Archive for October, 2009

In search of a broad-spectrum anti-viral agent

26 October, 2009

A guest blog from Gillian de Villiers in my lab – thanks, Gillian!

FGI-104: A broad-spectrum small molecule inhibitor of viral infection.
Am J Transl Res 2009; 1:87-98

THE PROBLEM:

Essentially there is a lack of treatment options for viral threats, especially should a new, hitherto unknown virus become prominent.  Vaccines take substantial time to develop, and most anti-viral agents are highly specific to only one virus and in targeting viral proteins place strong selection pressure on the viruses, causing resistance to evolve swiftly.

Since most viruses exploit the same small set of host molecules, and hijack these systems for their own replication, if such molecules could be targeted it could be a very effective approach and is known as “host-directed therapeutics”.  A development stage company called Functional Genetics, Inc. (FGI) is involved in research in this field.

One of the “housekeeping” molecules is TSG101, it usually escorts proteins that are to be degraded and is a positive regulator of the budding process.  After a virus has infected a cell, TSG101 moves newly formed viruses from the cell interior to the outer membrane.  This mechanism is shared by a broad range of virus families and therefore is a potential anti-viral target.

FGI selected a library of small molecule compounds with apparent ability to block TSG101 and checked for ability to block viral replication.  There is no detail on where this library came from, or what kind of molecules they are.  At http://www.faqs.org/patents/asn/32771 a number of patents by FGI are available, but as they generalise the family of compounds, on my brief reading I was unable to determine the chemical structure of the one molecule used in this study: FGI-104.  There is also no reference to any other scientific article in the paper that could give an idea what this compound is, but it would have been good to know.

CONCLUSIONS and COMMENTS

The authors were able to show that their compound FGI-104 has anti-viral activity against a broad range of viruses, although it did not have activity against influenza viruses (data not shown).  They do not make it clear what the actual structure is of compound FGI-104, or how the screened library of compounds was chosen.  They were able to show that the antiviral activity does not come from inhibition of viral nucleic acid replication.  This makes sense as the antiviral compound FGI‑104 was chosen for its action against cell protein TSG101 which moves viruses to the exterior in an infected cell.

They mention some work on viruses for which data is not shown, except for the Table, and more importantly, for which no information is given in the method section.  Their lethal Ebola mouse study is very striking, as it shows extension of life of animals exposed to a mouse Ebola when treated with the compound FGI‑104, where untreated controls died.  In addition when rechallenged the mice survived.  However the number of mice used is not made clear, there was an untreated control group (n unknown), and group of mice daily treated with FGI‑104 for 10 days (n unknown).  The article hints at a further group of mice treated less extensively, but this is almost pure speculation as the article has the same lack of detail as a cookbook from the 1500s.  I feel the methods section is therefore somewhat lacking.

What is significant, looking at the Table, is the variation in dose required for activity against the different viruses.  For example, while for HBV the EC50 (effective half-maximal concentration: concentration is half-way between baseline and maximum) is only 0.02uM, for Ebola it is 8uM.  The different cell lines also have different CC50 (concentration that kills 50% of host cells) levels.  What is also striking is that while, for example, for Ebola the CC50 is only 5X the EC50, making the safety margin quite narrow, it is clear that it is better to be a mouse treated with FGI‑104 (whether toxic at that dose or not) if you are a mouse unfortunate enough to be exposed to Ebola virus.  For most of the viruses however the safety margin was quite large, which is just as well as with further pharmaceutical testing through the various clinical trials, and even after approval, apparently new information tends to show the safety margin to be smaller than first calculated.  I do not know what is considered a “suitable” safety margin for a pharmaceutical.

The field of host-targeted therapeutics appears to be a promising angle in development of anti-virals with a reasonably broad-spectrum of activity.  Another article “Targeting inside-out phosptidylserine as a therapeutic strategy for viral diseases” (Nature Medicine 14:12, Dec 2008) by an unrelated group showed the use of a chimeric antibody against inside out phosphatidyl-serine molecules, a marker of viral infection on host cells, to be effective in controlling viral infection.  Both of these articles were cited in the New Scientist article “How to cure diseases before they have even evolved” (Issue 2720, Aug 2009).  These drugs may one day buy time before a vaccine is available for new virulent viruses, but even if the host molecules are being targeted, I am convinced that viral resistance will only be a matter of time…

Link to full report: Broadspectrum antiviral

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…and so it begins…

21 October, 2009

The northern hemisphere flu season, that is – with the imponderable being just how much of it will be due to the pandemic AH1N1 2009 strain.

And whether or not enough vaccine will be available, soon enough.

News links from today:

US, Mexico face shortage of H1N1 flu vaccine  (LA Times)

CDC: H1N1 vaccine behind schedule (CBS News)

…and how some people are scared of the vaccine, rather than of the flu:

H1N1 vaccine embraced, but also feared American Medical News

Some Parents Undecided about H1N1 Vaccine LocalNews8.com 

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AIDS vaccines in Paris

21 October, 2009

Because he was in Paris attending the AIDS Vaccine 2009 meeting, and because I asked him to, Dorian McIlroy from the University of Nantes has written an account of the presentation of the recent  Thailand HIV vaccine trial results.  Thanks Dorian!

Ed Rybicki.

Here in Paris, the initial results from the Thai ALVAC/AIDSVAX vaccine trial have just been presented. The first presentation was by Dr Supachai Rerks-Ngarm, who was followed by Colonel Nelson Michael (who gave his presentation in uniform). This was a big double-blinded RCT, with more than 16000 participants, about 8000 people in each arm of the study. I am not a methodologist, but this trial does appear to me to have been very well-designed, carried-out, and analyzed. So I think one should unreservedly treat the results as high-quality.

HIVimmunecells150The headline result – a 31% reduction in HIV transmission in vaccine recipients was reported in the press in September, but the difference between the vaccine and placebo recipients was only just statistically significant. So the big question was, are the data convincing enough to reject the null hypothesis? That is, could the difference in the number of HIV infections in the two groups just be down to chance, rather than vaccine efficacy?

Both presenting scientists involved in the study gave talks that were very scientifically rigorous, explaining the why the data was analyzed the way it was, and what conclusions can and cannot be drawn from the trial.

With regards to the first question, it was pointed out that the statistical analysis of the primary endpoint (new HIV infections in the two groups) was decided before the data were unblinded. That is, the statisticians who analyzed the data did not choose their technique to manipulate the interpretation in any way.

The main statistical approach applied was Kaplan-Meier analysis, looking at the number of people infected in each group over time. Differences between vaccine and placebo arms were tested by the log-rank test. However, there were three different ways of determining exactly which of the people enrolled in the trial were included in the analysis. These were intention-to-treat (ITT), modified ITT, and per protocol (PP).

The ITT definition was everyone who was HIV seronegative at study entry, and received at least one injection. The modified ITT excluded 7 individuals who were found to be positive for HIV infection by PCR at study entry. The PP definition was, everyone who received all of the vaccinations at the allotted times. Now this was a rather strict definition, because a person who got a vaccination one day later than the schedule was excluded from the analysis, leaving only about 6000 people per group in the PP analysis.

Kaplan-Meier curves for all three analyses (ITT, mITT and PP) looked pretty good, and showed more infections in the placebo arm than in the vaccine arm, although the difference was only statistically significant (p=0.04) in the mITT analysis. The reason why the ITT analysis did not show a statistically significative difference was because 5 of the 7 people who were infected (PCR postitive, but not seropositive) at entry into the trial were in the vaccine arm. So a net increase of just three more infections (5 in vaccine arm – 2 in the placebo arm) in the vaccine group changed the p-value from 0.04 to 0.08. However, excluding people who were infected before the beginning of the trial is entirely justified, and it is clear that the mITT analysis was preferable to the raw ITT.

The comparison of the mITT and PP results was more interesting. Although the same tendency was observed (more infections in the placebo arm) the Kaplan-Meier curves looked much more similar. There may be two explanations for this. Firstly, since the number of people in each group was decreased, the statistical power of the test also went down – so the same effect would not be statistically significant. Another factor, that was pointed out by Col. Michael, was that the PP analysis automatically ruled out patients who became infected during the vaccine protocol. That is, over the first six months of the trial. Looking back at the Kaplan-Meier curves from the mITT analysis, the main difference between the vaccine and placebo groups accrued during the first year of the trial. Afterwards, new infections occurred pretty much at the same rate in the two groups. Most of these infections were excluded from the PP analysis, resulting in a non-significant difference between the two groups.

This for me, is the key to the interpretation of the trial. In my opinion, there was a protective effect of vaccination in this study (so yes, the data are convincing enough to reject the null hypothesis) – but it seems to have been short lived. Indeed, Col. Michael also mentioned that innate immune responses (presumably induced by the viral ALVAC vector that was injected four times during the 6 months of the vaccination protocol) could be involved in protection. No empty virus vector was used in the placebo arm, (described here : http://www.fda.gov/OHRMS/DOCKETS/AC/04/briefing/4072B2_2.doc) only “a mixture of virus stabilizer, and freeze drying medium”. So more short-lived, non-specific innate immune responses could have been induced in the vaccine arm compared to the placebo arm. This is also consistent with the higher frequency of adverse reactions in vaccine recipients compared to placebo recipients that was also reported in Dr Rerks-Ngarm’s talk.

If the partial protection that was observed in the Thai trial does turn out to have been due to a transient induction of innate immune responses due to the ALVAC vector, then I’m afraid we won’t be able to say that the ALVAC/AIDSVAX candidate vaccine induced an adaptive immune response that is able to protect people from HIV infection.

Dorian McILROY

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HIV vaccines: some glimmer of hope??

19 October, 2009

Cells stimulated by HIV vaccines Copyright Russell Kightley Media

It has taken a while for me to get to this, because I have been waiting for the fallout / comment storm to settle a bit, so that I could get a good clear objective view.

And that is…that the recent Thai trial showed hints of promise, but was largely a failure.  At least it did no harm…!

First things first: Nature News’ Elie Dolgin had this to say on 24th September:

Vaccine protects against HIV virus [!!! sic – I had something to say about this, see Comments]

The largest HIV vaccine trial to date has shown moderate success at preventing infection by the virus.

The experimental vaccine — a combination of two older shots that failed to work on their own — reduced the risk of someone contracting HIV by nearly a third. Scientists, however, are still scratching their heads as to how the double-shot approach blocks the virus….

The US$119 million study involved more than 16,000 HIV-negative men and women from Thailand aged 18–30. The trial was launched in October 2003, conducted by the Thai health ministry and sponsored by the US Army Surgeon General. It tested a two-shot infection-fighting strategy using drugs made by Sanofi-Pasteur of Lyon, France, and VaxGen of Brisbane, Australia. Over the course of 24 weeks, participants received four doses of a ‘primer’ vaccine — a disabled bird virus [canarypox – Ed] containing synthetic versions of three HIV genes [ALVAC, subtype B env, gag and pro – Ed] — and two doses of a ‘booster’, which consisted of a protein called gp120 [AIDSVAX subtypes B/E – Ed], a major component of HIV’s outer coat.  [see here for link describing the components].   Clinicians tested for HIV infection every 6 months for 3 years….

Many HIV vaccine experts had previously criticized the approach as a waste of time because each of the vaccine components had a poor track record. The primer, called ALVAC, conferred little to no immune protection in multiple early-phase clinical trials, and the booster, called AIDSVAX, had flopped twice in high-profile, large-scale trials.

And here’s a thing: a high profile crew of scientists had, in 2004, written an open letter to Science magazine, stating in no uncertain terms that they thought the trial ought to be stopped.  In their words:

“Concerns are expressed by a group of AIDS researchers about the U.S. government’s plans to conduct a phase III trial of a combination HIV-1 vaccine in Thailand despite the cancellation of a trial of a very similar combination vaccine in the U.S.A. last year. One of the vaccine components, recombinant monomeric gp120, has already been shown to be ineffective in phase III trials in Thailand and the United States; the other component, a recombinant canarypox vector, is also poorly immunogenic. The scientific rationale that has been offered for the new trial in Thailand is considered by the authors to be weak.”

And now we have Dan Barouch – not a signatory to the 2004 letter, I note – quoted by Dolgin as saying:

“I don’t think anybody knows why this worked the way it did,” says Dan Barouch, an immunologist at the Beth Israel Deaconess Medical Center in Boston, Massachusetts. “It’s the largest step forward that’s ever occurred in the HIV-vaccine field, but there’s a tremendous amount of more work that will need to be done.”

But exactly what is it that people are hailing as a breakthrough here?  Dolgin again:

The two-pronged vaccine did not affect the amount of virus circulating in the blood of those who acquired HIV during the study. But it did show a protective effect — vaccinated individuals were 31% less likely to become infected. New infections occurred in 74 of the 8,198 people who received dummy shots, but only 51 of the 8,197 in the vaccine group [my emphasis – Ed], the researchers, led by Supachai Rerks-Ngarm of the Thai Ministry of Public Health’s Department of Disease Control, found.

Dorian McIlroy, a regular contributor to Viroblogy, had this to say on the 24th September in an email to me:

I just read the news story about the ALVAC/AIDSVAX trial results in Thailand.  From the numbers on this press release:

http://www3.niaid.nih.gov/news/newsreleases/2009/ThaiVaxStudy.htm

The significance level is extremely slim. For example, if you go to this site

http://www.statpages.org/ctab2x2.html

and type in the numbers you will find that p=0.048 by Fisher’s exact test.

If one more person in the vaccine arm had been infected, or if one less person in the placebo arm had been infected, the difference between the groups would not have been significant. [my emphasis – Ed]

None of the experts (Wayne Koff, Frances Gotch, for example) interviewed in different news stories seems to have noticed just how borderline the “statistical significance” really is, and seem to have accepted the bottom-line 30% reduction figure.

Ah well, I just thought I had to tell someone….

Dorian

Lecturer in Microbiology and Cell Biology,
University of Nantes

Others have also picked up on this – which shows just how desperately slim the hope is.  However, it does remain – although (pleasingly…B-) the pundits have been thrown into a state of confusion, as some strongly-held views have not been vindicated.  Another Nature News article – from Erika Check Hayden, on October 1st – has this to say:

As the dust settles from last week’s surprising announcement that an HIV vaccine combination may protect some people from the virus, scientists are talking about what else the vaccine trial might tell them.

On 24 September, leaders of a US$119-million study of 16,000 people in Thailand reported that the combination of two shots had reduced the risk of HIV infection by one-third …. Now, the vaccine’s fate will depend on whether scientists can figure out its ‘correlate of protection’ — in other words, what caused it to partially protect some people from HIV. The key does not seem to be anything scientists had predicted, which has led to much head-scratching — and some unease.

“It’s a humbling thing, because for the first time we got a positive signal and it doesn’t jump out at us as being related to any classical parameters you would expect from a successful vaccine,” says Anthony Fauci, head of the National Institute of Allergy and Infectious Diseases in Bethesda, Maryland, which supported the trial. “That tells us maybe we were not measuring the right thing.” [my emphasis – Ed]

Amen, brother Tony…a clearer proof of Clarke’s First Law I have yet to see.

So what ARE the things that fall out from this?  First, I would suspect, is that the value of a heterologous prime-boost combination seems to have been shown, albeit weakly.  Second, the use of a poxvirus vaccine in particular in combination with a protein may be a good thing to chase.  I note here that the South Africa / US joint Phase I human trial currently underway with the SAAVI DNA / SAAVI MVA (=modified vaccinia virus Ankara, a poxvirus) was almost certainly considerably more immunogenic in non-humanprimates than either of the ALVAC / AIDSVAX vaccines, so the gleam of hope may soon get brighter.

Third: take heed of Arthur C Clarke before you go sticking your neck out making predictions about HIV vaccines…B-)

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Phylogeography of HCV: slave trade spread the virus

19 October, 2009

Hepatitis C virus particles. Copyright Russell Kightley Media

Today a welcome guest blog by a PhD student in the lab, Aderito Monjane: this paper was presented by him in a recent lab journal club, and I thought it was interesting enough to get a wider airing.

Phylogeography and molecular epidemiology of hepatitis C virus genotype 2 in Africa

Peter V. Markov, Jacques Pepin, Eric Frost, Sylvie Deslandes, Annie-Claude Labbe´ and Oliver G. Pybus

Journal of General Virology (2009), 90, 2086–2096

Hepatitis C virus (HCV) is an important human pathogen. There are 170 million chronically infected people worldwide, and 2-4 million new cases of infection annually. The disease manifests itself late – liver cirrhosis and hepatocellular carcinoma – and in the USA alone 9000 people die of it each year.

HCV is quite diverse. Six genotypes have been identified, and each further classified into subtypes. Some of these subtypes are geographically localized and others are globally distributed. Endemic subtypes are found in the tropics (e.g. genotype 2 and 1 are found in west Africa; genotype 4 in central Africa and the middle East), whereas ‘epidemic’ subtypes are more widely distributed.

The case for the spread, genetic diversity and origin of HCV genotype 2 is very interesting. Phylogenetic studies using sequences sampled from individuals in a) west Africa (around Gambia, Senegal), b) and slightly more to the east of these countries (around Ghana, Benin), and c) central Africa (around Cameroon and Central African Republic) revealed interesting facts.

  • West Africa is the origin of HCV genotype 2 and this region has the greatest amount of viral diversity. This genetic diversity decreases as one moves further to central Africa
  • Sequences from west Africa are found in regions outside of west Africa, e.g. in central Africa, Madagascar and the Caribbean island Martinique, thus reaffirming that west Africa is the origin of HCV genotype 2
  • The proportion of HCV genotype 2 relative to other genotypes decreases from west to central Africa. This reaffirms that there is movement of HCV genotype 2 from west to east.

Phylogenetic and molecular clock trees showed that the oldest common ancestor to the HCV genotype 2 isolates in existence worldwide came into being in the year 1091 (actually, there is 95% confidence that it was between year 709-1228), and in 1470 the first HCV genotype 2 strains afflicting individuals in the African continent came into being.

The connection between these existing HCV genotype 2 strain, the transatlantic slave trade, and the use of mass vaccination or treatment of illnesses is interesting in that it shows the inadvertent spread of viruses globally by human activities.

Ghana was the major port for slave trade. So it is perhaps of no coincidence that HCV genotype 2 strains found in the Caribbean island Martinique (as well as most of its human population) resemble the strains found currently in the Ghana-Benin region. Movement of African troops under French colonial rule from Senegal and Mauritius during WWI has also resulted in the global spread of current epidemic HCV-2 strains. An insidious effect of mass-treatment campaigns is exemplified in the different ways HCV genotype 2 spread in Cameroon and Guinea-Bissau. In Cameroon, under French colonial rule, doctors treated European colonialists and African natives against illnesses such as syphilis and yaws using intravenous drugs, before there was any awareness of blood-borne viral transmissions. As a result, by the 60’s HCV cases were higher in Cameroon compared to Guinea-Bisau, where the Portuguese colonialists used intravenous drugs to treat the European colonialists and their immediate workers only.

In summary, this study shows that there is west to east movement of HCV genotype 2, and decreasing genetic diversity away from the origin of diversity.

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