Mystery Rays from Outer Space

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. [↩]

Herpesviruses are unusual¹ in their ability to establish a long-term latent infection in their hosts.  Another unusual² trait is that herpesviruses apparently all have genes that block MHC class I recognition by cytotoxic T lymphocytes.  (I suspect that we will also find that evasion of NK cells is also universal among herpesviruses, but that’s still a new and growing field.)  The other virus families that are known to evade MHC class I are human adenoviruses, which now turn out to establish true latency;³ HIV (which also has a true latent stage, though I don’t know if it’s relevant here); a handful of poxviruses, which as far as I know don’t do much in terms of latency; and some papillomaviruses⁴ which I don’t think have a real latent stage.

I’ve noted previously that these two unusual characteristics of herpesviruses may be linked, in that perhaps the herpesvirus MHC class I immune evasion is important for establishing latency.  A review in Nature⁵ points out that herpesviruses are also unusual in that they tend to have a lot of micro-RNAs, and suggests a similar concept: That herpesviruses use miRNAs to evade the immune system and this is related to their ability to maintain a latent infection.

It seems possible that the presence of miRNAs in herpesviruses is associated with the characteristic ability of herpesviruses to establish long-term latent infections. Avoiding the host immune response is particularly important during latent infection, and viral miRNAs not only have the advantage of not being recognized by the host immune system but also might be an ideal tool for attenuating immune responses by downregulating the expression of key cellular genes. ⁵

1. Though not quite unique[↩]
2. Though still not quite unique[↩]
3. Latent species C adenoviruses in human tonsil tissues. C. T. Garnett, G. Talekar, J. A. Mahr, W. Huang, Y. Zhang, D. A. Ornelles, and L. R. Gooding. J. Virol. doi:10.1128/JVI.02392-08 [↩]
4. High-risk human papillomavirus E7 expression reduces cell-surface MHC class I molecules and increases susceptibility to natural killer cells. Bottley G, Watherston OG, Hiew YL, Norrild B, Cook GP, Blair GE. Oncogene. 2008 Mar 13;27(12):1794-9.[↩]
5. Bryan R. Cullen (2009). Viral and cellular messenger RNA targets of viral microRNAs Nature, 457 (7228), 421-425 DOI: 10.1038/nature07757[↩][↩]

Silicosis (via OSHA)

A couple of years ago¹ it was discovered that the natural adjuvant uric acid stimulates immunity through an intracellular sensor called the Nalp3 inflammasome.  Last year, a bunch of groups showed the the commonly-used adjuvant alum — also kind of crystalline — acts through the Nalp3 inflammasome. ²  Then it turned out that silca causes the inflammation associated with silicosis via, right, the Nalp3 inflammasome. ³ (I talked about this in several places, including here.)

So it wasn’t a stretch to wonder if perhaps this was a general rule, and all (or at least most) particulate-type adjuvants act through the Nalp3 inflammasome.  And unsurprisingly, this turns out to be the case:

The ability of alum to enhance IL-1β secretion via NALP3 has been described, but our data indicate that this is not specific to alum but is a general property of all particulate adjuvants. ⁴

Not really a shock, I guess, at this point; but it’s nice to see things fitting neatly into a general model for a change.

1. Martinon F, Petrilli V, Mayor A, Tardivel A, Tschopp J (2006) Gout-associated uric acid crystals activate the NALP3 inflammasome. 440, 237 – 241 (11 Jan 2006), doi:10.1038/nature04516[↩]
2. Eisenbarth SC, Colegio OR, O’Connor W, Sutterwala FS, Flavell RA (2008) Crucial role for the Nalp3 inflammasome in the immunostimulatory properties of aluminium adjuvants. Nature  453, 1122-1126 doi:10.1038/nature06939

   Cutting edge: alum adjuvant stimulates inflammatory dendritic cells through activation of the NALP3 inflammasome. Kool M, Pétrilli V, De Smedt T, Rolaz A, Hammad H, van Nimwegen M, Bergen IM, Castillo R, Lambrecht BN, Tschopp J.J Immunol. 2008 Sep 15;181(6):3755-9.

   The Nlrp3 inflammasome is critical for aluminium hydroxide-mediated IL-1beta secretion but dispensable for adjuvant activity. Franchi L, Núñez G.  Eur J Immunol. 2008 Aug;38(8):2085-9.[↩]

3. Cassel, S.L., Eisenbarth, S.C., Iyer, S.S., Sadler, J.J., Colegio, O.R., Tephly, L.A., Carter, A.B., Rothman, P.B., Flavell, R.A., Sutterwala, F.S. (2008). The Nalp3 inflammasome is essential for the development of silicosis. Proceedings of the National Academy of Sciences DOI: 10.1073/pnas.0803933105[↩]
4. F. A. Sharp, D. Ruane, B. Claass, E. Creagh, J. Harris, P. Malyala, M. Singh, D. T. O’Hagan, V. Petrilli, J. Tschopp, L. A. J. O’Neill, E. C. Lavelle (2009). Uptake of particulate vaccine adjuvants by dendritic cells activates the NALP3 inflammasome Proceedings of the National Academy of Sciences, 106 (3), 870-875 DOI: 10.1073/pnas.0804897106[↩]

The Nervous System (Fritz Kahn (1888-1968))

Is “immunodominance” just what you get when you measure the wrong place?

Usually, when you look at T cell immune responses to a virus, they’re pretty strongly biased. That is, although the T cells are theoretically, and often observably, able to recognize a wide range of target peptides, the immune response is strongly focused on just a handful of these peptides, while the remaining pool of potential targets is either ignored altogether or given a cursory glance by a handful of T cells. This phenomenon is known as “immunodominance“, and it’s seen with  immune responses to all sorts of pathogens. In some cases — such as for HIV — it’s likely that a strongly immunodominant response is harmful, because it makes it easier for the infecting virus to mutate away from immune control. But in the vast majority of cases the immune response, be it never so immunodominant, does a perfectly good job of controlling the virus; which is why we’re able to easily control most of the viruses that we’re exposed to.

Usually when you measure an immunodominant response, you’ll take lymphocytes from the most abundant, easily-accessed place you can find. That would be blood, in humans; in mice you’d probably take a spleen or lymph nodes.  Some viruses like to hang out in these places, and these include some of the more popular research viruses.

But most of the viruses we’re exposed to don’t infect blood or secondary lymphoid organs; they infect the lungs, or the skin, or neurons, or some other tissue. When we measure the blood response, we believe we’re measuring a good approximation of the real response ongoing in the infected tissue, but that’s mostly been an assumption, not a demonstrated fact.

Recently there’s been some work starting to feel out how similar the tissue response is to the blood/lymphoid organ response. For example, I talked here about work establishing the timing of immune responses in the lungs, vs. the blood. In this case, the overall patterns were similar, though the details were somewhat different.

But that was only really looking at a fairly big picture — overall patterns. What about specifics of target recognition? In particular, is the immunodominance we measure in the blood what actually happens on the battlefield?

I’m only aware of a couple of studies that look at this at all, and those were mainly as asides, noticed in passing. Yewdell’s group has shown in a couple of paper that  infecting mice with poxviruses by different routes leads to differences in immunodominance:¹

The latter point is underscored by our observation that the ID hierarchy varies with the route of infection, the first observation of its kind to our knowledge. It will be of great interest to determine the underlying mechanism. ²

I’ve been told of unpublished data that show different immunodominant responses between lung and spleen, as well; also with a poxvirus.

But in those few examples, the epitopes were all known ones.  Known epitopes moved up or down a notch or two in the immunodominance hierarchy. A recent paper from Bob Hendricks’ group³ shows that T cells in the tissues can recognize things that are apparently not seen at all in the blood or spleen.

Electron tomogram of HSV4

Here they used herpes simplex virus (HSV) in C57BL/6 mice, which have long been believed to almost entirely focus their CD8 T cell response on a single peptide. Hendricks’ group has been looking at the immune response to HSV in the brain, where the virus sets up a latent infection  (I’ve talked about some of his findings here and here).  Contrary to more traditional concepts, it’s now becoming clear (from Hendricks’ work, and that of others) that T cells in the brain are important in controlling latent HSV infection.

In this paper, he found that the immune response in the brain is much more diverse, fairly strongly recognizing at least one  peptide other than the known dominant job.  Because the “normal” (that is, non-neuronal) immune response is so focused, this almost certainly means that the active immune response, down at the pointy end where the T cells are actually working, are responding to altogether different peptides.

It’s generally been assumed, as I say, that the easily-accessed blood or secondary lymphoid tissue is a reasonable approximation of what’s going on in the actual sites of action, in the peripheral tissues — in other words, the idea has been that there’s more or less equal flow of cells between the tissues and the blood and lymph. The recent work on timing and kinetics that I mentioned here sort of supported that assumption, but now we have to wonder whether in fact there’s some kind of filter that keeps some sets of T cells from entering, or staying in, the blood.

We also have to wonder if the whole “immunodominance” paradigm is what we think it is. Could immunodominance represent the filter between blood and tissues, rather than the actual formation of responses? I actually don’t think that would explain immunodominance in general (for one thing, we see strong immunodominance for viruses of lymphocytes, where the blood is the site of infection, so there shouldn’t be a filter) but it’s something to factor in.

1. D. C. Tscharke (2006). Poxvirus CD8+ T-Cell Determinants and Cross-Reactivity in BALB/c Mice Journal of Virology, 80 (13), 6318-6323 DOI: 10.1128/JVI.00427-06
   D. C. Tscharke (2005). Identification of poxvirus CD8+ T cell determinants to enable rational design and characterization of smallpox vaccines Journal of Experimental Medicine, 201 (1), 95-104 DOI: 10.1084/jem.20041912[↩]
2. D. C. Tscharke (2005). Identification of poxvirus CD8+ T cell determinants to enable rational design and characterization of smallpox vaccines Journal of Experimental Medicine, 201 (1), 95-104 DOI: 10.1084/jem.20041912[↩]
3. B. S. Sheridan, T. L. Cherpes, J. Urban, P. Kalinski, R. L. Hendricks (2008). Reevaluating the CD8 T cell response to HSV-1: Involvement of CD8 T cells reactive to subdominant epitopes Journal of Virology DOI: 10.1128/JVI.01699-08[↩]
4. Electron tomogram of a HSV nucelocapsid completing envelopment , from Baines, J. D., C. E. Hsieh, E. Wills, C. Mannella, and M. Marko. 2007. Electron tomography of nascent herpes simplex virus virions. J Virol 81: 2726-2735.[↩]

One of the reasons for epidemics and pandemics, is a virus that jumps from one species to a new one.  Among the original population (let’s call it the “natural host”), there’s a certain level of immunity . Individuals have been infected and survived, and walk away with some resistance to the virus.  That limits the virus’s ability to spread among the natural host.  If the virus can jump into a new host species, then none of the population will have ever seen that virus before, and there is the potential to burn through the population in a sudden, explosive pandemic.

Some obvious examples should jump to your mind: HIV jumping from non-human primates into humans; West Nile virus moving into North America. The best-documented example may be canine parvovirus entering the world-wide dog population in 1978, which I talked about here.

A more common scenario is for a virus to dip its toe into the new population, but not to establish a permanent, ongoing infection in that population.  Ebola periodically jumps into the human population from bats (probably) and causes serious problems for a while, but hasn’t moved into the larger human population yet.  SARS virus, ditto. Sin Nombre virus, Avian Influenza (so far!), canine distemper moving into seals and lions — these cause sudden but limited epidemics that burn out and don’t set up a long-term relationship in the new host.

How can we tell which route will be followed?  Did SARS in humans fizzle out because it wasn’t well adapted (even though it was visibly evolving to be human-adapted at a furious rate) or did the containment policies that were slapped on travel and so forth catch it before it has time? I have no idea how to generalize, and I don’t think anyone does.

Which means it’s probably a good idea to watch closely for any examples of species jumping, and to monitor it closely.

Right now, there’s apparently an example of a coronavirus (same virus type as causes SARS, by the way) that has moved from pigs into dogs. ¹ In fact, these viruses seem to be hydrids — recombinants that are part dog coronavirus, part swine coronavirus.  This is very reminiscent of the major changes in influenzaviruses that happens a couple of times per century, and that is the concern with avian flu.

So far, these new viruses don’t seem to be very virulent, at least on their own; they didn’t cause disease experimentally, though they were isolated from natural cases of sick dogs.  Something like this has apparently happened at least once before, about 10 years ago, with a previous canine/pig coronavirus recombinant,² and that one didn’t take off in the canine population.

I don’t know if it’s possible to draw general conclusions about new viruses and pandemics, but I am sure it’s worth trying.

1. N. Decaro, V. Mari, M. Campolo, A. Lorusso, M. Camero, G. Elia, V. Martella, P. Cordioli, L. Enjuanes, C. Buonavoglia (2008). Recombinant Canine Coronaviruses Related to Transmissible Gastroenteritis Virus of Swine Are Circulating in Dogs Journal of Virology, 83 (3), 1532-1537 DOI: 10.1128/JVI.01937-08[↩]
2. Wesley, R. D. 1999. The S gene of canine coronavirus, strain UCD-1, is more closely related to the S gene of transmissible gastroenteritis virus than to that of feline infectious peritonitis virus. Virus Res. 61:145-152[↩]

Jenner vaccinating a child

As I said last week, one of the biggest vaccine fiascos was the vaccine against respiratory syncytial virus (RSV) that was introduced in the 1960s. RSV is essentially a universal infection of children; it usually causes fairly mild respiratory disease, but because it’s so common the small fraction of cases that are more severe, end up being a leading cause of hospitalization for children. The vaccine was supposed to prevent that. As it happened, the vaccine itself didn’t cause any problems on its own; but children vaccinated with this RSV vaccine, who then later on were infected with RSV, actually had worse disease than those children who were uninfected. (Two children died.)

This enhanced respiratory disease (ERD) was really puzzling at the time, because the vaccine actually did induce a good, strong antibody response. But the antibody turned out to be non-protective. Just having an antibody response is not enough; the overall immune response needs to be involved and protective.

(I think we’re seeing some parallels to this concept now with T cell responses, where we are discovering that just having CD8 T cells doesn’t necessarily offer protection against things like HIV and hepatitis C virus, whereas the quality of the CD8 cells — now being measured as the range of cytokines they can produce — seems to be correlated with protection.)

The RSV vaccine turned out to trigger a TH2 type immune response. TH1/TH2 type responses are now a fundamental concept in immunology, but that hypothesis is a relatively new. Tim Mossman proposed it in 1986¹ and there was a significant lag before it was widely accepted. I think one of the findings that helped make TH1/TH2 accepted was the finding that the RSV vaccine triggered a strong TH2 immune response,² compared to the actual virus infection which mainly causes TH1-type immunity. This — to me, anyway — abruptly made the paradigm look less like a laboratory curiosity only seen in mice, and more like a real, clinically important phenomenon.

So the TH2 immune response seemed to more or less explain why the RSV vaccine caused disease. TH1 immune responses are generally protective against viruses, while TH2 immune responses are apparently more geared toward parasitic worms; TH2 responses tend to induce eosinophils and allergic-type responses, and that’s consistent with the clinical disease seen in the vaccinated children who got ERD.

But why did the vaccine induce a TH2 response? This is, of course, a huge question, especially if you’re trying to develop a new antiviral vaccine. One suggestion was the the vaccine screwed up the viral antigens too much. The vaccine used a formalin-inactivated virus, and the proposal was that the formalin alters the virus antigens and that directly caused the abnormal response³ If so, then this is a potential problem for any formalin-inactivated vaccine.

A new paper⁴ reaches a different conclusion. They say that formalin isn’t the main problem; rather, it’s the lack of adjuvant stimulation. Specifically, they say, you need to stimulate innate immunity via toll-like receptors (TLRs). Unless you do this, B cells don’t become completely activated, and though B cells produce antibodies the B cells don’t progress toward affinity maturation. That is, the normal process where antibodies are selected and shuffled to produce ultra-strong binders to their target antigens never gets underway. As a result, the vaccine induces low-affinity antibodies, and these low affinity antibodies are not protective.

It’s not clear — according to this model — whether the TH2 bias is actually the problem. Immune responses become biased to TH2 when there’s little innate immune stimulation, so the low affinity antibody and the TH2 response go hand in hand. Steve Varga (who has a nice commentary⁵ on this paper) has shown that some of the TH2 effects that were believed to be important in the pathogenesis of the ERD are not necessarily critical after all. Still, Varga and Delgado et al do seem to still feel that the TH2 shift is part of the disease.

The really exciting part of this finding is that it might actually be easy to fix. We now know a lot about TLR stimulation, and it should be possible to include TLR ligands along with the RSV vaccine:

These findings … open the possibility that inactivated RSV vaccines may be rendered safe and effective by inclusion of TLR agonists in their formulation. ⁴

Will this induce strong, protective immunity? Hopefully we’ll find out soon.

1. Mosmann TR, Cherwinski H, Bond MW, Giedlin MA, Coffman RL. Two types of murine helper T cell clone. I. Definition according to profiles of lymphokine activities and secreted proteins. J Immunol 1986; 136: 2348-2357[↩]
2. Priming immunization determines T helper cytokine mRNA expression patterns in lungs of mice challenged with respiratory syncytial virus. Graham BS, Henderson GS, Tang YW, Lu X, Neuzil KM, Colley DG. J Immunol. 1993 Aug 15;151(4):2032-40.[↩]
3. A potential molecular mechanism for hypersensitivity caused by formalin-inactivated vaccines. Moghaddam A, Olszewska W, Wang B, Tregoning JS, Helson R, Sattentau QJ, Openshaw PJ. Nat Med. 2006 Aug;12(8):905-7.[↩]
4. Maria Florencia Delgado, Silvina Coviello, A Clara Monsalvo, Guillermina A Melendi, Johanna Zea Hernandez, Juan P Batalle, Leandro Diaz, Alfonsina Trento, Herng-Yu Chang, Wayne Mitzner, Jeffrey Ravetch, José A Melero, Pablo M Irusta, Fernando P Polack (2008). Lack of antibody affinity maturation due to poor Toll-like receptor stimulation leads to enhanced respiratory syncytial virus disease Nature Medicine, 15 (1), 34-41 DOI: 10.1038/nm.1894[↩][↩]
5. Steven M Varga (2009). Fixing a failed vaccine Nature Medicine, 15 (1), 21-22 DOI: 10.1038/nm0109-21[↩]

The STEP HIV vaccine trial has been in the news a lot and it’s usually described as a “failed” trial. (I may even have described it that way myself.) It’s not really a failed trial, though; it’s a failed vaccine. The trial was successful in that exposed the failure of the vaccine, before the vaccine was widely deployed.

Most failed vaccines are caught this way, assuming they even make it to the clinical trial stage. Even in the old days, before the present clinical trial/licensing system,¹ most vaccines were highly effective and safe. I’ve shown stats about the truly spectacular effects of measles vaccine in the USA and in other first- and third-world countries, and I’ve shown what happens when anti-vaccine lies make people stop vaccinating (short answer: Children die).

Anti-vaccine loons use all sorts of distortion and outright lies to deny these effects. But that’s not to say that vaccines have been universally perfect. There’s no doubt whatsoever that vaccines have caused harm. All but the very safest vaccines do have a detectable complication rate. Some have a relatively high complication rate — the vaccine against smallpox, vaccinia virus, probably had the highest complication rate of any widely-used vaccine (somewhere around 1/100,000 – 1/300,000 vaccinees had significant complications), though most vaccines are far safer than that. The smallpox case makes the point obvious, though, that it’s a cost/benefit analysis. If vaccination can save a million lives, but costs ten lives, then most people would agree, however reluctantly, that vaccination is a good thing. ² These vaccines are still successes.

As an illustration, here’s a  chart showing overall effects of vaccination for a dozen or so diseases:³

However, there are several cases of true vaccine failures. They don’t seem to be widely known — outside of immunology circles, of course, where they’re well-known cautionary tales — so it’s worth mentioning them. For one thing, these stories flatly disprove one of the most common vaccine-loon claims. The conspiracy theories claim that even though vaccines cause harm, the harm is ignored. Yet in these cases, when vaccines did cause harm — even in small numbers — the problems were quickly spotted and the vaccine was forced out of use.

The three cases that jump to mind are the contaminated poliovirus vaccines, the RSV vaccine disaster, and the rotavirus vaccine. Of the three, the RSV vaccine was the worst. (A new explanation for this problem has just been published, and I’ll talk about it next post.)

The polio vaccine contamination thing was in the early days of virology, in 1959. To make a long story short, the polio vaccine virus was grown on monkey cells which turned out to have a virus of their own. This is now known as simian virus 40, SV40, and is a well-known virus now, but that was the first time it was identified. As it turned out, and mainly through luck, the SV40 contaminating the polio vaccine didn’t cause much, if any, disease in the recipients⁴ (though I’ve heard that immune-suppressed people, many years later, did have problems). It was a wake-up call to check much more thoroughly for unexpected passengers.

The rotavirus vaccine is a more recent example. This was mainly a rare-problem case: A small number of children who received the vaccine had serious problems (intussusception), but the incidence was so low (between 1/10,000 and 1/30,000 recipients) that the clinical trials didn’t have enough power to identify it as a problem. When put into widespread use in the general population, the risk was quickly spotted and the vaccine was withdrawn within a year.⁵ (This is the counter to the loon’s claim that vaccine risks are ignored, by the way.) The newer rotavirus vaccines that were recently introduced don’t have this problem.

Finally, that brings us to RSV, respiratory syncytial virus. This was the worst of the three, and the hardest to understand in terms of how the vaccine ever got licensed. To make this long story short, the RSV vaccine actually made the disease worse! RSV is a very common childhood virus, essentially infecting every child. Most have no problems, though because the disease is so common even rare complications in terms of percentages, turn out to be large in terms of actual numbers. But children previously vaccinated turned out to have much, much more frequent, and much more severe, complications. (Again, this was recognized and the vaccine was withdrawn, though I think it was not as quick as would happen today.) I’ll talk more about the RSV vaccine next time.

1. And I don’t know anything about the history of this, so if anyone does, please enlighten me[↩]
2. Of course, the lives saved are invisible — people who remain healthy — while the lives lost are apparent. That means the equation isn’t as simple as counting saved vs lost and going with the larger number. But it’s an equation, is my point.[↩]
3. Diptheria, Measles, Mumps, Pertussis, Polio, Rubella, Smallpox, Tetanus, Hepatitis A, Acute hepatitis B, Invasive Hib, invasive pneumococcal disease, and Varicella; Data are mostly from CDC, but include some other sources[↩]
4. Dev Biol Stand. 1998;94:183-90. Discovery of simian virus 40 (SV40) and its relationship to poliomyelitis virus vaccines. Hilleman MR.[↩]
5. Curr Opin Gastroenterol. 2005 Jan;21(1):20-5 Rotavirus vaccines and intussusception risk. Bines JE.[↩]

In 2008, I talked about maybe 100-200 papers here on Mystery Rays. Here are some of my favorite publications of 2008. I’m not saying these are the most important of the year, or anything like that; these are just papers that I thought covered something cool and did so in a nice clean way. As with last year, I managed to narrow it down to 12 subjects, but this time I couldn’t quite cut it down to 12 specific papers this year. Whatever, dude.  In no particular order:

1. Malaria vaccines. People have been working on effective malaria vaccines for decades, without much to show for it. In 2008, clinical trials with a new vaccine candidate showed encouraging, though not overwhelming, protection. (I talked about malaria eradication here and here.)
   

   Safety and immunogenicity of RTS,S/AS02D malaria vaccine in infants. N. Engl. J. Med. 359, 2533-2544. ¹
   Efficacy of RTS,S/AS01E vaccine against malaria in children 5 to 17 months of age. N. Engl. J. Med. 359, 2521-2532. ²

2. Mechanism of action of alum adjuvant. Alum has been the by far the most common adjuvant used in human vaccines, but until recently we didn’t know how it works. There were a bunch of papers in 2008 that at last offered explanations for alum’s effect: It acts through a particular branch of the innate immune sensor mechanism. (I talked about alum here and here.)
   

   Crucial role for the Nalp3 inflammasome in the immunostimulatory properties of aluminium adjuvants. Nature 453, 1122-1126. ³
   The Nlrp3 inflammasome is critical for aluminium hydroxide-mediated IL-1beta secretion but dispensable for adjuvant activity. Eur J Immunol. 2008 Aug;38(8):2085-9. ⁴
   Cutting edge: inflammasome activation by alum and alum’s adjuvant effect are mediated by NLRP3. J Immunol. 2008 Jul 1;181(1):17-21. ⁵

3. Immune evasion by tumor cells has been shown in a number of cases.  This year, it was shown that natural killer (NK) cells — or at least an NK cell receptor — in important in controlling tumors. (I talked about this paper here.)
   

   NKG2D-Deficient Mice Are Defective in Tumor Surveillance in Models of Spontaneous Malignancy. Immunity 28, 571-580. ⁶

4. T cells recognize latently-infected neurons. The most characteristic aspect of herpesviruses is their ability to become latent — to set up a long-term (often lifelong) infection in some cell type, without destroying the infected cells, and without being eliminated by the immune system. Herpes simplex viruses are the archetypal herpesvirus, yet in the past few years it’s become apparent that everything we thought we knew about herpes simplex latency is wrong. Herpes simplex latent infection turns out to be recognized and controlled, but not eliminated by immune T cells. (Here is my post on this paper.)
   

   Noncytotoxic lytic granule-mediated CD8+ T cell inhibition of HSV-1 reactivation from neuronal latency. Science 322, 268-271. ⁷

5. … What’s more, the view that HSV sets up a latent infection and then only rarely reactivates also turns out to be wrong. It seems that HSV is constantly reactivating from latent infection, but mainly in very short bursts. If you don’t monitor patients very, very closely — like swabbing them several times a day — you’ll miss most of the reactivations, so that it looks as if reactivation is rare.
   

   Rapidly cleared episodes of herpes simplex virus reactivation in immunocompetent adults. J. Infect. Dis. 198, 1141-1149 ⁸

6. … and even before latent infection, immune control may be critical. There seems to be a very narrow window of opportunity for immune responses to attack a herpes simplex infection before the virus slips up into neurons and establishes the latent infection. (More here.)
   

   CD8(+) T-cell attenuation of cutaneous herpes simplex virus infection reduces the average viral copy number of the ensuing latent infection. Immunol. Cell Biol. 86, 666-675 ⁹

7. Immunodeficiency virus immune escape. One of the major barriers to immune control of HIV is the virus’s ability to mutate and escape recognition by the immune system. This has been recognized for a long time, but techniques to measure and analyze this continue to improve. Here’s a representative paper looking in detail at immunodeficiency virus immune escape.
   

   Vaccination and Timing Influence SIV Immune Escape Viral Dynamics In Vivo. PLoS Pathog 4(1): e12 ¹⁰

8. Adenovirus evades NK cells. The first virus that was shown to have a way of blocking recognition by T cells was adenovirus, via its E3gp19k protein. Identified in the 1970s, there were a flurry of articles on the protein, and then it kind of languished, as people assumed the story was mined out. In 2008, though, it was shown that E3gp19k is much more potent that originally believed, because it also blocks recognition by natural killer (NK) cells. Another reminder that viruses are capable of much more subtlety than we give them credit for. (I talked about this here.)
   

   Adenovirus E3/19K promotes evasion of NK cell recognition by intracellular sequestration of the NKG2D ligands major histocompatibility complex class I chain-related proteins A and B. J. Virol. 82, 4585-4594 ¹¹

9. A new mechanism of immune evasion. One of the rather puzzling things about viral immune evasion is that even closely-related viruses often seem to have evolved their own, independent, approaches to the problem. Kalus Fruh showed that rhesus cytomegalovirus has a really weird way of blocking T cell recognition, a mechanism that so far seems to be unique to this virus.  In the past, weird virus immune evasion things have led to important advances in normal cell biology; maybe this will, too.
   

   Signal Peptide-Dependent Inhibition of MHC Class I Heavy Chain Translation by Rhesus Cytomegalovirus. PLoS Pathogens PLoS Pathog 4, e1000150. ¹²

10. Cancer vaccination remains tantalizingly close but just out of reach; clinical trials still show occasional spectacular successes coupled with more failures. One of the problems is to identify tumor antigens that are suitable targets for common vaccines. Endogenous retroviruses may, perhaps, be such a target. (More on HERVs and immunity here.)
    

    Regression of human kidney cancer following allogeneic stem cell transplantation is associated with recognition of an HERV-E antigen by T cells. J Clin Invest 118, 1099-1109 ¹³

11. Genome sequencing has been getting faster and cheaper at an amazing pace. We’re now entering an age when viruses can be tracked through epidemics by whole-genome sequencing, following through mutations and viral evolution throughout the epidemic and using the sequence to predict and analyze the stages of the epidemic. (More here.)
    

    Transmission pathways of foot-and-mouth disease virus in the United Kingdom in 2007. PLoS Pathog. 4, e1000050 ¹⁴

12. Meticulous tracking of T cells demonstrated last year that for CD4 T cells it is possible to count the tiny handful of naïve T cells for any one specificity, hidden though they are in the ocean of other T cells. This year Leo LeFrancois appled the same technique to cytotoxic T lymphocytes (CTL) and proposed that the number of naïve T cells is a major determinant of the entire downstream immune response. (Post is here.)
    

    Endogenous naive CD8+ T cell precursor frequency regulates primary and memory responses to infection. Immunity 28, 859-869. ¹⁵

1. Abdulla, S., Oberholzer, R., Juma, O., Kubhoja, S., Machera, F., Membi, C., Omari, S., Urassa, A., Mshinda, H., Jumanne, A., Salim, N., Shomari, M., Aebi, T., Schellenberg, D. M., Carter, T., Villafana, T., Demoitie, M. A., Dubois, M. C., Leach, A., Lievens, M., Vekemans, J., Cohen, J., Ballou, W. R., and Tanner, M. (2008). Safety and immunogenicity of RTS,S/AS02D malaria vaccine in infants. N. Engl. J. Med. 359, 2533-2544. doi:10.1056/NEJMoa0807773[↩]
2. Bejon, P., Lusingu, J., Olotu, A., Leach, A., Lievens, M., Vekemans, J., Mshamu, S., Lang, T., Gould, J., Dubois, M. C., Demoitie, M. A., Stallaert, J. F., Vansadia, P., Carter, T., Njuguna, P., Awuondo, K. O., Malabeja, A., Abdul, O., Gesase, S., Mturi, N., Drakeley, C. J., Savarese, B., Villafana, T., Ballou, W. R., Cohen, J., Riley, E. M., Lemnge, M. M., Marsh, K., and von Seidlein, L. (2008). Efficacy of RTS,S/AS01E vaccine against malaria in children 5 to 17 months of age. N. Engl. J. Med. 359, 2521-2532. doi:10.1056/NEJMoa0807381[↩]
3. Eisenbarth, S. C., Colegio, O. R., O’Connor, W., Sutterwala, F. S., and Flavell, R. A. (2008). Crucial role for the Nalp3 inflammasome in the immunostimulatory properties of aluminium adjuvants. Nature 453, 1122-1126. doi:10.1038/nature06939
   Cutting edge: alum adjuvant stimulates inflammatory dendritic cells through activation of the NALP3 inflammasome.
   Kool M, Pétrilli V, De Smedt T, Rolaz A, Hammad H, van Nimwegen M, Bergen IM, Castillo R, Lambrecht BN, Tschopp J.
   J Immunol. 2008 Sep 15;181(6):3755-9.[↩]
4. The Nlrp3 inflammasome is critical for aluminium hydroxide-mediated IL-1beta secretion but dispensable for adjuvant activity.
   Franchi L, Núñez G.
   Eur J Immunol. 2008 Aug;38(8):2085-9. [↩]
5. Cutting edge: inflammasome activation by alum and alum’s adjuvant effect are mediated by NLRP3.
   Li H, Willingham SB, Ting JP, Re F.
   J Immunol. 2008 Jul 1;181(1):17-21. [↩]
6. Guerra, N., Tan, Y. X., Joncker, N. T., Choy, A., Gallardo, F., Xiong, N., Knoblaugh, S., Cado, D., Greenberg, N. R., and Raulet, D. H. (2008). NKG2D-Deficient Mice Are Defective in Tumor Surveillance in Models of Spontaneous Malignancy. Immunity 28, 571-580. doi:10.1016/j.immuni.2008.02.016[↩]
7. Knickelbein, J. E., Khanna, K. M., Yee, M. B., Baty, C. J., Kinchington, P. R., and Hendricks, R. L. (2008). Noncytotoxic lytic granule-mediated CD8+ T cell inhibition of HSV-1 reactivation from neuronal latency. Science 322, 268-271. DOI: 10.1126/science.1164164[↩]
8. Mark, K. E., Wald, A., Magaret, A. S., Selke, S., Olin, L., Huang, M. L., and Corey, L. (2008). Rapidly cleared episodes of herpes simplex virus reactivation in immunocompetent adults. J. Infect. Dis. 198, 1141-1149. DOI: 10.1086/591913[↩]
9. Wakim, L. M., Jones, C. M., Gebhardt, T., Preston, C. M., and Carbone, F. R. (2008). CD8(+) T-cell attenuation of cutaneous herpes simplex virus infection reduces the average viral copy number of the ensuing latent infection. Immunol. Cell Biol. 86, 666-675; doi:10.1038/icb.2008.47[↩]
10. Loh, L., Petravic, J., Batten, C., Jane, Davenport, M., P., and Kent, S., J. (2008). Vaccination and Timing Influence SIV Immune Escape Viral Dynamics In Vivo. PLoS Pathog 4(1): e12. doi:10.1371/journal.ppat.0040012[↩]
11. McSharry, B. P., Burgert, H. G., Owen, D. P., Stanton, R. J., Prod’homme, V., Sester, M., Koebernick, K., Groh, V., Spies, T., Cox, S., Little, A. M., Wang, E. C., Tomasec, P., and Wilkinson, G. W. (2008). Adenovirus E3/19K promotes evasion of NK cell recognition by intracellular sequestration of the NKG2D ligands major histocompatibility complex class I chain-related proteins A and B. J. Virol. 82, 4585-4594. doi: 10.1128/JVI.02251-07.[↩]
12. Powers, C. J., and Fruh, K. (2008). Signal Peptide-Dependent Inhibition of MHC Class I Heavy Chain Translation by Rhesus Cytomegalovirus. PLoS Pathogens PLoS Pathog 4, e1000150. doi:10.1371/journal.ppat.1000150[↩]
13. Takahashi, Y., Harashima, N., Kajigaya, S., Yokoyama, H., Cherkasova, E., McCoy, J. P., Hanada, K., Mena, O., Kurlander, R., Abdul, T., Srinivasan, R., Lundqvist, A., Malinzak, E., Geller, N., Lerman, M. I., and Childs, R. W. (2008). Regression of human kidney cancer following allogeneic stem cell transplantation is associated with recognition of an HERV-E antigen by T cells. J Clin Invest 118, 1099-1109. doi:10.1172/JCI34409.[↩]
14. Cottam, E. M., Wadsworth, J., Shaw, A. E., Rowlands, R. J., Goatley, L., Maan, S., Maan, N. S., Mertens, P. P., Ebert, K., Li, Y., Ryan, E. D., Juleff, N., Ferris, N. P., Wilesmith, J. W., Haydon, D. T., King, D. P., Paton, D. J., and Knowles, N. J. (2008). Transmission pathways of foot-and-mouth disease virus in the United Kingdom in 2007. PLoS Pathog. 4, e1000050. doi:10.1371/journal.ppat.1000050[↩]
15. Obar, J. J., Khanna, K. M., and Lefrancois, L. (2008). Endogenous naive CD8+ T cell precursor frequency regulates primary and memory responses to infection. Immunity 28, 859-869. doi:10.1016/j.immuni.2008.04.010[↩]