Mystery Rays from Outer Space

Meddling with things mankind is not meant to understand. Also, pictures of my kids

July 2nd, 2009

Simple, obvious, and wrong answers

Macrophage and mycobacterium
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

Further reading
Tb family trees
Conspicuous consumption
Life & Death, pre-vaccination

MycobacteriaIn 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[][][][]
June 22nd, 2009

More symbionts and flight

Erigone atra
Erigone atra

Remember the paper I mentioned the other day, that showed how a symbiotic virus causes aphids to grow wings? Amazing at that may be, an equally amazing thing is that it’s not unique. A new paper1 points to a different species, and a different symbiont, that also flips the switch on flight.

There are some differences: the host is a spider, not an insect; the symbiont is a bacterium, not a virus; and the symbiont switches flight off, not on. But, you know, we’re talking about concepts, here, and conceptually this is remarkably similar.

The spider is Erigone atra, a “money spider”, an agriculturally important species (they control pests). They’re widespread and successful, and one of the reasons is their aeronautical ability. Like many other spiders, E. atra can travel many miles via “ballooning” — spinning a long, fine strand of silk that catches the breeze and takes them floating to a new habitat.

Also like many (if not all) arthropods, E. atra has a host of endosymbiotic bacteria. The most famous of the arthropod endosymbionts are the Wohlbachia family, which do utterly incredible things to to their hosts in order to spread their (the Wohlbachia’s) genes; but there are many other bacterial symbionts.

Ballooning baby lynx spider
Ballooning baby lynx spider

Treating the spiders with antibiotics (thus “curing” them of their endosymbionts — specifically, curing them of Rickettsia) changed their ballooning behavior: Treated, Rickettsia-free spiders were six times more likely to balloon. Ballooning is more than shooting out a web and hoping, it’s a complex behavior: “a stereotypical ‘tiptoeing’ posture, which is exclusively used for aerial dispersal (comprising leg stretching, abdomen raising and production of silk threads that are used as sails)“,1 and this behavior was strongly suppressed in Rickettsia-infected spiders.

Assuming that the bacteria are actually imposing this behavioral change on the spiders (the authors are appropriately cautious with their interpretation), why don’t the bacteria want to the spiders to balloon? Why are they the opposite of the densoviruses I mentioned earlier? 2

My own speculation here … One critical difference between the aphids and the spiders is that the spiders are much more versatile. Aphids grow on a tiny, constrained food source, and then have to fly to find a new food source (and, I believe, to breed) (Update: See comment #2 for more a informed explanation of aphid lifestyles). These spiders, on the other hand, can disperse locally as well as aeronatically; there are local communities of spiders that can be reached without ballooning, so they’re not dependent on flight to propagate the species. Ballooning takes the spiders to new, sparsely-settled regions, where there’s less competition for resources. But that’s also a region where there are fewer hosts for the bacteria. Building up a reasonably dense local spider population might be in the bacteria’s best interests, and suppressing ballooning might help do that.

  1. Microbial modification of host long-distance dispersal capacity.
    Sara L Goodacre, Oliver Y Martin, Dries Bonte, Linda Hutchings, Chris Woolley, Kamal Ibrahim, C.F. George Thomas and Godfrey M Hewitt.
    BMC Biology 2009, 7:32 doi:10.1186/1741-7007-7-32[][]
  2. In contrast to the densoviruses, it’s not a big stretch of imagination to imagine the bacteria doing this mechanistically. Bacterial endosymbionts do much more complex behavioral changes than this to their hosts.[]
June 19th, 2009

Tb family trees

BCG (Max Planck)
Macrophage engulfing Bacillus Calmette-Guérin

The oldest vaccine, as everyone knows, is the smallpox vaccine. 1 Another  old vaccine (though not in the same class as smallpox vaccine) is the tuberculosis vaccine, Bacillus Calmette-Guérin (BCG), developed in 1921.

There’s all kinds of interesting stuff about BCG. It’s a live vaccine, meaning that the vaccine is live Mycobacterium bovis (the bovine tuberculosis bacterium). Albert Calmette and his assistant/colleague Camille Guérin, at the Institut Pasteur, repeatedly passaged a virulent isolate of M. bovis on a particular type of medium, noticed that some colonies looked different, and determined that these were less virulent for lab animals; after further passages, they announced that the variant was “inoffensif” in humans,2 and by the late 1920s BCG was being used around the world as a vaccine against tuberculosis. Overall, it’s probably around 50% effective as a vaccine, with geographical location accounting for most of the variability. 3 That’s very low as vaccine efficacy goes, and it means that vaccination is really only useful where there’s lots of disease, not so much where there’s a moderate incidence; which pretty much matches how BCG has been used worldwide.  On the other hand, the vaccine seems to be pretty innocuous, with a low complication rate.

BCG Genealogy
BCG genealogy 4

This much, I was more or less aware of,5 but there’s much more to the story than that, as I’ve recently learned. 4,6 One fascinating question is, exactly what is the vaccine? This is an old vaccine, it was never homogenous (that is, it was basically a crude culture consisting of a wide range of minor variants around a central genome), and it’s been passaged independently all over the world for 85 years. There’s no such thing as “the” BCG any more; there are dozens of different BCGs, some of which can be traced directly back to the original Pasteur stock, others of which have bounced around and been sub-cultured from sub-cultures. There are at least 6 widely-used vaccine strains of BCG,7 not to mention older strains,  trial strains, and so on; and most of them behave differently in more or less subtle ways.  (See the genealogy of some of the more prominent strains, to the right.)  Even the “same” strains have clearly changed over time; for example, somewhere between 1926 and 1934, the Pasteur strain of BCG became drastically less virulent:8

Watson also stated that the strains he had received from Calmette between 1924 and 1927 were potentially virulent for animals, whereas the BCG strains he received and tested between 1929 and 1932 were no longer virulent.9

We don’t have the original BCG strain any more, let alone the virulent M. bovis isolate from which it was derived, but some of the history can be inferred from genome analysis. Unsurprisingly, the different sub-strains have slight variants in their DNA sequences:

These results are best understood from an evolutionary perspective and indicate that BCG has continued to change with in-vitro passage. All BCG strains are lacking deletion region 1, a genetic deletion that may correspond with the altered morphotype observed by Calmette and Guérin. Subsequently, strains obtained before 1926 and maintained on different continents have the 2-IS6110/mpt64+ genotype, which was likely the genetic composition of early BCG. … With such documented genetic change it would be surprising if there has not been phenotypic change over 1173 passages in-vitro, a notion supported in numerous reports describing ongoing attenuation after 1921. … In conclusion, we have demonstrated that through DNA fingerprinting, it is possible to verify the micro-evolution of attenuated Mycobacterium bovis over about one thousand passages. 4

So: We have an old, widely-used vaccine strain, that has a long history of variation. (This is similar, by the way, to smallpox vaccine — vaccinia virus was also widely distributed and showed extensive variation in its characteristics.) The BCG strains used today have all been more or less selected — either deliberately, or empirically — to be safe and effective vaccines; they’ve also been selected for a wide range of other factors (stability, ease of processing, rapid growth, and so on), some of which we know about, some of which we don’t.

Could strain variability account for the wild variation in tested vaccine efficacy? Well. historically, perhaps not — different strains of BCG haven’t correlated with the differences in efficacy that have been seen, most of which (as I said) follow geography.

But I’ll leave you with this: It seems that the BCG vaccines used today are generally less effective than they were back in the 1920s.10 Could that be linked to some of these genomic variations?

In early clinical studies, the live tuberculosis vaccine Mycobacterium bovis BCG exhibited 80% protective efficacy against pulmonary tuberculosis (TB). Although BCG still exhibits reliable protection against TB meningitis and miliary TB in early childhood it has become less reliable in protecting against pulmonary TB.11

  1. Even if we take into account the mysterious shift from cowpox to vaccinia virus as the vaccine strain[]
  2. A. Calmette and C. Guérin, Nouvelles recherches expérimentales sur la vaccination des bovidés contre la tuberculose. Ann. Inst. Pasteur. 34 (1920), pp. 553–560.[]
  3. Efficacy of BCG vaccine in the prevention of tuberculosis. Meta-analysis of the published literature.
    JAMA. 1994 Mar 2;271(9):698-702
    Colditz GA, Brewer TF, Berkey CS, Wilson ME, Burdick E, Fineberg HV, Mosteller F.[]
  4. Behr, M. (1999). A historical and molecular phylogeny of BCG strains Vaccine, 17 (7-8), 915-922 DOI: 10.1016/S0264-410X(98)00277-1[][][]
  5. As I’ve pointed out before, I’m not a bacteriologist, so I mainly picked up this much by osmosis[]
  6. Development of the Mycobacterium bovis BCG vaccine: review of the historical and biochemical evidence for a genealogical tree.
    Tubercle and Lung Disease(1999) 79(4), 243–250
    T. Oettinger, M. Jørgensen, A. Ladefoged, K. Hasløv, P. Andersen[]
  7. Connaught, Danish, Glaxo, Moreau, Pasteur, and Tokyo[]
  8. Watson E A. Studies on bacillus Calmette-Guérin (BCG) and vaccination against tuberculosis. Can J Med Res 1933;9:128.[]
  9. Development of the Mycobacterium bovis BCG vaccine: review of the historical and biochemical evidence for a genealogical tree.
    T. Oettinger, M. Jørgensen, A. Ladefoged, K. Hasløv, P. Andersen
    Tubercle and Lung Disease(1999) 79(4), 243–250  []
  10. Correlation between BCG genomics and protective efficacy.
    Scand J Infect Dis. 2001;33(4):249-52.
    Behr MA


    Has BCG attenuated to impotence?
    Marcel A. Behr1 & Peter M. Small
    Nature 389, 133-134 (11 September 1997) doi:10.1038/38151[]

  11. Reducing the Activity and Secretion of Microbial Antioxidants Enhances the Immunogenicity of BCG
    Sadagopal et al
    PLoS ONE 4(5): e5531. doi:10.1371/journal.pone.0005531[]
September 30th, 2007

Tuberculosis [hearts] HLA-B?

Mycobacterium tuberculosisA paper in the latest issue of PLoS Pathogens1 makes a provocative suggestion, summarized in their title: Immunodominant Tuberculosis CD8 Antigens Preferentially Restricted by HLA-B.2 This is another paper that offers reasonably exhaustive mapping of T cell targets of a particular pathogen — a genre that’s becoming more and more common.

Just a few days ago I commented on a similar paper that mapped influenza epitopes. In this case, the pathogen is Mycobacterium tuberculosis, and they screened overlapping peptides from eight Mtb proteins. (It’s still, of course, prohibitively expensive and time-consuming to test the entire Mtb genome, the way some viral genomes are being screened.) There’s not much new or different about this paper, for the most part (it’s a useful technical contribution). There’s the obligatory comment on epitope prediction, which should seem familiar to anyone who’s been reading my earlier posts:

Because much work on human CD8+ T cell responses to Mtb has relied upon the use of HLA prediction algorithms, as each epitope was defined we asked whether or not the epitopes would have been predicted by these approaches. Given the prevalence of HLA-B alleles and 10-mer and 11-mer epitopes, it is perhaps not surprising that many of these epitopes were not ranked strongly (unpublished data)

So I’ll skip over almost all their results and move on to the “provocative statement”:

All but one of the epitopes that have been mapped to date are restricted by HLA-B molecules. … we speculate that Mtb antigens may preferentially bind to HLA-B molecules, that Mtb preferentially interferes with HLA-A processing and presentation, that infection with Mtb leads to selective upregulation of HLA-B, or that HLA-B is preferentially delivered to the Mtb phagosome.

They identified 12 epitopes, and 11 of them were restricted to HLA-B (various alleles). They take this as evidence of skewing toward HLA-B as opposed to HLA-A, and speculate as to the cause of the skewing. Yeah, well. Maybe. But I’d like to suggest some other possibilities.

Mtb genome
M. tb genome

First of all, there are lots of non-HLA-B-restricted Mtb epitopes in the literature. My databases3 contain 24 human Mtb epitopes, of which 9 are HLA-A restricted. That’s far from definitive, because many of those come from experiments specifically screening HLA-A2, but it certainly demonstrates that there’s no hard and fast effect.

Second, there are two possible explanations they didn’t mention:

  1. Chance. Even if the epitopes are “really” distributed evenly between HLA-A and HLA-B,4 an 11/12 or 12/12 distribution (either way — HLA-A or B) will appear about 0.625% of time. 5 The appearance of strong skewing (10 or more out of 12) will appear ~4% of the time by chance. Both of those are under the tradition 5% cutoff — but that’s only for hypothesis testing! The skewing was not an a priori hypothesis, it was a post facto observation, and these sorts of p-values are not applicable. It’s probable that something odd would appear in their results, whether it’s the number of epitopes that have alanine in P2 or whatever. You can’t focus on one oddity after the fact and declare that it’s significant.
  2. Peptide length. They commented specifically on how many long peptides they found in their output (6 of the 12 epitopes are longer than the canonical 9 amino acid epitope length). Well, they screened with 15mers. If the HLA-B alleles they were dealing with are more likely to bind long peptides,6 then they’re skewing to HLA-B right there.

My bet is that this HLA-B skewing is purely chance, and that further epitope mapping in Mtb will find a bunch of HLA-A-restricted epitopes — revert to the mean. That’s not to say their suggestions are biologically implausible. Several of the viral immune evasion molecules, for example, preferentially target either HLA-A or HLA-B, though the effects are usually not black and white — which is consistent with what they’re seeing here. Still, I really think the likeliest explanation is simply chance.

For the record, here are the human and mouse MTb epitopes I know of:

Lewinsohn et al
Epitope MHC Allele Source (Accession) Reference (PMID)
LLDAHIPQL HLA-A*0201 O53692 17892322
AEMKTDAATL HLA-B*44 P0A566 17892322
AVINTTCNYGQ HLA-B*1501 O50430 17892322
RADEEQQQAL HLA-B14 P0A566 17892322
ASPVAQSYL HLA-B*3514 O50430 17892322
TAAQAAVVRF HLA-B*3514 P0A566 17892322
ELPQWLSANR HLA-B*4102 P31952 17892322
AEMKTDAATLA HLA-B*4501 P0A566 17892322
AEMKTDAA HLA-B*4501 P0A566 17892322
EMKTDAATL HLA-B*0801 P0A566 17892322
AAHARFVAA HLA-B*0801 O53692 17892322
NIRQAGVQY HLA-B*1502 P0A566 17892322
Previously published
Epitope MHC Allele Source (Accession) Reference (PMID)
GLIDIAPHQI HLA-A*0201 15607482 12010981
RLPLVLPAV HLA-A*0201 581380 11035787
GLPVEYLQV HLA-A2 29027587 12519392
KLIANNTRV HLA-A2 29027587 12519392
VLGRLDQKL HLA-A*0201 15608832 12972510
ALEAFAIAVA HLA-A*0201 15608832 12972510
LVVADLSFI HLA-A*0201 15608832 12972510
LLSVLAAVGL HLA-A*0201 15607267 12972510
SGVGNDLVL H-2-Db 840827 15153510
RPREATIIY HLA-B*07 15609960 15762882
IPRDEVRVM HLA-B*3501 15608599 15762882
KPRDDAAAL HLA-B*53 15607810 15762882
RPKIDDHDY HLA-B*53 15608779 15762882
RPKPDTETY HLA-B*3501 15610825 15762882
RPKPDYSAM HLA-B*3501 15610514 15762882
RPKVEGLEY HLA-B*53 15609319 15762882
RPRLDSITY HLA-B*3501 15608420 15762882
RPRYEIFVY HLA-B*53 15609613 15762882
IPKLRQGSY HLA-B*53 15609803 15762882
KPGCDAPAY HLA-B*53 15610603 15762882
RPGCDAPAY HLA-B*3501 15609082 15762882
SPKETWLRL HLA-B*53 15610850 15762882
GAPINSATAM H-2-Db 15607267 16113299
VLTDGNPPEV HLA-A*0201 X07945 9725236
RADEEQQQAL HLA-B*14 AF004671 11123322
AEMKTDAATL HLA-B*44 AF004671 11123322

  1. Since the PLoS papers just continually conveyer-belt out their papers, do they really have “issues”?[]
  2. Immunodominant Tuberculosis CD8 Antigens Preferentially Restricted by HLA-B. Lewinsohn, D. A., Winata, E., Swarbrick, G. M., Tanner, K. E., Cook, M. S., Null, M. D., Cansler, M. E., Sette, A., Sidney, J., and Lewinsohn, D. M. (2007). PLoS Pathog 3, e127. []
  3. Just compilations of the curated on-line databases and a couple other sources[]
  4. HLA-C is kind of the red-headed stepchild of classical antigen presentation, and we’ll leave it out of the question[]
  5. I think.[]
  6. I don’t know if they are or not; in my databases the HLA-B alleles they used, and HLA-B in general, are not so biased, but the numbers are small[]