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Integrating the enemy

23 November, 2010

Ever since I first discovered them as a student, sometime in 1976, I have found retroviruses fascinating.  Not quite as fascinating as Ebola, possibly, but captivating nonetheless.  The whole concept of a virus that converts a perfectly ordinary mRNA into dsDNA, then  inserts it into the host chromosome as a provirus in a eukaryotic version of lysogeny – was truly wonderful.

And as the years have gone by, I have seen no reason to lessen the feeling of wonderment: other

The Retroid Virus Replication Cycle

viruses – now called pararetroviruses, including both hepadnaviruses and plant viruses – whose replication  starts at a different position in the  cycle have been found; these and retroviruses have been integrated into a whole family of “reverse transcribing elements” – retrons – which include prokaryote transposons; HIV burst in on the scene, and suddenly we know so much about how the immune system works, because a virus messes with it so well.

But the actual mechanics of one particular process have consistently escaped elucidation – until now.  The 11 November issue of Nature contains, apart from only the second SF short-short story by a South African (kudos, Anand!), a Letter of great interest.

The mechanism of retroviral integration from X-ray structures of its key intermediates
Goedele N. Maertens, Stephen Hare & Peter Cherepanov
Nature 468,326–329 (11 November 2010) doi:10.1038/nature09517

To establish productive infection, a retrovirus must insert a DNA replica of its genome into host cell chromosomal DNA. This process is operated by the intasome, a nucleoprotein complex composed of an integrase tetramer (IN) assembled on the viral DNA ends. The intasome engages chromosomal DNA within a target capture complex to carry out strand transfer, irreversibly joining the viral and cellular DNA molecules. Although several intasome/transpososome structures from the DDE(D) recombinase superfamily have been reported, the mechanics of target DNA capture and strand transfer by these enzymes remained unclear. Here we report crystal structures of the intasome from prototype foamy virus in complex with target DNA, elucidating the pre-integration target DNA capture and post-catalytic strand transfer intermediates of the retroviral integration process. [my emphasis – Ed] The cleft between IN dimers within the intasome accommodates chromosomal DNA in a severely bent conformation, allowing widely spaced IN active sites to access the scissile phosphodiester bonds. Our results resolve the structural basis for retroviral DNA integration and provide a framework for the design of INs with altered target sequences.

Basically, these folk have managed to freeze-frame several different stages of the process in crystals, by clever use of synthetic DNA targets – and then solved the structures.  NOT trivial, and the pictures are absolutely superb.  So are the movies…but you need to subscribe to Nature to see those.

Harking back to a previous post – Entrance, Entertainment and Exit, anyone? –  the more we know about viruses, the more we can mess with them.  And this is a VERY good step along that road.

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Posted in General Virology, HIV | 1 Comment »

Sendai don’t do it like they said

13 October, 2010

It is a sad fact of virological life that quite a lot of what we see, in the experiments we do, is artefactual: that is, the way we do experiments leads us to see results that do not necessarily reflect reality, but rather, the scenario we inadvertently selected for.

And it is electron microscopy that is at once our friend and our foe in this regard: over the last thirty years I have revised several aspects of my teaching on how virus particles interact with cells in particular, as what was once considered common knowledge has subsequently been proved to be false.  This is usually a consequence of having to use large numbers of virus particles – or high multiplicities of infection – and cultured cells, which may lead to rare events being selected for simply because they may be easier to detect.  An important example of this was the revelation that poliovirus (and presumably other picornaviruses) almost certainly enters cells via receptor-mediated endocytosis, rather than via some mysterious direct passage mechanism as is often depicted in textbooks (or here).

 


Paramyxovirus: image by Linda M Stannard

 

One of the long-time models for entry of enveloped viruses into animals has been Sendai paramyxovirus: this ss(-)RNA virus was supposed to fuse its membrane with that of the host cell, and uncoat via diffusion of its envelope glycoproteins into the host membrane, and deposit of virion internal components into the host cell cytoplasm.

Except, it turns out, that this is probably wrong: in a Journal of Virology Minireview published in July of 2010, Anne Haywood of the University of Rochester (NY, USA) describes how Sendai virions uncoat via a “connecting structure” that largely preserves the virion envelope.

Membrane Uncoating of Intact Enveloped Viruses
Anne M. Haywood
JOURNAL OF VIROLOGY, Vol. 84, No. 21, Nov. 2010, p. 10946–10955
Experiments in the 1960s showed that Sendai virus, a paramyxovirus, fused its membrane with the host plasma membrane. After membrane fusion, the virus spontaneously “uncoated” with diffusion of the viral membrane proteins into the host plasma membrane and a merging of the host and viral membranes. This led to deposit of the viral ribonucleoprotein (RNP) and interior proteins in the cell cytoplasm. Later work showed that the common procedure then used to grow Sendai virus produced damaged, pleomorphic virions. Virions, which were grown under conditions that were not damaging, made a connecting structure between virus and cell at the region where the fusion occurred. The virus did not release its membrane proteins into the host membrane. The viral RNP was seen in the connecting structure in some cases. Uncoating of intact Sendai virus proceeds differently from uncoating described by the current standard model developed long ago with damaged virus. A model of intact paramyxovirus uncoating is presented and compared to what is known about the uncoating of other viruses.

Interesting: a whole model for entry of viruses into cells was predicated upon the interactions of a  laboratory-derived virus strain which produced damaged particles.

Haywood presents a new model for virus entry, based upon the observation that “early harvest” virions differ substantially form the “late harvest virions” previously used, in that “…the RNP is regularly folded parallel to the long axis of the virions…”, while  late-harvest particles “…have RNP strands that are randomly distributed in the virus rather than regularly arranged in relation to the membrane”.

She goes on to review a qualitatively very different alphavirus – Sindbis virus, an enveloped ss(+)RNA virus – for which similar things had been claimed, and shows that virus particles that have been gently treated also make a connector.  Moreover, she says that:

“…there is a structure that has no electron-dense material and is released from the cell. It is identified as viral by antibodies conjugated with gold beads. This release of an empty viral membrane has not been noted before, but the use of labeled antibodies meant such a structure would be revealed. If the envelope membrane disengages from the cell instead of merging with the host membrane, then not only would the cell not have viral proteins on its surface until the virus replicates but the released membrane pieces could serve as immunologic decoys.” [my emphasis]

Interestinger and interestinger…so enveloped viruses may have an entry mechanism which serves to hide them more effectively than we knew – by keeping their membranes intact, and possibly even using them as releasable decoys?

I note that in the case of HIV – possibly the best-studied single organism on the planet just recently – it has also recently been shown that virions probably enter cells via endosomal vesicles.

I hear the grinding sound of a shifting paradigm, folks: time for a relook at some other cherished models, possibly??

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Vaginal gel works against HIV

20 July, 2010

In a major press release at the International Aids Conference in Vienna today, published simultaneously in Science, South African researchers claimed a significant advance in prevention of HIV infection using a microbicide.

From the Commentary in Science Express by Jon Cohen:

HIV and its life cycle

For the first time ever, a vaginal gel has unequivocally blocked the transmission of HIV.
In a trial that involved nearly 900 South African women, those who received a vaginal gel that contains an anti-HIV drug had a 39% lower chance of becoming infected by the virus than those who received a placebo. …
More than 30 randomized controlled studies of microbicides, vaccines, and drugs to date have failed to thwart sexual transmission of HIV or have yielded such marginal success that researchers wound up hotly debating the data for years after the trials were complete. But there’s no ambiguity about the data from this new microbicide study reported today online in Science and in a presentation at the 18th International AIDS Conference in Vienna: Of the 444 women who received a placebo gel, 60 became infected with HIV versus 38 infections in the 445 women who received the microbicide. The result was statistically significant, and no serious side effects occurred.  “It’s a moment we’ve been waiting for 2 decades,” says epidemiologist Quarraisha Abdool Karim, who, with her husband, Salim Abdool Karim, headed the study, known as CAPRISA 004.

Published online 19 July 2009; 10.1126/science.329.5990.374

This truly is good news – both for the HIV/AIDS research and treatment community, who have needed a shot in the arm recently, and for women in developing countries who often have little choice in how or when they have sex.

From the paper in Science:

Effectiveness and Safety of Tenofovir Gel, an Antiretroviral Microbicide, for the Prevention of HIV Infection in Women

Quarraisha Abdool Karim et al., Published Online July 19, 2010
Science DOI: 10.1126/science.1193748

The CAPRISA 004 trial assessed effectiveness and safety of a 1% vaginal gel formulation of tenofovir, a nucleotide reverse transcriptase inhibitor, for the prevention of HIV acquisition in women. A double-blind, randomized controlled trial was conducted comparing tenofovir gel (n = 445) with placebo gel (n = 444) in sexually active, HIV-uninfected 18- to 40-year-old women in urban and rural KwaZulu-Natal, South Africa. HIV serostatus, safety, sexual behavior, and gel and condom use were assessed at monthly follow-up visits for 30 months. HIV incidence in the tenofovir gel arm was 5.6 per 100 women-years, i.e., person time of study observation (38/680.6 women-years), compared to 9.1 per 100 women-years (60/660.7 women-years) in the placebo gel arm (incidence rate ratio = 0.61; P = 0.017). In high adherers (gel adherence >80%), HIV incidence was 54% lower (P = 0.025) in the tenofovir gel arm. In intermediate adherers (gel adherence 50 to 80%) and low adherers (gel adherence <50%), the HIV incidence reduction was 38% and 28%, respectively. Tenofovir gel reduced HIV acquisition by an estimated 39% overall and by 54% in women with high gel adherence. No increase in the overall adverse event rates was observed. There were no changes in viral load and no tenofovir resistance in HIV seroconverters. Tenofovir gel could potentially fill an important HIV prevention gap, especially for women unable to successfully negotiate mutual monogamy or condom use.

Note my bolding above: while the results were encouraging overall, they were especially good where adherence to the protocol was high.  Moreover, it appears as though tenofovir administered externally does not get into the blood in sufficient amounts to cause infecting virus to develop resistance to any noticeable degree.  As an added bonus, the treatment appeared to reduce the incidence of herpevirus as well.

To paraphrase someone far more famous, this is only the end of the beginning of the fight against HIV and AIDS: this is not the answer; it is merely an indication that this is a strategy that may work – in the absence of a vaccine – to protect people from infection.

But so nice that it came from South Africa….

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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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Posted in General Virology, HIV, Vaccines: General | 2 Comments »

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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First African-developed HIV vaccine goes to trial

21 July, 2009

Finally, finally, a product of our 10-odd-year-old South African HIV vaccine development programme goes into Phase I human trial, in South Africa!

I say “our” because I was part of the overall team; however, the two vaccines which comprise the SA AIDS Vaccine Initiative (SAAVI) / HIV Vaccine Trials Network (HVTN) trial – designated SAAVI 102/HVTN 073 – were designed and developed by others.

The vaccines consist of a DNA component, consisting of an artificial “polygene” dubbed Grttn (for Gag-RT-Tat-Nef) and a truncated Env (gp150) cloned separately into vector plasmids, and the same genes recombined under the control of different promoters into the genome of a poxvirus (Modified Vaccinia Ankara, MVA).  These and their testing in mice and non-human primates have been described in published work: see here and here for relevant journal articles on the MVA and DNA components.

From the July 20th SAAVI press release:

The test vaccines – called SAAVI MVA-C and SAAVI DNA-C2 – have shown promising results in animal testing. The SAAVI DNA-C2 vaccine was constructed in South Africa using a plasmid backbone provided by the Dale and Betty Bumpers Vaccine Research Center (VRC) of NIAID, while the MVA vaccine was designed by the team at UCT and constructed and manufactured in the USA.

“Reaching this important milestone of translating our discoveries in the laboratory to testing in humans would not have been possible without the support of a large team of people from the University of Cape Town, together with national and international collaborations.  An effective vaccine against HIV/AIDS remains a top global health priority and it is our hope that the evaluation of these vaccines in clinical trial will provide some important answers that will bring us closer towards this goal,” says Prof. Anna-Lise Williamson, leader of the vaccine development team and joint staff member of UCT’s Institute for Infectious Disease and Molecular Medicine, and the National Health Laboratory Services (NHLS).

The SAAVI DNA-C2 was constructed in South Africa and manufactured in the US by Althea Technologies. The MVA vaccine was manufactured by Therion Biologics, USA. The vaccines will be tested in a prime-boost approach where the SAAVI DNA-C2 vaccine will be given to prime the immune response and the SAAVI MVA-C vaccine to boost or enhance the immune response.

National and international press got hold of the story in a big way – unsurprisingly, given as there is the 5th IAS Conference on AIDS Pathogenesis going in in Cape Town at the same time, which incidentally has its own live blog feed.

The University of Cape Town is obviously pleased with the press release (see here); however, the launch had its fair share of controversy: Associated Press reporter Michelle Faul posted a story yesterday entitled “South Africa begins AIDS vaccine trial, cuts funds“, which has been taken up by a wide spectrum of especially foreign media.  According to Faul:

“South Africa launched a high-profile trial of an AIDS vaccine created by its own researchers Monday, a proud moment in a nation where government denial, neglect and unscientific responses have helped fuel the world’s worst AIDS crisis.

After a government official lauded the project at a ceremony at Cape Town’s Crossroads shantytown, the scientist leading the research said state funding had been halted.

The contrast between Monday’s hopeful vaccine launch and the revelation of funding cuts raised questions about whether the government was backsliding on its pledge to combat AIDS.

Anna-Lise Williamson, an AIDS researcher at the University of Cape Town, told The Associated Press the clinical trial would continue with U.S. money. But she said South Africa’s Department of Science and Technology had pulled its funding in March, while the project’s other sponsor, the state electricity utility Eskom, did not renew its contract when it expired last year.

Neither government spokesmen nor Eskom immediately returned calls seeking comment about funding cuts.”

In the midst of light, there is darkness…frequently, thanks to ESKOM! 

I have blogged on my personal view elsewhere; suffice it to say that bad decisions were made, and  9 years worth of momentum has effectively been lost – along with a number of very experienced personnel, and many years worth of accumulated and very relevant experience.

For an illustration of the product pipeline which existed behind the current trial offerings – and which may now never be developed – click here  for published descriptions of our plasmid- and BCG-vectored and virus-like particle (VLP) subunit vaccines.

But who knows, this current trial may even show promise – and then it will all have been worth it.  Let’s live in hope!

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Posted in HIV, Vaccines: General, Viruses | 3 Comments »

Guest Blog: HIV Vaccines

18 March, 2009

You remember we had a competition, end of last year?  Well, the runner-up of same – Dorian McIlroy – has claimed his prize by writing a guest blog.  Clayton, that means you have to do two…?!

And Dorian writes on a subject close to our collective hearts, here in the Subunit Vaccine Group at Univ of CT: HIV vaccines, and how T-cell vaccines in particular may not be down and out after all.  I note that he refers to two published papers that were topics of talks at the recent AIDS Vaccine Conference here at UCT recently, which was covered here in ViroBlogy: always useful to have your material on the verge of being published when you talk…!

HIV vaccine candidates – The return of the recombinants…

Towards the end of 2007 Merck interrupted a large scale clinical trial of a candidate HIV vaccine based on recombinant adenovirus. As well as showing no protective effect, there was a worrying tendency for vaccinees with serum antibodies to the vector – serotype 5 adenovirus (Ad5) – to show greater susceptibility to HIV infection than volunteers receiving placebo injection. This looked like the end of the road for adenovirus-based vaccines, and maybe even for any strategy based on recombinant viruses. However, two recent papers indicate that there may yet be considerable mileage in this approach.

The first, from Dan Barouch’s team in Boston, shows how recombinant adenoviruses could be made considerably more effective. One of the big drawbacks with these recombinant viruses is that repeated injections do not give an immunological booster effect. That is, even though the immune response to a single injection can be strong, there is not much increase after a second, or a third injection of the same recombinant virus. This happens because as well as inducing an immune response to the target antigen (SIV gag, for example), the recombinant virus vector induces an immune response against itself. So when the recombinant virus is injected a second time, it is neutralized by the antibodies induced by the first injection. 

The Barouch paper shows that it is possible to get round this problem by using two recombinant adenoviruses with different serotypes, so that the second injection can provide an effective boost. Giving macaques two injections of SIV gag-recombinant Ad5 gave a good response to the first injection, but no boost. Using an SIV-gag recombinant Ad26 for the first injection, then using the Ad5 recombinant for the second, gave a 9-fold higher T-cell response measured by IFNgELISPOT than two doses of Ad5 recombinant.

So far, so good – but does this immune response translate to protection from infection?

Well, yes and no. After intravenous challenge with highly pathogenic SIVmac251, all animals in all groups were infected, so none of the animals were completely resistant to SIV. Peak viral load in the group vaccinated with Ad26/Ad5 was 1.4 log lower than in the control group, and about a log lower in the group vaccinated with Ad5/Ad5. Set-point viral load was also much lower in the Ad26/Ad5 group compared to the control group, so replication of the challenge virus was controlled to some extent in the animals that received the most effective vaccination protocol.

Overall there are two messages here. The first is that it is possible to do much better than the strategy that failed in the Merck trial, that employed three injections of Ad5 recombinant viruses. That’s the good news. The bad news is that even with a much more effective vaccination, in terms of the T-cell response, the protection against infection, although significant, was relatively modest. This means that vaccine candidates based on T-cell immunity alone are never going to work……. or does it?

Which brings me to the second paper, from Louis Picker’s group in Oregon. The big novelty here is the generation of recombinant rhesus macaque cytomegalovirus (CMV) expressing SIV proteins, that are tested as vaccine candidates in the SIV Mac model.

Why on earth would anyone want to try out CMV, when other recombinant (adeno- and pox-) viruses have been so disappointing? The difference is in the lifestyles of the viruses involved. Both adeno and poxviruses provoke acute infections, whereas CMV both in humans and apparently, in macaques, causes a lifelong infection, in which active viral replication is held in check largely by a robust T-cell response. When the immune response is compromised, as in AIDS patients, or transplant recipients receiving immunosuppressive drugs, CMV infection often reactivates, which can cause serious illness, and even be life-threatening. Because of the persistent nature of both the virus and the cellular immune response, a high-level of effector-memory T-cells (TEM, not to be confused with Transmission Electron Microscopy) specific for CMV are maintained in CMV+ individuals (who probably make up about half of the world population, in case you were wondering).

TEM have two interesting characteristics from a HIV vaccine point of view. Firstly, they are armed and dangerous. If they see their cognate viral antigen, they can either kill the infected cell, or secrete cytokines. Secondly, they migrate to mucosal sites, rather than lymph nodes, so they are in the right place to stop HIV infection after sexual transmission. More sedate central-memory T-cells (TCM), on the other hand, hang around the blood and lymph nodes, and do not immediately have anti-viral effector functions. Their response to HIV infection might be too late to be any use, as the first rounds of productive viral infection occur in the mucosa, not the draining lymph nodes.

Picker’s group points out that most memory T-cells that remain months after a recombinant adenovirus vaccination are TCM, not TEM, and set out to test the hypothesis that using a recombinant virus (CMV) that naturally gives a strong TEM response might be more effective in protecting against SIV infection at a mucosal site. So they generated recombinant, replication competent macaque CMV carrying genes for SIV gag, a Rev-Tat-Nef fusion protein, and an intracellular form of Env. Cells infected with these viruses expressed high levels of the recombinant proteins, and animals inoculated subcutaneously with them became persistently infected – just like wild-type CMV infection.  As predicted, high levels of SIV-specific TEM were found in the blood, and in broncho-alveolar lavage (which is a relatively convenient way to obtain mucosal T-cells). More than one year after vaccination, animals were submitted to a mucosal challenge (intrarectal, if you really want to know) with SIVmac239, which is the same strain as that used to produce the recombinant CMV.

The details of the viral challenge are interesting. In order to simulate a real infection, a low viral dose was used, and this means that not all control animals get infected. So the challenge was repeated weekly until infection (detected by plasma viral load) was observed. I have turned the data from the paper into Kaplan-Meier curves, and used the log-rank test to compare the two groups (BTW: if Louis Picker is reading this, that’s the test you should have used). With p=0.03, survival without infection was significantly prolonged by vaccination, and 4 out of 12 vaccinated macaques were resistant to mucosal challenge. These four animals did not have a latent or cryptic infection, as no SIV DNA or RNA was detectable in CD4 T-cells in blood or lymph nodes, and CD8+ T-cell depletion did not result in viral rebound.

recombinantviruses_032009_html_48846d22This was without any neutralizing antibodies, so for the first time a T-cell vaccination strategy has been shown to confer protection against infection (not just better control of viral load after infection, as with recombinant adenoviruses) in at least some vaccinated animals. Although 33% protection is not enough, at least things are going in the right direction.

So what’s the catch? Well, there are two really. Firstly the SIV sequences in the vaccine and challenge virus were identical. The vaccination strategy may not be so effective against a heterologous challenge, that would be more representative of the real-world. Secondly, the vaccination provokes a persistent infection with a genetically-modified virus, that (unlike other recombinant viruses and gene therapy vectors) remains infectious, so it’s hard to see how this kind of vaccine could be licensed for clinical trials.

Nevertheless, I think this paper is telling us – at last – what kind of T-cell response vaccines should be aiming to induce. Now all we need to do is solve the neutralizing antibody problem, and we’ll really be cooking with charcoal….

Dorian McIlroy
Dorian.Mcilroy@univ-nantes.fr

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AIDS Vaccine 2008: Cape Town

26 November, 2008
I was a presenter and rapporteur at this, one of the biggest of this series I have been to – over 900 delegates – held in the Cape Town International Convention Centre (CTICC) in October 2008.  This represents the first time that the organisers, the Global HIV Vaccine Enterprise, have held one of these annual conferences outside of North America or Europe – and certainly the first time in such a high HIV prevalence area (~11%).
The Abstract Book of the conference is now online, courtesy of the journal AIDS & Human Retroviruses, which prompts my publishing the written version of my rapporteur’s report, on Vaccine Concepts and Design.  The oral version had a lot more pictures and Star Wars references in it, but this one is more serious.  A more condensed version will also appear as part of a combined rapporteur’s report in the journal Human Vaccines sometime soon.

 

 

AIDS Vaccine 2008, Cape Town – Vaccine Concepts and Design

Ed Rybicki, Institute of Infectious Disease and Molecular Medicine, University of Cape Town

The fallout cloud from the failed STEP and Phambili clinical trials of the Merck adenovirus 5-vectored vaccines cast a long shadow over the conference, and especially over the area of vaccine concepts and design.  Inevitably, there was debate over whether or not T-cell response-based vaccines should ever be tested on a large scale again; and there appeared to be an intense and renewed interest in broadly-neutralising antibodies, and how to elicit them.  It was understandable, then, that those of whose stock in trade is T-cell vaccines were a little apprehensive going into this meeting: however, there was much to excite and much to enthuse, and in particular, several lines of evidence suggesting that T-cell vaccines are not dead and should still be vigorously pursued.

The conference opening was memorable for a number of reasons: among these was the Sizophila Choir of HIV+ folk from Cape Town, who moved many to tears with their amazing harmonies and hymns to ARVs.  Another, very important reason was the presence of South Africa’s new Minister of Health, Barbara Hogan: for the first time in years in a major forum, a senior member of the SA Government affirmed that HIV causes AIDS, and that the search for a vaccine was of paramount importance to SA and the rest of the world.

Arthur C Clarke’s Third Law states that “When an elderly and distinguished scientist says something is possible, he is almost certainly correct”: it was a pleasure, therefore, to hear the certainly distinguished Stanley Plotkin (Sanofi Pasteur / Univ Penn., PA) close the evening with a calm and reasoned explanation of why he thinks vaccines against HIV are possible.  He noted that HIV is not the only vaccine to see major difficulties in its development – and cited measles and CMV as object examples.  He suggested that multivalent vaccine(s) and regular boosters may be necessary; that the immune response needs neutralising Ab and CD4+ and CD8+ cells, in blood and mucosa – and pointed out that these are feasible to produce for other vaccines, so why not for HIV?

The most important Keynote/Plenary talks from the point of view of T-cell vaccines were those by Julie McElrath (Fred Hutchinson Cancer Res Inst, Seattle, WA; Plenary Session 1) on immune responses in the STEP trial; Tony Fauci (NIAID/NIH; Special Keynote) on future strategy, and Bruce Walker (Mass Gen / Harvard U; Plenary 2) on correlates of protective T-cell immunity.  Julie McElrath’s analysis of the STEP data was sobering, and potentially depressing, but there was a positive message: she said that T-cell epitope recognition as a result of the Merck Ad5 vaccine was inadequate, so we needed to use different strategy – such as a protein vaccine, which should almost certainly be adjuvanted for increased immunogenicity.  Tony Fauci summed up current strategic thinking very well, with his analogy of a radio dial, with Discovery and Development at opposite sides: he said that the failure of all large-scale vaccine trials to date meant we should turn the dial back to Discovery, with more focus on innate immunity, animal models and adjuvants, before any more large-scale trials were done.  Bruce Walker’s message, after an exhaustive analysis of “elite controllers”, was that these people have weaker CD8+ T-cell responses to HIV antigens – but they are significantly more Gag-focussed, and that stronger Env responses are correlated with increased risk of progression to AIDS.  His most important comment was that the Merck vaccine / STEP trial result was a failure of product, not of the concept, and that we are not barking the wrong tree with T-cell vaccines.

A novel introduction at the Conference was Special Session 02, Innovations in AIDS Vaccine Discovery: this was chaired by Wayne Koff (IAVI, NY), and had the objective of highlighting novel strategies for vaccine development.  K Reed Clark (Nationwide Children’s Hospital, Columbus, OH) presented a case for “reverse immunisation”, or using a DNA construct to express a humanised neutralising mAb: he used rAAV1 DNA to express scFv-h-C2-C3 IgG2 constructs for sustained delivery of neutralising Ab in macaques.  Sterilising immunity was achieved following NAb gene transfer in the face of a pathogenic SIV challenge, and he achieved sustained (1 yr) circulating levels of 200-400 ug/ml.  As a possible downside, there was an idiotypic anti-NAb response in animals which became  infected.  Sanjay Phogat (IAVI, NY) spoke on the use of immune complexes as vaccines: he used neutralising and non-neutralising MAb complexed to gp120 with an adjuvant (AdjuplexLAP) to generate quick and durable neutralising antibody responses against the Env protein, with immune sera neutralising 6 out of the 10 clade B viruses tested – far better and at much higher titre than adjuvanted gp120 alone.  Clayton Beard (Carolina Vaccine Inst, UNC, NC) had as his goal the use of a chimaeric live alphavirus (VEE) to create a simple self-replicating entity that presents the major antigens of HIV in vivo until an appropriate immune response suppresses its growth, leaving the recipient immune to HIV.  His almost complete redesign of VEE resulted in a virus expressing SIV/HIV Env and a SIV Gag modified to bind the VEE genomic encapsidation  signal, which replicates to titres of ~106/5 ml culture in Ghost cells.  All in all, this session was a welcome addition to the programme, and very well received.

Session OA02 – T-Cell Vaccines and Animal Models – contained several interesting approaches to T-cell vaccines.  Brad Jones (Univ Toronto) opened with a description of how T-cells specific for LINE-1 (long interpersed nuclear element) retrotransposon proteins were effective at eliminating HIV-1 and HIV-2-infected cells: apparently APOBEC-3 family proteins inhibit LINE-1 transposition, and HIV Vif interference with APOBEC allows aberrant LINE-1 expression in HIV-infected cells, which leads to MHC presentation of the LINE-1 proteome, and CTL killing of the affected cells.  A LINE-1-specific T-cell clone recognised, and killed within 2 hours, cells infected with 42 HIV isolates (37 of them primary isolates) from all subtypes, and HIV-2 isolates.  He argued that LINE-1 proteins represented a novel, stable vaccine target as they lacked variability, and speculated that anti-LINE-1 responses could be a part of natural control of HIV, as their T-cell clone was derived from an elite controller.  David Garber (Emory Univ, GA) spoke on the optimisation of modified vaccinia virus Ankara (MVA) to reduce expression of irrelevant antigenic targets: his group had essentially reduced the vector to immediate-early expression only outside of cells used for propagation, as well as lessening its immune evasion capacity by targetted deletions.  Modified vectors with gag and env genes performed 3-5 fold better than MVA in macaques, and it was possible to tune responses for better CD8+ or multifunctional responses.  Tomáš Hanke (Univ Oxford) presented a “universal T-cell vaccine”, HIVconsv: this was a DNA vaccine encoding a spectrum of T-cell epitopes separated by junction regions, derived from the HIV-1 proteome, concentrating on Gag and Pol, with some Env and Vif epitopes.  The vaccine potentially had 270 of a documented 1100 possible HIV-1 CD8+ T-cell epitopes.  T-cells from HIV-infected subjects were stimulated by vaccine epitopes: 11 of 12 subjects reacted to 2 or more peptide pools (covering, indicating good coverage.  Macaque immunisation resulted in a strong, broad response as assessed by ELISpot assay.  His hope was that the vaccine would redirect responses compared to natural infection, so as to negate immunodominance of one or a few epitopes.

Symposium 03 – Next Generation Vaccine Vectors – was a highlight of the Conference, with a number of excellent presentations.  Dan Barouch (Beth Israel Deaconess Med Ctre, Harvard) gave a tour de force talk on what amounted to a rerun of the Merck Ad5 vaccine efficacy trial in macaques, with a gag-only heterologous Ad26/Ad5 or Ad35/Ad5 vaccination regime.  The Ad26/Ad5 combination was best, 2x the Ad35/Ad5 response, which was 2x the Ad5/Ad5 response.  The Ad26/Ad5 regime gave long-term (500 day) durable partial protection against challenge, with a 3x greater breadth of epitope responses than to Ad5/Ad5.  The 26/5 regime elicited a good memory Gag-specific response, and similar to what Bruce Walker had said for elite human controllers, there was a significant correlation of the height and breadth of the Gag–specific response, and reduction of viral load.  Dan repeated Walker’s earlier comment, with some significant evidential weight to his iteration: the STEP trial was a failure of product, not of concept.

Louis Picker (Oregon Health & Science Univ, OR) discussed how a kinetic mismatch between replication and development of T-cell clones at the site of infection could result in infection taking hold – and further, that live attenuated SIV vaccines elicited mainly effector memory (EM) cells, whereas prime-boost vaccine regimes elicited mainly central memory (CM) cells.  His group used rhesus CMV – known to elicit mainly EM-dominated responses, and which can infect and reinfect monkeys, which remain infected lifelong – to vector SIV rev, nef, tat and gag genes into macaques.  In contrast to the CM response of Ad5-vectored genes, these elicited EM responses, enriched in bowel and lung and other mucosa.  Very weak Ab responses with no NAb were seen.  Protection against infection was seen in macaques challenged by repeat low-dose intrarectal SIV, with control groups infected at a median of two doses, and vaccinees taking 8: replication of virus was eliminated or controlled very early in infection, apparently by a CD8+ T-cell independent mechanism.  His message was that the CMV vaccine and the EM cell response drastically cut down transmission.

Anna-Lise Williamson (IIDMM, Univ Cape Town) closed out the session with an account of the vaccine development efforts in Cape Town under the auspices of the SA AIDS Vaccine Initiative (SAAVI).  Her group has brought a DNA and an MVA-vectored heterologous prime-boost multigene HIV vaccine combination to the point of human trial after successful broad-spectrum immunogenicity trials in baboons; however, they are also developing M bovis Bacillus Calmette-Guerin (BCG) auxotrophs and the limited host range Lumpy skin disease capripoxvirus (see also P16-02) as vectors, with very promising baboon and macaque immunogenicity results with HIV genes.  Additionally, the group has gone a long way in developing Pr55Gag and chimaeric Gag virus-like budded particles (VLPs) as vaccines, with good evidence of significant T-cell response boosts by VLPs of DNA-or BCG-primed immunity in mice and baboons.

An important sub-theme at the Conference was DNA vaccines: there has been a lot of disparaging talk in recent years concerning their potential efficacy; however, in the post-STEP era, the usurping adenovirus vectors have lost some popularity, and it seems the original genetic vectors have a new lease on life. 

George Pavlakis (NCI, Frederick, MD) in talk OA05-01 gave a masterly account of how electroporation of optimised DNA vaccines hugely enhanced humoral and mucosal responses.  He made the point forcefully that DNA vaccines had the advantage that there was no immune response to the vector, and that their preparation was rapid, scalable and safe – and that “increased expression improves the DNA vaccine result”.  He noted that different forms of antigen affect the immune response that electroporation as a means of delivery increases both antigen expression and immunogenicity; that natural cytokines delivered as DNAs were effective molecular adjuvants (eg: IL-12, IL-15), and that heterologous combinations such as DNA+protein, or DNA+viral vectors could be very effective.  In macaque vaccination experiments using gag, pol, nef and vif genes in combination with chemokine fusions, the group was able to get very high (30 000 sfu/106 cells) Ifnγ ELISpot results, with ~0.3% of total circulating T-cells being Ag-specific, and high serum Ab response.  They got a balance of central memory and effector CD4+/CD8+ cells, which is a shift of the type of response (central memory CD4+) obtained with previous DNA vaccines.

David Weiner (Univ Pennsylvania, PA) spoke in S03 on a very similar theme – electroporation and molecular adjuvants – and reiterated that optimisation of DNA yielded excellent results.  He also added manufacture as an optimization parameter, noting that it was now possible to get ~15 mg/ml of plasmid DNA: this allowed much higher, less dispersed doses of DNA.  He also noted that electroporation (EP) changes the phenotype of the response.  They tested an SIV DNA vaccine with IL-12, IL-15 or RANTES DNA as adjuvants in macaques: challenge showed IL-12, RANTES gave viral loads 2 log less than IL-15 DNA adjuvanted or DNA vaccine alone.  Concentrated DNA was as good an immunogen as Ad5, and much better when used with IL-12 DNA: there was increased magnitude of individual responses and increased polyfunctionality.

Fiona Tanzer (IIDMM, Univ Cape Town) in OA02-02 gave an excellent example of how to improve a DNA vector.  She used elements from Porcine circovirus (PCV) to significantly enhance expression from and immunogenicity of an already good DNA vector, increasing HIV Ag-specific Ifnγ ELISpot scores by 3 – 5-fold in mice using only 172 bases from the capsid gene promoter of PCV inserted upstream of a HIV-1 polygene vaccine construct. 

Taken together, these talks give an indication that DNA vectors for T-cell and other vaccines are alive and kicking, and on the verge of another growth phase in their deployment.  Improvements in manufacturing, antigen expression levels and DNA delivery, and parallel advances in the use of co-expressed molecular adjuvants, all herald a new era in heterologous prime-boost studies for HIV and other vaccines.

Two posters – one an oral abstract – stood out for me as vaccine design highlights of the very rich and well-attended sessions.  Darrin Martin (IIDMM, Univ Cape Town) spoke briefly on P19-08: designing a recombination-proof HIV vaccine.  His bioinformatic approach identifies “cold spots” for recombination in the HIV-1 genome, and suggests targets for polyepitope-based vaccines.  Champiat et al. (P12-15) found that APOBEC is a T-cell target in HIV+ people: this further extends the range of invariant, HIV-induced potential T-cell vaccine targets. 

A few plant production-related HIV vaccine posters caught the eye, as my group has been involved in this for years, and it is a field with much promise but as yet, no few realised achievements.  Cherni et al. (P02-03) presented interesting data on gp41 MPR on Hepatitis B virus core particles made in plants; Andersson et al. (P12-08) demonstrated that transgenic Arabidopsis expressing p24 is orally immunogenic; Meyers et al. (P12-10) showed that plant-produced vaccine-relevant HIV Ag boosted DNA primed T-cell responses; Regnard et al. (P18-08) showed that plant production of HIV antigens could be significantly increased by used of a replicating geminivirus-derived vector.

 As for other posters, Welte and Walwyn (Univ Witwatersrand, SA) in P01-01 demonstrated elegant mathematical modelling of acute infection and vaccine design – and Guerbois et al. (LB-32) had truly excellent expression of budded Gag-ΔV1V2Env particles from a measles-vectored vaccine. 

All in all, then, the HIV vaccine enterprise is battered but still functional.

 

 

 

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Nobel Virology 2008

7 October, 2008

It gives me great and unalloyed pleasure, as someone acquainted with one of the new Nobellists, and who has followed the science behind the awards ever since the beginning, to feature the three virologists who were jointly awarded the Nobel Prize in Medicine for 2008.

http://www.nytimes.com/2008/10/07/health/07nobel.html?hp

Discoverers of AIDS and Cancer Viruses Win Nobel Prize – NYTimes.com via kwout

To quote the NY Times article, written by Lawrence K Altman:

“The Nobel Prize in Medicine was awarded Monday to three European scientists who had discovered viruses behind two devastating illnesses, AIDS and cervical cancer.

Half of the award will be shared by two French virologists, Françoise Barré-Sinoussi, 61, and Luc A. Montagnier, 76, for discovering H.I.V., the virus that causes AIDS. Conspicuously omitted was Dr. Robert C. Gallo, an American virologist who vied with the French team in a long, often acrimonious dispute over credit for the discovery of H.I.V.

The other half of the $1.4 million award will go to a German physician-scientist, Dr. Harald zur Hausen, 72, for his discovery of H.P.V., or the human papilloma virus. Dr. zur Hausen of the German Cancer Research Center in Heidelberg “went against current dogma” by postulating that the virus caused cervical cancer, said the Karolinska Institute in Stockholm, which selects the medical winners of the prize, formally called the Nobel Prize in Physiology or Medicine.

His discovery led to the development of two vaccines against cervical cancer, the second most common cancer among women. An estimated 250,000 women die of cervical cancer each year, mostly in poor countries.”

The news is all the more welcome, because I am very familiar with the entire history.  The HIV pandemic has paralleled most of my career: I remember vividly my then Honours student – now a distinguished Professor in her own right – coming to me in 1984 to tell me that “…they have found the virus that causes acquired immune deficiency syndrome”.  Again, it was greatly of interest when Harald zur Hausen initiated the work that would lead to his award, as it was some of the first hard evidence that viruses were implicated in cancer – which suddenly made learning and teaching Virology a whole lot more sexy.  Especially in view of the mode of transmission of the viruses concerned…I like to think I may have put more people off casual sex by talking about viruses like herpes, HPV and HIV and what they can do to you, than any ten school guidance counsellors – but I digress.

The news is also welcome because I now work with both HPV and HIV: thus, reward for the people who invented our main field of endeavour is especially pleasing.

 But as ever, the Nobel awards are not without controversy.  Altman again:

“In 1983, Dr. Montagnier and Dr. Barré-Sinoussi, a member of his lab at the Pasteur Institute in Paris, published their report of a newly identified virus. The Karolinska Institute said that discovery led to blood tests to detect the infection and to anti-retroviral drugs that can prolong the lives of patients. The tests are now used to screen blood donations, making the blood supply safer for transfusions and blood products.

The viral discovery has also led to an understanding of the natural history of H.I.V. infection in people, which ultimately leads to AIDS and death unless treated.

H.I.V. is a member of the lentivirus family of viruses. The French scientists were cited for identifying a virus they called L.A.V. (now known as H.I.V.) in lymph nodes from early and late stages of the infection.

“Never before has science and medicine been so quick to discover, identify the origin and provide treatment for a new disease entity,” the Karolinska Institute said.

…Nobel Foundation rules limit the number of recipients of its medical prizes to a maximum of three each year, and omissions often create controversy.

The dispute between Dr. Gallo and the French team spanned years and sprawled from the lab into the highest levels of government. Dr. Gallo, 71, now at the University of Maryland in Baltimore, worked for many years at the National Cancer Institute in Bethesda, Md.

While in Bethesda in 1984, a year after the French team’s report, Dr. Gallo reported finding a virus that he called H.T.L.V.-3 and that was later shown to be nearly identical to the French L.A.V. After additional studies, Dr. Gallo said cultures in his laboratory had accidentally become contaminated with the French virus.

In 1986, Dr. Gallo and Dr. Montagnier shared a prestigious Lasker award, given in the United States; Dr. Montagnier was cited for discovering the virus and Dr. Gallo for determining that it caused AIDS.

In 1987, President Reagan and Prime Minister Jacques Chirac of France signed an agreement to share royalties and credit for the discovery.

But Maria Masucci, a member of the Nobel Assembly, told Reuters on Monday that “there was no doubt as to who made the fundamental discoveries.”

Dr. Gallo told The Associated Press on Monday that it was “a disappointment” not to have been honored with the French team. Later, Dr. Gallo issued a statement congratulating this year’s Nobel Prize winners and said he “was gratified to read Dr. Montagnier’s kind statement this morning expressing that I was equally deserving.” “

We’ve been waiting for this for a long time…and the result is interesting indeed, for many of us virologists.  Satisfying too….  I remember wondering at the time how the US team could blithely rename a virus that appeared very similar to one described a year earlier – and was even more fascinated to see how the story unfolded, with LAV becoming HTLV-III becoming HIV, as eventually sense and taxonomy overtook hubris.

The HPV award seems not to be controversial at all, and Professor zur Hausen is seen by everyone I have spoken to as a most worthy recipient.  Now, just to get that vaccine into people who need it….

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HIV: roots run deeper than we knew

2 October, 2008

I have previously posted a number of articles on “molecular archaeology” of viruses, and how one can use extant sequences, archived tissue samples, or even blood of pandemic survivors to speculate on the origins of specific viruses, of viruses generally, or on the nature of old pandemic strains.Now HIV falls under the spotlight – again – as the 2nd October issue of Nature publishes three articles (one letter, a commentary on it and an independent commentary) on the origins of HIV-1 pandemic strains.I picked up on the first news – evidence for an older-than-previously-thought origin for HIV-1 – via our local paper this morning. Now this is VERY impressive; they usually keep science news for a slow day, and here they were telling us about a Nature paper on the day it was published! Accessing Nature brought up the Nature News commentary by Heidi Ledford, titled “Tissue sample suggests HIV has been infecting humans for a century”. Essentially, the commentary summarises the findings of Michael Worobey of the University of Arizona and his colleagues, who managed to amplify and sequence HIV-specific cDNA and DNA from a paraffin-embedded lymph node biopsy dating from 1960 from a woman in Léopoldville (now Kinshasa) in what is now the DR Congo. To quote Ledford:

“Their results showed that the most likely date for HIV’s emergence was about 1908, when Léopoldville was emerging as a centre for trade.”

Their findings added credibility to an earlier demonstration of HIV-1 in a 1959 sample, also from Kinshasa. What was interesting was that the sequences of the two viruses differed by 12%: this indicates that there was already significant divergence in the HIV-1 strains infecting people as early as 1960, pointing to a longer history of human infection than the previous estimate of the 1930s.Which led on to the Comments section, where one finds gems like this:

“This is one of the most stupid discovery I have ever heard. You will blame every single human plague on Africa, This is against all the Theories of evolutionary biology where The descents of the people that lived in the area might have developed a kind of resistance instead of being vulnerable to a new strain of the Virus.”

And:

“HIV is older than your great-grandparents, uh-huh! And I’ll bet that the US bio weapons effort is just ecstatic about this deflection. So now these members of science play to the bio-jackboot population controllers with this ‘revelation’ that those sex-crazed Africans of course just couldn’t stop themselves from pulling chimpanzees (I thought the original scientific theory was “green monkeys”) out of the trees for a quickie.”

I couldn’t take this, so I replied:

“It continues to amaze me, as a teacher of virology who tells big classes every year where HIV comes from, how every year some clique of students takes the African origin of HIV personally, as a direct affront. I echo the correspondent above: it is a virus, people. Viruses infect animals, they infect people, and sometimes spread from one to the other – and back, if you are a zoo animal and catch something from your handler. The AIDS pandemic is an accident of sociology, demography, access to high-speed, long-distance travel – and truck routes, and truck drivers. It happens that it originated in Africa. So did the human race – only a lot longer ago. Inevitably, as humans encroached on apes, things get passed across. And don’t spread, much, until…someone puts a road through the village.Why don’t people get more exercised about the origins of HTLV, another retrovirus that almost certainly jumped from monkeys to humans? Except that happened many thousands of years ago, and in south-east Asia, not Africa. And for the same reasons: people eat monkeys and great apes. For that matter, it is speculated that chimpanzees got SIV-CPZ from vervet (I HATE the term “green”) monkeys – and that it may have caused a population bottleneck, some 100 000 years ago. I note that chimpanzees are known to eat vervets, incidentally – so they caught the virus the same way we did.

Ah, well…. In any case, Paul Sharp of the University of Edinburgh – and phylogenetics guru – and the godmother of HIV/SIV diversity, Beatrice Hahn of the University of Alabama (from whom I got the chimp-vervet virus link), have an independent commentary in the same issue, wherein they speculate on “The prehistory of HIV-1”. They make this very interesting comment:

“If the epidemic grew roughly exponentially from only one or a few infected individuals around 1910 to the more than 55 million estimated to have been infected by 2007, there were probably only a few thousand HIV-infected individuals by 1960, all in central Africa. Given the diverse array of symptoms characteristic of AIDS, and the often-long asymptomatic period following infection, it is easy to imagine how the nascent epidemic went unrecognized.”

They also make the important point that the findings of the Worobey group were replicated – with similar but non-identical virus sequences being found – by another group working independently with the same tissue sample. This is important because it nails down the findings more firmly, as HIV sequences within an individual do differ, and:

“…the distance along the evolutionary tree from the group M ancestor to the ZR59 or DRC60 sequences is much shorter than those between the ancestor and modern strains, consistent with the earlier dates of isolation of ZR59 and DRC60, and confirming that these viruses are indeed old”.

a, The HIV-1 genome fragments that were successfully amplified from DRC60 (red) and are available for ZR59 (black). The numbering for the HIV-1 sequences corresponds to the HXB2 reference sequence (Supplementary Table 1). b, The A/A1 subtree from the unconstrained (in which a molecular clock is not enforced) BMCMC phylogenetic analysis. 1960.DRC60A is the University of Arizona consensus sequence, and 1960.DRC60N is the Northwestern University consensus sequence (that is, the sequences independently recovered in each of the two laboratories). The DRC60 sequences form a strongly supported clade with three modern sequences also sampled in the DRC.

Reproduced with permission from Nature Publishing Group (RightsLink License No 2041420001096) from:
Direct evidence of extensive diversity of HIV-1 in Kinshasa by 1960
Michael Worobey, Marlea Gemmel, Dirk E. Teuwen, Tamara Haselkorn, Kevin Kunstman, Michael Bunce, Jean-Jacques Muyembe, Jean-Marie M. Kabongo, Raphaël M. Kalengayi, Eric Van Marck, M. Thomas P. Gilbert & Steven M. Wolinsky
Nature 455, 661-664(2 October 2008) doi:10.1038/nature07390

So where did the virus infecting humans come from? The best guess, from the paper and the commentaries, is that it originated – as do the extant chimpanzee virus supposed to have descended from the common origin – in chimpanzees somewhere in southeast Cameroon.How did it get into people? Sharp and Hahn again:

“The simplest explanation for how SIV jumped to humans would be through exposure of humans to the blood of chimpanzees butchered locally for bushmeat.”

No sex, no weird practices…just eating our cousins.  And how and why did it get to Léopoldville? Trade…and in those days before widespread truck routes, that would have been via rivers – which, Sharp & Hahn point out, drain from southeast Cameroon into the Congo River, which flows past what is now Kinshasa. The Worobey paper has some interesting history in it, documenting times of founding and rates of growth of cities in equatorial west Africa: Léopoldville/Kinshasa was and probably still is by far the fastest-growing of these, and was the earliest founded (in 1885). All that was needed to seed a pandemic, then, was that people infected by a virus as a result of butchering chimpanzees, moved some 700 km down natural trade routes to an emergent trade centre – and settled, and passed it on.Then, of course, it is the same old story, told so well by Jared Diamond in “Guns, Germs and Steel“: increased human population density and breakdown in social structure leads to increase in rate of transmission and incidence / prevalence of a disease agent, until it reaches the threshold necessary to break out. It is interesting that it took so long to become noticed – but then, HIV is passed on considerably less efficiently than Hepatitis B virus, so the pace of the epidemic was necessarily slow.But very sure….

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