Comment by reidrubsamen | Hacker News Reader

Comment by reidrubsamen | Hacker News Reader

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Hi there, Reid from Immunity Project. Thanks for the great question! Would love to talk with you off line about your research, by the way.

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.

Hi Reid, as I said before, I wish you all the best but I simply don't understand how your experiment 2 is going to work. Did I understand correctly that you will immunize mice and then isolate CD4 T cells from the spleen. Then infect those cells and see if cells from immunized mice die slower/less/not at all and don't express p24 in high levels. I'm afraid this experiment will not work this way. CD4 T cells by itself can not be immunized against HIV. The immunizing effect comes from memory B cells that produce antibodies. These antibodies bind to HIV infected CD4 T cells and as such label them as evil to be killed by other cells. CD4 T cells alone will not show any effect in regards to susceptibility to HIV no matter how well your immunization works. As rgejman pointed out, it would be way better to infect the mice with HIV and do a CD4 T cell count there every week. This would be so much easier and it might actually show you something relevant.

I think that they will incubate CD8 and CD4 T cells from the same mouse together. The CD8 (cytotoxic) T cells, if they recognize the antigen, should kill the infected CD4 cells.

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.

#1 suffers from the critique I mentioned above. The peptide(s) you are using are designed for H2d. Do you have evidence that their human analogs* are immunogenic?

#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

Ian from the Immunity Project team 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.

That's an interesting approach.

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!

Thanks for this thread. I really enjoyed seeing the science discussed. I've been interested in computational vaccine design and HIV evolutionary dynamics for a long time. I kind of see the whole thing as a dynamical system and a hidden Markov model. Essentially since the copy mechanism is leaky and produces multiple variants, and the immune system produces a selective response, how do you know that the single epitope you vaccinate against will be sufficient? I would expect their to be multiple stable populations and hence a need multiple vaccine epitopes (including populations that are stable under specific drug regimes).

This is what makes rgejman's phage display comments so interesting. We already know two major populations (Hiv-1 and Hiv-2 exist).

Great comments! We expect the beneficial targets to be HLA restricted. We need to do more work to understand if any one individual needs to hit two targets - we think right now that a "master vaccine" would need to contain multiple peptides to cover multiple HLA type individuals yielding at least one "valid" target per individual.

This sounds really interesting! Let me talk to my team. I would love to discuss this with you further. Please email me at team@immunityproject.org

The role of the TLR-4 and TLR-9 agonists; Are they essentially acting as adjuvants for your vaccine when delivered in conjunction with the peptide on the surface of the same microsphere?

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.

Thanks for the question! PLGA is the one of the most commonly implanted biodegradable materials in the world - primarily because it is used is used in dissolving suture (check out this link to very popular PLGA based suture material).

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.

Got it, thanks and all the best! I for one will be watching closely so keep the updates coming.

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