Ectromelia mouse (from McFadden)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 infected wth vaccinia
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.[]