1. We have successfully gotten an immune response with memory (by elispot) after a single dose of the PLGA microspheres containing one or two peptides when combined with TLR-4 (MPLA) and TLR-9 (CpG) agonists. We used H2d restricted epitopes to do the study in C57BL/6 mice – this study is done and in the PRJ review process. We are being careful about posting the results in the event we need to re-submit to a journal who would get heartburn if they saw the data posted.
2. We are raising money now to essentially repeat the experiment (on a smaller scale because we think we understand the optimal formulation configuration now) in C57BL/6 NOG mice (with human PBMCs grafted in from a donor with known HLA type). The new experiment starts off looking like the experiment in (1) above: intradermal tail injection, wait 14 days, sacrifice, get the spleen, confirm immune response to peptide target by flurospot. The next part is new: separate CD8 from CD4 cells (from the mouse spleen), infect the CD4 cells with HIV, see if the vaccination attempt was good enough to see p24 antigen suppression and/or a change in the CD4 counts (all in-vitro). We can finish this study in a couple months, assuming the crowd funding keeps going well and we start in a couple weeks. We will use a different mechanism to publish our paper around this experiment so that we can get it out right away in a fashion that will lead to peer review and open access (We just learned about peerj.com – thoughts on that?).
Looking forward to hearing your thoughts on our approach.
A caveat is activation/co-stimulation. The CD8 T cells should already be activated due to the vaccination and no longer need co-stimulation from DCs... but this depends on the vaccine having delivered enough peptide to stimulate a true immune response in a human/humanized immune system. That's the (first) big unknown here.
#2 I assume now you are using the human version of the peptide? I'm not sure why you're doing this in vitro when you can now do it in vivo. Why not (a) infect the mouse with HIV, (b) look at viral titers w/ and w/o pre-vaccination, (c) look for specific CD8 T cells that have developed against peptide-HLA complexes.
I'm not sure I understand your in vitro experiment. Are you planning on mixing CD4 and CD8 T cells together in vitro from vaccinated/non-vaccinated mice and looking for increased killing of the CD4 cells (as estimated by decreased p24)?
*I assume it's mostly anchor changes here
Thanks for the follow up!
No evidence yet that human analogs are immunogenic. Doing new experiment to find out.
Yes! Using human version of the peptide for the new experiment. The NOG mouse model we are using will not infect with HIV - hence the in-vitro arm - I realize that humanized mice do exist that we could infect - we may try that later. Looking to take CD8/CD4 cells from vaccinated mice. Infect the (isolated) CD4 cells, combine and follow p24.
Re not knowing if the human analogs are immunogenic yet: An alternative way to to answer your question would be to identify a T cell receptor (TCR) clone that recognizes your peptides of interest. There are several ways of identifying TCR clones that match your peptide of interest. Probably the best way is via phage display. The advantages here are manifold:
1. By identifying a human TCR you immediately prove that your peptide is recognized by the human immune system (immunogenic)--you still need to do a bit more work to show that the immune system will produce this antibody in response to your peptide--but that's a pretty good start.
2. You could perform phage display separately on TCRs from elite controllers and more susceptible patients. The differences in these TCRs would likely make for a very interesting academic publication.
3. You could make a TCR-like antibody based on this TCR. If your vaccination strategy failed, hey at least now you have an antibody you can take into trials!
This is what makes rgejman's phage display comments so interesting. We already know two major populations (Hiv-1 and Hiv-2 exist).
Also, did you consider using nanospheres? Why PLGA?
EDIT: This is the bit I want some clarification on, from your white paper:
>These microspheres make the very small targets look large and threatening to the immune system, provoking an immune response after a single dose
EDIT2: Got my answer in the white paper, yes they are adjuvants. Still would like to know why PLGA and what the mechanism of how they appear more "threatening" is.
Aston S, Rees T. Vicryl Sutures. Aesthetic Plastic Surgery 1977;1:5.
The "threatening point" is just a way of describing what TLR-4 (MPLA) and TLR-9 (CpG) do. In particular, MPLA "looks like" bacterial cell wall and CpG "looks like" bacterial DNA to the immune system.
Steinhagen F. TLR-based immune adjuvants. Vaccine, 2011: 12.
We did not consider using nano spheres because we wanted to get as much payload as possible into the antigen presenting cells without requiring multiple phagocytosis events per cell.
Let's not overlook the decades of prior data! The field is littered with names like VRC01, Camelid and so many other players. I know, I know, antibodies all versus antigen in this case. But keep reading ...
HIV is a master at developing resistant forms of itself. What if it changes the epitope and evades the vaccine induced antibody(ies), again!?! (Edit: Don't forget the long-term viral reservoirs that survive against the best antiretrovirals known to us!)
This is something I care about personally: What is their strategy against resistance? (Or, like everyone else before them, they will build that bridge when the water is above their nose!?! Not being snarky, just very concerned.)
And might I also ask: Any concerns about accidentally inducing an autoimmune disorder with that antigen/epitope? Why/why not?
Added: I'd love to see these guys succeed and really want to see a rationale and approach that goes beyond what has been tried and not worked so far. Also, kudos for the open-science approach!
The targets we are trying to immunize with are believed to be beneficial by a statistical analysis similar to what is described here: Mothe B, Anuska L, Ibarrondo J, Daniels M, Miranda C, Zamarreno J, et al. Definition of the viral targets of protective HIV-1-specific T cell responses. Journal of Translational Medicine 2011;9(208):20.
The guys who generated the peptides we are using have specifically asked us not to talk about the sequences because they are trying to publish now. We hope that these targets will be conserved to the point that resistance is limited (as may be the case in some HIV controllers) - won't know without clinical data.
I am not asking about the peptide sequence, just the protein it is derived from. But, if you cannot share that right now, that's OK too. I'll look out for the publication.
However, assuming I am making an educated guess about the parent protein, I'd go with gp120/gp41 envelope (env). I may be totally wrong there and you guys may have another ace up your sleeve (do hope so, fingers crossed!). Good luck!
You first need to make humanize mice (i.e. mice with human immune systems). Luckily we can make those by taking NOD/SCID mice (an [expensive] immunodeficient mouse line) and then colonize them with human hematopoietic stem cells from a human donor to reconstitue the immune system. Then you have to do a lot of experiments to make sure your humanized mice can mount immune responses. Then, you can test your peptide and see if your humanized mice will make CD8 T cells to your peptide... Finally, you can infect your mice with HIV (now that you have a humanized immune system) and see how your vaccinated vs. unvaccinated mice fare.
I have no idea what the downsides of this approach are because I don't work on HIV or with humanized mice--but mice ain't people. So after all that work you'd have shed limited light on what would happen in real people.
I would argue that a better approach is to see whether HIV patients have actually made CD8 T cells against these antigens. This can be done more inexpensively and would give you pretty good evidence that your peptide is immunogenic in real people.
This vaccine relies on inducing immunity to an HIV peptide* (i.e. a ~9 amino acid cleavage product of an HIV protein) that is predicted to bind to HLA molecules. At first blush, this sounds like a great idea--let's immunize people to the very thing that the immune system is supposed to recognize as bad! Unfortunately, this has been tried lots of times and usually doesn't work very well, if at all. It's been tried in cancer, it's been tried in HIV, it's been tried in practically any system you can imagine. I'm not saying that peptide vaccines don't ever work (hell, my lab has worked on one)--I'm just saying that they have had lots of promise and limited successes. In fact, animals and patients have been immunized to many cleavage products of many cancer associated antigens to little, if any, effect—or effects in animals but not in humans. We can hem and haw all day about what the specific studies show, but suffice it to say that peptide vaccines are an old idea. Perhaps Immunity will get lucky here, but I'd be surprised if there isn't a lab somewhere that has tried immunizing animals to every single breakdown product of HIV proteins already.
Immunity's white paper says that their putatively immunogenic human peptide cannot be tested in animal models because it will only bind to human HLA. So they are planning to use a mouse-analog of the human peptide to do their animal testing (or have already done this?). That is, they are planning to use (or maybe have already used?) a completely different peptide (probably 1-3 amino acids different from the human version) in animal studies than what they would use in patients. This may be a significant difference... or it might not be. There's no way to tell a priori. A more classical and IMO better approach would be to identify T cell receptor clones from patients with HIV (or without HIV) that bind to these peptides (using tetramer staining, phage display or some other high-throughput technology), demonstrate that humans are capable of creating T cells that recognize their putatively immunogenic peptide followed by T cell killing assays on cells presenting their peptide of interest. There are other approaches, but that is a commonly used one in the field.
* in some places the white paper says "peptides" and in other it says "peptide" so it's not clear if we are talking about 1 peptide or multiple. I think they are mostly talking about multiple peptides, because they want to immunize people no matter their HLA type. Different HLA types bind to different peptides. So if you have HLA A02 you will bind different peptides than if you have HLA A24... and so on for all the different HLA types.