Mystery Rays from Outer Space

Macrophage phagocytosing mycobacteria

Sometimes the simple, obvious answer is right, and sometimes it’s completely backwards.

Tuberculosis was a terrifying, ubiquitous killer in the 19th century, but is relatively rare today (at least, in developed countries). The reason for the drop in Tb deaths isn’t entirely clear; it started with social factors probably including accidental or deliberate isolation of Tb patients, antibiotic treatment also knocked the disease back, and in some areas the vaccine (known as BCG) made a difference as well.

BCG is one of the oldest vaccines still in wide use; it was developed in the 1920s when a strain of Mycobacterium bovis (tuberculosis of cattle, contagious to humans) spontaneously lost virulence in culture. This avirulent strain of the bacterium was sent around the world and cultured independently, resulting in many distinct vaccine strains in different places and times. These strains are not only distinct genetically, but also phenotypically — they look different in culture, or grow differently, or whatever.

Over time, the vaccine has changed functionally, as well. Very early on the vaccine abruptly became even less virulent. More gradually, it seems that BCG has also become less effective; it’s no longer is able to protect against pulmonary Tb (although it’s still protective against other forms of the disease). Why is this?

At first glance this seems unsurprising. The bacterium has been grown in culture — outside of any animal host — for nearly 100 years. It’s had no selection to maintain its ability to grow in animals, or to avoid their immune responses, so of course it’s going to lose its ability to grow in animals.

But a recent paper¹ suggests that exactly the opposite happened. Whether randomly, or because of some unexpected type of selection, the BCG strain has actually amplified an immune evasion function. This modern variant of the vaccine strain isn’t simply passively failing to induce an immune response; it’s actively suppressing the immune response.

Specifically, the authors argue that normal (wild, virulent) Mycobacterium secretes antioxidants as an immune evasion mechanism; that modern BCG also secretes lots of antioxidants; and that this is related to genomic duplications in some BCG strains:

Some BCG daughter strains exhibit genomic duplication of sigH, trxC (thioredoxin), trxB2 (thioredoxin reductase), whiB1, whiB7, and lpdA (Rv3303c) as well as increased expression of genes encoding other antioxidants including SodA, thiol peroxidase, alkylhydroperoxidases C and D, and other members of the whiB family of thioredoxin-like protein disulfide reductases.¹

In other words, the long-term culture of BCG has yielded variants that are less immunogenic, because they are more actively suppressing the immune response. If their reasoning is correct, then reducing the antioxidant secretion from BCG should increase its immunogenicity. They took a BCG strain and deleted the duplicated antioxidant gene sigH (as well as the overexpressed SodA), and sure enough, the deleted version was more immunogenic and more protective in mice. “By reducing antioxidant activity and secretion in BCG to yield 3dBCG, we unmasked immune responses during vaccination with 3dBCG that were suppressed by the parent BCG vaccine.”¹

As a possible explanation, they note that their deletion variant also grows more slowly in culture than the “wild-type” BCG, and especially under certain culture conditions, and that this has led, coincidentally, to the reduced immunogenicity:

The practice of growing BCG aerobically with detergents to prevent clumping may have increased oxidant stress to cell wall structures and selected for increased antioxidant production. Then with each transfer the bacilli making more antioxidants represented a slightly greater proportion of the culture until they became dominant. In vivo, these mutations caused the vaccine to become less potent in activating host immunity. In effect, we believe that as BCG evolved it yielded daughter strains with an increased capacity for suppressing host immune responses. ¹

If this turns out to be generally true, then there’s a relatively straightforward handle for converting BCG back into a more effective, and safer, vaccine; whereas if the reduced immunogenicity was because of over-attenuation, it’s not so simple — you’d be trying to make a vaccine more virulent, which is a tricky tightrope to walk.

Incidentally, I frequently complain about the terrible, terrible quality of press releases about scientific advances  (and therefore the terrible quality of much “science reporting”, which is basically regurgitating the terrible press releases) so I want to give props to the person at Vanderbilt University Medical Center who put together the release for this paper — it’s a clear, simple, interesting, and as far as I can tell accurate account of the finding, background, and observation.  It can be done well — I wish it was done this well more often.

1. Sadagopal, S., Braunstein, M., Hager, C., Wei, J., Daniel, A., Bochan, M., Crozier, I., Smith, N., Gates, H., Barnett, L., Van Kaer, L., Price, J., Blackwell, T., Kalams, S., & Kernodle, D. (2009). Reducing the Activity and Secretion of Microbial Antioxidants Enhances the Immunogenicity of BCG PLoS ONE, 4 (5) DOI: 10.1371/journal.pone.0005531[↩][↩][↩][↩]

Researchers have used the mouse extensively as a model organism to study the pathogenesis of human infections and found that it imperfectly recapitulates many aspects of infectious disease as seen in patients. ¹

Humanizing a mouse

That strikes a chord with me because I just sent off a grant application explaining that mice are not suitable models for viral immune evasion.  However, my application may show a failure of imagination (or courage), because what Coers et al.¹ are driving toward is humanizing mice to make them better models for human disease, whereas I am merely proposing a different animal model.

What causes species specificity in pathogens?  That is, why is it that many pathogens infect humans very nicely, but don’t infect mice to any extent?  (And, of course, conversely, why do other pathogens cause disease in mice and not in humans.)

Chlamydia trachomatis in human cells

In some cases, a viral pathogen may simply be unable to get into the appropriate cell in the wrong species. An example is poliovirus, which normally doesn’t infect mice at all. But if you make a transgenic mouse² that expresses the (human) poliovirus receptor³, then the virus infects mice, and causes disease in them, perfectly well.   In this case, the receptor is the critical determinant of species specificity.  As a natural example of the same concept, SARS virus at least partly adapted to infecting humans by modifying its receptor-binding protein⁴ to improve interaction with the human version of the protein.

But there are also lots of cases where the virus can get into cells from the other species, yet doesn’t manage to replicate well or cause disease.  I’ve talked about mouse cytomegalovirus (MCMV) and its inability to infect humans here; it turned out that MCMV can’t infect human cells well because its normal ability to disarm the programmed cell death (apoptosis) pathways only works against the mouse versions of the pathway.  There are similar stories with HIV and its primate-infecting cousins; these viruses are limited to infecting hosts in which they (the viruses) can eliminate the APOBEC retrovirus-destroying proteins.  And the poxvirus myxomavirus is at least partly restricted to infecting rabbits because it can only inactivate the interferon pathway in rabbit cells. ⁵

You may notice two things about these examples: First, the non-receptor examples are generally immune evasion stories.  That is, these viruses are often apparently restricted to infecting a limited number of species because their immune evasion arsenal is limited to those species; take away their immune evasion by putting them in the wrong species, and they’re enfeebled.  Second, these examples are viruses.  The reason for that is just that I’m used to dealing with the crisp, clean mountain air of virology, and I don’t usually descend into the fetid swamps of bacteriology.⁶

But it turns out that at least in some cases the principles seem to be the same.  The Coers et al. paper¹ I cited at the top here makes some very familiar points: The receptor half of the story (”Colonization often relies on species-specific interactions of microbial ligands with host cell receptors“) applies to some bacterial pathogens (”Transgenic mice expressing human E-caherin in the small intestine, on the other hand, are susceptible to oral infections with L. monocytogenes and develop enteropathogenicity and systemic infections“).  And the immune evasion half also applies to some bacterial pathogens (”Additionally, host restriction may be caused by the failure of pathogens to deter immune assaults in the non-typical host“).

Even the nature of the immune evasion targets is familiar. Interferon pathways are frequent targets of bacterial immune evasion, as they are of viral immune evasion.  The details are different, in that the instances Coers et al. describe target a different branch of the interferon induction pathway, but the pattern is the same:

… the mouse-adapted strain Chlamydia muridarum, but not its close relative C. trachomatis, can specifically evade IRG-mediated⁷ host resistance … The divergent counterimmune mechanisms employed by the human pathogen C. trachomatis and the mouse-adapted pathogen C. muridarum clearly reflect the differences in the IFN? responses of their respective hosts. ¹

They finally discuss the possibilities of “Mus homunculus”, humanized mice, tailored to each pathogen, that would make more authentic models of infectious disease.  “Though the creation of humanized mouse models for infectious disease will require substantial effort and resources, the long-term benefits of these new models would undoubtedly be enormous.” ¹

1. Coers, J., Starnbach, M., & Howard, J. (2009). Modeling Infectious Disease in Mice: Co-Adaptation and the Role of Host-Specific IFN? Responses PLoS Pathogens, 5 (5) DOI: 10.1371/journal.ppat.1000333[↩][↩][↩][↩][↩]
2. Hi, Vincent![↩]
3. Transgenic mice expressing a human poliovirus receptor: A new model for poliomyelitis.
   Ruibao Rena, Frank Costantinib, Edward J. Gorgaczc, James J. Leeb and Vincent R. Racanielloa
   Cell 63:353-362 (1990) [↩]
4. Li W, Zhang C, Sui J, Kuhn JH, Moore MJ, et al. (April 20, 2005) Receptor and viral determinants of SARS-coronavirus adaptation to human ACE2. EMBO J 24.:1634-43.
   Sheahan T, Rockx B, Donaldson E, Sims A, Pickles R, et al. (March 2008) Mechanisms of zoonotic severe acute respiratory syndrome coronavirus host range expansion in human airway epithelium. J Virol 82.:2274-85.[↩]
5. Wang F , Ma Y , Barrett JW , Gao X , Loh J , Barton E , Virgin HW , McFadden G (2004) Disruption of Erk-dependent type I interferon induction breaks the myxoma virus species barrier. Nat Immunol 5: 1266-1274[↩]
6. In other words, I don’t know much about bacteriology.[↩]
7. IRG is part of an interferon-induction pathway[↩]

Simulation of hA3G-mediated HIV-1 evolution.

One of the host defenses against HIV¹ is “APOBEC3G” and related proteins.  These proteins force HIV to hypermutate, killing its ability to replicate in the next cell it infects.  On the other hand, it looks as if low-level mutation by APOBEC3G over the decades has driven HIV evolution:

We have found that hA3G activity acting on prior generations of virus has left detectable footprints in the HIV-1 genome. ²

HIV can only infect human cells because it has a defense against APOBECs: the viral protein “vif” causes APOBECs to be destroyed, and the virus is able to replicate without being hypermutated. 

So let’s say we develop an antiviral drug that blocks vif.  APOBECs would drive hypermutation of the virus.  This hypermutation would include the gene encoding vif.  Would this mutation, in a kind of molecular judo, drive rapid evolution of vif, so that it becomes resistant to the drug?

According to some experiments ²  both in cells and in silico, perhaps not:

However, since the predicted effect on resistance to standard antiviral drugs is likely to be small, we propose that concerns over increased resistance mutations should not impede development of HIV-1 Vif as a candidate drug target. ²

I’m not quite convinced this paper was actually modeling the phenomenon they say they’re modeling — is vif that’s shut off by deliberate mutations the same as vif that’s blocked by a (hypothetical) drug? But it’s a useful start, anyway.

1. and other lentiviruses; as well as quite a few other virus types[↩]
2. Patric Jern, Rebecca A. Russell, Vinay K. Pathak, & John M. Coffin (2009). Likely Role of APOBEC3G-Mediated G-to-A Mutations in HIV-1 Evolution and Drug Resistance PLoS Pathogens, 5 (4) DOI: 10.1371/journal.ppat.1000367[↩][↩][↩]

We know that the immune system can destroy tumors. We also know, unfortunately, that by the time we see a tumor, immunity probably won’t destroy the tumor. There are lots of reasons for that. One is that tumors are essentially part of the normal body, so it’s normal for the immune system to ignore them. It looks as if you need to have immunity that’s just right to get rid of a tumor.

Tumors arise from normal self cells,¹ that the immune response has been programmed to ignore. Now, the process of becoming a tumor is not normal, and so tumors are not entirely normal self any more — meaning that there are likely to be some targets in most if not all tumors. But in all but the most reckless tumors the differences between abnormal and normal are relatively small, compared to, say, a virus-infected cell that contains many potential targets.

There’s actually a long list of known tumor antigens; the T-cell tumor peptide database lists many hundreds of them. But most are not truly specific for the tumor.  The’re actually normal self antigens; they’re derived from proteins that are overexpressed in tumors, or that are differentiation antigens or “cancer-germline” antigens that are normally also found in self tissues. What’s more, these normal self antigens are the most interesting tumor antigens, as far as clinical utility is concerned. Mutations can make brand-new, non-self targets for the immune system, but they’re going to be sporadic targets, often unique to individual tumors — not something you can prepare for. The normal antigens, though, are likely to be predictable, common targets; it’s conceivable that tumor vaccines can be prepared in advance.

Human melanoma cell

If these antigens were common (which they are, in some tumor types — like melanoma), and they were good targets for the immune system, then we wouldn’t see much cancer. We do see melanomas quite often, and part of the reason may be that the immune system generally responds quite weakly to these antigens.  Why is that? And, more to the point, how can we make the immune system respond more strongly? A recent paper in the Journal of Experimental Medicine² offers answers for both of these questions.

From work in the past couple of years, we now have decent estimates of how many T cells there are that can react with any particular target. (See here and here for my discussion of the earlier papers.) A reasonably strong immune response to a non-self epitope might originate from maybe 100 or so precursor T cells. There’s a rather wide range of frequency for these precursor cells, say from 20 to 1000; and to some extent, the fewer T cells there are the weaker (the less immunodominant) the immune response.

We expect T cells against normal self targets to be less common, because they should be eliminated as they mature in the thymus. Some may survive, though, and we would count on these survivors to attack the normal (albeit overexpressed, or abnormally present) target in the cancer cells. But just how rare are they?

Rizzuto et al say they’re really rare (this was in mice, by the way); at least ten times less abundant than T cells against non-self antigens.  If you look at the range I gave for “normal” precursors, that could mean there are fewer than 5 or 10 precursors.  If the average is “fewer than five”, then quite possibly some mice have only two, or one, or no precursors.  You can’t have much of a response with no precursors.

So there’s a weak anti-tumor response because there aren’t many T cells in the body that can respond to the normal self targets in the tumor. That’s not really a surprise, but it does raise the question, What if there were more of the T cells? To ask that question, Rizzuto et al. tried transferring more of these precursor T cells into tumor-bearing mice — starting at around the normal level for a precursor to non-self antigen, and going up from there — and then vaccinating with the appropriate target.

The effects were pretty dramatic. With no supplemental T cells (that is, with the natural, very low, level of T cell precursors) the mice all died of the tumor quickly. At the middle of the range, almost all of the mice rejected the tumor. And at the highest levels of transfers? The mice all died again. Having enough T cells to respond was protective, but putting in too many made them useless.

These results identify vaccine-specific CD8+ precursor frequency as a remarkably significant predictor of treatment and side-effect outcome. Paradoxically, above a certain threshold there is an inverse relationship between pmel-1 clonal frequency and vaccine-induced tumor rejection.²

Mouse melanoma cell

(My emphasis) This paradoxical effect is probably because the numerous T cells started to compete with each other so that none of them were properly activated; they only saw effective-looking polyfunctional T cells at the lower transfer levels.

In other words, if you’re going to transfer T cells to try to eliminate a tumor, more is not necessarily better. Quality and quantity are both important factors, and quantity helps determine quality.

One question I have is how this relates to tumor immune evasion. Many tumor types  acquire mutations, as they develop, that block presentation of antigen to T cells. Are these mutations perhaps only partially effective — giving the tumors sufficient protection against the tiny handful of natural precursors they “expect” to deal with, but not against a larger attack after, say, vaccination — or are they more complete, and protective even if the optimal number of T cells are transfered? I’d guess that it would depend on the tumor, but it looks as if it might be a relevant question and it would be nice to have more than a guess.

Our results show that combining lymphodepletion with physiologically relevant numbers of naive tumor-specific CD8+ cells and in vivo administration of an effective vaccine generates a high-quality, antitumor response in mice. This approach requires strikingly low numbers of naive tumor-specific cells, making it a new and truly potent treatment strategy.   ²

1. I’m ignoring here crazy things like the contagious tumors of Tasmanian Devils and dogs[↩]
2. Rizzuto, G., Merghoub, T., Hirschhorn-Cymerman, D., Liu, C., Lesokhin, A., Sahawneh, D., Zhong, H., Panageas, K., Perales, M., Altan-Bonnet, G., Wolchok, J., & Houghton, A. (2009). Self-antigen-specific CD8+ T cell precursor frequency determines the quality of the antitumor immune response Journal of Experimental Medicine, 206 (4), 849-866 DOI: 10.1084/jem.20081382[↩][↩][↩]

Hierarchical clustering of breast carcinomas1

Something that’s puzzled me for years is why the same kinds of tumors tend to have the same kinds of immune evasion mechanisms. And I’m not going to give an answer, just trying to share the confusion a little.

What I mean is this:

It has been demonstrated that human tumors of distinct histology express low or downregulated MHC class I surface antigens … The distinct frequency of MHC class I abnormalities is caused by total HLA class I antigen loss, HLA class I down-regulation as well as loss or down-regulation of HLA class I allo-specificities. However, the frequency and mode of these defects significantly varied between the types of tumors analysed and could be associated in some cases with microsatellite instability. ²

(My emphasis) As I’ve noted here several times (most specifically here) tumors very often evade the immune system as they mature. Cytotoxic T lymphocytes (CTL) can control tumors in the tumors’ eary stages, but by the time we detect a tumor clinically the tumor is almost always resistant to the immune system. They do this in various ways, including inducing regulatory T cells, but also by mutating themselves to make themselves invisible to CTL (and other components of the immune system, but let’s keep it simpler for the moment).

There are a myriad ways for a tumor to become invisible, at the molecular level.  The MHC class I antigen presentation pathway is long and complex, and for any partiuclar tumor there are likely to be many different bottlenecks, points of attack.  Since tumors are all independent events³, so at first, and even second, glance, there’s no obvious reason why tumors of the same type should find a similar approach.  That is, just because two colon carcinomas look the same histologically in two different individuals, there’s no link between them.  ⁴ Why should colon carcinomas avoid CTL using one set of mutations, while, say, breast cancers use a different set of mutations? Yet apparently, that’s what tends to happen; for example:

Mutations or deletions in β2-m were detected in colon carcinoma (21%), melanoma (15%) and other tumors (<5%). So far, no mutations in β2-m have been found in RCC lesions, bladder and laryngeal tumors despite MHC class I loss or downregulation. … haplotype loss was found in head and neck squamous cell carcinoma (HNSCC) with a frequency of 36%, whereas in renal cell carcinoma (RCC) LOH only occurs in approximately 12% of tumor lesions analyzed. ²

If we saw these patterns only with virus-associated cancers, such as cervical carcinomas and even hepatic carcinomas, there would at least be a common link, but these tumors are not (as far as we know) caused by viruses in humans.

Part of the answer may be that the particular oncogenes associated with different tumor types lead to particular transcriptional hot-spots, and being a transcriptional hot-spot makes the region a mutational hot-spot as well, but at least as I understand it that’s not enough to account for the trends.

So why are particular MHC abnormalities linked to tumor type?  Anyone?

1. Turashvili et al. BMC Cancer 2007 7:55   doi:10.1186/1471-2407-7-55[↩]
2. Seliger, B. (2008). Molecular mechanisms of MHC class I abnormalities and APM components in human tumors Cancer Immunology, Immunotherapy, 57 (11), 1719-1726 DOI: 10.1007/s00262-008-0515-4[↩][↩]
3. barring such weird things as canine transmissible venereal tumor and Tasmanian Devil facial tumors; see here for more on those[↩]
4. The comparison is, of course, viruses.  A herpesvirus of chickens, and one of humans, may both use immune evasion mechanisms, but they have a common ancestor even if it’s a couple of hundred million years ago.[↩]

It’s well known that HIV mutates rapidly in infected patients in order to escape from the immune system. The mutations in HIV track with the peptides that bind to MHC class I in any particular patient. When the virus is transmitted to a new patient, though, those mutations don’t help it much, because MHC is so variable between individuals that the new infected person will very likely have a different MHC class I pattern. (In fact, the mutations the virus developed in the first patient, are likely to be actively harmful to the virus.) The virus has to start all over again and discover a new path toward immune escape. Over a long enough time, the virus may be able to slowly accumulate mutations that allow it to escape from the worst of the MHC class I alleles (see here for a possible example), but it’s very difficult, simply because MHC is so diverse.

But MHC class I itself is only the final stage of a longish pathway of antigen presentation — the route by which peptides are produced, modified, transferred into the right location, bind to the right proteins, all that stuff. (If it’s slipped your memory a little, I made a summary page for MHC class I antigen presentation here.) Within that pathway, at least in humans, it’s only the MHC class I heavy chain itself that’s wildly diverse; the other steps are pretty similar between any two individuals. So why doesn’t the virus mutate to avoid one of these monomorphic steps, and then not have to worry about re-mutating all over again after the next transmission?

Putting that less teleologically, why don’t mutations in HIV, that allow it to escape from the monomorphic steps in antigen presentation, persist in each new individual and accumulate within the population? Those mutations should be just as beneficial to the virus in the new infected person as in the original infectee.

Rob de Boer’s group  asked this question recently,¹ and found that

… within hosts, proteasome and TAP escape mutations occur frequently. However, on the population level these escapes do not accumulate … ¹

TAP structure2

(My emphasis) And the reason is the same reason other immune escape mutations don’t easily accumulate in the population: MHC is too diverse. If I follow the argument correctly, because the other components of the system are monomorphic, they have a very broad specificity for peptides, whereas MHC itself has a fine specificity. The virus can’t mutate every possible sequence in its genome that would interact with, say, TAP, because there would be thousands of them. If a mutation that prevents TAP binding does arise in one host, it’s selected because it prevents recognition of a particular MHC class I-binding peptide, and when it moves into a new host that peptide is no longer relevant for immune escape, so it’s not selected any more.

That means that, even taking the whole antigen presentation pathway into account:

The total number of predicted epitope precursors and CTL epitopes in a large population data set of HIV-1 clade B sequences is not decreasing over time. ¹

I am a little cautious about accepting this paper completely, because it’s heavily based on database analysis without a lot of testing; we don’t actually know whether the escape mutations they identify for TAP actually do escape TAP, for example. They make a number of arguments, in passing, for the accuracy of the epitope prediction programs out there; I am slowly backing in to some acceptance of the notion that the predictive programs are getting pretty good, which wasn’t my position a couple of years ago, but I still am not convinced they’re as good as they say here.

But the conclusion is fairly simple and straightforward, and it leads to an interesting suggestion:

… we speculate that only one of the steps in the antigen presentation pathway has to be polymorphic to prevent pathogens from adapting to any step in the pathway. The mechanism functions best when the polymorphy occurs at the most specific step in the pathway, as that increases the fraction of epitope precursors that is not under selection pressure. While in humans it is the MHC class I molecules that are highly polymorphic and specific, other solutions do appear to exist. The TAP molecules of rats are more specific than the human TAP, and have a limited functional polymorphism, and the TAP and MHC genes of chickens are equally polymorphic on the nucleotide level ¹

Chicken MHC is an interesting case, and is very strongly linked to resistance to some pathogens. But the reason for the tight linkage to resistance isn’t really known; there’s no obvious reason at the level of the MHC. It might be interesting to look at TAP as part of the resistance, as well.  I have some chicken stuff in the lab, and I should see if we can test that.

1. Schmid, B., Kesmir, C., & de Boer, R. (2008). The Specificity and Polymorphism of the MHC Class I Prevents the Global Adaptation of HIV-1 to the Monomorphic Proteasome and TAP PLoS ONE, 3 (10) DOI: 10.1371/journal.pone.0003525[↩][↩][↩][↩]
2. Structural arrangement of the transmission interface in the antigen ABC transport complex TAP.
   Oancea G, O’Mara ML, Bennett WF, Tieleman DP, Abele R, Tampé R.
   Proc Natl Acad Sci U S A. 2009 Mar 18  doi: 10.1073/pnas.0811260106[↩]

Any time a species meets some kind of barrier, there’s going to be selection to overcome that barrier.  In the case of pathogens, one major barrier they have to hurdle is their hosts’ immune systems.  What’s more, this isn’t a simple, static barrier.  Immune systems change on a day-to-day basis; and immune systems also change on a population basis, as the individuals in the host population are in turn selected by the pathogen.

Last week I talked about an example where a population — frogs in the UK, in this case — are apparently being selected by a pathogen.   The relatively recent introduction of frog virus 3 into the UK has caused large-scale die-offs of frogs there, and Teacher et al.¹ have just shown evidence that the survivors have been selected for a particular MHC class I type.

MHC class I is often associated with resistance to viruses, because it’s responsible for recognition by antiviral T cells.  What probably happens is that viruses sweep through a population, infecting (and imposing a selective pressure on) most members of the population.  A few individuals that happens to have some particular MHC class I type are relatively resistant to the virus, and have a selective advantage; that MHC allele becomes more frequent in the population; and the population as a whole becomes relatively resistant to the virus.  Of course, this now presents a new barrier to the original virus, and there’s selective pressure on it; virus mutants that are resistant to that particular MHC type do better; the virus sweeps through the new population; and a new minority with a different MHC type has a new selective advantage.

This is the most popular model (”frequency-dependent selection”), but it’s been hard to definitively show examples of it because things are happening on a evolutionary timescale. Even with the very rapid (as evolution goes) change in the UK frogs’ MHC, we don’t have all the pieces.  We see that frogs in the UK have a different set of MHC alleles than those frogs that haven’t been exposed to FV3, but we don’t have the population frequencies of these alleles over the time since the virus was introduced.  And we don’t have examples of  the virus accommodating itself to the new MHC; we’d see that as virus sequences changing over time.

Last week I ended the frog story by saying:

Some people may wonder if this frog virus story has any real relevance to humans. Well, apart from the pure scientific interest of tracking a potential frequency-dependent selection event in real time, one of the clearest links between an MHC class I allele and resistance to a viral infection is in humans, where the MHC class I alleles HLA-B27 and HLA-B57 are linked to resistance to HIV and HCV. Is it possible for HIV to adapt at the population level, so that the dominant strains of HIV in the world are no longer contained by HLA-B57? More generally, if we succeed in developing a T cell-based vaccine against HIV, it will probably have strong allele-dependent effects — will HIV adapt to this vaccine?

Astute readers² may have guessed that I wasn’t just guessing wildly, and indeed I had already seen the paper from Kawashima et al.,³ on exactly this topic.

Even though HIV is generally incredibly good at ripping through human immune responses without being controlled, there are some people who are long-term non-progressors (LTNP); they’re infected with HIV, yet they manage to control the virus pretty well, without antiviral treatment, for long periods. Many of these people, it turns out, have a particular subset of MHC class I types; they’re much more likely than the general population to have the HLA-B51, HLA-B57, or HLA-B27 MHC class I alleles.

HIV normally mutates very rapidly within infected individuals, so that as an antiviral immune response arises the virus may be temporarily controlled, but the new mutations that arise escape from the immune control and continue to replicate.  It seems that this immune escape is less likely to happen when the individual has one of the LTNP-associated alleles, and that’s probably because the immune target associated with HLA-B51 (etc) is essential for the virus’s survival.  When HIV mutates the immune target, the virus can’t replicate properly.  The only way HIV can escape immune control by people with these MHC alleles is to make multiple mutations at the same time, compensating for the escape mutation with several other changes.  These multiple mutations are exponentially less probable than single mutations, so the virus is essentially controlled, for a long time.

Humans are today a very large, highly mixed population, and it would take a vast plague, even worse than HIV, to rapidly cause frequency changes that we could measure in the brief period since HIV become common.⁴  But that hasn’t always been true; humans historically have included relatively small and isolated populations subject to intense disease selection, and we believe we see the outcome of that today in that different human populations  have different frequencies of HLA-B51, B57, and B27 — the equivalent of frogs in the UK vs. elsewhere.

What’s happening to HIV in those areas where HLA-B51 is common?  The prediction is that viruses that have managed to make the mutations that give resistance to HLA-B51 should have a selective advantage in those areas that isn’t seen elsewhere.  That’s precisely what Kawashima et al. saw.

… the frequency of these epitope variants (n = 14) was consistently correlated with the prevalence of the restricting HLA allele in the different cohorts (together, P < 0.0001), demonstrating strong evidence of HIV adaptation to HLA at a population level.  ³

HLA-B51 complexed with an immunodominant HIV peptide

Immune escape isn’t the only selective pressure on HIV.  There’s the ability to spread from one individual to another, for example, which isn’t necessarily linked to immune escape.  In principle, some of the other selective factors may counteract immune escape selection.  And in general, some (though not all) of the mutations that allow a virus to escape immune control by on individual are harmful to the virus. That means that some of the escape sequences will quickly revert back to the generic HIV sequence. If HLA-B51 is rare in the population, the virus will constantly be reverting back to generic sequences and the HLA-B51-resistant strain will not particularly accumulate.  But if HLA-B51 is common, even these reverting sequences will build up in the population.

As anticipated, non-reverting variants such as I135X accumulate at the population level, but even rapidly reverting mutations such as T242N can accumulate, if the selection rate exceeds the reversion rate ³

Perhaps as a result, formerly-protective MHC alleles are no longer protective in some areas:

Data here suggest that, whereas 25 years ago HLA-B*51 was protective in Japan, this is no longer the case. The apparent increase in I135X⁵ frequency in Japan over this time supports the notion that HLA-B*51 protection against HIV disease progression hinges on availability of the HLA-B*51-restricted TAFTIPSI⁶  response. However, whether this is the case remains unknown. ³

Any effective anti-HIV vaccine will probably rely on antiviral T cells, and will therefore rely on MHC class I presentation.  What this paper suggests is that HIV is likely to be a moving target.  Even if an effective vaccine is developed, it is possible that the virus will gradually evolve resistance to the vaccine.

Thus, the data presented here, showing evidence that the virus is adapting to CD8⁺ T-cell responses, … highlight the dynamic nature of the challenge for an HIV vaccine. … The induction of broad Gag-specific CD8⁺ T-cell responses may be a successful vaccine strategy, but such a vaccine will be most effective if tailored to the viral sequences prevailing, and thus may need to be modified periodically to keep pace with the evolving virus.  ³

Since we still don’t have any vaccine that protects against HIV at all, this is pretty much a hypothetical worry.  Still, it’s something to think about for the future.

1. Amber G. F. Teacher, Trenton W. J. Garner, Richard A. Nichols (2009). Evidence for Directional Selection at a Novel Major Histocompatibility Class I Marker in Wild Common Frogs (Rana temporaria) Exposed to a Viral Pathogen (Ranavirus) PLoS ONE, 4 (2) DOI: 10.1371/journal.pone.0004616[↩]
2. The only kind I have, I’m sure[↩]
3. Yuka Kawashima, Katja Pfafferott, John Frater, Philippa Matthews, Rebecca Payne, Marylyn Addo, Hiroyuki Gatanaga, Mamoru Fujiwara, Atsuko Hachiya, Hirokazu Koizumi, Nozomi Kuse, Shinichi Oka, Anna Duda, Andrew Prendergast, Hayley Crawford, Alasdair Leslie, Zabrina Brumme, Chanson Brumme, Todd Allen, Christian Brander, Richard Kaslow, James Tang, Eric Hunter, Susan Allen, Joseph Mulenga, Songee Branch, Tim Roach, Mina John, Simon Mallal, Anthony Ogwu, Roger Shapiro, Julia G. Prado, Sarah Fidler, Jonathan Weber, Oliver G. Pybus, Paul Klenerman, Thumbi Ndung’u, Rodney Phillips, David Heckerman, P. Richard Harrigan, Bruce D. Walker, Masafumi Takiguchi, Philip Goulder (2009). Adaptation of HIV-1 to human leukocyte antigen class I Nature DOI: 10.1038/nature07746[↩][↩][↩][↩][↩]
4. Someone who knows about evolution could probably attach some numbers to this.[↩]
5. I135X is the HIV sequence that’s escaped HLA-B51 control[↩]
6. TAFTIPSI is the original HIV sequence that’s controlled by HLA-B51[↩]

HCV protein associated with lipid droplets Torsten Schaller, Research As Art)

Although there are quite a few viruses that infect and then persist in the infected animal for a long time, most of these viruses don’t cause a lot of problems during the persistent state. Herpesviruses, adenoviruses, and several other families can stick around for a long time (life-long, in the case of most herpesviruses) and although you’ll sometimes see occasional recurrence, and occasionally there can be serious disease from the reactivation (examples being shingles, from recurrent varicella-zoster virus, or the very rare cancers associated with Epstein-Barr virus) — for the most part these are really unusual outcomes. Mostly we can stroll around with our complement of viral passengers and we’re perfectly fine with it.

There are a handful of exceptions, in humans and in other animals, where viruses that cause chronic infection also cause chronic, severe disease. With these viruses we’d like to know why they become chronic in the first place (why doesn’t the immune system eliminate them?), and we’d like to know why they cause disease (why aren’t they like our friendly neighborhood herpesvirus)? We don’t have good answers for either of these questions. In fact, I don’t think we even have a good sense if the answers are the same for different viruses, or whether each of them manages to persist and cause chronic disease through their own unique factors.

Two of the most prominent chronic virus diseases in humans are HIV and HCV (hepatitis C virus). A recent paper¹ suggests that these guys may have at least something in common in the way they escape immune control, and in the way the immune system controls them.

HCV protein distribution in infected hepatoma cells (Mark Harris lab)

It’s pretty well known that one way HIV escapes immune control is that it mutates so fast, the immune system can’t get a grip on it. In particular, the cytotoxic T lymphocytes (CTL) that are specialized to control virus infections need to recognize a stretch of about 9 amino acids (a peptide, in other words), and if that sequence changes the CTL may no longer recognize the virus. (I’ve talked about that here and here.) It’s also become clear that HCV does the same thing, although perhaps less dramatically than does HIV, and that this mutational immune escape is one reason HCV can persist and continue to cause disease (see here for more).

With HIV, there are a number of “elite controllers” who seem to be resistant to the virus’s ability to mutate away from immune control. In many cases this seems to be because the CTL in those individuals are focused on a particular critical stretch of amino acids that simply can’t mutate without severely damaging the virus (reducing HIV’s replicative fitness). The reason these elite controllers select that critical peptide is that they have a MHC class I allele that specifically binds to that peptide sequence. The HLA-B27 and HLA-B57 alleles seem to be particularly likely to find critical peptides, and people with those MHC alleles are more likely to be elite controllers.

The new HCV paper¹ shows that HLA-B27 is also protective against HCV infection ², and for the same reason — the HCV peptide that binds to HLA-B27 is a critical sequence that can’t mutate much without severely damaging the virus. (When HCV does mutate away from HLA-B27-mediated control, it’s because it has developed multiple mutations in the peptide, not just one, and it’s exponentially³ harder for the virus to make two mutations vs. one.)

Is it just a coincidence that HLA-B27 is involved in both cases, or is there something specially magical about HLA-B27? I’d be inclined to say it’s just coincidence except that HLA-B27 is such a special molecule⁴ already. It’s involved in all kinds of disease risks, both reducing the risk of some infectious diseases like HIV and HCV and dramatically increasing the risk of autoimmune diseases like ankylosing spondylitis and many others. And off the top of my head, I think it’s one of the very ancient and highly diversified groups of HLA molecules. So maybe there is something about it that manages to focus on critical viral peptides, or that makes it a particularly strong stimulator of CTL, and that gives it a selective advantage that outweighs its increased risk of autoimmune disease.

1. Eva Dazert, Christoph Neumann-Haefelin, Stéphane Bressanelli, Karen Fitzmaurice, Julia Kort, Jörg Timm, Susan McKiernan, Dermot Kelleher, Norbert Gruener, John E. Tavis, Hugo R. Rosen, Jaqueline Shaw, Paul Bowness, Hubert E. Blum, Paul Klenerman, Ralf Bartenschlager, Robert Thimme (2009). Loss of viral fitness and cross-recognition by CD8+ T cells limit HCV escape from a protective HLA-B27–restricted human immune response Journal of Clinical Investigation DOI: 10.1172/JCI36587[↩][↩]
2. This part was already known[↩]
3. or maybe geometrically, I don’t know[↩]
4. Unicorns have HLA-B27! Well-known fact![↩]

I know all my regular readers¹ are expecting me to talk about the bombshell announcements that NK cells have memory, but I’ll put that off for a bit and instead quickly note a very cool advance on a story I’ve mentioned a few times before.

Interferons are among the most critical early warning and protective cytokines, and they’re so effective that just about any successful virus of vertebrates has strong defenses against them. Without those defenses, the virus is essentially dead. For example, in some cases the the virus’s interferon blocker only works in one species, and that limits the virus to infecting that species only; if you get rid of the interferon response that the virus can’t deal with, then it’s perfectly capable of infecting other species.² 

Influenza viruses are no exception; they possess a gene (NS1) that protects them against the host interferons. (Some strains of influenza have especially effective NS1 functions, and it’s been suggested that those with the most effective interferon blockers are the most virulent pathogens — like the 1918 flu, or avian influenza). I’ve talked about NS1 before here.

When you eliminate NS1 from influenza, the virus is — as you’d expect — greatly weakened, and infection with these defective viruses cranks up interferon and thereby, in turn, cranks up the rest of the immune response. That gives you a highly attenuated, highly immunogenic virus, which is exactly what you want to use for a vaccine; and indeed, NS1-defective influenza viruses are apparently effective and safe vaccines.³ (I’ve previously mentioned a vaccinia virus vaccine technique that follows a similar approach, with similar results.)

So NS1 is clearly an extremely important molecule for influenza, and without it, the virus is basically harmless. What if you could block NS1 after an infection? Would it do the same thing — in other words, would an NS1-blocker be an antiviral treatment?

As it turns out: Yes, it would. Daniel Engel’s lab developed a group of compounds that inhibit influenza NS1. These things inhibit influenza growth in cells, and (although NS1 has functions other than blocking interferon) the effect was dependent on the interferon response. ⁴

A couple of viral immune evasion molecules have already been targeted for antiviral therapy — for example,  Luis Sigal  has shown that a poxvirus immune evasion molecule is a good vaccine target⁵ — but as far as I know this is the first antiviral compound that’s specifically been developed to inactivate an immune evasion molecule, and it offers the potential for a brand-new class of antivirals. Of course there are still huge barriers between these particular compounds and actual therapy in infected animals, but it’s encouraging that they work at all, and I’m interested in seeing what arises from it.

1. Hi, Mom![↩]
2. Wang F , Ma Y , Barrett JW , Gao X , Loh J , Barton E , Virgin HW , McFadden G (2004) Disruption of Erk-dependent type I interferon induction breaks the myxoma virus species barrier. Nat Immunol 5: 1266-1274[↩]
3. Live Attenuated Influenza Viruses Containing NS1 Truncations as Vaccine Candidates against H5N1 Highly Pathogenic Avian Influenza. John Steel, Anice C. Lowen, Lindomar Pena, Matthew Angel, Alicia Solórzano, Randy Albrecht, Daniel R. Perez, Adolfo García-Sastre, and Peter Palese. Journal of Virology, February 2009, p. 1742-1753, Vol. 83, No. 4 doi:10.1128/JVI.01920-08 [↩]
4. D. Basu, M. P. Walkiewicz, M. Frieman, R. S. Baric, D. T. Auble, D. A. Engel (2008). Novel Influenza Virus NS1 Antagonists Block Replication and Restore Innate Immune Function Journal of Virology, 83 (4), 1881-1891 DOI: 10.1128/JVI.01805-08[↩]
5. The orthopoxvirus type I IFN binding protein is essential for virulence and an effective target for vaccination. Xu RH, Cohen M, Tang Y, Lazear E, Whitbeck JC, Eisenberg RJ, Cohen GH, Sigal LJ. J Exp Med. 2008 Apr 14;205(4):981-92. doi:10.1084/jem.20071854 [↩]

Adenovirus infecting HeLa cells

I’ve quoted before that “the stupidest virus is smarter than the smartest virologist”. Adenoviruses are far from the stupidest viruses, and even after 55 years of study, and nearly 40,000 papers in PubMed, adenoviruses still throw surprises at us on a regular basis. Last week, while talking about herpesviruses, I added that “the other virus families that are known to evade MHC class I are human adenoviruses, which now turn out to establish true latency”. True latency has been hinted at for a while, but Linda Gooding’s group has added much more support for it in a paper in press at J Virol.¹

Adenoviruses are very, very common viruses, both in humans and in many other species. In humans there are over 40 different adenovirus “species”, mostly causing cold-like symptoms and mild gastrointestinal disease. Occasionally, and perhaps with increasing frequency, there’s a more or less widespread outbreak of moderate disease, but in general these guys are not huge problems as far as mortality in immune-competent people.

As well as being common, many adenovirus species are pretty easy to grow in cultured cells, and so it’s not surprising that they were isolated and described a long time ago. In fact, the first identification of adenoviruses was as an accidental isolation in 1953, when Rowe et al noticed that the tonsil cell cultures they were growing were showing signs of viral infection.² Since then, adenoviruses have been used as models for a huge number of cell biology functions (the Nobel on splicing came from work on adenoviruses, just as one example), as well as cancer biology and lots of other things; adenoviruses have also been used as workhorses for the viral vector field, moving into gene therapy as well.

Tonsillectomy (Greenfield Sluder: 1923)

If you look in lots of tonsils, you’ll find lots of adenovirus; something like 80% of samples turn up positive. ³ That’s much higher than the disease rate, of course, and it’s also much higher than any plausible incidence rate — that is, those can’t all be new infections, in the past week or so, that simply haven’t been cleared by the immune system. That means that adenoviruses must be able to persist for some fairly significant period — months to years — after their initial infection, without causing symptoms.

This long-term persistence by adenovirus is one of its most characteristic, um, characteristics, but it’s been completely mysterious. (If you don’t believe me, here’s what Bill Wold and Marshall Horwitz say in the latest version of the authoritative Fields Virology: “We really do not understand whether and how adenovirus persists at very low levels in humans … “)

For adenoviruses, usually the term “persistence” is used, rather than latency, because “latency” has a specific meaning: Basically, a latent virus is still present at the genome level, but isn’t capable of forming a new virus particle. Herpesviruses are the main viruses that are known to do this. Some retroviruses set up latent infection by intergrating into the host genome, but that’s a little different story. Herpesviruses can maintain their own genomes in the latently-infected cell, but in a form that’s independent of the host genome. As I’ve noted before, this latency may be tightly linked to immune evasion functions. The other option is mere “persistence”, where presumably the virus would remain in a replicating form, and would be capable of forming a new virus, perhaps at a very slow turnover rate of replication.

A handful of papers have suggested that adenoviruses might establish truly latent state⁴, but this new paper from Gooding’s lab¹ is the most convincing I’ve seen yet. They compared infectious virus to the amount of viral DNA — that is, to viral genomes — and concluded that “only a small amount of viral DNA is present as infectious virus, even in samples with large amounts of viral DNA. … time in culture also appears to activate latent virus in the tissues, which was detected by transferring “activated” lymphocyte-derived virus onto permissive A549 cells.” (This is exactly how you detect latent infection by herpesviruses, in general — you transfer latently-infected tissue onto other cells, and over time the virus reactivates from latency to become infectious virus, and shows up on the permissive cells.) They showed that on initial exam the virus wasn’t making any transcript, again a requirement for true latency, and that over time transcripts began to appear as the virus reactivated.

As a side note, Gooding notes dryly that sloppy technique may be part of the reason adenoviruses were so often found in tonsil explants, a suggestion I hadn’t heard before.

Reports of infection of laboratory workers in the early adenovirus groups suggest that some exogenous contamination might have elevated the frequency with which live virus was found in these studies.

Finally, Gooding re-emphasizes the same point I’ve made here:

Furthermore, like all DNA viruses that form latent or persistent infections, human species C adenoviruses encode a variety of gene products, primarily within the E3 transcription unit, that function to counteract host anti-viral defense mechanisms. We have previously reported that the E3 promoter is up-regulated when cells are exposed to signals that activate T lymphocytes. Hence, it appears likely that the immune evasion strategies of these viruses are directed toward protecting the T lymphocyte from destruction during the period of viral activation from latency.

(My emphasis) As far as that goes, it’s particularly interesting to me that only human adenoviruses, and not even all of them, have MHC class I immune evasion functions. Does that mean that that particular function is less critical for latency, or does it mean that non-human adenoviruses don’t establish true latency (even in these humans, they really only found group C adenoviruses to be latent, though that may just reflect tissue preferences), or what?

1. C. T. Garnett, G. Talekar, J. A. Mahr, W. Huang, Y. Zhang, D. A. Ornelles, L. R. Gooding (2008). Latent species C adenoviruses in human tonsil tissues Journal of Virology DOI: 10.1128/JVI.02392-08[↩][↩]
2. Rowe WP, Huebner RJ, Gillmore LK, et al. Isolation of a cytopathic agent from human adenoids undergoing spontaneous degeneration in tissue culture. Proceedings of the Society for Experimental Biology and Medicine. 1953;84:570-573. [↩]
3. Garnett CT, Erdman D, Xu W, et al. Prevalence and quantitation of species C adenovirus DNA in human mucosal lymphocytes. J Virol 2002;76:10608-10616. [↩]
4. Neumann R, Genersch E, Eggers HJ. Detection of adenovirus nucleic acid sequences in human tonsils in the absence of infectious virus. Virus Res 1987;7:93-97. [↩]