Mystery Rays from Outer Space

Melanoma blood vessel

Designing tumor vaccines presents a bunch of problems that anti-pathogen vaccines don’t. One of those problems is identifying an appropriate antigen. There’s been a lot of interest in finding tumor antigens that cytotoxic T lymphocytes will recognize, and in fact hundreds have been identified. The database of tumor antigens at Cancer Immunity lists some 750 of them, divided into various categories:

• Unique antigens result from point mutations in genes that are expressed ubiquitously. They are unique to the tumor of an individual patient or restricted to very few patients;
• Shared tumor-specific antigens are expressed in many tumors but not in normal tissues;
• Differentiation antigens are also expressed in the normal tissue of origin of the malignancy;
• Overexpressed antigens are expressed in a wide variety of normal tissues and overexpressed in tumors

Overall, shared tumor-specific antigens may be the ideal target. Because they’re found in multiple tumors, a vaccine can be pre-designed and go through a time-consuming optimization and validation process; because they’re only found in tumors, there’s less concern about safety. That is, the risk of the vaccine precipitating an autoimmune reaction to normal cells is low.

MHC expression in eye before and after vaccination

The problem with this class is that there just aren’t all that many tumor-specific antigens. The database lists 20-odd such antigens, and many of them are only found in a limited subsets of tumors (mostly melanomas). What’s more, I think it’s not merely that the targets are out there yet haven’t been identified. More likely, there simply are not many shared tumor-specific antigens.

The next-best category, as far as safety and effectiveness is concerned, is the unique antigens. These may be great as far as safety and effectiveness are concerned, but there are major technical problems in identifying them in a clinical context. Because they’re unique, you can’t pre-design the vaccine; you need to customize the antigen to each patient. And (at least with present techniques) by the time there is enough tumor available to look for unique antigens, the disease is likely to be pretty far advanced (and advanced tumors are more likely to be resistant to vaccination, for several reasons). There’s a lot of interest in making preparations of tumors that would contain unique antigens, without the trouble of identifying the antigen, but as far as I know that hasn’t made it very far into clinical trials yet.

So that leaves overexpressed and differentiation antigens. These are both, by definition, found in normal cells, and that means either the immune system is already tolerized to the antigen, or that targeting these antigens with a vaccine risks triggering an autoimmune reaction.

In fact, clinical trials using these kinds of vaccines against melanomas have found that successful tumor attack is almost invariably associated with an autoimmune effect, usually manifesting as vitiligo (de-pigmented patches on the skin).

It is expected that immune responses to such peptides will be compromised by self-tolerance or, alternatively, that stimulation of effective immune responses will be accompanied by autoimmune vitiligo. ¹

I believe the first record of this goes back to 1964.² Vitiligo is not, as autoimmune diseases go, a terrible problem, and certainly one would be delighted to trade melanoma for vitiligo. However, there are more serious potential problems as well, and one of them was recently reported by Nick Restifo’s group. ³ In this case, a highly active anti-melanoma vaccine not only killed the tumor, it also triggered severe autoimmune destruction of the eye; and the more effective the vaccine, the worse the autoimmune disease:

Thus, in the present model, the efficacies of the antitumor immune therapies were directly correlated with the induction of autoimmunity in the eye. … Our data suggest that, as tumor immunotherapies improve, these autoimmune manifestations may become more prevalent.

(My emphasis.) The autoimmune disease in these melanoma patients is still probably manageable (they mention using 30 months of steroid treatment in one of the most severely affected patients), and even if not, again the tradeoff is one most people would probably take (blindness vs. death). But other tumors may make the decision much more difficult:

Although the autoimmune side effects of melanocyte/melanoma-targeted therapies have been manageable, the unintended autoimmunity of therapies targeting colorectal, brain, or lung cancer might prove more severe.

1. Antigens derived from melanocyte differentiation proteins: self-tolerance, autoimmunity, and use for cancer immunotherapy. Engelhard VH, Bullock TN, Colella TA, Sheasley SL, Mullins DW. Immunol Rev. 2002 Oct;188:136-46. [↩]
2. Vitiligo In A Case Of Vaccinia Virus-Treated Melanoma. Burdick Kh, Hawk Wa. Cancer. 1964 Jun;17:708-12.[↩]
3. Palmer, D.C., Chan, C., Gattinoni, L., Wrzesinski, C., Paulos, C.M., Hinrichs, C.S., Powell, D.J., Klebanoff, C.A., Finkelstein, S.E., Fariss, R.N., Yu, Z., Nussenblatt, R.B., Rosenberg, S.A., Restifo, N.P. (2008). From the Cover: Effective tumor treatment targeting a melanoma/melanocyte-associated antigen triggers severe ocular autoimmunity. Proceedings of the National Academy of Sciences, 105(23), 8061-8066. DOI: 10.1073/pnas.0710929105 [↩]

Animation of biological function doesn’t really contribute much to the research side of things (despite the claims of some press releases); but the movies can be good teaching tools.  Now there’s a site (http://www.molecularmovies.com/) that has aggregated a number of the animations that are out there.  Many of the movies can be freely used for teaching. I haven’t seen any mention of this in the blogworld, which surprises me: it seems like a useful resource.

A couple of years ago, Jurg Tschopp’s group showed that uric acid crystals acted as inflammatory agents (and probably, also as adjuvants) by stimulating the Nalp3 inflammasome.¹ A month ago, Richard Flavell’s group showed that alum adjuvant — also (sort of) crystalline — also acts through the Nalp3 inflammasome.² And now, a paper just out in PNAS says that crystalline silica causes silicosis by acting through, you’ll never guess, the Nalp3 inflammasome. ³ Could a trend be showing up?

I don’t know much about silicosis, other than the obvious stuff (it’s an inflammatory lung disease caused by inhaling crystalline silica) and to be honest I had never much wondered why silica would cause lung disease; I assumed that it’s toxic, caused cell damage because of direct cytotoxicty, and the cell damage led to an inflammatory response. It turns out that that’s partly right; silica is cytotoxic and does cause cell damage, but the cell damage per se is not the cause of the inflammation, because Nalp3-knockout mice still had the same amount of cell death, but didn’t have the inflammation.

Nalp3 is an intracellular sensor, which raises an obvious question⁴ about all these crystals: How do they get into the cell to stimulate the response? As far as I know, this is the first of the papers to look at this question, and the answer is a little disappointing in that it seems fairly simple: It’s just endocytosis.

… endocytosis of silica by macrophages is needed to activate the Nalp3 inflammasome in response to silica for the resultant processing and secretion of proinflammatory cytokines.

I was at least half expecting a much more complicated and exciting answer, but this does make sense.  (I don’t know, actually, if silica, alum, and uric acid crystals are about the same size, or if for some other reason endocytosis is not a plausible explanation for the other two.)  That still leaves the issue of how the crystals get out of the endosome and into the cytosol, but we do know that in macrophages and dendritic cells there’s a poorly-characterized pathway by which some substances — proteins that are cross-presented, in particular — can exit the endosome and gain access to the cytosol, so we’re not adding any new mysteries, anyway.

The next candidate is probably asbestos; at this point, I think it’s fairly likely that asbestosis is also mediated by the Nalp3 inflammasome.

The only one of these crystalline substances that’s physiological is uric acid — monosodium urate crystals, a natural adjuvant that’s believed to act as a danger signal for cell death. I wonder if all these things are mimicking MSU crystals, or if there’s some other reason they act through the same receptor.

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[↩]
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. Brought up by Kay, last time I talked about it[↩]

Avian influenza has a terribly high mortality rate in the humans it infects — perhaps as many as 80% of infected people die. Why is avian flu so lethal, while other strains of influenza rarely cause serious damage in young, healthy people?

One explanation has been cytokine storms. According to this hypothesis,¹ avian influenza causes a massive innate immune response, leading to the release of large amounts of cytokines. It’s the resulting inflammation that is lethal, not the damage that the virus causes directly.

The problem with this hypothesis was that it apparently didn’t help with treatment. If cytokine storms are responsible for mortality, then suppressing cytokine responses should reduce the death rate; and that didn’t happen, according to a paper² published last year. What’s more, although cytokine levels are associated with increased mortality, they’re also associated with virus levels. That is, cytokine levels may be an indicator of the amount of virus present, rather than a direct risk factor.

Because cytokine inhibition does not protect against death, therapies that target the virus rather than cytokines may be preferable. ²

For this reason (and others) I said at the time that “I think the evidence for this is pretty weak – and for avian flu in particular, it’s been shown that cytokines are probably not the culprits at all.”

But I’m always happy to have my mind changed, and a new paper that came out a couple of weeks ago³ has made me reconsider (even though it’s mouse work, not tested yet in humans). This is from Kwok-Yung Yuen’s group in Hong Kong. (Yuen is probably best known for his work on SARS — another disease that’s been claimed to act via cytokine storms — but he has worked on on avian influenza for a long time as well.) Their trick was to use triple therapy: a dual blockade of cytokines, plus antiviral treatment. None of these treatments worked alone. If you suppress viral replication while allowing the cytokines to persist, the mice die; if you shut down the cytokines while allowing the virus to continue replicating, the mice die. But if you block cytokines and virus — then most of the mice survive.

Actually, the antiviral alone does work pretty well if you start treatment almost immediately after infection. Of course, that’s not practical in humans; humans arrive at the hospital days after they’ve been infected, and by then viral replication and cytokine levels are already roaring ahead. The exciting part about Yuen’s triple therapy is that it gives decent survival — again, in mice, not yet humans — even if you don’t start treatment for 2 days after infection. This makes it a practical treatment for humans, and since the anti-inflammatory drugs they used are not particularly expensive or obscure, I’m sure we will see this treatment tried out in humans sooner rather than later.

1. Chan, M. C., Cheung, C. Y., Chui, W. H., Tsao, S. W., Nicholls, J. M., Chan, Y. O., Chan, R. W., Long, H. T., Poon, L. L., Guan, Y., and Peiris, J. S. (2005). Proinflammatory cytokine responses induced by influenza A (H5N1) viruses in primary human alveolar and bronchial epithelial cells. Respir Res 6, 135.
   and
   de Jong MD, Simmons CP, Thanh TT, Hien VM, Smith GJ, Chau TN, Hoang DM, Chau NV, Khanh TH, Dong VC et al. (2006) Fatal outcome of human influenza A (H5N1) is associated with high viral load and hypercytokinemia. Nat Med 12:1203-1207. doi:10.1038/nm1477[↩]
2. Salomon, R., Hoffmann, E., and Webster, R. G. (2007). Inhibition of the cytokine response does not protect against lethal H5N1 influenza infection. Proc Natl Acad Sci U S A 104, 12479-12481. [↩][↩]
3. Zheng, B., Chan, K., Lin, Y., Zhao, G., Chan, C., Zhang, H., Chen, H., Wong, S.S., Lau, S.K., Woo, P.C., Chan, K., Jin, D., Yuen, K. (2008). Delayed antiviral plus immunomodulator treatment still reduces mortality in mice infected by high inoculum of influenza A/H5N1 virus. Proceedings of the National Academy of Sciences, 105(23), 8091-8096. DOI: 10.1073/pnas.0711942105[↩]

Vaccination is one of the (if not the most) important medical advances in history. The problem today is that most of the easy diseases already have vaccines available, and now we’re trying to develop vaccines against the hard ones. Fortunately, I think we’re entering a new golden age of vaccine development, as we begin to understand why immunization works at the molecular level, to the point where we may soon be able to deliberately tweak them for optimal efficacy.

Back in the dark ages, when I was first working with vaccines,¹ adjuvants were a witches’ brew of newts’ eyes and frogspawn, and the ones that worked, just sort of … worked. No one really knew why. But around the time I backed away from vaccines, (partly because of this empirical adjuvant stuff) new theoretical frameworks were being developed that began to explain how and why adjuvants work, and now — some 20 years later — we are at the point where theory is moving solidly toward practice.

I’ve commented several times on the issue of immunodominance. T cell responses to antigens aren’t smoothly distributed over all the possible targets in the antigen; instead, a handful of targets get the lion’s share of the T cell response. Sometimes this is a good thing (for example, it’s a way of getting a screaming hot response to a target, instead of having a bunch of wimpy little responses); sometimes it’s bad (if it’s a moving target, as with rapidly-mutating viruses such as HIV, then your screaming hot response may be to a target that no longer exists, whereas having a bunch of targets at least nearly guarantees that you’ve got something to shoot at.)

In spite of its importance, though, the underlying mechanisms that drive immunodominance aren’t well understood. For example, one possible explanation is that the T cell that ends up becoming dominant, started out as the most abundant clone originally. A paper last year² (I talked about it here) supported that possibility, but a more recent study³ that I talked about earlier this week suggested that while clonal abundance is one factor, there must be other, equally important, influences on the response.

That fits with another paper that came out in May,⁴ looking at the effects of different adjuvants on the immune response. Of course this has been done many times in a quantitative way — which adjuvant gives the biggest response? — but Malherbe et al. asked the question qualitatively: What exactly happens to the T cell response? That is: We know that different adjuvants can cause higher or lower responses to an antigen; but are the different responses made up of the same CTL⁵, or do different adjuvants crank up different sets? Can we drive a T cell response that is qualitatively, as well as quantitatively, better?

I, for one, (and I think most of the field) would have said “No”; no matter what your adjuvant is, the response would be qualitatively the same. Why would one particular CTL precursor clone be stimulated better or worse by a particular adjuvant? That’s the answer that would be predicted from the first study, that suggested that immunodominance is determined mainly by the precursor frequency: You can’t really affect the precursor frequency (that’s set during thymic development), so no matter what you do with your antigen you should get the same relative response (even though the total response may be higher or lower, it would contain the same proportion of T cell clones).

In fact, that’s not what happens. Malherbe et al. compared five different adjuvants, mixed with the same antigen. The adjuvants are known to act through different mechanisms. (That is, while they all act by stimulating innate immune recognition molecules, they stimulate different innate receptors — different TLR molecules, or [as we now know⁶ ] pattern recognition receptors that are different from TLRs altogether.) Then they assessed the subsequent immune response by comparing the immunodominance hierarchies that came out of the immunization. The different adjuvants drove expansion of different T cell clones, so that the response was qualitatively different.

In particular, adjuvants drove expansion of higher-affinity clones:

…adjuvants regulate clonal composition by using a mechanism that alters initial TCR-based selection thresholds and that relies most heavily on blocking the propagation of antigen-specific clonotypes expressing low-affinity TCR. … Thus, adjuvant formulation can modify the TCR-based selection threshold that regulates Th cell clonal composition in response to protein vaccination.

How adjuvants do this remains unknown. It wasn’t related to the antigen dose (which has previously been shown to affect the TcR affinity). Possibilities include differential dendritic cell maturation, altering local antigen contentration (the “depot” effect that has been the classic explanation for alum’s mechanism of action — though that explanation is at least partly rendered obsolete by the recent paper⁷ from Richard Flavell’s group), and direct stimulation of T cell clones — but who knows.

Assuming this holds up for different antigens (they’ve only looked at one, so far) the key thing, in clinical terms, is that it’s possible to alter immunodominance without changing the antigen. We need to understand how this works, because it may be a much simpler way of improving immune responses than altering the antigen itself.

1. It looks as if I may be doing so again; our proposal for a Vaccine Center here has been funded, at least for a few years; although I’m only a small part of the group[↩]
2. Naive CD4(+) T Cell Frequency Varies for Different Epitopes and Predicts Repertoire Diversity and Response Magnitude. Moon JJ, Chu HH, Pepper M, McSorley SJ, Jameson SC, Kedl RM, Jenkins MK. Immunity. 2007 Aug;27(2):203-13.[↩]
3. Obar, J., Khanna, K., LeFrancois, L. (2008). Endogenous Naive CD8+ T Cell Precursor Frequency Regulates Primary and Memory Responses to Infection. Immunity, 28(6), 859-869. DOI: 10.1016/j.immuni.2008.04.010[↩]
4. Malherbe, L., Mark, L., Fazilleau, N., McHeyzer-Williams, L., McHeyzer-Williams, M. (2008). Vaccine Adjuvants Alter TCR-Based Selection Thresholds. Immunity, 28(5), 698-709. DOI: 10.1016/j.immuni.2008.03.014

   Commentary at:
   Immunity 28:602-604 (16 May 2008) doi:10.1016/j.immuni.2008.04.008
   Preview: Taking a Toll Road to Better Vaccines
   Sharon Celeste Morley and Paul M. Allen[↩]

5. CTL: Cytotoxic T lymphocytes[↩]
6. Eisenbarth, S.C., Colegio, O.R., O'Connor, W., Sutterwala, F.S., Flavell, R.A. (2008). Crucial role for the Nalp3 inflammasome in the immunostimulatory properties of aluminium adjuvants. Nature DOI: 10.1038/nature06939[↩]
7. Eisenbarth, S.C., Colegio, O.R., O’Connor, W., Sutterwala, F.S., Flavell, R.A. (2008). Crucial role for the Nalp3 inflammasome in the immunostimulatory properties of aluminium adjuvants. Nature. DOI: 10.1038/nature06939[↩]

Immunodominance is one of the many critical, yet poorly understood, phenomena associated with antiviral immunity. Why is it that one particular viral peptide may be recognized by as many as 1% of all the cytotoxic T lymphocytes (CTL) in the body, while a different epitope may be recognized only by 0.001%? There are obvious implications for vaccine design and development; yet we really have little idea of the causes. People have proposed all kinds of explanations — kinetics of peptide presentation, kinetics of T cell response, number of T cell clones, amount of peptide presented — and each of the suggestions has some support but doesn’t seem to explain every instance.

One of the problems is the technical difficulty involved. Accurately quantifying the minute, highly localized amounts of peptide involved, or the tiny handful of cells that could respond, has simply been out of our reach; until very recently.

About a year back, Marc Jenkins’ group described a new technique for measuring very small numbers of T cells in mice¹ and came up with some interesting numbers. They looked at three epitopes (for CD4, T helper, T cells, not CTL; but guesses have been that the two groups of T cells have similar numbers of precursors) and concluded that the epitopes had 190, 20, and 16 precursor T cells specific for them. What’s more, the more T cell precursors there are, the higher the ultimate T cell response to the epitope — the more immunodominant that epitope is.

I commented on the paper at the time and said “It’s an interesting suggestion, and their data certainly are suggestive. I’m sure there will be more epitopes examined by this technique over the next little while, so we’ll see how well it holds up.” Now, a year later, we’re seeing the first followup, and it turns out to hold up pretty well; although there are, not surprisingly, some added complexities.

The followup is from Leo Lefrancois’s group in UConn.² I will skip over their controls, except to say that they did a bunch of ingenious controls to demonstrate that they really were looking at what they claim to be.

First, they looked at a half-dozen known epitopes and asked how many precursor CTL there were for each. Their numbers were in the same ballpark as the CD4 precursors measured earlier; they came up with a range from 80 to 1200 CTL (average, 120-160) specific for their various epitopes. This is somewhat larger than the Moon et al. estimates I mentioned earlier, but I think that these epitopes were all, or almost all, fairly abundant to start with, so it’s pretty consistent.

They also used the technique for an extremely cool experiment. They infected mice with viruses, and then tracked through the number of CTL present each day. This way they were able to ask the exciting question, When is the immunodominance hierarchy set?

Moon et al. last year suggested that immunodominance hierarchies are set on day 0; that the number of T cell precursors present determines the size of the response to an epitope. I was a little dubious about that, saying “I think it’s equally likely that while the size of the naive precursor pool is one factor, you can also get different T cell responses out of the same number of precursors, for any of a variety of reasons.”

Here’s Obar  et al.’s conclusion:

Although the M45:Db- and VSV-N:Kb-specific responses differed kinetically, they were of similar overall magnitude, even though their initial precursor frequencies differed on average by 4-fold… These data suggested that interclonal competition for resources (i.e., APC interactions, growth factors, or costimulatory molecules) prior to the peak of the response was important in modulating overall clonal expansion.

(My emphasis)

So the bad news, I guess, is that there may not be a single simple explanation for immunodominance, at least for CTL. However, precursor frequency does seem to be one factor — and an important one — in setting CTL immunodominance hierarchies, and knowing the timing of other factors (hierarchies are set around day 3, Obar et al. determined) should be a big help in narrowing down possibilities.

1. Naive CD4(+) T Cell Frequency Varies for Different Epitopes and Predicts Repertoire Diversity and Response Magnitude. Moon JJ, Chu HH, Pepper M, McSorley SJ, Jameson SC, Kedl RM, Jenkins MK. Immunity. 2007 Aug;27(2):203-13.
   Commentary on the paper, by Mark Davis, here: The T Cell Repertoire Comes into Focus. Davis MM. Immunity. 2007 Aug;27(2):179-80. [↩]
2. Obar, J., Khanna, K., LeFrancois, L. (2008). Endogenous Naive CD8+ T Cell Precursor Frequency Regulates Primary and Memory Responses to Infection. Immunity, 28(6), 859-869. DOI: 10.1016/j.immuni.2008.04.010[↩]

… our analyses suggest that parasite lifetime fitness would be maximized at an intermediate replication rate. Importantly, at this level of replication, the parasite causes considerable virulence to the host. … natural selection in this system favors parasite genotypes that cause significant damage to their hosts based on a trade-off between virulence and transmission.

–de Roode JC, Yates AJ, Altizer S (2008) Virulence-transmission trade-offs and population divergence in virulence in a naturally occurring butterfly parasite. Proc Natl Acad Sci U S A 105:7489–7494. DOI:10.1073/pnas.0710909105

Spleen 3 days after immunisation. B cells (red), CTL (green) and dendritic cells (blue

I noted some time ago that, despite the name, it’s clear that cytotoxic T lymphocytes (CTL) are more than just cytotoxic. They can limit or eliminate virus infections by killing infected cells, sure; but as well, they can  produce cytokines, like interferons, that also shut down viruses.

So which of these abilities makes for an effective CTL response? What should we be aiming for when we design vaccines? What should we be measuring, to test whether our vaccines are doing something useful?

The recent failure¹ of the STEP anti-HIV vaccine trial makes this an especially timely question:

… the disappointing outcome of this study highlights one particular point: our understanding of T cell-mediated efficacy remains limited. Without such knowledge, vaccine design strategies will remain largely empirical, and further failures are likely. ²

(My emphasis.)

The latest Nature Medicine has a review² and a perspective³ looking at this issue.

In their review, Appay et al. emphasize that not all CTL are equal. Frequency of CTL isn’t a good correlate with control of viruses (at least with HIV) or of tumors. Part of this is undoubtedly because of immune escape –that is, the virus or tumor are likely to have mutated to evade recognition by the most frequent CTL –  but in this review Appay et al. are looking at the other side of the equation: What’s wrong with the CTL, that they can be escaped from, or otherwise can’t control the virus? And how to decide if your vaccine has a chance of working? If raw CTL numbers are not useful, is there some characteristic of CTL that does correlate with control?

They talk about “polyfunctional” CTL (CTL that are able to produce several kinds of cytokines on recognizing their target — a relatively new concept⁴ that I’ve been a little skeptical about in the past, but my skepticism is slowly waning) as well as “functional avidity”, clonal senescence, and some other factors. Essentially, they conclude, the ideal CTL would be highly sensitive to antigen; would have multiple responses to draw on; and would be able to replicate and maintain itself over a long period.

So how can we drive the development of such highly effective CTL with vaccines? They make several comments:

• Low antigen concentration is more likely to induce highly effective CTL. “… it should be borne in mind that recurrent immunizations and boosting with high-dose antigen, conducted with the aim of achieving maximum immunogenicity as determined by solely quantitative measures, may have adverse effects; specifically, effective vaccine-induced T cells could become exhausted through the loss of replicative capacity and apoptotic deletion.”
• Because low levels of antigen may not drive any immune response whatsoever, we need to think about effective co-stimulation to kick-start the immune response. “… the level and type of costimulation may compensate for low antigen abundance and play a considerable part in boosting T cells with high antigen sensitivities. ” This includes working through innate immune receptors, a very active field of research.

• Dendritic cells (red) in a lymph node

  As part of improved co-stimulation, we need to think about inducing CD4 (helper) T cells along with CTL. CD4 T cells are important in activating and licensing the dendritic cells that then in turn activate CTL. However, inducing CD4 T cells has the risk of stimulating a regulatory (TReg) type response, which might reduce CTL functions. We need to understand this better; it’s another very active field.

• Route of vaccine administration may be more important than previously thought. Different antigen-presenting cells are present in different tissues, and the likelihood of active versus regulatory responses is strongly affected by these cells.
• Vaccine antigens may not be optimal. “… for practical reasons, vaccine formulations often contain consensus or optimized antigens … it seems that vaccine-induced T cells generally recognize the native antigen less efficiently and are therefore less effective in the face of their real targets.”

All of these points are the subject of research (some more than others). Hopefully, the outcome of the STEP trial will lead to a better understanding of underlying principles, and so eventually to much more effective vaccine development.

The failure of the Merck STEP trial represents a turning point for the field of vaccination. However, far from embodying the end of the T cell vaccine strategy, it heralds a new era in vaccine research based on comprehensive immunomonitoring.⁵

1. Though see Richard Jeffrey‘s comment in Nature (Vaccine failure is not a ‘crisis’ for HIV research. Richard Jefferys.  
Nature 453, 719-720 (5 June 2008) | doi:10.1038/453719d) as to the meaning and implication of “failure” here[↩]
2. Appay, V., Douek, D.C., Price, D.A. (2008). CD8+ T cell efficacy in vaccination and disease. Nature Medicine, 14(6), 623-628. DOI: 10.1038/nm.f.1774[↩][↩]
3. Nature Medicine 14, 617 – 621 (2008) doi:10.1038/nm.f.1759. David I Watkins, Dennis R Burton, Esper G Kallas, John P Moore & Wayne C Koff. Nonhuman primate models and the failure of the Merck HIV-1 vaccine in humans[↩]
4. Betts MR, Nason MC, West SM, De Rosa SC, Migueles SA, Abraham J, Lederman MM, Benito JM, Goepfert PA, Connors M et al. (2006) HIV nonprogressors preferentially maintain highly functional HIV-specific CD8+ T cells. Blood 107:4781–4789.[↩]
5. CD8+ T cell efficacy in vaccination and disease. Victor Appay, Daniel C Douek & David A Price. Nature Medicine 14, 623 – 628 (2008) doi:10.1038/nm.f.1774[↩]

Individual viruses just like individual  people or any other concrete objects cannot be “defined”; they can only be named in a manner  reminiscent of baptism or they can be pointed at ostensively.  … Since species are actually fuzzy sets, i.e. polythetic classes, creating and delineating a virus species is more a matter of opinion and convenience rather than of logical necessity.

–Van Regenmortel MH (2003). Viruses are real, virus species are man-made, taxonomic constructions. Arch Virol 148:2481–2488. DOI:10.1007/s00705-003-0246-y

One of the interesting things about the retrovirus family is that it contains some of the most lethal viruses we know of, and also some of the least harmful. For obvious reasons, the lethal family members (HIV) get lots of recognition, while their harmless cousins get little more than an occasional mention. There are more — far more — articles on HIV in the past two weeks, than there have been on the spumaviruses in the past 50 years. In fact, it’s only very recently that people have demonstrated why the spumaviruses are actually harmless. ¹

Spumaviruses, or foamy viruses, infect many species — though not, apparently, humans,² even though they’re common infections of old world monkeys and apes. Normally I’d assume that the lack of human spumaviruses was simply because nobody has looked hard enough, but I think in this case that simple answer isn’t true; lots of groups have been looking and haven’t found any evidence. Perhaps they’re only found in a limited number of tissues, or under certain circumstances, but I believe the work has been pretty thorough. Indeed the work has been thorough enough to turn up people infected with spumaviruses, but only because of cross-species infections from apes and so on³ — even though the detection methods are apparently adequate, there’s no evidence for human to human transmission.⁴

Anyway, spumaviruses infect many species, and in all of them appear to be completely harmless. ⁵ Yet in tissue culture, these viruses are deadly, rapidly killing infected cells.⁶ I’ve always assumed that the reason spumaviruses are harmless in an authentic infection is that the host immune system controls the virus, preventing it from replicating so that its host cells don’t get killed by the virus. This turns out not to be the case.

You may be wondering, by the way, why anyone should care why they’re harmless. Why not accept that they are harmless, and concentrate on dangerous pathogens? Apart from being interesting in their own right, I think part of the answer is probably obvious — is there some underlying mechanism that can be applied to a different pathogen, to make it apathogenic?

But another part of the question is viral vectors. There’s intense interest in developing viruses as gene therapy vectors, and one of the obstacles to many of the present candidate vectors — adenoviruses, adeno-associated viruses, lentiviruses, and so on — is that the vector is intrinsically harmful. To use these vectors to treat disease you need to render the virus harmless, first and foremost; and almost ineviatably, this reduces the virus’s ability to infect, which is why you want it in the first place. If you can start with a virus that’s already harmless, then you’re halfway to your goal from the start. There’s increasing interest in developing spumaviruses as gene therapy vectors,⁷ and so we really would like to know why they are harmless.

It turns out, surprisingly (to me, anyway) that spumaviruses are probably just as cytopathic in their natural host, as they are in cultured cells. However, they strictly limit their replication to expendable cells, that are going to die anyway: epithelial cells that are about to be sloughed off and shed anyway.

… FV replication is limited to cells at a very late stage of epithelial cell differentiation, when the cells are about to be shed from the tissue. … The most superficial terminally differentiated cells in the epithelial cell layer turn over in about 3 hours. Thus, the limitation of viral replication to a relatively expendable, superficial cell type could account for the innocuous character of FV infection in vivo. … The contrasting in vitro situation, in which FV infection spreads rapidly and is cytopathic to the entire culture, may be an artifact of culture systems that do not represent the differentiated cell types or cell turnover rates within mucosal epithelial tissues.

My first reaction is that this isn’t very encouraging for spumaviruses as gene therapy vectors, but that’s not necessarily true; the viruses can infect many other tissues, though probably latently, and that may well be perfectly adequate for gene therapy.

I’ve pointed out in the past that pathogens don’t necessarily evolve toward reduced virulence; rather, pathogens evolve toward efficient transmission, which may or may not involve a healthier host. This is a lovely example of efficient transmission coupled with reduced virulence:

This mechanism of viral replication promotes efficient virus transmission via shed cells while limiting viral replication to a superficial site and thus minimizing host tissue damage.

The authors make a comparison to some other virus families that do something quite similar — herpesviruses and papillomaviruses, which as a group often infect one cell type latently but undergo lytic, active replication in differentiated skin cells. They say

… herpesviruses and papillomaviruses, with similar replication patterns, are also ancient viruses. Differentiation-specific viral activation within differentiated cells of the oral mucosa or the skin arose independently in these diverse virus families. … Indeed, in the majority of infections, none of these viruses leads to pathogenic sequelae.

What they mean by “ancient” here, I think, is that they’ve co-evolved with their hosts for a long time, and are well adapted. Herpes and papillomaviruses are more often pathogenic than are spumaviruses, I think, but it’s an interesting suggestion.

1. Murray, S.M., Picker, L.J., Axthelm, M.K., Hudkins, K., Alpers, C.E., Linial, M.L. (2008). Replication in a Superficial Epithelial Cell Niche Explains the Lack of Pathogenicity of Primate Foamy Virus Infections. Journal of Virology, 82(12), 5981-5985. DOI: 10.1128/JVI.00367-08[↩]
2. The so-called human foamy virus that was isolated in the 1970s is actually a chimp virus that was probably acquired through interspecies transmission.[↩]
3. Calattini S, Betsem EB, Froment A, Mauclere P, Tortevoye P, Schmitt C, Njouom R, Saib A, Gessain A (2007) Simian foamy virus transmission from apes to humans, rural Cameroon. Emerg Infect Dis 13:1314-1320.[↩]
4. Boneva RS, Switzer WM, Spira TJ, Bhullar VB, Shanmugam V, Cong ME, Lam L, Heneine W, Folks TM, Chapman LE (2007) Clinical and virological characterization of persistent human infection with simian foamy viruses. AIDS Res Hum Retroviruses 23:1330-1337.[↩]
5. It’s possible that they are associated with very rare disease and because humans aren’t involved there isn’t the intense search for cause that human disease often leads to; but the virus is really very common, infecting at least a third of cats, cattle, and primates in the wild; at that frequency of infection, even rare diseases would probably be picked up in cats or cattle.[↩]
6. Saib A (2003) Non-primate foamy viruses. Curr Top Microbiol Immunol 277:197-211.[↩]
7. For example, Bastone P, Romen F, Liu W, Wirtz R, Koch U, Josephson N, Langbein S, Lochelt M (2007) Construction and characterization of efficient, stable and safe replication-deficient foamy virus vectors. Gene Ther 14:613-620.
   Also, reviewed in Rethwilm A (2007) Foamy virus vectors: an awaited alternative to gammaretro- and lentiviral vectors. Curr Gene Ther 7:261-271.[↩]