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.

Tametomo’s force driving away the gods of smallpox. Yoshitoshi Taiso, 1890

Following up to my last post , about the Gates Foundation’s call to eradicate malaria, I thought I would talk about historical experience with eradication of infectious diseases. Here is the list of diseases that have been eradicated throughout all of recorded history:

1. Smallpox

I’ll pause so you can write that down.

OK, there are a couple of other diseases that are, hopefully, on their way to eradication (notably poliovirus), and there are a bunch of others whose incidence has been spectacularly reduced through vaccination (such as measles, diphtheria, and rubella),¹ sanitation (such as guinea worm), and even antibiotics (leprosy). But only smallpox has been eradicated. ²

Why was smallpox eradicated, where four other global eradication campaigns³ failed? What was special about smallpox and its vaccine? What are the factors that allowed this disease to be reduced from millions of cases per year, to none? And, most to the point, what aspects of smallpox eradication are applicable to malaria?

In fact, most of the special aspects of smallpox that allowed it to be eradicated are not particularly true for malaria. Smallpox …

• Has no animal host. If you can eradicate the disease in humans, it won’t re-emerge from a mouse, or monkey, or bat reservoir — compare to yellow fever, for example.
• Has no persistent phase. Smallpox either kills people, or they recover completely and eliminate the virus. In either case, if there are no clinical cases over a reasonable period, then you can be confident that there is no more virus.
• Induces long-term immunity in survivors.
• Was a fearful enough disease that the political will to eradicate it lasted through the campaign. Smallpox vaccination continued throughout civil wars and other upheavals.

Vaccinating the poor. Sol Ettinge, Jr., 1872

And the smallpox vaccine (vaccinia virus) is also exceptional in that it …

• Induces very long-term immunity with a single dose. Vaccinia virus induces a memory, and probably protective, immune response for an extraordinarily long time — response have been shown for up to 60 years.
• Is relatively stable and easy to transport and deliver. With large-scale vaccination campaigns, logistics become the limiting factor, especially as the campaign progresses and the final reservoirs of disease may be in remote, third-world areas.
• Leaves a marker of treatment. Vaccinated people usually had a small scar at the site of scarification, so that it was possible to identify susceptible people and protect them.

The smallpox vaccine is also exceptional in its frequency and severity of adverse effects. I think that for no disease today would the risks of smallpox vaccine be tolerated — back to the fourth point above, that smallpox was such a terrible disease that people were willing to take the risks of vaccination. ⁴

There were also a vast number of technical and logistic components that, I think, are mostly applicable to any eradication program (for example, the cost per dose of a vaccine is much less if the vaccine can be prepared in large, multi-dose vials; but that means you need to use the vial up all at once, which means in turn organizing large numbers of vaccination on a single day; and that in turn implies an efficient communication network and so on), and which I won’t talk about here. There’s a fascinating review in Henderson, D. A. (1987). Principles and lessons from the smallpox eradication programme. Bull World Health Organ, 65(4), 535-546. if you want to learn more.

“A much greater change — not apparent but real — was produced by the introduction of vaccination in 1798. It was computed, that, in 1795, when the population of the British Isles was 15,000,000, the deaths produced by the small-pox amounted to 36,000, or nearly 11 per cent. of the whole annual mortality. Now, since not more than one case in 330 terminates fatally under the cow-pox system, either directly by the primary infection, or from the other diseases supervening; the whole of the young persons destroyed by the small-pox might be considered as saved, were vaccination universal, and always properly performed. This is not precisely the case, but one or one and a half per cent. will cover the deficiencies; and we therefore conclude, that vaccination has diminished the annual mortality fully nine per cent. After we had arrived at this conclusion by the process described, we found it confirmed by the authority of Mr Milne, who estimates, in a note to one of his tables, that the mortality of 1 in 40 would be diminished to 1 in 43-45, by exterminating the small-pox. Now this is almost precisely 9 per cent.”
Combe, George. 1847. The Constitution of Man and Its Relation to External Objects. Edinburgh: Maclachlan, Stewart, & Co., Longman & Co.; Simpkin, Marshall, & Co., W. S. Orr & Co., London, James M’Glashan, Dublin.It’s important to point out that eradication of a disease is possible when not all of these factors are matched — poliovirus, which is almost eradicated (and could have been eradicated altogether with a bit more political help) is different in several ways. But it does offer a checklist for known success. How does malaria match up?

Not so well, actually. Malaria …

• May have an animal reservoir. Apes can be infected experimentally, and are sometimes naturally infected. This is not a practical issue today, where the animal reservoir is negligible, but if human infection is reduced an animal reservoir might serve as a source for reinfection.
• Does have a persistent phase. This is especially a concern since partially-immune people (common in endemic areas) can be infected and trasmit the disease without showing clinical symptoms — again, a potential reservoir of re-infection.
• Does not consistently induce protective immunity.
• Is a terrible scourge, but one to which the world has become accustomed. Is there the will to take on the cost of eradication? The last attempt at malaria eradication — which failed — cost a billion dollars. As Melinda Gates pointed out, the cost of the disease in perpetuity is greater than the cost of eradication, but the costs come from different places.

Since there are no effective malaria vaccines as yet, we can’t very well compare them to the smallpox vaccine. I don’t know enough about the irradiated vaccine that will enter trials next year, but the “RTS,S/AS02D” vaccine in phase I/II trials⁵ requires multiple doses and apparently offers relatively low protection — certainly better than nothing, if this holds true through phase III trials, but it’s hard to imagine that it’s sufficient for eradication.

So vaccines are probably going to be an important component of malaria eradication (if it happens) but the nature of the disease means that they’re not likely to be sufficient. Melinda Gates said in her eradication speech that “This is a long-term goal; it will not come soon,” and she focused on four “intervention points”:

To eradicate malaria, you have to end transmission — and there are multiple points where you can intervene. Reduce the number of infected mosquitoes. Keep mosquitoes from biting people. Keep people who are bitten from getting infected. Keep people who are infected from transmitting malaria back to mosquitoes.

Vaccines are good candidates to help with the last two points, and may help with the first. But overall, this is a more complex problem than smallpox. Nevertheless, smallpox eradication has plenty of lessons for malaria, as well.

1. A great review, with dramatic incidence tables is: Roush, S. W. & Murphy, T. V. (2007). Historical comparisons of morbidity and mortality for vaccine-preventable diseases in the United States. JAMA, 298(18), 2155-2163. I have adapted their numbers to make a table here [↩]
2. It is probably true that there are stocks of the virus around as well as the official stocks. However, there have been no cases of “wild” human smallpox since 1977.[↩]
3. Henderson, D. A. (1999). Lessons from the eradication campaigns. Vaccine, 17 Suppl 3, S53-5. [↩]
4. Belongia, E. A. & Naleway, A. L. (2003). Smallpox vaccine: the good, the bad, and the ugly. Clin Med Res, 1(2), 87-92.[↩]
5. Aponte, J. J., Aide, P., Renom, M., Mandomando, I., Bassat, Q., Sacarlal, J. et al. (2007). Safety of the RTS,S/AS02D candidate malaria vaccine in infants living in a highly endemic area of Mozambique: a double blind randomised controlled phase I/IIb trial. Lancet, 370(9598), 1543-1551.[↩]

I’m marking final exams for the grad immunology class I teach, so I don’t have a lot of time to blog. But I do want to point to a really amazing, ambitious, and potentially world-changing initiative that doesn’t seem to have got the attention it deserves in the blog-world. A couple of months ago, Melinda Gates made a speech in which she said:

Bill and I believe that these advances in science and medicine, your promising research, and the rising concern of people around the world represent an historic opportunity not just to treat malaria or to control it-but to chart a long-term course to eradicate it.

I don’t need to give figures, I think, on what a devastating disease malaria is. The WHO fact sheet is filled with dismal stats (”A child dies of malaria every 30 seconds.“) And I’ve previously blogged about the track record of malaria vaccines, which have been encouraging but unsuccessful for forty years. Gates’ proposal really is (as she herself says) audacious, but I think she presents three excellent reasons for aiming for eradication:

• the human cost of malaria
• the financial cost. “If we plan only to control malaria, we will never eradicate it.“
• history, which tells us that any malaria control is just temporary: “the ability of the parasite to develop resistance to insecticides and medicines tells us that no set of control strategies can control malaria for very long.“

Is it possible? I have no idea, myself. It’s been tried before (the poster at the top is from 1958) without success, and we are certainly a long, long way away from that aim at the moment. I do think it’s a worthy goal. And there are some new glimmers of hope. A Lancet article¹ that came out about the same time as Gates’ talk shows that a new malaria vaccine is safe and at least moderately effective.

Is “moderately effective” good enough? We don’t really know yet how effective the vaccine is; this study (which wasn’t designed to test effectiveness per se) found around a 65% level of protection — low for a vaccine in general; high for a malaria vaccine. A commentary on the paper in the same issue of Lancet² says that “Some experts have predicted that the effect of the introduction of a partly protective vaccine will be reduction in morbidity and mortality in the first years of life, with negligible effect on transmission.” If so, then this is more a step toward control than eradication.

Still, it’s a step, and if in fact vaccination can reduce malaria at all then it’s a very promising step. Other vaccines are on their way, and the experience with this one will help in developing more and more effective approaches. As Epstein’s commentary³ says, “The next 5-10 years will probably be the most exciting in the long journey to bring a malaria vaccine to the developing world.“

1. APONTE, J., AIDE, P., RENOM, M., MANDOMANDO, I., BASSAT, Q., SACARLAL, J., MANACA, M., LAFUENTE, S., BARBOSA, A., LEACH, A. (2007). Safety of the RTS,S/AS02D candidate malaria vaccine in infants living in a highly endemic area of Mozambique: a double blind randomised controlled phase I/IIb trial. The Lancet, 370(9598), 1543-1551. DOI: 10.1016/S0140-6736(07)61542-6[↩]
2. What will a partly protective malaria vaccine mean to mothers in Africa?Judith E Epstein The Lancet 370, 3 November 2007-9 November 2007, Pages 1523-1524 [↩]
3. What will a partly protective malaria vaccine mean to mothers in Africa? Judith E Epstein. The Lancet 370, 3 November 2007-9 November 2007, Pages 1523-1524 [↩]

As everyone knows, the incidence of allergies and asthma has exploded over the past 50-odd years. As lots of people also know, while the reasons for this explosion isn’t known (and are probably complex) one of the popular concepts explaining this is the “hygiene hypothesis”. This was originally proposed way back in 1989:¹

These observations . . . could be explained if allergic diseases were prevented by infection in early childhood, transmitted by unhygienic contact with older siblings, or acquired prenatally . . . Over the past century declining family size, improved household amenities and higher standards of personal cleanliness have reduced opportunities for cross-infection in young families. This may have resulted in more widespread clinical expression of atopic disease.

In the nearly 20 years since, this hypothesis hasn’t been proved or disproved. There are quite a few interesting correlations, and the underlying biology seems to make a lot of sense, but as least as far as I know there’s been no smoking-gun study that makes an undisputable link. A Nature Medicine paper² from a couple of weeks ago adds a little more support to the hypothesis, and this one also holds out a distant hope of some kind of intervention as well. It’s long been known that parasitic worms — now rare in the West, but until recently a normal part of the human condition — induce an immune response that is broadly similar to a lot of allergic responses.

Melendez et al show that one class of parasitic worms make a protein that inhibits the anti-parasitic immune response. The protein, ES-62, does this by binding the pathogen pattern receptor molecule TLR4, thereby blocking a signalling pathway that ultimately leads to mast cell activation. This is presumably a parasite immune evasion molecule, analogous in concept to the many viral proteins that block TLR pathways. (The reason I say this is “presumably” a immune evasion molecule is that the other possibility is that the response is driven by the host — that this is more analogous to the way rodents develop a regulatory T cell response to persistent viruses, reducing harmful inflammatory diseases but allowing long-term infection with the virus.)

As the authors say, this is an exciting observation for two reasons. First, while not a smoking gun (that’s a rhetorical question in the title, OK?), it offers a mechanistic explanation for at least part of the hygience hypothesis. Second, the protein offers a handle for therapy of allergic diseases:

Suppression of mast-cell function by ES-62 offers a new explanation for the reason why people harboring worms of at least the filarial nematode type show reduced incidence of allergy, in spite of their elevated serum IgE. … By inhibiting mast-cell effector function, ES-62 offers a new potential therapeutic approach for diseases such as asthma, a medical problem of enormous importance in the developed world. Although ES-62 per se is unlikely to be used for treatment, enough is known about its structure and function to allow one to envisage the development of small, presumably phosphorylcholine-based, derivatives as drugs.

1. Strachan, D. P. (1989). Hay fever, hygiene, and household size. BMJ 299, 1259-1260.[↩]
2. Melendez, A. J., Harnett, M. M., Pushparaj, P. N., Wong, W. S. F., Tay, H. K., McSharry, C. P., and Harnett, W. (2007). Inhibition of Fc[epsi]RI-mediated mast cell responses by ES-62, a product of parasitic filarial nematodes. Nat Med 13, 1375-1381.[↩]

Malaria parasites in mosquito midgut

Why is it so hard to come up with a disproof (or a proof) of overdominant selection in MHC?

I’m starting kind of in mid-sentence here, because this is a continuation of a series of posts on MHC diversity. Briefly: The major histocompatibility complex region in vertebrates is extraordinarily diverse — a hundred times more variable (more alleles) than the average genomic chunk. Even populations that are otherwise inbred and lack diversity throughout their genome, rapidly evolve, or maintain, MHC diversity. There is clearly powerful evolutionary selection for this diversity, and there are several different explanations as to what this driver might be. The two most plausible explanations are frequency-dependent selection (in which rare alleles are selected simply because they are rare, and pathogens haven’t adapted to them) and overdominance, or heterozygote advantage (where individuals with diverse MHC regions, containing many alleles, are selected because they are more resistant to pathogens).

Overdominance was (as far as I know) the first mechanism put forward to explain MHC diversity. ¹ The concept is a simple one: If one MHC allele protects against disease by binding a certain set of peptides, then two alleles should protect against more diseases by cumulatively binding a larger set of peptides. A heterozygous individual should be more resistant to pathogens than individuals that are homozygous for either single allele.

This simple concept, though, turns out to be very difficult to test rigorously. Most importantly, several of the different predictions between overdominance and frequency-dependent selection depend on how the population evolves, over time; but when trying to test predictions, we are usually looking, more or less, at a static snapshot of evolution. In the static state, it’s much harder to differentiate between the two possibilities: Is the allele diversity we see a stable diversity (consistent with overdominance) or is it a dynamic diversity, with different alleles gaining and losing advantage as they become more or less frequent (consistent with frequency-dependent selection)?

True overdominance is explicitly not dependent on allele frequency. ² There are conditions (that are not true overdominance) in which heterozygotes will have a selective advantage over homozygotes, where the advantage is strictly dependent on allele frequency. Therefore … ³

overrepresentation of HLA heterozygotes among individuals with favorable disease outcomes (which we term population heterozygote advantage) need not indicate allele-specific overdominance. On the contrary, partly due to a form of confounding by allele frequencies, population heterozygote advantage can occur under a very wide range of assumptions about the relationship between homozygote risk and heterozygote risk. In certain extreme cases, population heterozygote advantage can occur even when every heterozygote is at greater risk of being a case than either corresponding homozygote.

There are a fair number of studies on more or less wild populations that have claimed to show evidence for overdominance, but few (if any) deal with the frequency problem. For that reason, most of the claims in the literature are at best consistent with overdominance, but are not proof of it.

A second complication is that individuals heterozygous at the MHC are quite likely to be heterozygous generally. How can specific effects of MHC heterozygosity be distinguished from a general heterozygote advantage? Again, this makes studies on wild populations hard to interpret cleanly.

There’s a third complication: Overdominance is most likely to be a factor in infections with more than one pathogen. ⁴

MHC-mediated resistance to a single pathogen is inherited as a dominant trait. This means that there will be no differences in susceptibility between a homozygote MHC allele or haplotype and a heterozygote carrying the focal allele plus a different one. Therefore, heterozygote advantage is difficult to detect in single pathogen challenges.

An exception to this might be when there’s technically a single pathogen, but it’s highly antigenically diverse — the obvious example being HIV, which mutates rapidly and regularly throws out antigenic variants during the course of infection. It’s interesting, then, that HIV infection is one of the situations where heterozygote advantage has been observed, ⁵ though I don’t think population allele frequency was taken into account in these studies. On the other hand, malaria is antigenically diverse as well, but experiments have not shown overdominance in that case.⁶

So we’re left with the difficult situation where you need to have fairly large numbers and multiple generations, in order to detect selection; yet you probably can’t use most natural populations to strictly the test the theory. Setting up a large and relatively diverse, yet well-controlled, lab animal population, and then infecting with multiple pathogens; or following a reasonably well-controlled field population; is a daunting task.

In the next post in this series I’ll mention a few cases where this has been done.

1. Doherty, P. C., and Zinkernagel, R. M. (1975). A biological role for the major histocompatibility antigens. Lancet 1, 1406-1409.[↩]
2. At least, that’s how I understand it; and I repeat that I’m not an expert. Anyone knowledgeable about this, feel free to jump in.[↩]
3. Lipsitch, M., C. T. Bergstrom, and R. Antia. 2003. Effect of human leukocyte antigen heterozygosity on infectious disease outcome: the need for allele-specific measures. BMC Med Genet 4: 2. [↩]
4. Wegner, K. M., M. Kalbe, H. Schaschl, and T. B. Reusch. 2004. Parasites and individual major histocompatibility complex diversity–an optimal choice? Microbes Infect 6: 1110-1116. [↩]
5. For example, Carrington, M., G. W. Nelson, M. P. Martin, T. Kissner, D. Vlahov, J. J. Goedert, R. Kaslow, S. Buchbinder, K. Hoots, and S. J. O’Brien. 1999. HLA and HIV-1: heterozygote advantage and B*35-Cw*04 disadvantage. Science 283: 1748-1752. [↩]
6. Wedekind, C., Walker, M., and Little, T. J. (2005). The course of malaria in mice: major histocompatibility complex (MHC) effects, but no general MHC heterozygote advantage in single-strain infections. Genetics 170, 1427-1430.[↩]

A couple of months ago, I talked about a paper from the Sigal lab,¹ that demonstrated a role for cytotoxic T lymphocytes (CTL) in protecting mice against ectromelia infection very early after infection. A recent paper in Nature Medicine² makes a remarkably similar observation with malaria.

I’m sure I don’t have to run through the human costs of malaria here, or the enormous benefits that an effective malaria vaccine might bring. In fact, we are on the verge of having a vaccine against malaria, just as we have been on the verge of a vaccine for the past 40 years, because that’s about how long it’s been known that irradiated sporozoites confer strong protection against malaria.³

But I’m not here to be snarky about malaria vaccines, especially since I really don’t know much about the subject. Instead, let’s focus on the similarities between this parasite and the poxvirus ectromelia. Both establish an initial infection in the skin — the parasite, through a mosquito bite; the virus, through superficial cuts and scratches. They both then trickle out slowly from this initial site and seed the liver, where they both replicate extensively. Both then spread through the blood (viremia or parasitemia).

Of course, there are plenty of differences, too. For example, ectromelia is usually fatal during the liver stage, while for Plasmodium the liver is merely a staging spot. Another difference is the route they take to the liver; ectromelia is believed to reach the liver mainly through the lymph, whereas for Plasmodium sporozoites the lymph is apparently a dead end⁴ and sporozoites infect the liver via the blood.⁵ (The beautiful image to the right is not, in fact, a late Pollock, but is an image of sporozoite motility in the dermis from Amino et al. )

That second difference leads to a critical difference in interpretation. Sigal’s group observed ectromelia-specific CTL in the draining lymph node very early (3 days after infection). Because ectromelia travels through the lymph to the liver, they conclude that the CTL were probably protecting the mice by killing ectromelia-infected cells in the lymph nodes.

In contrast, Chakravarty et al find malaria-specific CTL in the draining nodes very early (2 days after infection). Because malaria travels through the blood to the liver, the CTL in the lymph nodes presumably aren’t blocking traffic to the liver. Instead, they reached a different conclusion: The draining lymph nodes are the site in which CTL are primed against sporozoite proteins, and the primed CTL then travel to the liver and provide protection there:

We show that a protective anti-sporozoite CD8+ T-cell response, however, originates early in lymphoid tissues linked not to the liver but to the cutaneous infection site. … In fact, we did not find appreciable evidence for CD8+ T-cell priming in the CLN, although the target antigen in this model (circumsporozoite protein) is expressed by sporozoites in early phases of liver infection.

I said (about the ectromelia paper): “One caveat I have is that I’m not convinced that the draining lymph node is the critical spot. At least in this paper, it’s shown that CTL are active there, but not that CTL activity in the node is essential. It’s also possible that the virus spreads through some other route, and that other route is also blocked by CTL.” Luis⁶ tells me he agrees that’s a possibility and was already working on ways to test it. The malaria paper might strengthen that possibility, but I think the diseases actually are different enough that it’s still not clear how applicable the one is to the other. Both papers, though, give a new appreciation for the power and flexibility of CTL.

1. Xu, R. H., Fang, M., Klein-Szanto, A., & Sigal, L. J. (2007). Memory CD8+ T cells are gatekeepers of the lymph node draining the site of viral infection. Proc Natl Acad Sci U S A, 104(26), 10992-10997. [↩]
2. CD8(+) T lymphocytes protective against malaria liver stages are primed in skin-draining lymph nodes. Chakravarty, S., Cockburn, I. A., Kuk, S., Overstreet, M. G., Sacci, J. B., and Zavala, F. (2007). Nat Med 13, 1035 - 1041. [↩]
3. Nussenzweig RS, Vanderberg J, Most H, Orton C. Protective immunity produced by the injection of x-irradiated sporozoites of Plasmodium berghei. Nature. 1967 Oct 14;216(5111):160-2. [↩]
4. Yamauchi, L. M., Coppi, A., Snounou, G., & Sinnis, P. (2007). Plasmodium sporozoites trickle out of the injection site. Cell Microbiol, 9(5), 1215-1222. [↩]
5. Amino, R., Thiberge, S., Shorte, S., Frischknecht, F., & Menard, R. (2006). Quantitative imaging of Plasmodium sporozoites in the mammalian host. C R Biol, 329(11), 858-862. [↩]
6. Rolling his eyes at this statement of the obvious[↩]