Mystery Rays from Outer Space

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 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.[↩]

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.[↩]

A couple weeks ago I was having a chat with a friend about cancer immunity (as one so often does) and he asked if the Holy Grail of cancer immunity would be to identify tumor antigens. Not at all. There are hundreds of tumor antigens known. (The journal Cancer Immunity hosts a database that lists many of the known ones.) The problem is if anything the opposite; there are too many antigens, and many are one-offs, unique to one or a handful of tumors and of no use to most patients. A better Holy Grail would be a single target that many tumors have in common.

Our genomes are littered with the withered corpses of ancient retroviruses. Everyone has them. These human endogenous retroviruses (HERVs) are defective, and their proteins are usually not expressed, or are expressed at low levels. Because they’re not normally expressed much, they don’t necessarily tolerize the immune system. At least hypothetically, if there are pathologic conditions in which HERVs become expressed, they might form targets for immunity.

As it happens, there may be several such conditions. It’s been suggested (though not, to my inexpert eye, all that convincingly) that HERVs might represent targets in autoimmunity. More usefully, Douglas Nixon’s group showed some evidence, last fall, that HIV infection upregulates HERVs, offering a target for CTL that (unlike HIV itself) isn’t constantly mutating.¹ And it’s been suggested for quite a while that HERVs might be immunogenic in tumors.

For example, over ten years ago it was shown that patients with certain kinds of tumors, which consistently show high-level HERV activation, often have antibody responses to HERVs.² However, in general, antibodies are not particularly effective against tumors, and as far as I know, nothing much arose directly from the antibody findings.

On the other hand, T cells are (at least sometimes) more effective against tumors; and T cell immunity was linked to HERVs first (as far as I know) in 2002,³ with the observation that a melanoma tumor antigen was derived from a HERV. Some similar work has followed.⁴

So: HERVs are potential antigens; they are more or less immutable; they can be upregulated in some tumors; and they can trigger an immune response by antibodies and by T cells. These are interesting observations, but is this at all relevant for tumor treatment?

The next step in answering that question came out recently, in J Clin Invest. ⁵ Here we see not just reactive T cells (that is, T cells specific for HERV peptides) but a potent immune response that actually cleared a metastatic tumor. The response was due to an allogeneic bone marrow transplant, and when they tracked down the target peptide for the immune response, it was directed against a HERV peptide:

The genes encoding this antigen were found to be derived from human endogenous retrovirus (HERV) type E and were expressed in RCC cell lines and fresh RCC tissue but not in normal kidney or other tissues.

It’s still far from clear how universal a target HERVs might be. This group identified a HERV target in one of their patients, but they treated 74 patients, saw at least partial responses in 29 of those patients, sought to identify targets in four of the responders, and found the HERV target in just one of the four. Some of the other targets were apparently the more standard mutated proteins, specific to the individual tumor.

This peptide target, by the way, is from a group E HERV; most of the previous work has focused on group K HERVs, which tend to be more active and are expressed to some extent in normal tissue. HERV-E generally are pretty quiescent, so if tumors do upregulate HERV-E, it would be a more specific target. The authors did check, and found that most of that particular type of tumor expressed HERV-E. Interestingly, this is the kind of tumor that is most likely to be responsive to immunotherapy:

A histological review of the RCC⁶ cell lines and fresh RCC tissues used in experiments presented in this article showed all to be clear-cell carcinomas, with more than half expressing HERV-E transcripts. Furthermore, limited preliminary data from an ongoing study of fresh tumors suggest that this HERV-E may have transcriptional activity limited to the clear-cell variant of kidney cancer (unpublished observations), which is intriguing given the track record for this tumor being the immunoresponsive subtype of RCC.

It would be a very useful discovery if this turns out to be a common antigen among these tumors. That said, there are some other known common tumor antigens — such as tyrosinase in melanomas — and immunization hasn’t proven a silver bullet in those yet. But it’s early days, still.

1. 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., and Nixon, D. F. (2007). T Cell Responses to Human Endogenous Retroviruses in HIV-1 Infection. PLoS Pathog 3, e165. [↩]
2. Boller, K., Janssen, O., Schuldes, H., Tonjes, R. R., and Kurth, R. (1997). Characterization of the antibody response specific for the human endogenous retrovirus HTDV/HERV-K. J Virol 71, 4581-4588.[↩]
3. Schiavetti, F., Thonnard, J., Colau, D., Boon, T., and Coulie, P. G. (2002). A human endogenous retroviral sequence encoding an antigen recognized on melanoma by cytolytic T lymphocytes. Cancer Res 62, 5510-5516.[↩]
4. Rakoff-Nahoum, S., Kuebler, P. J., Heymann, J. J., E Sheehy, M., M Ortiz, G., S Ogg, G., Barbour, J. D., Lenz, J., Steinfeld, A. D., and Nixon, D. F. (2006). Detection of T lymphocytes specific for human endogenous retrovirus K (HERV-K) in patients with seminoma. AIDS Res Hum Retroviruses 22, 52-56.[↩]
5. Takahashi, Y., Harashima, N., Kajigaya, S., Yokoyama, H., Cherkasova, E., McCoy, J.P., Hanada, K., Mena, O., Kurlander, R., Abdul, T., Srinivasan, R., Lundqvist, A., Malinzak, E., Geller, N., Lerman, M.I., Childs, R.W. (2008). Regression of human kidney cancer following allogeneic stem cell transplantation is associated with recognition of an HERV-E antigen by T cells. Journal of Clinical Investigation DOI: 10.1172/JCI34409[↩]
6. RCC: “Renal cell carcinoma.” IY[↩]

We’ve been promised that as genome sequencing becomes faster and simpler, we’ll start seeing practical dividends as well as parlour tricks like sequencing Watson’s genome. Some of the dividends are already paying out, as a paper in the latest PLoS Pathogens¹ shows.

Probably most of you remember the outbreaks of foot-and-mouth disease in Britain in 2001, and again last year. FMD is a virus that affects many hooved animals; it’s not usually fatal, but causes productivity loss. FMD outbreaks are economically devastating, because aside from the productivity loss many countries, that are free of the disease, will refuse to take meat or other agricultural products from outbreak areas. The goal of FMD management, then, is to keep it away, and if it ever hit, to contain it and slaughter all infected and potentially-infected animals.

The 2001 outbreak in Great Britain came from outside the country. The 2007 outbreak, though, was clearly from a local source: The FMD research lab in the Institute for Animal Health (IAH), Pirbright, Surrey. The latest paper discusses the epidemiology of that outbreak, and how they used whole-genome sequencing to track and predict sites of FMD.

(This is timely, because the US is planning to move the sole American FMD research center, now on Plum Island, to the mainland. There’s obvious concern that the virus could escape from containment within research labs and infect neighboring animals, causing the first American FMD outbreak since 1929. I am not particularly knowledgeable about the field, but I have to think that, at best, the timing of the planned move is unfortunate.)

FMD is caused by a picornavirus, the same broad family as polio and cold viruses. Like those viruses, FMD mutates rapidly, traveling around as a quasispecies cloud. The clouds can be easily divided into 7 broad groups, and within the most common serotype (O) there are 8 distinct subgroups (see the map² to the right [click for a larger version] for their geographical distribution).

The FMD genome is 8134 nucleotides long, and the sequence analysis that has been used for epidemiology like the 7 different topotypes has been based on no more than 8% of that length — the VP1 gene, usually. That’s enough to track high-level changes, because of FMD’s rapid mutation rate:²

the rate of evolution is approximately 1% per year …. If the concept of a constant evolutionary rate is accepted and there are no constraints on virus evolution then it would expected that new topotypes could arise in approximately 15 years. In reality, this extent of evolution probably takes much longer. For example, FMD viruses belonging to the Asia 1 serotype, first identified in samples from Pakistan in 1954 … have not yet exceeded 15% nucleotide difference …

But 8% of the genome is not nearly enough to track changes within a single epidemic, like the one in Surrey last year; it simply isn’t long enough to pick up the handful of variations. It was known in the previous outbreak, in 2001, that the information was there in the genome (”virus recovered from closely housed animals can differ by 1 to 2 nucleotides and is likely to pass through a “bottleneck” on passage between farms”).³ The issue was a practical, technological one — being able to sequence entire virus genomes quickly enough to pass back information to people in the field.

By 2007, the technology was there. The people at the IAH were able to sequence genomes from viruses isolated in the outbreak with a fine enough comb to track changes throughout the spread, and fast enough pass information back to the field within 24-48 hours. Their sequencing confirmed that the virus was in fact a lab escapee, because it was almost identical to a couple of lab strains but was different from circulating viruses. ⁴

The 40-odd viral genomes yielded a fair bit of useful information (see the figure to the left for a summary). For example,

The small number of nucleotide substitutions observed between viruses from source and recipient IP suggests that there has been direct transmission without the involvement of other susceptible species, e.g. sheep or deer.

It’s obviously useful to know if there’s a wild-animal reservoir of disease, but an even more important insight came from this work as well.

the virus from IP3b was nine nucleotides different from the virus from IP1b … This is a high number of changes for a single farm-to-farm transmission … and we predicted that there were likely to be intermediate undetected infected premises between the first outbreaks in August and IP3b. … Serosurveillance of all sheep within 3 km of the September outbreaks revealed another infected premises (IP5), on which it was estimated that disease had been present for at least two, and possibly up to five weeks. As Figure 2B shows, IP5 is a likely link between the August and September outbreaks.

I would be interested in hearing from the people on the ground just how useful this information was — for example, were they impelled to search more for an intermediate source based on this information, or did they already suspect it from other, classical ways? But in any case, it’s clear that genomics is capable of pushing epidemiology a lot further in the future.

1. Cottam, E.M., Wadsworth, J., Shaw, A.E., Rowlands, R.J., Goatley, L., Maan, S., Maan, N.S., Mertens, P.P., Ebert, K., Li, Y., Ryan, E.D., Juleff, N., Ferris, N.P., Wilesmith, J.W., Haydon, D.T., King, D.P., Paton, D.J., Knowles, N.J. (2008). Transmission Pathways of Foot-and-Mouth Disease Virus in the United Kingdom in 2007. PLoS Pathogens, 4(4), e1000050. DOI: 10.1371/journal.ppat.1000050[↩]
2. Samuel, A. R., and Knowles, N. J. (2001). Foot-and-mouth disease type O viruses exhibit genetically and geographically distinct evolutionary lineages (topotypes). J Gen Virol 82, 609-621.[↩][↩]
3. Cottam, E. M., Haydon, D. T., Paton, D. J., Gloster, J., Wilesmith, J. W., Ferris, N. P., Hutchings, G. H., and King, D. P. (2006). Molecular epidemiology of the foot-and-mouth disease virus outbreak in the United Kingdom in 2001. J Virol 80, 11274-11282.[↩]
4. As far as I know, it’s not yet known how exactly the virus escaped from the IAH. I’ve read what seems to be informed speculation that it may have come from the drains, as decontamination systems designed to prevent that weren’t properly maintained; but I don’t know if that’s true, an educated guess, or mere rumor and guesswork.[↩]

“Human cytomegalovirus infected human endothelial cells” by Joerg Schroeer

There’s a famous picture in Field’s Virology¹ showing how ectromelia (mousepox virus, a model for smallpox) infects a new host, spreads within the mouse, and then is transmitted to a new host. The figure is below² (click for a larger version). Simplified, ectromelia initially infects the skin through small cuts; it replicates at the site, then spreads through blood and lymph to organs (spleen, liver) where it replicates further. The progeny virus from this replication then spreads again through the blood, this time back to the skin, where it replicates once again (now vastly amplified from the initial infection) to form the classic “pock” lesions, which shed virus that can infect a new victim.

It’s generally accepted that this is a common pattern of pathogenesis for many of the viruses that go systemic; not necessarily all viruses, because certainly some remain localized or only spread through, say, direct contact, but for something that spreads through the entire host, it should be a reasonably accurate model.

Human and mouse cytomegaloviruses certainly spread throughout the entire host, and infect many cell types within the body — endothelial cells, lymphoid cells in the spleen and elsewhere, liver, and probably other tissues as well. However, Ulrich Koszinowski’s group now suggests that in spite of this, it isn’t following the ectromelia pattern; replication within some of the organs (liver) is a dead end, that doesn’t help disseminate the virus. ³

Koszinowski has a habit of constructing very cool systems for analyzing his pet virus; sometimes so fancy that I wonder if he makes them a little baroque just because he can (and I know⁴ that he has occasionally been bitten by his elaborate systems). Here he used a Cre/lox recombination system, with the flox in the CMV and the Cre in the mouse under cell-specific promoters, so that the virus genome gets modified only when it replicates in the particular organ. Don’t worry about the details, the point is that the virus is tagged as soon as it replicates in a particular organ, so you can look at viruses throughout the whole mouse and identify whether their ancestors ever replicated in one particular organ. You can also work out timing of replication, and a few other things.

The unexpected bottom line is that what happens in the liver, stays in the liver. There is more MCMV in the liver than anywhere else in the body, but it’s a dead end; once it’s in the liver it doesn’t spread to other organs.⁵ Instead, the relatively small amount of virus that replicates in endothelial cells (and perhaps in the spleen) seems to be a major source for further spread within the body and for transmission.

The results challenge the concept that organs that produce the bulk of infectious virus during acute infection necessarily also play a major role in dissemination.

Surprisingly, to me anyway, even suppression of the adaptive immune system didn’t change this; the virus still hung out in the liver. (The door is still open for innate immune restriction of spread.)

This shows how little we really know about what goes on in authentic viral infections. So much of our understanding of virology is based on tissue culture, but it’s harder than it looks to extrapolate a simple in vitro observation to the complicated interactions within the body, and it’s dangerous to extrapolate from one virus to another.

Why does CMV replicate in the liver if it’s a dead end? Is this simply a matter of indifference to the virus (replicate anywhere you can and hope you’re in the right spot to spread) or is it doing something specific to modulate the host in some way? My bias is that this is something the virus is doing for a reason, but I don’t know what.

1. It’s adapted from a figure in Fenner’s Viral Pathogenesis, which I haven’t read:
   Fenner F, Buller RM. Mousepox. In: Nathanson N, Ahmed R, Gonzalez-Scarano F, et al., eds. Viral Pathogenesis. Philadelphia: Lippincott-Raven; 1997:535-553.
   and is based on old research from Fenner:
   Fenner, F. (1949). Mouse-pox; infectious ectromelia of mice; a review. J. Immunol. 63, 341-373.[↩]
2. I think this qualifies as fair use[↩]
3. Sacher, T., Podlech, J., Mohr, C. A., Jordan, S., Ruzsics, Z., Reddehase, M. J., and Koszinowski, U. H. (2008). The major virus-producing cell type during murine cytomegalovirus infection, the hepatocyte, is not the source of virus dissemination in the host. Cell Host Microbe 3, 263-272.[↩]
4. That is, “I have heard rumors that … “[↩]
5. Even though the virus in the liver is actually perfectly competent for replication in other tissues, if taken from the liver and used to infect other mice.[↩]

CD1 is a fascinating molecule, but it hasn’t traditionally been associated with antiviral protection. Viruses, however, seem to disagree.

CD1 is (actually, CD1 are, since these are a family of related molecules) members of the MHC class I family, with many of the traditional MHC class I features — binding to β₂-microglobulin, a “groove” made of two alpha-helices on top of a beta-pleated sheet ( (in classical MHC, the peptide-binding groove: the “bun” of the peptide’s “hot dog”).

I previously wrote a field guide to the MHC family that shows these features across a wide range of the MHC family, but here are some comparisons of CD1d (PDB number 2GAZ) to classical MHC (HLA-A2; PDB number 2GTW). Here I’m showing the “heavy chain” of the complex in red, the ligand that fits in the binding groove is green, and β₂-microglobulin in blue. (The “top view” is looking “down” at the molecule more or less as a T cell would “see” it; the ribbon view lets you see the ligand interactions a little more easily; the “ligand only” view shows the thing that goes in the groove with all the MHC or CD1 resides removed.) (Click on a molecule for a larger version.)

	Side view	Top view	Top view (ribbon)	Ligand only
CD1d
HLA-A2

The similarities are pretty obvious, but it’s the difference that makes CD1 particularly interesting. Classical MHC molecules present peptide ligands; CD1d presents, instead, very hydrophobic molecules: lipids, glycolipids, and lipopeptides. These sorts of things are typically found in mycobacteria (as with phosphatidylinositol mannoside, shown in the images here), and I’ve thought of CD1 as an example of how our physiology has been shaped by pathogens — this whole branch of the immune system, devoted to detection and elimination of tuberculosis and leprosy.

My view started to slip in around 2000, with Frank Chisari’s observation that NKT cells may be involved in control of hepatitis B virus in his transgenic mouse model.¹ (NKT cells are the main lymphocytes that recognize CD1 molecules.) I’ve talked about Chiasri’s HBV mouse model before — it’s so artificial that I always am hesitant to extrapolate from it. That said, his findings in that model have all (as far as I know) held up in more natural systems, and the NKT observation is no exception; several other groups have seen similar things. ²

What really confirmed to me that CD1 can be antiviral, though, was the virus’s side of the story. Viruses employ an arsenal of anti-immune molecules, presumably targeting whichever immune components that are especially dangerous to the particular virus. Over the past few years, there’s been an increasing number of sightings of viruses that block CD1-mediated presentation. The first (that I know of) was HIV,³ and since then vaccinia virus⁴ and herpes simplex⁵ have also been shown to block CD1-mediated antigen presentation. The latest addition to the list is human cytomegalovirus.⁶ These viruses (HIV, poxviruses, and herpesviruses) are particularly good at blocking classical MHC class I presentation as well; I don’t know if this dual blockade is typical, or if people have mainly looked in those viruses most renowned for immune evasion — in other words, maybe we’re seeing this double action because people are looking under the streetlamps.

It’s interesting that HSV and HCMV (though not HIV, which blocks both classical MHC class I and CD1 with the same protein, nef) have apparently developed separate systems to block CD1 and classical MHC. The molecules responsible for their CD1 blockade are not yet identified, but they don’t seem to be the same as the ones that block MHC class I. If CD1 blockade is the main function of these genes (and not a side-effect of blocking some other aspect of immunity, say), the implication is that CD1 is an important-enough player in controlling these viruses that they have had to maintain distinct pathways to escape from it.

I wonder what it’s doing.

1. Kakimi, K., Guidotti, L. G., Koezuka, Y., and Chisari, F. V. (2000). Natural killer T cell activation inhibits hepatitis B virus replication in vivo. J Exp Med 192, 921-930.[↩]
2. For example, Grubor-Bauk, B., Simmons, A., Mayrhofer, G., and Speck, P. G. (2003). Impaired clearance of herpes simplex virus type 1 from mice lacking CD1d or NKT cells expressing the semivariant V alpha 14-J alpha 281 TCR. J Immunol 170, 1430-1434. [↩]
3. Shinya, E., Owaki, A., Shimizu, M., Takeuchi, J., Kawashima, T., Hidaka, C., Satomi, M., Watari, E., Sugita, M., and Takahashi, H. (2004). Endogenously expressed HIV-1 nef down-regulates antigen-presenting molecules, not only class I MHC but also CD1a, in immature dendritic cells. Virology 326, 79-89.[↩]
4. Webb, T. J., Litavecz, R. A., Khan, M. A., Du, W., Gervay-Hague, J., Renukaradhya, G. J., and Brutkiewicz, R. R. (2006). Inhibition of CD1d1-mediated antigen presentation by the vaccinia virus B1R and H5R molecules. Eur J Immunol 36, 2595-2600.[↩]
5. Sanchez, D. J., Gumperz, J. E., and Ganem, D. (2005). Regulation of CD1d expression and function by a herpesvirus infection. J Clin Invest 115, 1369-1378.
   and
   Yuan, W., Dasgupta, A., and Cresswell, P. (2006). Herpes simplex virus evades natural killer T cell recognition by suppressing CD1d recycling. Nat Immunol 7, 835-842.[↩]
6. Raftery, M.J., Hitzler, M., Winau, F., Giese, T., Plachter, B., Kaufmann, S.H., Schonrich, G. (2008). Inhibition of CD1 Antigen Presentation by Human Cytomegalovirus. Journal of Virology, 82(9), 4308-4319. DOI: 10.1128/JVI.01447-07[↩]

My first foray into research, when I started grad school, was to work on a vaccine against bovine adenovirus type 3.