight of pathogens and parasites, and death by hyperimmunity, eaten alive by our own defenses. It’s amazing that our immune system can be tuned so precisely as to recognize anything foreign, yet ignore the vast antigenic universe of our own normal self.

Of course, sometimes the immune system fails, in both directions. We often hear about deaths from pathogens, and autoimmune diseases in general are pretty common. There are many ways by which (it’s believed) the immune system can become self-reactive, but a very common observation is that there are both genetic and environmental predisposing causes to autoimmunity. That is, you may have the genetic makeup to be autoimmune, but until you’re exposed to some environmental trigger, autoimmunity never develops. So, for example, if your identical twin has an autoimmune disease, you are much more likely than someone in the general population to develop the disease; but you still have a good to excellent chance of never getting the disease.

In many cases the neither the environmental triggers nor the genetic factors are well understood. The most likely environmental trigger, though, is some kind of microbe. In some cases, this may be because of “molecular mimicry” — the microbe has an antigen that looks like self antigen; the self antigen is normally ignored, because the immune system needs some kind of “danger” signal before it becomes activated; the microbial antigen is seen in the context of microbial “danger” signals; an immune response forms against the microbial antigen; the immune response cross-reacts with the self antigen; self cells are damaged by this immune response; the dead cells release more danger signals along with self antigen; and a positive feedback loop drives a full-fledged autoimmune disease.

That’s the model, but there aren’t many, if any, diseases where the whole process has been tracked through step by step; in fact, I think that there has been so much difficulty getting clear molecular connections between microbes and autoimmunity that there’s a robust search for other mechanisms. However, in the latest issue of Cell Host and Microbe, Albert Bendelac’s group shows a series of links between bacterial infection and the autoimmune disease human primary biliary cirrhosis (PBC).¹ (There’s also a helpful, if rather dry, commentary² by Sebastian Joyce and Luc van Kaer in the same issue.) Rather than trying to cover everything today I’m going to give background here, and then talk about the specific findings in a few days.

One interesting thing about Bendelac’s paper is that they link CD1 to the disease, through NKT cells. CD1 is an MHC class I family member; I talked about it back here, and that’s its mug shot to the left here (click for a larger version). CD1, like many members of the MHC class I family, has a “groove” in its “top” side. MHC class I proper binds peptides in that groove, but CD1 has a much more hydrophobic groove that binds to greasy things like lipids, glycolipids, and lipopeptides. These kinds of molecules are typically found in some kinds of bacteria — especially mycobacteria, like tuberculosis and leprosy, but also other kinds of bacteria such as the commensal microbe Sphingomonas.

MHC class I molecules, with their peptides, are recognized by cytotoxic T lymphocytes (CTL),³ but CD1 molecules and their lipids are recognized by a specialized subset of T cells, “natural killer-like” T cells (NKT cells). The function of this CD1/NKT system really isn’t all that clear. The early guesses that this was a branch of the immune system specialized for dealing with mycobacteria has been weakened as NKT cells have been linked to resistance to various viruses, and also as various viruses have been shown to block CD1 — suggesting that CD1 and NKT cells would otherwise eliminate them.

OK, enough for now. In my next post I’ll talk more about the disease itself, and then try to spell out the process by which, according to Bendelac, NKT are central to the autoimmune reaction; as well as how this abnormal reaction suggests some of the normal functions of NKT and CD1.

1. Mattner J, Savage PB, Leung P, Oertelt SS, Wang V, Trivedi O, Scanlon ST, Pendem K, Teyton L, Hart J et al. (2008) Liver Autoimmunity Triggered by Microbial Activation of Natural Killer T Cells. Cell Host & Microbe 3:304-315.[↩]
2. Joyce S, Van K, Luc (2008) Invariant Natural Killer T Cells Trigger Adaptive Lymphocytes to Churn Up Bile. Cell Host & Microbe 3:275-277.[↩]
3. And natural killer cells, but let’s not go into that now[↩]

I’m not so much an antibody guy, but of course I’ve heard about catalytic antibodies. Catalytic antibodies bind, with the very high affinity that’s typical of many antibodies, to transition state molecules, stabilizing the transition state and facilitating the chemical reaction. They’ve been around for quite a while (I think the first, or at least first widely-announced, catalytic antibody¹ was described in the mid-1980s) and a fair number of them have been created.

But as far as I know, catalytic antibodies remain a curiosity — a fascinating curiosity, but one without a lot of practical application. Although they can be custom-made and can have great specificity, as enzymes they tend to be really crappy (as you might expect), acting thousands or millions of times slower than “genuine” enzymes. Again, I’m no expert, but it seems that in spite of decades of promise, there hasn’t been much payoff; which is a shame, because the concept is so cool they deserve to make it big.²

Recently, though, there was a paper³ offering a catalytic antibody that actually was therapeutic in a real live animal model. Is this the one where promise actually follows through to reality?

The antibody was raised against a virulence factor, urease, of Helicobacter pylori, the ulcer-causing bacterium. (Urease helps neutralize the stomach acid, so it helps H. pylori colonize stomachs.) The antibody degraded urease reasonably well; and more remarkably, the light chain alone of the antibody was also able to degrade H. pylori urease. I don’t know how common this is for catalytic antibodies,⁴ but it strikes as potentially useful, since the light chain can be more readily synthesized and is probably more stable on its own.

The interesting part was that they treated mice with this — I think the first time catalytic antibodies have been used in vivo. They infected the mice with H. pylori, and then treated them with either the catalytic light chain in buffer, or with buffer alone (they should really have used a control light chain in buffer, though — in general this paper doesn’t strike me as terribly well-controlled when is comes to the animal work, but part of that may be the poor English throughout). The treated mice had about a third as many bacteria in their stomachs as did control mice, suggesting that the antibody actually did some good. Presumably it degraded urease in vivo as it does in vitro, and by inceasing stomach acidity helped reduce the bacterial survival.

This is still far, far from any kind of useful treatment, I think, but it’s a step forward.

1. Pollack SJ, Jacobs JW, Schultz PG (1986) Selective chemical catalysis by an antibody. Science 234:1570-1573.[↩]
2. If I’m wrong, by the way, and catalytic antibodies have achieved a toehold somewhere in medicine or industry, please let me know. As I say, I don’t follow this field all that closely.[↩]
3. Hifumi, E., Morihara, F., Hatiuchi, K., Okuda, T., Nishizono, A., Uda, T. (2007). Catalytic Features and Eradication Ability of Antibody Light-chain UA15-L against Helicobacter pylori. Journal of Biological Chemistry, 283(2), 899-907. DOI: 10.1074/jbc.M705674200[↩]
4. It’s not unique, because the authors say they had the same thing for a previous catalytic antibody they made[↩]

The amount of HIV diversity within a single infected individual can exceed the variability generated over the course of a global influenza epidemic, the latter of which results in the need for a new vaccine each year.

–Walker BD, Burton DR (2008) Toward an AIDS vaccine. Science 320:760–764.

(See my previous posts here and here for more explanation.)

The other day I was talking about immune recognition of human endogenous retroviruses (HERVs) in tumors. (HERVs are the husks of ancient retroviruses, now trapped in our genomes. Some of them still express various proteins, either under normal conditions or when stimulated, as in tumors.) One of the reasons this is an interesting finding, is that HERVs may offer a relatively constant antigen, even though the tumors themselves may be highly variable.

There are other, rather obvious, scenarios in which we would like to have a constant antigen in the face of an antigenically-variable disease. For example, HERVs have been proposed to be useful vaccine targets in HIV infection.

One of the many obstacles to overcome in developing a vaccine against HIV is the virus’s rapid mutagenesis. Because of its error-prone replication, HIV can readily escape a lot of immune recognition. Especially when cytotoxic T lymphocytes (CTL) recognize a limited number of antigenic targets, all the virus needs to do to escape immune control is mutate a single amino acid. Usually once the virus does this and escapes immune control, new CTL arise and once again shut down the virus, but only to have new escape variants arise and replicate. Over the multi-year course of an HIV infection, there may be dozens or hundreds of major HIV variants, each escaping temporarily from CTL and destroying T cells during their limited period of freedom.

There are several strategies aimed at reducing the effectiveness of immune escape: targeting multiple HIV antigens, so that the virus would have to simultaneously find many mutations at once; targeting regions in the virus that are so essential that they can’t tolerate mutation; and so on. But wouldn’t it be nice if there was an antigen that wouldn’t change?

HIV and HERVs are distant cousins, both retroviruses, so it seems reasonable that HIV infection might turn on sleeping HERVs. In fact, for nearly 10 years there have been intermittent studies suggesting this might be the case; first based on antibody responses in HIV patients¹, and recently with more specific evidence of reactivation of HERVs both in patients² and in the lab, in infected cells.³

Last fall Douglas Nixon’s group took this to the next step.⁴ Although the antibody responses¹ had suggested that HERVs were immunogenic when turned on by HIV, antibodies aren’t believed to be terrible important in control of HIV; rather, CTL are thought to be critical.⁵ Nixon’s group showed that in HIV-infected people, there were often functional CTL responses to HERVs; what’s more, the higher the anti-HERV response, the lower the HIV plasma load, implying that the anti-HERV CTL might actually be controlling HIV. (See the figure to the left; click for a larger version.)

As endogenous retroviral sequences are fixed in the human genome, they provide a stable target, and HERV-specific T cells could recognize a cell infected by any HIV-1 viral variant. HERV-specific immunity is an important new avenue for investigation in HIV-1 pathogenesis and vaccine design.

Let’s go back to a paper⁶ I mentioned last year, where a group looked at genomic variation linked to disease progression in HIV. They found three genomic regions that were linked to viral set-point; one is an RNA polymerase, one is in the MHC region and affects levels of the MHC class I gene HLA-C, and the third … well, the third is a HERV, called HCP5.

The authors pointed out that HCP5 might not be the actual factor involved, because it might be riding along with HLA-B*5701, an MHC class I allele that’s associated with HIV resistance (and I noted that natural killer ligands MICA and MICB are also close by). Still, they clearly like the idea that HCP5 is itself directly involved. They suggested that it might act by an antisense mechanism or something, but I think it might be very interesting to look at CTL responses to HCP5 proteins.

1. Stevens RW, Baltch AL, Smith RP, McCreedy BJ, Michelsen PB, Bopp LH, Urnovitz HB (1999) Antibody to human endogenous retrovirus peptide in urine of human immunodeficiency virus type 1-positive patients. Clin Diagn Lab Immunol 6:783-786.[↩][↩]
2. Contreras-Galindo R, Kaplan MH, Markovitz DM, Lorenzo E, Yamamura Y (2006) Detection of HERV-K(HML-2) viral RNA in plasma of HIV type 1-infected individuals. AIDS Res Hum Retroviruses 22:979-984.[↩]
3. Contreras-Galindo R, Lopez P, Velez R, Yamamura Y (2007) HIV-1 infection increases the expression of human endogenous retroviruses type K (HERV-K) in vitro. AIDS Res Hum Retroviruses 23:116-122.[↩]
4. Garrison, K.E., Jones, R.B., Meiklejohn, D.A., Anwar, N., Ndhlovu, L.C., Chapman, J.M., Erickson, A.L., Agrawal, A., Spotts, G., Hecht, F.M., Rakoff-Nahoum, S., Lenz, J., Ostrowski, M.A., Nixon, D.F. (2007). T Cell Responses to Human Endogenous Retroviruses in HIV-1 Infection. PLoS Pathogens, 3(11), e165. DOI: 10.1371/journal.ppat.0030165[↩]
5. Because of the way CTL recognize their targets, by the way, it doesn’t matter if the HERVs produce defective proteins — even a truncated protein that is unstable and rapidly destroyed might be a good CTL target.[↩]
6. Fellay, J., Shianna, K. V., Ge, D., Colombo, S., Ledergerber, B., Weale, M., et al. (2007).A whole-genome association study of major determinants for host control of HIV-1. Science, 317(5840), 944-947.[↩]

One of the genes I’m interested in is an aminopeptidase called “ERAP1″¹ that’s involved in antigen presentation.² ERAP1 is a member of a family of closely-related genes, roughly 50% identity, that in humans and many other mammals are adjacent on a chromosome. That is, on human chromosome 5 (figure to the right), there are three genes side by side: ERAP1; ERAP2; and LNPEP, all of which are clearly the result of gene-duplication events at some point. Several other mammals have the same arrangement. The neighbouring (non-ERAP-related) genes are the same, also, for that matter.

In mice and rats, however, there’s a different arrangement. ERAP1 is on chromosome 13, LNPEP is on chromosome 17, and there’s no ERAP2.

So it seemed that the most likely scenario was the in the rodent lineage, there was a chromosome break right between ERAP1 and LNPEP, right in the middle of ERAP2, that destroyed ERAP2. However, it was also possible (though I thought less likely) that there was a fusion — that having ERAP1 and LNPEP on different chromosomes was the basal situation, but that the chromosomes merged in the non-rodent lineage, with a gene duplication arising from the chromosome fusion. Or something like that.

Fish have members of the ERAP family, but I wasn’t confident in assigning them to ERAP1, ERAP2, or LNPEP — the mammalian genes are too similar for me to be confident in my feeble bioinformatic understanding.

But the platypus genome that just come out gives a lot of support to the idea that rodents split their chromosome from the basal patterns. To the left (click for a larger version) is the appropriate platypus chromosome. ERAP1 (here called “similar to type I tumor necrossis factor receptor shedding aminopeptidase regulator”) and LNPEP (here called “similar to leucyl/cystinyl aminopeptidase”) are on the same chromosome, and in between them is “LOC10081647″ — something the annotation called a pseudogene, which it may well be, but it’s ERAP2. I haven’t had time to do any careful alignments, but it aligns more closely with mammalian ERAP2 than anything else.

So almost certainly mice and rats lost ERAP2, though it may have been a pseudogene at that point, and other species either kept a functional ERAP2, or somewhere along the line turned a pseudogene into a functional enzyme.

This is not a big deal and likely won’t lead anywhere, but it’s an answer to a piece of trivia that’s been bothering me a little on and off for about 5 years. So yay for the platypus genome.

1. by us, anyway: York IA, Chang SC, Saric T, Keys JA, Favreau JM, Goldberg AL, Rock KL (2002) The ER aminopeptidase ERAP1 enhances or limits antigen presentation by trimming epitopes to 8-9 residues. Nat Immunol 3:1177–184. [↩]
2. For example, York IA, Brehm MA, Zendzian S, Towne CF, Rock KL (2006) Endoplasmic reticulum aminopeptidase 1 (ERAP1) trims MHC class I-presented peptides in vivo and plays an important role in immunodominance. Proc Natl Acad Sci U S A 103:9202–9207.[↩]

I’ve talked several times about Charlie Janeway’s “dirty little secrets“, and the insights into fundamental immunity that arose from the concept. I’ve also mentioned a couple of potential clinical advances arising from it. Here’s another one, that I find particularly elegant for its use of the weak to conquer the powerful. ¹

As a very quick reminder: Janeway’s insight² was that an immune response wouldn’t start unless there were signals present, indicating that a hazardous situation was at hand. Janeway proposed that the immune system would be on the alert for molecular patterns that are generic to many pathogens. Without such patterns the immune system would ignore “foreign” antigen; when pathogen-associated molecular patterns (”PAMPs”) appear, the immune system kicks on and starts looking for trouble. (By the way, sorry about all the acronyms in this. I usually try to avoid using too many, but it’s unavoidable this time. There’s a glossary in the footnote here if you need it.)³

Janeway, and subsequently many others, went on to identify some of the PAMP receptors; first the toll-like receptors (TLRs) and then several other types. There are quite a few — maybe a dozen TLRs, maybe a couple dozen other types, in mice or humans. The different PAMP receptors recognize different subsets of PAMPs, and we have relatively recently reached the point where we understand enough about the receptors to make occasional predictions: Researchers can analyze a virus, say, and say with some confidence that a certain PAMP receptor is likely to recognize it.

Immune recognition of mousepox virus
Hubertus Hochrein’s group is interested in smallpox, the archetypal poxvirus, and they’re using ectromelia (mousepox) as their model for smallpox. Poxviruses are large DNA viruses that are remarkably versatile in their dealings with the immune system; as a group, and as individual viruses, they have evolved molecules that evade multiple components of the immune system. One of those components is the TLR system, apparently, because at least some poxviruses encode molecules that block TLR signalling. ⁴

There’s an interesting general question, by the way, about how to interpret immune evasion molecules in viruses. If we find that vaccinia virus encodes blockers of TLR signaling, do we argue that TLRs must be important in protecting against vaccinia virus? Or do we instead say that TLRs must not be important, because the virus has defenses against them? In this case, at any rate, Hochrein’s group guessed that TLRs are important, and further guessed that TLR9 might be important.

TLR9 recognizes DNA, both viral and bacterial, but until now there haven’t been any instances of virus recognition that’s strictly dependent on TLR9. Ectromelia, however, turned out to be the first; immune activation by ectromelia is almost entirely dependent on TLR9 signaling, and mice lacking TLR9 were highly sensitive to ectromelia infection:

The in vivo relevance of this TLR9-only dependence for ECTV⁵ recognition was clearly illustrated by our in vivo studies that revealed that the lack of TLR9 rendered mice more than 100-fold more susceptible to infection with ECTV. … We calculated an LD₅₀⁶ of 19 TCID₅₀⁷ for the TLR9-deficient mice and an LD₅₀ of about 2,120 TCID₅₀ for the WT mice.

Cells (actin cytoskeleton in green) infected with vaccinia virus (red)

Broader recognition of a weakened poxvirus
Does TLR9, and only TLR9, recognize poxviruses in general? Ectromelia is a highly virulent virus even as poxviruses go. There are plenty of more benign viruses, such as vaccinia virus; and even within vaccinia viruses there is a wide range of virulence. Probably the least virulent vaccinia virus is a semi-artificial version of it called “Modified vaccinia Ankara” (MVA). ⁸ MVA has lost about 13% of its genome compared to its more virulent ancestor, and many of its remaining genes are damaged as well.⁹

Like ectromelia, TLR9 drove an immune response to MVA. Unlike ectromelia, that isn’t the whole story; even without TLR9, the immune system recognizes MVA.

This is almost certainly an immune evasion function that has been lost in MVA. That is, both wild-type vaccinia virus and ectromelia virus seem to have a gene (or genes) that blocks recognition by PAMP receptors other than TLR9, whereas the massively defective MVA has lost this gene and is recognized by both TLR9 and this other, unknown, receptor.

Overriding blindness
So if immune activation by ectromelia is partially blocked by its immune evasion function, would we reduce its virulence by artificially activating the immune system after ectromelia infection? Ideally, of course, we’d want to only activate the components that are involved in protecting against poxviruses. Like, for example, the aspects that the poxvirus MVA activates.

You see where this is going. Can MVA act almost like an adjuvant, turning on the immune components that ectromelia virus has blinded? And the answer is yes. If you infect mice with a lethal dose of ectromelia, and then superinfect them with MVA, they survive:

MVA given at the same time or immediately after challenge with a high lethal dose of ECTV of 1 × 10⁵ TCID₅₀ completely protected WT mice against death, whereas all control mice died with the 10-fold-lower dose of 1 × 10⁴ TCID₅₀.

You wouldn’t normally think that two viruses would be better than one; and you wouldn’t normally think that the dainty little MVA could override its brutally virulent cousin’s lethality. But at least in mice, it seems that therapeutic infection worked.

1. Samuelsson, C., Hausmann, J., Lauterbach, H., Schmidt, M., Akira, S., Wagner, H., Chaplin, P., Suter, M., O’Keeffe, M., Hochrein, H. (2008). Survival of lethal poxvirus infection in mice depends on TLR9, and therapeutic vaccination provides protection. Journal of Clinical Investigation, 118(5), 1776-1784. DOI: 10.1172/JCI33940[↩]
2. And Polly Matzinger’s[↩]
3. PAMP: Pathogen-associated molecular pattern;
   TLR: toll-like receptor;
   ECTV: ectromelia virus;
   LD₅₀: Dose of virus that kills half the recipients;
   TCID₅₀: 50% tissue-culture infectious dose - more or less, the number of infectious particles of virus;
   MVA: Modified vaccinia Ankara[↩]
4. Bowie A, Kiss-Toth E, Symons JA, Smith GL, Dower SK, O’Neill LA (2000) A46R and A52R from vaccinia virus are antagonists of host IL-1 and toll-like receptor signaling. Proc Natl Acad Sci U S A 97:10162-10167.[↩]
5. ectromelia virus[↩]
6. LD₅₀: Dose of virus that kills half the recipients.[↩]
7. TCID₅₀: 50% tissue-culture infectious dose - more or less, the number of infectious particles of virus[↩]
8. MVA was produced by repeatedly passing a wild vaccinia virus (Ankara strain) through chicken cells more then 570 times. In the process of becoming chicken-adapted, it lost its mammalian adaptations and barely replicates in mammalian cells. Since it’s so enfeebled, there’s interest in using it as a vaccine, since the standard smallpox vaccine is quite dangerous as vaccines go.[↩]
9. Meisinger-Henschel C, Schmidt M, Lukassen S, Linke B, Krause L, Konietzny S, Goesmann A, Howley P, Chaplin P, Suter M et al. (2007) Genomic sequence of chorioallantois vaccinia virus Ankara, the ancestor of modified vaccinia virus Ankara. J Gen Virol 88:3249-3259.[↩]

After spending a week¹ trying to find a bug in XPlasMap, I just realized that “[-\d]+” should have been “[-\d\.]+”

Sheesh.

1. Well, a half-hour every other night for a week[↩]