Archive for September, 2011

Virus Origins II

28 September, 2011

I have updated the blog on virus origins quite considerably – new pictures, more detail, more speculation!

Pathways on information flow for RNA viruses

The game of structures

27 September, 2011

You just have to love it: harnessing all the creative energy that otherwise goes to entropic waste in obsessively playing computer games, for good.

And in this case, a good structure for the monomeric form of a retroviral protease, that was proving next to impossible to solve by any other means.

An article in Nature Structural & Molecular Biology from the 18th of September has a very interesting author list: it consists not only of David Baker and other actual scientists from a variety of respectable institutions, but also the “Foldit Contenders Group” and the “Foldit Void Crushers Group“, who are acknowledged as follows:

“…the F.C.G. and F.V.C.G. contributed through their gameplay, which generated the results for this manuscript”

The paper in question is entitled “Crystal structure of a monomeric retroviral protease solved by protein folding game players“, and I will let its summary tell the story.

Following the failure of a wide range of attempts to solve the crystal structure of M-PMV [Mason-Pfizer monkey virus] retroviral protease by molecular replacement, we challenged players of the protein folding game Foldit to produce accurate models of the protein. Remarkably, Foldit players were able to generate models of sufficient quality for successful molecular replacement and subsequent structure determination. The refined structure provides new insights for the design of antiretroviral drugs.

I note Foldit now has two Nature papers under its belt – not bad for a bunch of gamers who, in the words of the NSMB paper,

“…have little or no background in biochemistry [and] were able to solve protein structure refinement problems in which backbone rearrangement was necessary to correctly bury hydrophobic residues…”

The Foldit Home Page

From the Foldit site:

“…a protein causing AIDS in rhesus monkeys that hadn’t been solved for 15 years was resolved by Foldit players and confirmed by x-ray crystallography.

…The crystal structure will be released to the Protein Data Bank soon:

We are so proud of everything that you Foldit players have accomplished already, and we hope that this article will show the world the power of citizen science! This is the first instance we are aware of in which online gamers solved a longstanding scientific problem  and we want to thank you all for your amazing work on this and everything else.”

Power to the gaming fingers, guys!

HIV Vaccines From Bangkok – 4, and final….

22 September, 2011

Thursday morning started with three parallel oral sessions – and I chose Symposium 07, Characterization of Breakthrough Viruses.  The second talk – by Morgane Rolland, in the US Military HIV Research Program – detailed a study of the sieve analysis of breakthrough viruses in the RV144 Thai trial.  They wished to see whether or not the vaccine could block infection of specific variants, and thought they might see that viruses in vaccinees were evolutionarily distant from the insert in the vaccine, relative to the placebo arm.

HIV and its life cycle

The saw no differences in virus diversity over 10 sequences per person, in 121 people,  71 of whom were in the placebo arm.  They did note, however, that linked transmissions showed less diversity in the env gene than normal – 1 vs 10%.  Over 75% of cases had a single founder virus, in both placebo and vaccine arms.  There was no significant divergence from the vaccine sequence in either group in anything but the Pro aa sequence – with some non-significant evidence for Env variation.

When they looked for Env sites under selection in gp120, they saw 4 in the placebo group at positions 181, 208, 327 and 359 – with less variation in vaccine than placebo recipients.  Rolland speculated that this could be to do with entry being more restrictive in vaccinees?  4 different sites in the vaccine group were under selection: they found that for MHC I epitopes there was a greater distance for vaccine than placebo groups, with a result that was not significant for MHC II epitopes.

There was a trend toward longer Env V2 loop sequences in vaccinees at later times, and a reduced number of cysteines in Env among vaccinees – this was seen also in the VAX004 trial.

Phil Berman – formerly of VaxGen, which made the gp120 for RV144 and earlier trials – mentioned that there was lower variance in Env than in the unsuccessful VAX 003 trial.  Jerome Kim noted that men seroconverting had a much higher incidence of HCV infection – which could be associated with undeclared IV drug use.

Katharine Barr of Univ Alabama spoke next, on the increased incidence of multiple variant transmission of HIV in VAX003 injection drug users.  She noted that this efficacy trial was of gp120 in IV drug users, while VAX004  was in MSM and high-risk women: they speculated that differences if any could be due to transmission route – as in, IV route vs sexual.  She further noted that in RV144, the best (non-significant) effect was in low-risk heterosexuals.

Something that was a little disturbing to me, given HIV transmission in our part of the world is overwhelmingly by heterosexual sex, was that the IV route is responsible for 10% of world infections.  They had looked at transmitted founder viruses – the ones going in and replicating in recipients.  They predicted that consensus of a low diversity lineage is the sequence of the founder virus – and that several founders would give multiple low variance lineages.

She noted that 80% of heterosexual infections are established by single viruses, so there exists a window of opportunity of viral vulnerability when vaccine-induced immunity could block infection.  However, with MSM, the multiple infection goes up to 40%; while injection drug users (IDUs) are less studied, multiplicity goes up  60% in one study and 31% in another….

Looking at Vax003 results, they had asked how high a barrier there had been for placebo infections, and whether in vaccinees there were more or fewer founder viruses?  While they had found that there was an 44% incidence of multiple variant transmission in the  placebo arm, and  22% in the vaccinees, this was unfortunately not significant, given the low numbers.  There was a median of 1.8 viruses per transmission vs 1.3, but this too was not significant.  However, it could mean there is a higher bar for vaccine protection among IDUs, which has important implications for which groups to use in vaccine trials.

Katherine incidentally gave the best answer yet heard to a long and detailed question: “I think that’s a really good question but I have zero data to address it…” = I don’t know.

Which prompted thoughts of new conference drinking games: take a shot every time you hear a speaker say “I would like to thank the organisers for inviting me…”, or “Our hypothesis [generally pronounced hy-PAH-the-sis] was…”, or a question which starts with either “…really good talk / great data” or “So – ummmm – when you/we did…”.

Paul Edlefsen (Fred Hutchinson Cancer Res Ctr) described a sieve analysis of RV144 [and started: “So…umm…” = another shot!].  He repeated the finding that observed correlates of risk generated two hypotheses; namely, that high IgG response to Env protected from HIV infection while a high IgA response interfered with protection.  Additionally, analysis of the antibody response using scaffold V region showed that a high V2 response correlated with a lower infection rate.  He noted that the STEP trial results showed a distinct difference in Gag between vaccine and placebo groups.  He noted further that were only 110 usable subjects in RV144, so they could only detect large sieve effects in their study of CTL and Ab epitope responses.


There were 2 sites of evidence for sieving – aa positions 169 and 181 in the Env V2 loop, in the middle of a region identified by Ab binding array data.  There was also some evidence of covariation among pairs of aa residues in the V2 loop for vaccinees only.

After a long and complicated structural question, he gave the second-best answer of the conference: “I could say that I do, or that I don’t – but I have so little expertise in this area…(laughter)”.  And after long rambling statement: – “I’m sorry, was there a question in there?”

Brandon Keele (National Cancer Inst, MD) described work on NHPs which they had extended to studying human transmission of HIV, on transmitted/founder viruses.  NHP studies show multiple founders because doses are high generally, in order to get 100% infection rates.  One study using very low dose multiple intrarectal exposures to see if one can immunise macaques showed that one virus could do it.  Animals followed up from early times stayed with one evolving variant.

He noted that the consensus sequences in humans posited to have had one transmitted variant are average in  neutralisation susceptibility.  These viruses are all functional in vitro and in vivo and one can get full length viral clones ex NHPs which recap original founder viral load and pathogenicity.  All such viruses use the CCR5 coreceptor.  All HIV clones replicate in CD4 T-cells but not in  macrophages.  The transmission signature is to increase Env processing and infectivity.

They now mix cloned viruses with tags so can follow them in NHP challenge experiments, as most challenge studies have used virus with <1% diversity, which represents a clone in any one epitope – which he felt to be non-reflective of the real world .

The closing plenary session was opened by IAVI‘s Wayne Koff, who remarked that he had heard someone say “The  airport….”, in answer to the session name “Where do we go next?”….

Jeffrey Boyington (Vaccine Res Ctr) described some very impressive work on using structure of Env for rational immunogen design, specifically to target the CD4 binding site as a good target for broadly neutralising Ab.  They used crytallographic data to make proteins best mimicking the struc and then used them as immunogens.  They had used stabilised resurfaced gp120 with mutations around the binding site and isolated dozens of Abs with them from several infected subjects.  Part of the process involved stabilising flexible regions by bolstering cysteine content, removing glycans from the site of interest and adding them to immunodominant sites, and using Chikungunya virus VLPs to multimerise spike proteins for maximal immunogenicity.  Boyington noted that there were 80 native trimers on the surface of the VLPs, and that one can put the Outer Domain of gp120 on the tip of each monomer.  They get good Ab back for gp120 and get CD4 binding site Ab in rabbits.  In rhesus monkeys primed with gp140 trimers they got good boosting and better Abs to the CD4 BS.

Altogether a very impressive account – and one which advances to possibility of other opportunities for the design of other good broad-binding vaccine epitopes.

Rick King of IAVI followed, with an account of the current status and future directions of vector-based HIV vaccines.  He stated that most HIV vaccines now involve vectors – so there is a wealth of data that can be efficacious, so how to use it?  He thinks that we want the next generation of vectored vaccines to block infection and control virus load – meaning a combination of Ab and cellular responses.He noted that in NHPs, SIV protection is possible, and that it requires Env in the vaccine – and that the mechanism of protection is under intense investigation right now.

He further noted that in a DNA prime MVA boost vaccine regime, protection is associated with the avidity of the Abs.  Thus, a major goal is to improve the response to Env, by identifying the nature of the protective response, and enhancing and using native Envs to do it.  He stated in this context that there were only two vaccine regimens using native spike protein – and that one of them is the SA AIDS Vaccine Initiative (SAAVI) vaccine.

It was possible to engineer Env to bind a broader array of broadly neutralising Ab and to incorporate it into vesicular stomatitis virus (VSV) instead of the native G protein spike, or into canine distemper virus (CDV, a measles relative), which replicates in lymphoid tissue.  One could also bias processing of Env in CDV to get better cleavage and presentation.  The rCDV could be put into ferrets and shown to replicate.

He said that while the RV144 vaccine did not control viral load, vaccines can control SIV replication, so we need to have those components in HIV vaccines.  For instance, recombinant live cytomegalovirus (CMV) expressing the whole proteome of SIV could control the virus, this was associated with CD8 effector memory T-cells.

He thought we need to capitalise on information on mechanisms of control, and to increase immunity by use of replicating vectors and heterologous prime/boost combos, and deal with diversity by broadening the response.  The reason for replicating vectors was because live attenuated virus works for SIV – preventing infection and controlling replication.  Possibilities were vaccinia, measles, VSV, Sendai, CMV, AdV, CDV and VSV-HIV chimaeras.  As for diversity, one could increase the number of epitopes by using mosaics, and direct responses using conserved epitopes, as Tomas Hanke has demonstrated in IAVI-funded trials using chimpanzee Ad as prime then MVA as a boost with his HIVCONS Ag.

Finally, there was what I consider to have been the best talk of the conference – simply because it was much wider in scope than the rest: Steven Reed of the Infectious Disease Res Inst, Seattle, described new generation adjuvants for use with HIV.  He started by noting that adjuvants were necessary for lots of things; eg: for T-cell vaccines for TB and leishmania; for Ab response-broadening (Cervarix, HPV vaccine); Ag dose sparing (eg flu); to combat immune sensescence, and for vaccine therapy.

They had focused on a toll-like receptor (TLR4) agonist as an adjuvant, following work that showed that the well-known MPL was a TLR4 agonist ,and vaccines including TLR agonists had been used unknowingly since 1885.

He thinks the ideal adjuvant should have no effect on lymphocytes, no systemic effects, no non-specific B or T cell responses, should elicit potent long-lived responses, should redirect ongoing immune responses, and should be safe and effective in all age groups.  They had accordingly designed GLA – based on lipid A – to bind TLR4: this was purely synthetic, and induces Th1 CD4 helper cells and a broad humoral immunity.  They used a hexaacyl chain length that was preferred by human TLR4, which is restricted to macrophages and dendritic cells, has transient local effects, and reduces inflammation so as to get better central memory.

They can also formulate it differently for different vaccines and can get very different effects thereby.  For example, emulsion alone stimulates Th2 responses while GLA stimulates Th1 even in combo with an emulsion, which helps in leishmania and TB vaccines.

He noted that alum-based adjuvant stimulated mainly a Th2 response, while adding GLA gives a Th1 response with the same antigen.  They get good Ab diversity with GLA and expansion of it with the malaria vaccine – and Ab diversity leads to better neutralisation (eg transl med 2011).

GLA increases and broadens the haemagglutination-inhibtion (HAI) Ab response to the influenza vaccine Fluzone, which contains lots of inactivated virions.  He noted one gets a better protective response against “drifted” viruses – which have evolved away from the vaccine strains – with GLA.  Baculovirus-made H5N1 vaccine requires 30x less vaccine to get the same response with GLA.

It is also possible to get mucosal immunity by IM vaccination with HIV gp140, according to Robin Shattock’s results.

Reed noted that intradermal adjuvants are very rare – and that this looks good with flu vaccines delivered this way.  They were in the process of optimising the adjuvant formulation for intradermal delivery to increase vaccine potency, get mucosal immunity, and CD8+ T-cell responses.  Dermal dendritic cells have a wider range of TLRs than Langerhans cells – so Sanofi target them with ID delivery, and GLA works well to amplify the response.  It was impressive that they could protect ferrets with a single ID vaccine shot of flu vaccine.  It was also interesting that they are working with Medicago Inc., who have one of the most successful plant-produced influenza virus vaccine candidates, presently in human trial.

Thereafter, closing remarks from the conference organiser were as one would expect; people were honoured for their present and long-term contributions – notably Jose Esparza – and the venue of the next conference was announced to be Boston, with Dan Barouch as Organising Chair.

It was a good conference, with all of the high-intensity interactions and presentations one would expect from such a loaded topic.  However, it possibly suffered from over-emphasis of the “RV144 results” – which weren’t that impressive, in my opinion – as part of an effort to keep up perceived momentum from announcement of the RV144 success (small as it was) from the previous meeting.  For me, the highlights were the envelope antigen design talks, and what I managed to catch of the actual virology, and especially analysis of diversity by massively parallel sequencing.

We still don’t have an effective HIV vaccine – but we’re getting closer. 

HIV Vaccines From Bangkok – 3

20 September, 2011

HIV: a retrovirus. Courtesy of

The Wednesday morning agenda for the conference followed a somewhat bemusing Tuesday evening entertainment: one day I will learn NOT to involve myself in anything that involves getting onto a fleet of buses in the company of several hundred other people, and especially not in Bangkok!  It took us one-and-a-half HOURS to go from the venue to the Navy Yard for a reception and supper – but the first half an hour was spent just going around the block, such is the traffic density at rush hour.  There followed the standard fare for a conference in any country with any sort of culture: local entertainment (drummers and folk running around with bolts of cloth in this case), together with so-so food with very little choice, too much noise, and no possibility of being heard more than one person away.  But thankfully, only a twenty minute ride back!

“New Prevention Strategies” was the theme for the second set of plenaries – which were opened (unexpectedly; she was second on the programme, but No 1 overslept) by our very own Carolyn WIlliamson (IIDMM, UCT), speaking on implications for combination prevention strategies from HIV pre- and post-infection studies.  Carolyn noted again a point first raised by Pontiano Kaleebu on the first evening, that future vaccine efficacy trials should as as a matter of ethics offer preventions – eg ARVs – as a minimum standard of care, which will affect size and expense as well as endpoints like acquisition and disease progress.

She pointed out that 80% of infections with HIV were due to single viruses – but 20% were due to multiple infections, influenced by dose, IV injection, MSM transmission, inflammatory genital tract infections and the like.  The lesson from study of the Phambili and STEP trial breakthrough infections by sieve analysis showed vaccination had had a selective effect on T-cell pressure.  Phambili got 277 sequences from 43 people, in vaccine and placebo arms.  The Merck vaccine had no effect on the transmission bottleneck.  Scanning sites across the genome showed 2 sites of selection in Gag and 1 site in Nef were significantly different in the two arms; one in the region p6 of Gag looks like an epitope escape.  There was a weaker signal in Phambili than in STEP, however: this was due in part to the lower number of participants (the trial was stopped before recruitment was complete), the fact that there were men and women involved vs mainly men in STEP, among other factors.

It is worth remembering that the Phambili and STEP trials were stopped in 2007, and reported on at a very gloomy AIDS Vaccine Conference in Cape Town (covered here in ViroBlogy).

There was also no effect of pre-exposure Tenofovir on the transmission bottleneck, or evidence of immunity in highly exposed uninfected individuals using the gel – despite the evidence for “chemovaccination” in macaques, due to abortive infection checked by ARVs in target cells.  Thus, chemovaccination did not enhance the of impact microbicides and preinfection immune responses would not interfere with vaccine monitoring by this assay.

Tenofovir did impact early Ifn-g Gag-specific CD4+ responses post infection – indicating that possibly the drug prevents the initial destruction of CD4 cells in the gut, which would be a very valuable result.

Carolyn finished by noting that the implication for combination of preventive therapies is that it will increase the complexity of trials, make them cost considerably more, and make them longer.  However, microbicides that reduce inflammation may dramatically reduce infections, ARVs may increase the barrier to infections, and also increase the time for the effects of vaccination to kick in and increase post infection immunity, and combination of multiple partially effective interventions may have significantly greater impact than any alone.

Helen Weiss (London School of Tropical Medicine and Hygeine) was the late riser: she spoke on lessons from male circumcision for other prevention strategies.  It was interesting to many of us that it was a study in Nairobi in 1989 that showed the effect first – circumcision protected to some extent against in infection even in the presence of genitourinary diseases (GUDs).  A metastudy combining 15 studies subsequently showed reduced risk in all, to a 60% protection level.  Accordingly, three studies had been set up in Uganda, Kenya and SA  in 2005-2007 to directly study the effect.  They saw 50% efficacy in all locations, and all were stopped early as there was an obvious effect, with all participants being offered circumcision.  The studies saw an overall 58% protective effect, and the  effect in the Uganda trial persists up to 5 years post trial.

As for why this should be, Helen said that some studies say that the inner foreskin has a greater density of Langerhans and T-cells compared to the outer – and there is some evidence the inner is more easily infected in explant studies.  HIV infections also induce retention of Langerhans cells within the epidermis of the inner foreskin.  There is evidence that the inner foreskin facilitates efficient entry and translocation of cell-associated HIV, retention of Langerhans cells, and the incidence of infection is greater in men with larger foreskin area.

The conclusion was that one should offer circumcision in HIV prevention studies where heterosexual contact is the mode of transmission.  Among MSM, habitual penile inserters show some effect of protection, while habitual accepters are obviously not protected.

She closed by commenting that scaling up circumcision to 80 % coverage of adults and newborns by 2014 could save US$ 40 billion US: however, the reality was that uptake was slower than planned, with only 2.6% done by 2010.  However, there was obvious buy-in with a fourfold increase in circumcisions between 2009-2010.

While I thought she oversold the intervention rather – it is decidedly less simple than drug or microbicide interventions after all, benefits only one partner directly, and the lesson in South Africa is that even communities with a high circumcision rate can have very high prevalences of HIV infection – there is no doubt that circumcision in combination with pre- and post-exposure ARVs and microbicides cannot do other than have an additive effect in protection, and possibly even a synergistic one in some cases.

For me that was the morning; I missed three very worthy parallel late morning oral sessions while dealing with nagging emails – but started fresh again in the post-lunch period (an aside: best conference coffee break munchies and light lunches I have ever seen…B-), with Oral Session 11 – Mucosal Immunity.

Anthony Smith introduced us to a fascinating study of transcriptional “imprints” correlating with protective immunity in macaques following vaccination with the live attenuated SIV-∆-Nef virus, done by microarrays on RNAs from the cervix.  Smith noted that live attenuated viruses offer some of the best protection available in monkeys, and that SIV Mac239-∆-Nef was one of the best.  They isolated total RNA ex the cervix of rhesus macaques post-challenge with a heterologous virus at 140 days with native virus and tested unvaccinated and vaccinated samples with an Affymetrix rhesus chip.  There was 103-fold less viral RNA in vaccinated animals, and very little overlap of gene expression – only 1% (eg 5 genes) – of 405 vs 246 unvaccinated to vaccinated samples.  There was greater expression of inhibitors of innate immunity and inflammation in vaccinees; MIP3alpha expression was higher in unvaccinated monkeys – this brings in effector cells, including CD4+ T-cells, which would enhance infection.  Unvaccinated monkeys get a signalling cascade of cytokines which cause an inflammatory response – vaccinees get a short circuit in this signalling by mucosal conditioning with mutant virus.  There were important differences in humoral responses too, which were not reported here.  In light of this one could almost wish that the proposed trial in humans in the 1990s of the natural Nef deletant HIV-1 found in Sydney and associated with long-term non-progression had gone ahead – but only almost, as people with the virus did eventually start to progress to AIDS.

Steve Reeves then spoke on mucosal natural killer (NK) cells in SIV infected monkeys in chronic infection: he noted that NK cells respond early in infection in a variety of tissues.  They act to suppress viral replication in vitro, and are linked to disease control in vivo.  while much of what he said was straight over my head – I really do not have much truck with cytokine signalling cascades and lymphoid cell types and subtypes – it is becoming increasingly evident that not only are NK cells actively involved in controlling HIV infections, but that there are hitherto unsuspected variations among them, and often evidence of specificity in their interaction with infected cells.  Expect to hear much more about these fascinating guys in the future….

There followed a slightly disappointing talk by Shari Gordon, on the use of Human papillomavirus (HPV) pseudovirions (PsV), made in cell culture from co-expression of transfected HPV L1 and L2 capsid proteins and a replicating plasmid vaccine, to immunise mice vaginally.  The idea was to use a mucosa-infecting agent to make a vaccine which induces T-cells and antibody responses at mucosal sites to prevent HIV infection, during the window of opportunity where founder infections are being established.  HPV naturally infects the disrupted vaginal mucosa via interactions of L1 and L2 with receptors on basal keratinocytes – thus it was necessary to disrupt the vaginal epithelium by administration of progesterone and the known inflammatory agent nonoxynol-9 in order to infect.  They used HPV-16 PsV vectoring a SIV gag gene, then boosted with non-cross-reacting HPV-45 PsVs, both expressing red fluorescent protein (RFP) as a marker for in vivo fluorescence tracking.

They got a good response to HPV, and see anti-Gag IgA in vaginal secretions and IgG in serum.  They also see recruitment of T-cells to the site of infection in mucosa – both CD4 and 8 and activated cells.  T-cell responses assayed by intracellular cytokine staining  (ICS) showed that they get CD4+ and 8+ cells in tissue and in blood, which waned over time.  They then set up an experiment to see if systemic priming and mucosal HPV PsV boosting could protect macaques, using a regime consisting of sequential HPV-16, -45 and -58 PsV administration, with and without ALVAC + gag, and gp120 administered with the PsV 45 and 58.  They saw the same gp120 titres at the end of the regimen, with or without ALVAC priming.  They got a Gag-specific response, which expands and recruits T-cells in the genital tract but was lower in blood.  The response was better with ALVAC priming.  There was a primarily monospecific response of both CD4 and 8 T-cells, and primary effector memory.  Upon SIV challenge they saw a similar rate of acquisition despite the immune responses – however, they were only in mid-experiment, and still hoped to see viral control.  She noted the vaccine does not exacerbate the SIV infection rate.

While this was all good science, it was disappointing for a number of reasons.  First, they did not do or report the obvious control, of using DNA only in parallel with PsVs.  Second – in the opinion of my resident HIV/HPV vaccine expert, Anna-Lise Williamson – such vaginal immunisation using PsVs in humans would be a complete non-starter, because it is not ethically acceptable to use agents like nonoxynol-9, which is known to increase HIV infection rates, in a vaccine regimen.  Third, the vaccine did not seem to be very good, despite the supposed advantage of using particles to deliver a DNA vaccine: this is a subject close to my heart, given an interest in both HPV VLPs and DNA vaccines, and I think that oral or intranasal immunisation would have been a far better idea.  Fourth, and although this was not stated, the PsVs are made in immortalised 393TT cells expressing significant amounts of an oncogenic viral protein (polyomavirus T antigen) to enable replication of the vector plasmid – all of which I am sure would be a stern no-no for use in humans.

H Li spoke on the use of recombinant adenovirus vectors in monkeys: he noted that effector memory cells were induced by replicating viruses while non-replicating induced primarily memory cells in blood.  However, people had not looked at mucosal responses.  Accordingly, they used single or double recombinant Ad26 immunisations and showed one could get mucosal T-cells.  With a heterologous Ad5/26 prime/boost they get a potent and widely distributed T-cell response, which they have followed for 4 yrs and still see the responses 2.5 yrs post boost.  Mucosal T-lymphocytes are persistently activated.  They looked at T-cells in PBMC vs colon, duodenum and vaginal tissue: the latter were activated while PBMC were not, so there was only transient activation here.  Memory phenotype shows Tem (effector memory) to Tcm (core memory) evolution in the periphery.  Mucosal T-cells show a persistent Tem1 phenotype.

Ming Zeng revisited the attenuated live SIV vaccine, and its mucosal protective properties.  Live attenuated vaccines offer the best protection yet in monkeys against homologous or heterologous virus challenge – and understanding the correlates would help understand design principles for human vaccines.

They inoculated monkeys with SIV-∆-Nef intravenously, and challenged with repeated intravaginal inoculation.  He showed evidence of a fascinating vaccine-induced Ab concentration at the mucosal border of the monkey cervix, correlated with limited spread and prevention of infection.  They cannot see significant challenge viral growth at portal of entry in vaccinees from 20 weeks post vaccination.  Tissue-associated IgG is concentrated at the port of entry at 20 wk in the cervix and vagina: distribution of the IgG shows one gets plasma cells at the cervix, but also IgG-staining cells especially just underneath the epithelial cell layers.  The cells are epithelial reserve cells and enrich IgG inside cells, presumably by uptake mediated by the neonatal IgG receptor expressed on their surfaces.  This can be shown in vitro by incubating plasma cells with a filter-separated layer of epithelial cells from the female reproductive tract (FRT).

In challenge phase they noticed Ab concentration increased rapidly after challenge in situ.  All genes involved in Ab synthesis were upregulated in challenged monkeys in FRT and germinal centre cells see a dramatic local expansion of plasmablasts after challenge – presumably of memory B cells.

They think the SIV-∆-Nef vaccine converts the FRT to an inductive site for B cell expansion and maturation.  They get 5-10x the amount of IgG produced vs IgA.  They think it is both local recruitment of B cells and activation of local cells that results in the IgG production – which is total and not just HIV-specific IgG.

Again, this is a fascinating result obtained using a controversial vaccine candidate – and one which is not going to go away.

Late afternoon Wednesday was the turn of Symposium Session 02: Recent Advances in B Cell & Protective Antibody Responses – and two talks that took the prize as far as I was concerned were one by Peter Kwong and the following one by Pascal Poignard, both from Scripps in San Diego.

I couldn’t pretend to do justice to the Kwong talk: the graphics were so good, and there was so much detail, that it was like watching a great big complicated shiny machine in motion.  It was very beautiful, but I couldn’t tell you exactly what it is that he did.  Suffice it to say that he introduced us to the concept of mining the “antibodyome” by using structural bioinformatics to get solutions for vaccines by deep sequencing.  A consequence of this was that they could follow the maturation path of specific clones of cells making antibodies binding specific Env epitopes.  An important thing to come out of his talk was a possible reason for why strongly-binding broadly-neutralising antibodies are so rare: they found that, for their preferred target of the CD4 binding site, initially-produced antibodies were of very low affinity and needed a lot of maturation to become strongly neutralising and broadly reactive – which, of course, meant the producing cells were generally selected against and did not make it to being memory B-cells.  Knowing what was possible, however, and being able to make antigens to stimulate those antibodies specifically, would make for a rational vaccine design strategy.

Pascal Poignard described something that has been much in the news lately: the recent discovery of many strongly-binding broadly-neutralising monoclonal antibodies in people living with HIV.  He detailed how the IAVI protocol G search screened 1800 donors, mainly from Africa, for “elite neutralisers”.  They took the top four and did high throughput screening of memory B cells with antigen, and rescued the Ab gene sequences from selected wells, triaged them, and ended up with a selection of potent neutralising MAbs.  These were mostly broadly neutralising, but some were very potent – tenfold better than the previous best.  One group of 5 MAbs – all from the same individual – bind at various sites around the V1 and 2 and 3 loops of Env; another group of 3 from different individuals bind glycans and the V3 loop.  Data suggest protection needs 100x the IC50 value – which was very low for most of them, meaning they could be highly efficacious at low concentration, and synergise each other’s effective in mixtures.  Certain combinations of MAbs would give better protective coverage than others – especially if they did not neutralise the same spectrum of viruses.

Sprawling Bangkok - from the 37th floor

The work raises all sorts of very interesting possibilities, including mimicking the structures bound so well by these MAbs in order to elicit them more frequently, as well as using them therapeutically or in prevention regimes.  As far as antibodies are concerned, it is apparent that we are in a new era of sophistication as regards the potential for both exploiting the natural “antibodyome”, and even designing our own.

There followed a most enjoyable “Faculty Dinner” – my wife got me invited – on the 54th floor of the Centara Grand Hotel, followed by an even more enjoyable sojourn with pleasing beverages on the open deck of the 55th floor, overlooking Bangkok.

Until it rained, anyway.

HIV Vaccines From Bangkok – 2

16 September, 2011

Big News Day: HIV Vaccine Conference, Tuesday 13th September
The first plenary session of the conference had as its theme “Novel approaches in clinical evaluation through global collaboration” – and it was graced by the presence of no fewer than three scientists in full military dress uniform complete with medals, from the USA and Thailand (Nelson Michael and Jerome Kim and Punnee Pitisuttithum), reflecting the significant involvement of both countries’ military in the RV144 efficacy trial.

It was probably fitting, however, that it was led off by Pontiano Kaleebu of the MRC/UVRI Research Unit on AIDS from Uganda, on Africa’s contributions to AIDS vaccines.  He said that Africa had been crucial to the endeavour for a number of good scientific and societal reasons, but principally because most infections are there:  some countries are up to 15% of total population being HIV+ and sub-Saharan Africa contributed 17% of global infections in 2010.  Factors influencing vaccine efficacy that were unique were the great diversity of viruses, the mainly heterosexual transmission of viruses, diverse HLA alleles and significant preexisting vector immunity.

Small Buddha used for offerings, temple of the Reclining Buddha

South African scientists – largely drawn from the University of Cape Town, he says, modestly – had been responsible for the only vaccines designed in Africa, which were now in clinical trial.  Africa had been part of much work on epidemiology and variation of HIV-1.  Africa and Africans had contributed to understanding transmission events, mechanisms of early viral control and immune escape, and had helped in the addition of new broadly neutralising MAb derived from patients in African cohorts.

The first vaccine trial in Africa was done in 2000 in Uganda, and there have been many since:  30 trials or 17% of all trials have been done in Africa, mainly by  IAVI (13)  and the HVTN (11).

He noted that financing had declined and that the  reduced vaccine pipeline was a challenge, as many well-established sites have no vaccine to trial.  Another challenge was that new prevention successes means lower viral incidence, so trials have to be bigger – and may be impossible in certain cohorts.  There was also a challenge in the up and downstream HIV research imbalance in Africa – where there was no research infrastructure in many centres so sampes got shipped out, while clinical trials were large and well serviced.

His conclusion was that Africa had made significant contributions to vaccine research and development, but that challenges such as those mentioned could threaten further work.

Dr Punnee Pittisutithum of Mahidol University in Bangkok described how Thailand had a national plan established as early as 1993, and revised in 2006, to transfer technology, and to collaborate with a variety of institutions and countries, in HIV vaccine research and development and prevention efforts.  A collaboration in 1997 with Japan had used recombinant BCG as a subtype E vaccine prime, boosted with live vaccinia virus.  There had since been14 preventive trials including 2 efficacy trials.  In 1997 they had established a plan to monitor circulating virus – and now 1765 Thai viruses had been sequenced, and they had a very good idea of variation and currently circulating viruses.
They had an impressive infrastructure to set up and monitor clinical trials, which accounted for the success of the Thai trials over the years.  The partially successful RV144 efficacy trial had resulted in a study of correlates of protection involving 35 investigators from many institutes, including in Thailand.  She also made a point – as many others did subsequently of thanking the 16 402 Thai men and women who participated in the RV144trials.

Jerome Kim of the Walter Reed Army Institute of Research and Deputy Director of the US Military HIV Research Program spoke next, on correlates, sieve analysis and clinical development of the RV144 trial.

His first news was that there was a correlation of high Ab concentration to Env in vaccinees with a low risk of infection – resulting from 4000-odd samples analysed in the last two years for correlation. The work was the result of 35 investigators from 25 institutions collaborating on samples gathered during the trial.

A unique finding was that the gp120 and ALVAC vaccines were novel immunogens – the gp 120 from Vaxgen (also used in previous trials) used a N-terminal 11 aa replacement from a HSV gD glycoprotein epitope, which may have affected presention of Env HIV epitopes: its presence changed the binding of mAbs directed against gp120 up to 10-fold better for conformation-specific epitopes in the V2 and V3 loop epitopes, but not for linear epitopes.
There had been a good reaction to V2 peptides by intracellular cytokine staining (ICS) assays, with the response by CD4+ T-cells mainly.  One unfortunate finding was that gp120 Ab binding dropped 1.5 logs from 12 to 24 weeks post the last vaccination.

Sieve analysis – a new term to me, but denoting analysis of breakthough HIV or other infections in vaccine trials for selection pressure by the vaccine – looked at the gp120 V2 loop sequence in placebo and vaccine arm infectees – and saw selection.

It was left to the ununiformed Barton Haynes – Duke Human Vaccine Institute – to actually break the big news on correlates in the RV144 case control study.  Bart gave a lesson in the careful exposition of a complex topic of potentially huge significance in the HIV vaccine world, by starting with an explanation of what they had discovered – correlates of risk rather than of protection – and exactly what it meant.

He explained that a correlate of risk may be causal, or that it could be a surrogate marker.  Their team had looked at 41 infected and  205 uninfected vaccinees and 40 placebo recipients,  followed for 3 yrs.  There were only 41 cases, so the statistical study was only powered to pick up strong correlates and could miss weak ones.

Pilot work noted that most Ab responses were directed to the gp120 V2 loop.  In brief, their work found that there were two correlations associated with infection rate: the first was that serum IgG binding to a scaffolded V2 loop correlated inversely with infection rate.  The second was that Env binding of serum IgA correlated positively with infection rate: the statistical analysis showed a 43% reduction in infection rate associated with high serum IgG to the V2 region and a 54% increase in infection rate with high serum IgA binding.  Their hypothesis to explain this result was that high V2 IgG Ab levels were protective and low plasma IgA was associated with protection.

Ongoing analysis wase focused on looking at 9 PBMC-produced cytokines – and medium to high cytokine levels seemed to be correlated with protection.  Epitope mapping of IgA binding find C1 peptide response was correlated with infection, so maybe plasma monomeric IgA can block antibody dependent cytotoxicity (ADCC) caused by IgG binding?  He noted that only monomeric plasma IgA was collected in this trial, and mucosal dimeric IgA was to be collected next.

Haynes thought the way forward is to see if correlates of risk are causal correlations, by new clinical trials, or by having the trial Abs tested in non-human primates (NHPs) for passive protection.  They were presently testing the IgA association with risk by looking at mAbs in macaques binding all sorts things in Env, to see if IgA inhibited other Ab binding.

While the result was undoubtedly interesting – and unexpected – I was not convinced that it was as significant as it was made out to be, for a number of reasons – and a number of people I spoke to at the conference agreed.  Thus, in my slightly jaundiced opinion, the relatively weak correlations with risk applied for just one region in Env for this one vaccine combination with a unique monomeric gp120 product, for one subtype of HIV-1, in one population, for a trial in which the efficacy was only 30-odd percent, and which even then was only just significant.  They also tested only plasma antibodies, which may not give the full picture, and got next to no cellular response, which could correlate with the total absence of virus control seen in both placebo or vaccine recipients who became infected.

Accordingly, I think the massive obsession with analysing the results of the trial may turn out in retrospect to have been a bit of a waste, compared to testing new products which have markedly better responses in preclinical trials.  Another view can be seen here.

Giuseppe Pantaleo of the Centre Hospitalier Universitaire Vaudois in Lausanne closed out the session, with a presentation on poxvirus vector-based vaccines beyond the RV144 trial.    He pointed out that modified vaccinia Ankara (MVA) was the result of 571 passages in chick embryo fibroblast (CEF) cells; that the ALVAC canarypox vaccine resulted from 200 such passages, and vaccinia Copenhagen had had the deliberate deletion of 18 ORFS to result in the NYVAC vaccine.  The two former vaccines did not replicate in humans; however, replication competent poxviruses give appropriate innate immune cytokine responses and CD4 help.  To this end, NYVAC is known to grow in human primary keratinocytes, is highly attenuated, has no effect on dendritic cell (DC) maturation, and one gets higher levels and longer persistence of expressed antigens, cross presentation of Ag by MHC I and II receptors and stimulation of memory T-cell responses.

They tested replication competent and a replication deficient NYVAC and DNA expressing the same Ag and compared them, with  a boost of type C gp120.  They compared the effect of DNA priming or not, and scarification or intramuscular injection for NYVAC, with DNA and NYVAC both expressing Env and a Gag/Pol polyprotein.  The gag gene in constructs makes particles and a trimeric secreted gp140.  Pantaleo noted that the DNA plus regime elicited much more cellular immunity and a predominantly Gag/Pol response, while NYVAC alone gives 70-80% response to Env.  In the DNA+ group there was a balanced CD4 and CD8 T-cell response.  High Elispot results get long term and durable response.  There was no difference between scarification and im immunisaiton, and no increase of immune response with protein boost.  There was also no difference between rep and non rep NYVAC.  In the no DNA group the rep virus was lots better.

Most HIV-1 neutralising Ab response was in the DNA- groups and SHIV neutralisation was restricted to the DNA- group.

In ADCC assays for the DNA+ group there was no advantage in boosting with protein, and response decayed later with some animals being negative; in the DNA- group responses were considerably higher, there was an advantage to boosting,and all animals were positive.  In cross-type titring assays there was good cross-binding of IgG, with the DNA- groups being better.

The lesson from this was that a greater magnitude of T-cell responses do not necessarily correlate with neutralising (NAb) responses.

For plasma IgA responses they see the same distribution as for IgG.  In the DNA- groups they get very little response up to 3 months, then good responses 8-9 months, which then wane after 12.  Their response would be to boost with poxvirus plus Env at 12 months.  Pantaleo thinks we need compressed regimens to reduce the time of reduced protection, that we should try Env-only regimens, and that we should tailor vectors for optimal Ab responses.

My opinion on this is that one should try for Env-specific Ab responses AND Gag- and other protein-specific T-cell responses, elicited at the same time by immunisation in different limbs.

There were 6 Tuesday afternoon sessions, in two sets of three, so some judicious choices were needed.  I went with Oral Sessions 3 and 5, entitled Novel Immunogens and Inserts, and Acute Infection/Viral Diversity, respectively.

Oral 03: Novel Immunogens and Inserts
Two stand-out talks for me were one by A Flamar, and another by M Zhou – with a third on my favourite virus-like particles, by L Yang.

The Flamar talk reported targetting to CD40 receptors of five 19-32 aa peptides containing a string of known highly conserved CD4 and CD8 T-cell epitopes from Gag, Nef and Pol covalently linked to a lipid tail for antigen presenting cell (APC) uptake.  These have been tested and found to be therapeutic already.  The targetting is done using a MAb targetting CD40 with the HIV5 pep attached to the heavy chain C-terminus. The epitopes are 2 from Gag, 2 from Nef and one from pol.  The MAb is a humanised one with mouse Vh and Vkappa portions, which binds monocytes and APCs specifically.  The immunogen expands HIV peptide-specific CD4 and 8 T-cells from HIV+ patients.  They get broad peptide-specific responses and CD4 and CD8 polyfunctional responses.  The latter are CTL-characteristic and can kill target cells as well as suppressing HIV replication in vitro.

They are presently humanising the V region for clinical manufacture and testing in mice and NHP.

The Zhou talk discussed the use of mimotopes – peptide sequences mimicking native epitopes – displayed via phage surfaces, which mimic a membrane-proximal or MPER gp41 epitope, and bind Ab from an elite controller of virus load.  The M13 display library is made by env-specific PCR and fragmentation followed by cloning, and is bound by immobilised IgG.  “Panned” phage is eluted and amplified.  They get epitopes localised to gp41, inclusing the MPER region, using EC26-2a4 Ab.  The core epitope overlaps the known binding site of the broadly-neutralising MAb 2F5 but is distinct: the sequence is  NEQELLELDK.  They used this as an immunogen after a env DNA prime as phage plus adjuvant x3 – and got neutralising Ab back.  This is a genuinely exciting result, as it builds on much speculation regarding just how good Abs directed against this region are at neutralising a wide rtange of HIV variants – and answers some of the questions about how difficult they are to make.  These two sorts of immunogen – one aimed at T-cell responses, the other at neutralising Ab response – may yet be a valuable adjunct to other vaccines containing more conventional ingredients.

The third talk by Yang was very useful in that it demonstrated the possibility of using insect cells – which can be cultured very reliably at large scale, and are already used to make a major human vaccine (GSK’s anti-HPV Cervarix) – to make genuine HIV virus-like particles, with a Gag shell inside a membrane, studded with processed Env spikes.  I was especially interested as we have already used the same technology to make Gag-only VLPs, which are a real possibility as a subunit vaccine.  However, routine production of VLPs is complicated by the fact thats VLP are usually produced in low quantity, there is poor cleavage of Env, there is often recombinant baculovirus contamination, and poor batch consistency.

They used VLPs Drosophila S2 cells that had been stably transfected with plasmids encoding HIV-1 Gag and Env.  S2 cells are good as they grow up to high density and can easily be cultured in suspension – in a WAVE bioreactor in this case, which is scalable up to hundreds of litres.  They get glycosylated Env which undergoes appropriate cleavage and ends up as spike protein on budded Gag-containing VLPs.  They get 23 million cells per ml, and 8 mg of gp120 / litre – which is an excellent yield for VLPs.  Appropriate Mabs bind the spikes, indicating correct conformation.  Upon immunisation with a DNA prime and VLP plus CpG boost, they get a good Ab response which is weakly neutralising.  An ADCC test was also positive.  The T-cell response was a relatively poor CD4 but good CD8 cell.  The result is not entirely new – there were two posters at the conference describing the same thing, and our group has used stably transfected insect cells to make baculovirus-free VLPs – but they have investigated production at scale, and have shown appreciable and appropriate immunogenicity for what may be a valuable future component of heterologous prime-boost regimens.

Oral 5: Acute Infection/Viral Diversity
While I probably should have been more interested in acute infections, and there were several most worthy talks on this, I am inexorably drawn as a result of my history in virology and with HIV, to studies of virus diversity and especially of virus evolution over time and between individuals.  So there were really only two talks in it….

L Yin presented a fascinating account of the use of deep pyrosequencing to look at the evolution of viral diversity in peripheral blood cells in single individuals over time.  The study used pyrosequencing of cell-associated virus – which of course, reflects the whole history of the individual’s infection as integrated DNA – to look at diversity and both real and inferred longitudinal variation, given multiple blood samples from the individuals over time.  They used samples from children infected at birth, for time spans of 18 months to 6 years.  In six children, 4 of the 6 showed big differences in virus populations, while 2 did not not.  The biggest diversity was for R5 coreceptor binding sequences, illustrating immune selection of viruses.

Their conclusion was that deep sequencing was a robust method for evaluation of complexity and population structure and for evaluation of the virus historical record in an individual.  It was also easily possible to compare cell-associated and plasma-isolated virus, as DNA and cDNA respectively.

V Novitsky from the Essex lab presented on the dynamics of changes in Gag sequences in the global epidemic; how they change over time and the probable age of HIV subtype C in particular.

They sampled databases for sequences of 500 bases and up for gag, and found only 1800 -odd suitable sequences: these were mostly from South Africa, and Zambia, Malawi and Botswana.  The sequences were reduced for dating purposes to 433 by criteria such as <10 per year per country, while  966 were used for diversity.  They arbitrarily defined 9 groups of about 150 viruses over 20 years from 1983.  Interestingly, there was no clustering by year of sampling, or extinguishing of lineages.  SA had profound founder effects for 2 groups of viruses;  for diversity of Gag over time, one could see significant increase over time.  The p17 C terminus had the highest changes for this protein, p24 less so and spread throughout the sequence, with the most changes in the rest of Gag (p15).  Only 20 aa positions over 500 show consistent selection pressure changes, meaning the consensus sequence of Gag fom Subtype C HIV-1 remains pretty much same over more than 25 years.  They estimated the time of viral diversification from other subtypes to have been around 1959 – with a hefty uncertainty.

While the results may not seem exciting – and indeed, some people said “What’s new?” – the fact that Gag consensus sequences have essentially been stable over a protracted period is interesting; so too is the fact that lineages do not seem to have disappeared as one sees with influenza viruses with immune selection.  A very interesting virus, HIV-1….

HIV Vaccines From Bangkok – 1

14 September, 2011

Given that I am presently at the HIV Vaccine 2011 Conference here in Bangkok, I thought (belatedly) that I might blog on the proceedings, given Dorian McIlroy’s previous excellent example on CROI in recent months.

Reclining Buddha, Bangkok

Yesterday morning a Crown Princess of the Kingdom of Thailand was opening the first proper session of the oral proceedings: I was not there, as I needed breakfast after handling an email overload and didn’t feel like wearing a suit, so I missed an important performance by a Thai orchestra. Close call, that…!

We were there on Monday night, though, when a lineup of dignitaries presented in an opening plenary session.  First up was Pratap Singhasivanon, the Conference chair from Thailand. He introduced for the ignorant the long history and impressive list of Thailand’s achievements in the world of HIV vaccinology and prevention. It was sobering to hear that 40% of injecting drug users and 33% of men who have sex with men (MSM) were HIV+, despite that history.

Josè Esparza, acting head of the HIV Vaccine Enterprise, came next.  He was of the opinion that this is the Golden Age of HIV vaccines – an age of unprecedented successes and great promise, and that an HIV vaccine to end the pandemic is within reach. He told us that UNAIDS says that behaviour modification and testing is bringing down infection rates worldwide, which is another encouraging development. He thought that we Need increased and sustained financial support for the vaccine effort, however, including for a greater number of trials with short timelines so as to better test a wide range of possible vaccines.

Stanley Plotkin of Univ Pennsylvania is a luminary of the vaccine world, having helped as an industry insider to develop rubella and pentavalent rotavirus vaccines, among others: his job was to tell us how the success of other vaccines could inform the development of HIV vaccines. He said he had thought of saying “There are no lessons!” and sitting back down, but on reflection he had better not.

What he did share was that he thought that antibody response is king, but that it must be functional. A second lesson was that Ab at mucosal surfaces can give sterilising immunity. As an example, injected inactivated poliovirus vaccine (IPV) does not prevent shedding virus in gut while the live oral OPV does as it is much better at eliciting mucosal imm – but interestingly, at the pharynx both work.  A lesson from human papillomavirus vaccination was that while low Ab concentration did not prevent binding of the virus to the first receptor, it did prevent binding to the second – so entry of the virus into susceptible cells was prevented. Another lesson from polio was that high challenge dose can overwhelm immunity, and that IPV was a lot less good at protecting against high challenge doses. It was important that one could still get protection from disease in the presence of infection: for example, Rotateq rotavirus vaccine prevents disease very well, but vaccinees often get infected.

Ab- and cell-mediated immunity can also synergise: with smallpox it was found that both B and T cells are necessary for survival from vaccination, but on secondary exposure to infection in vaccinees, only Ab was necessary to prevent infection.

An important lesson for HIV was that several diseases required vaccine boosters in later life to maintain protection: with diphtheria, immunity in vaccinees declined dramatically while in those naturally infected it did not. Pertussis too needed boosters in children, and several more in ones lifetime to maintain functional immunity.

It was also important to revaccinate where pathogens changed significantly through time and with place – eg rotavirus was much more varied in Africa than elsewhere, as is HIV-1, and strains changed with time in one place, as do HIV and influenza viruses.

An important societal lesson was that vaccination of adolescents and high risk groups may not be accepted: Eg HPV vaccine coverage in the USA in adolescents was only 27% for all 3 doses, despite a very intensive campaign promoting the vaccine. HBV vaccination in high risk adults was also only at 50% and incidence only decreased when adolescents were vaccinated.

Herd immunity was also essential for public health success: eg pneumococcal vaccination of children protected old people indirectly as they were no longer exposed to the live pathogen in familial or sociatal settings.

His conclusions for HIV vaccines were that:

  • one needed a protective Ab response;
  • that IgA or IgG at mucosal surfaces may prevent transmission;
  • strong cellular responses will help control viral replication;
  • there is a good chance that we will get herd immunity;
  • the vaccine composition may have to change envelope component with time and or region;
  • regular boosters will probably be necessary;
  • public health may require universal vaccination of adolescents rather than only of high risk groups.

Sanjay Gurunathan of Sanofi Pasteur gave an industry view of how to move forward from the partially successful Thai RV144 vaccine trial, also reported here in Viroblogy. He observed that the traditional vaccine development model has large volume purchase in developed countries as the main driver, with industry doing R and D and clinical trials and the public sector doing purchase and delivery, with a trickle down to developing countries over time. He thought that HIV needs novel technology, and needs parallel development for 1st and developing worlds – with partnerships being of paramount importance together with guaranteed volume and price to some extent.

He noted that we must realise that for HIV vaccines failure will preceed success in an iterative process, that successes may be population-specific, that we may need multicomponent regimens, that we need to address developing country infrastructure – and that no company, NGO or even country can do it alone.

In this vein, he described a new partnership which was extending RV144 – this was P5, or the Poxvirus Protein Public Private Partnership, of the US NIAID, Gates Foundation, the HIV vaccine Trials Network, the US Military, Sanofi Pasteur and Novartis. This had in mind a broad poxvirus based protein boost regimen to further exploit the surprising success of the regimen in RV 144.

An important result from RV144 was that it was most efficacious at 12 months (60% efficacy) but that protection had dropped >30% by two years, indicating that boosting may significantly and positively impact level and durability of protection.

P5 want to increase efficacy to at least 50%, which would give a big impact for regional epidemics. There is historical precedent for this with cholera and meningococcal vaccines, neither of which is very good but which do impact public health. Their strategy will use a common regimen of poxvirus prime and a recombinant HIV gp120 boost, and will test MSM in Thailand and heterosexuals in South Africa. They planned to use MF59 or similar adjuvant to increase immune responses, unlike the earlier trial. Another new development was that they planned parallel development and clinical tracks, with a research arm in S Africa on NYVAC vaccinia plus protein and adjuvant and a DNA-poxvirus-protein combination.

An interesting evening – with promises of a major announcement to come the following day….