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.

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.[↩]

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.[↩]

We know that HIV can be controlled by an appropriate immune response. Cytotoxic T lymphocytes (CTL) are capable of very effectively suppressing HIV; in fact, in a standard HIV infection, the virus typically spends most of its early phase being controlled by a T cell response. In most people, unfortunately, the control is temporary; since HIV replication is sloppy, the virus throws off mutants at regular intervals, and eventually one of the mutants will be invisible to the dominant CTL response. That mutant replicates rapidly (probably damaging the immune response as it does so) until a new CTL response brings that virus under control, only for other variants to arise again.

Some people are apparently able to hold the virus under control for very long periods — the long-term non-progressor HIV patients. Some of these people seem to have T cell responses against part of the virus that has very precise sequence requirements; if the virus mutates away from CTL recognition, the virus is crippled and can’t replicate effectively. Other people seem to have a broad T cell response, one that recognizes several parts of the virus at once. The odds of successfully mutating all of the targeted areas simultaneously are exponentially lower than of mutating a single region.

Obviously, either of these are states that vaccine designers want as outcomes. That’s not all that easy. People are variable, and there don’t seem to be general rules that you can use to force an immune response to the target of one’s choice. ¹ Wouldn’t it be nice if there was a way of bypassing the whole messy immunization step, and just moving straight on to the desired finale of CTL specific for the target of one’s choice?

A paper in the March ‘08 issue of Journal of Virology² does just that.

When you induce T cell-mediated immunity, whether through a vaccine or a real infection, what you’re actually doing is expanding a pool of T cells whose receptor recognizes your special antigen. There are a huge number of potential T cell receptors (TcRs); under normal conditions, any particular antigenic target might have only 20 or 100 T cells that can recognize it, scattered among the millions of T cells with irrelevant specificities. Once a T cell finds its antigen, though,³ that T cell clone divided and expands enormously, as much as 100,000 times. The next time that antigen rides through town, it finds hundreds of sheriffs awaiting it, not just one or two.

If the TcR is all you need for specific recognition, can you bypass the whole annoying specific recognition and expansion step? Why not take the TcR from a previous clone, that you already know is useful (perhaps one from another individual altogether) and swap it into generic, non-specific T cells? In fact, that’s been done in a number of cases, and it actually seems to work.⁴

Joseph et al. tried this with a TcR specific for a HIV antigen. They swapped this known TcR into ordinary generic T cells from a normal blood donor, and turned those boring old plain T cells into CTL that specifically killed HIV-infected cells.

OK, their system is very artificial, involving transformed target lines and a Rube Goldbergesque mouse system to test “in vivo activity”, so it’s not really possible to draw any conclusions about clinical potential. In an actual infection, you’d presumably want to do this with multiple TcRs simultaneously, to target many HIV antigens at once and reduce the risk of immune escape (otherwise, just putting in one chimeric TcR is not different from getting a strong CTL response to HIV — which we know is not sufficient in the long run). I don’t think we know what would happen in that situation; would there be competition between the different TcRs to the point that most would be outcompeted and swamped, ending up with a de facto single target after all? ⁵

Another question I have is whether the original TcRs might cause mischief — if the T cell has two TcRs, stimulation through one might lead to reactivity with the other, and if the other, original, TcR happens to react with a self antigen you might get the mother of all autoimmune diseases. So my guess is that this is mostly a cute idea that will never go anywhere (for HIV; I think it has much more potential in tumor treatment).

Still, it really is a neat concept, and I hope some of my questions get addressed.

1. There are some approaches that can do this, but they also have drawbacks.[↩]
2. Joseph, A., Zheng, J.H., Follenzi, A., DiLorenzo, T., Sango, K., Hyman, J., Chen, K., Piechocka-Trocha, A., Brander, C., Hooijberg, E., Vignali, D.A., Walker, B.D., Goldstein, H. (2008). Lentiviral Vectors Encoding Human Immunodeficiency Virus Type 1 (HIV-1)-Specific T-Cell Receptor Genes Efficiently Convert Peripheral Blood CD8 T Lymphocytes into Cytotoxic T Lymphocytes with Potent In Vitro and In Vivo HIV-1-Specific Inhibitory Activity. Journal of Virology, 82(6), 3078-3089. DOI: 10.1128/JVI.01812-07[↩]
3. assuming appropriate conditions for activation and so forth[↩]
4. E.g. for tumors; Morgan, R. A., Dudley, M. E., Wunderlich, J. R., Hughes, M. S., Yang, J. C., Sherry, R. M., Royal, R. E., Topalian, S. L., Kammula, U. S., Restifo, N. P., Zheng, Z., Nahvi, A., de Vries, C. R., Rogers-Freezer, L. J., Mavroukakis, S. A., and Rosenberg, S. A. (2006). Cancer regression in patients after transfer of genetically engineered lymphocytes. Science 314, 126-129.[↩]
5. Some models for immunodominance predict this, in fact[↩]

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.

What about increasing viral virulence? What could drive that?

There’s at least one fairly well-documented example of that. The increase in virulence is probably because of a change in the virus’s environment that  forces the virus to become more virulent in order to continue to transmit efficiently. Ironically, the environmental change is vaccination.

As far as I know — I want to put this up front, to forestall the vaccine loons — there’s no instance where this has happened with a vaccine used for humans. ¹ I’m talking about a chicken vaccine, for Marek’s Disease.

Marek’s Disease Virus (MDV) is an extraordinarily interesting virus. It’s a herpesvirus of chickens; it causes, among other symptoms, tumors. MDV was a relatively minor problem when chicken farming was a backyard industry. When very large, intensive commercial chicken farms arose, the virus was able to sweep through flocks and cause truly enormous losses. The first Marek’s Disease vaccine, introduced in the 1960s, reduced losses by some 99%. (Incidentally, this was the first vaccine ever to prevent cancer.)

But the 99% protection rate didn’t last long. Losses began to creep up once again, as more virulent viruses arose. New vaccines have been introduced a couple times; each time losses dropped, but then once again new and increasingly-virulent viruses arose. Marek’s Disease viruses isolated today are far more virulent than the relatively benign viruses of the 1960s and early 1970s; the original vaccine is essentially useless against them.

The figure at right² (click for a larger version) shows the virulence of virus strains isolated over a ten-year period — although there’s a lot of variability, there’s a pretty clear upward trend. (This chart — and all the others I could find — only shows changes relatively late in the story, skipping the interesting periods in the 1970s and early 1980s when the first changes in virulence were noted. I think this is a technical issue of having the appropriate strains available for comparison. However, see: Increased virulence of Marek’s disease virus field isolates. Witter RL. Avian Dis. 1997 Jan-Mar;41(1):149-63. doi:10.1016/j.tvjl.2004.05.009 for a more detailed analysis of MDV strain virulence over the years.)

This evolution is actually very reminiscent of the myxoma/rabbit co-evolution story I’ve talked about, here and here. Australian rabbits have evolved to become much more resistant to myxoma virus than their European cousins. In this case, MDV is more analogous to the rabbits than to myxoma — evolving mechanisms to persist and replicate in the face of a lethal challenge (for the rabbits, myxoma virus; for Marek’s Disease virus, the vaccine-derived immunity).

Before rabbits could evolve resistance, there had to be some survivors of myxoma infection. In that case, myxoma virus itself evolved to become somewhat less virulent (70-90% lethal, instead of 98%). In the Marek’s Disease story, a key factor is that the vaccines all suck³ in their ability to actually prevent infection; they prevent the disease, but viruses can still infect vaccinated birds, although the virus replicates slower (which reduces transmission).

This is a recipe for virulence. Viruses in general evolve toward improved transmission. The MDV vaccine reduces, but doesn’t eliminate, transmission. Increasing replication in the face of the vaccine increases transmission. Increasing viral replication also increases viral virulence.⁴

This probably isn’t the whole story (there’s some evidence that the virus was already evolving toward increased virulence even before the vaccine was introduced — perhaps related to changes in its environment brought about by factory farming), and the mechanisms underlying the changes in virulence are not known, but the solution would seem to be clear: Develop a Marek’s Disease vaccine that will induce sterilizing immunity, as do most vaccines used against human viruses. That way, there’s no survivor virus that can act as a seed for evolution of virulence.

Unfortunately, of course, herpesviruses like MDV are notoriously difficult to vaccinate against. There’s still no commercial vaccine against herpes simplex virus, in spite of decades of research. Feline herpesvirus vaccine, which is universally used among pet cats, is like Marek’s in that it prevents symptoms but doesn’t prevent infection. (There is an effective vaccine against varicella-zoster virus [chicken pox] which does seem to effectively prevent infection — an exception to the rules.) So the chicken world is forced to stick with the non-sterilizing vaccines, even though “MD vaccines also appear to have a malign influence on the continued evolution of the pathogen itself.” ²

1. I’m not saying there’s no such instance, but I don’t know of one.[↩]
2. Nair, V. (2004). Evolution of Marek’s disease — a paradigm for incessant race between the pathogen and the host. The Veterinary Journal DOI: 10.1016/j.tvjl.2004.05.009[↩][↩]
3. Note rigorous technical terminology[↩]
4. This is not a universal equation; virus virulence isn’t necessarily linked to increased replication, for example.[↩]

Sand rat, Psammomys obesus

Although a lot of viruses have ways of blocking recognition by T cells and NK cells, there’s not much known about the importance of these mechanisms in actual infections. That’s because the best-studied viruses in this class tend to be highly species-specific. So, for example, we don’t have good animal models for the human herpesviruses human cytomegalovirus, Epstein-Barr virus, varicella-zoster virus, or Kaposi’s sarcoma herpesvirus. Herpes simplex virus does infect mice, but its immune evasion molecule ICP47 doesn’t work well in mice, so we’re no further ahead.¹

Immune evasion by all these viruses has been studied pretty extensively in cultured cells, but because they essentially only infect humans we only have circumstantial evidence for a role in vivo. Similarly, porcine, bovine, and equine herpesviruses encode immune evasion molecules, but pigs, cattle, and horses are not very convenient models for basic research either.Another major group of viruses, besides the herpesviruses, that are noted for immune evasion are the adenoviruses. However, only the human (and primate) adenoviruses contain the classical E3gp19k immune evasion molecule. There’s an animal model for human adenoviruses (the cotton rat) but as I pointed out the other day, there’s little evidence for an important function of CTL immune evasion in this model.

So what we need are small animal, and preferably lab mouse, models for infection with adenoviruses or herpesviruses that include immune evasion molecules. As far as we know, mouse adenoviruses don’t have T cell or NK cell immune evasion properties. That leaves us with mouse herpesviruses. Of the hundreds of known herpesviruses, six are known to be murid-specific (see the table at right), and three of those infect lab mice. One of those is totally obscure (there’s very little known about mouse thymic herpesvirus), leaving us with mouse cytomegalovirus and mouse herpesvirus 68 (MHV68). I’ve already commented on immune evasion by mouse CMV. The bottom line is that removing all known T cell evasion molecules from MCMV makes almost no difference to infection or latency; the one difference is that the virus persists longer and at higher levels in salivary glands. That may be important in virus transmission, but lacks a little oomph.

That leaves MHV68, and I’m pleased to say that there is actually some evidence that T cell immune evasion is important for this guy. (I’ve mentioned this in passing earlier, but it deserves its own post.) MHV68 uses a gene “mK3″ to attack MHC class I (MHC class I is recognized by cytotoxic T lymphocytes). ² In 2002, Philip Stevenson and Stacey Efstathiou made a mutant of MHV68 lacking mK3,³ and tested its ability to infect mice:

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 γ-herpesvirus. Nature Immunology DOI: 10.1038/ni818

In cultured cells, where there’s no immune system, the mutant virus grows exactly as well as wild-type MHV68. As well (more surprisingly) there was no difference in the initial virus clearance; the mutant virus and the parent were both cleared from the lungs of infected mice at the same rate, and were undetectable after about 13 days. However (finally!) there was a big difference in the amount of latency. The figure to the right shows latent wild-type (left panel) and mutant (right panel) MHV68, the black dots, in spleens of mice infected 13 days previously. What’s more, during the latent phase there was a better immune response to mutant MHV68; mice infected with the mutant virus had about twice as many CTL specific for MHV68.

The association of higher virus-specific CTL frequencies with lower viral loads suggested that CTLs were responsible for the elimination of DeltaK3 viruses during latency amplification.

Eliminating CTL from the mice removed the difference; in the absense of CTL, the mK3 knockout virus established latency just as well as the wild-type. This shows that effects on CTL, and probably not some other unknown function of mK3, are responsible for the difference.

So with the two and a half authentic models of infection (I count the herpes simplex virus one as a half because it’s more contrived than a natural infection) we have immune evasion molecules helping to establish latency (MHV68), helping to reactivate from latency (herpes simplex) and helping with persistence (MCMV) . In no case is there much effect on acute infection.

1. However, swapping in a different immune evasion molecule, that does work in mice, helps HSV reactivate from latency; see my previous post here. [↩]
2. mK3 is so named because it’s highly similar to a Kaposi’s sarcoma herpesvirus gene called “K3″; K3 is one of two KSHV genes that target MHC class I, but we don’t know much about KSHV infection. MHV68 probably isn’t a great model for KSHV, in spite of using a similar protein in immune evasion.[↩]
3. This virus was still able to down-regulate MHC class I to some extent, though, so there may be still other immune evasion genes in MHV68[↩]

I’ve lost an old friend. Apparently it’s bee