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.
MUCH MIND-NUMBINGLY BORING STATISTICAL METHODOLOGY FOLLOWED…sorry, Paul!
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.