Posts Tagged ‘Thailand’

HIV Vaccines From Bangkok – 3

20 September, 2011

HIV: a retrovirus. Courtesy of http://www.rkm.com.au

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….

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

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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