Best in Class

Adding to the now lengthy list of broadly neutralizing antibodies is the recently discovered antibody N6. This one tops the list in terms of neutralization breadth—knocking out 98 percent of HIV isolates—and also packs a potent anti-viral punch.

By Kristen Jill Kresge

Antibody researchers are experiencing a windfall. They continue to isolate HIV-specific antibodies that can neutralize a broad swath of HIV isolates, so-called broadly neutralizing antibodies (bNAbs), at a rapid clip even though they are made by only about 20 percent of HIV-infected individuals. From what was a meager handful of bNAbs just seven years ago, researchers have amassed hundreds of these powerful infection-fighting proteins to guide vaccine design, aid in HIV prevention more broadly, serve as a potential long-acting HIV treatment, or even be part of an eventual cure strategy.

One of the more recent antibodies isolated by researchers at the US National Institute of Allergy and Infectious Diseases (NIAID) is getting top billing for its unprecedented ability to knock out 98 percent of HIV isolates, and to do so at a low dose, meaning it is potent as well (Immunity 45, 1108-1121, 2016). This antibody, dubbed N6, targets the CD4 binding site on the virus—the critical location where HIV attaches to CD4+ T cells, its primary targets (see image, below). Several other antibodies researchers have isolated recently also target this spot on the virus, the most well studied of them being the antibody VRC01 that was isolated back in 2010 by scientists at the Vaccine Research Center (VRC) at NIAID. But none of them so far can match the breadth or potency of N6, which are just some of the attributes that make this antibody so intriguing to vaccine researchers. Another factor is that N6 is even active against the vast majority of virus isolates that are resistant to other CD4 binding-site antibodies. Taken together, these characteristics make N6 an attractive antibody from which to design vaccine immunogens.

In addition to helping guide vaccine design, these bNAbs have other applications. In animal studies, passively administered or vector delivered bNAbs can prevent infection (Curr. HIV/AIDS Rep. 13, 31–37, 2016). Clinical trials are now testing whether passive administration of VRC01 (given during regularly scheduled intravenous infusions) can block HIV infection in humans (https://clinicaltrials.gov/ct2/show/NCT02716675). An antibody like N6, which researchers note is five to ten times more potent than VRC01, is an attractive candidate for passive administration because less antibody would need to persist for the protective effect to be sustained. Still, given HIV’s unprecedented ability to mutate to avoid immune responses, researchers suspect an ideal recipe for passive administration would be a cocktail of the best bNAbs targeting multiple sites of vulnerability on the virus. N6, the latest and greatest in the class of CD4 binding site antibodies, would definitely be a prime candidate for this.

As Devin Sok of IAVI and Dennis Burton of IAVI’s Neutralizing Antibody Center at The Scripps Research Institute in La Jolla, CA, suggest (Immunity 45, 958-960, 2016), “Not only will the continued isolation of bNAbs like N6 contribute to vaccine efforts, but antibodies with such levels of breadth and potency and minimal autoreactivity are also being championed for potential use as prophylactic and therapeutic agents.”

Managing Editor Kristen Jill Kresge spoke recently with Mark Connors, chief of the HIV-specific immunity section at NIAID whose laboratory isolated N6, about this all-star antibody and its unique mechanism of action.

Could you start by explaining how N6 was isolated?

The method of isolation we use is micro-culture of peripheral blood B cells. The strength of that is that it allows you, without any sort of prior knowledge, to ask the question of what in this patient is mediating neutralization. The patient from whom N6 was isolated had particularly broad and potent serum. That technique has allowed us to isolate novel antibodies that maybe wouldn’t have been expected. In this case we had some inkling that at least one of this patient’s specificities would have been a CD4 binding site antibody, and that is what we pulled out. The remarkable thing in initial testing was that it has pretty remarkable breadth and potency, but also that it can neutralize the VRC01-resistant isolates quite potently. So we thought that this antibody might have a novel mode of binding and that we could learn some things from this.

Is it unusual among the antibodies isolated to date to show such potency and breadth at the same time?

Yes, it has been so far. Some of the most potent antibodies like PGDM1400 and PGT16 tend to be a bit less broad. And some of the most broad antibodies, such as 10E8, are considerably less potent. N6 combines both of those features into one antibody.

The potency has become more and more of a focus in addition to breadth because it increases the likelihood, from a practical standpoint, that you can use the antibody for passive administration for prophylaxis. Combining some of these antibodies with a long-acting mutation that enhances the half-life potentially could offer the possibility—if the antibody’s potent enough—that you could administer it subcutaneously on the order of months apart between doses.

When the idea of passive administration was first introduced it seemed that people described it as a way to show that bNAbs could indeed protect against infection and therefore would be reasonable vaccine targets. Now it’s considered an implementable prevention strategy. Was there a switch in how passive administration is viewed?

Well I think that the incorporation of some of these mutations that alter the half-life are really what changed people’s minds with regard to that, as well as the increases in potency we see with some of the more recently isolated antibodies. Once we got to the threshold where you could potentially administer a dose subcutaneously and that dose is going to last for months, then that made this prevention approach feasible. In earlier years we had neither of those two things: the antibodies were not especially potent and they had not been combined with some of these binding mutations that would enhance half-life.

Can you describe how N6 uniquely neutralizes HIV?

Well it binds the CD4 binding site similarly to other antibodies, but there’s an important twist there, and that is that its flexibility allows it to shift the light chain out of the way. One of the constraints of CD4 binding site antibodies is that they require the short CDRL3 [light chain complementarity-determining region 3] in order to get out of the way of changes in the V5 loop of the virus. Meanwhile, the virus co-evolves to change V5 and put up bulky groups there that potentially can keep VRC01-like antibodies from binding. N6 sort of rotates the light chain out of the way and so it enables it to bind some of these highly resistant isolates that have changes at the V5 loop, and therefore N6 maintains its potency against some of those viruses.

Another thing is that N6’s binding seems to be more spread out across the various binding regions of the antibody, so it is better able to tolerate point mutations. Even if HIV makes changes in individual amino acids, N6 is better than other CD4 binding site antibodies at tolerating the loss of some of those contacts.

Those two features together really dramatically improve the breadth of the N6 antibody and its ability to neutralize resistant isolates.

What are the next steps for N6 and optimizing it for testing in animal studies or in humans?

Our lab is more involved in discovery of new antibodies and trying to stimulate specificities similar to N6, but we have collaborators either in large pharma or at universities, as well as at the VRC, who are pursuing many of these avenues of research. The pharmacokinetic studies in macaques have already been done. And Dan Barouch has done some studies of N6 in therapy in macaques… There are quite a few different groups who are pursuing this for either therapy or prophylaxis in macaques, and then ultimately in humans.

What advantages, if any, do CD4 binding site-specific antibodies offer to vaccine researchers?

Well, number one is that this is a highly conserved site. I think that most people now feel that we’re going to need to induce neutralizing antibodies to at least a couple of different sites, and so the CD4 binding site, in addition to MPER, and possibly some of the apex-directed antibodies, are a few possible sites that are areas of vulnerability, or so-called vulnerability for HIV. The research that we, Dennis Burton, John Mascola [director of the VRC], Michelle Nussenzweig [Zanvil A. Cohn and Ralph M. Steinman Professor in the laboratory of molecular immunology at the Rockefeller University], and others in the field that have discovered these antibodies have done has now put us in a position where we could potentially use that information. The answer that we’ve gotten back is that here are the areas of HIV vulnerability, and I think that probably it’s wise to target those areas that are relatively conserved. Clearly the human immune response can mediate broad and potent neutralization through those areas of vulnerability. So, I think the human immune system has now told us what the critical vaccine targets are.

So do you foresee a future passive administration trial involving multiple antibodies?

Yes. It’s just getting started now with single antibodies before using multiple antibodies in animal models, but it’s just a matter of time. The fraction of isolates that aren’t sensitive to N6 are smaller than for other antibodies, and it’s possible that combining these antibodies that are increasingly difficult to mutate around together might be the best possible combination to move forward with, but that has not yet been demonstrated in animal experiments.

One important feature that I and others have done a lot of work on is the resistance pattern after passive administration of antibodies. It remains to be determined what the relative advantage that antibodies like N6 have with regard to the selection of resistance, but it’s another property in addition to potency and breadth that needs to be considered.