A paper published yesterday in Nature has stirred considerable excitement and widespread media coverage. Led by the laboratory of Michael Farzan at The Scripps Research Institute, the research involves a newly designed inhibitor of HIV named eCD4-Ig. The inhibitor is designed to bind the HIV envelope at sites that attach to CD4 and CCR5 molecules on CD4 T cells; blocking this interaction prevents HIV from gaining entry and infecting the cell. In laboratory experiments, eCD4-Ig showed activity against an unprecedentedly broad array of HIV and SIV variants, including viruses known to be resistant to even potent neutralizing antibodies. In addition to the greater breadth, activity was generally achieved at lower concentrations than has been documented with the broadly neutralizing antibodies (bNAbs) described to date. Due to similarities between HIV envelope binding to CCR5 and CXCR4 co-receptors, eCD4-Ig also proved capable of neutralizing CXCR4-dependent virus isolates. These results prompted the researchers to explore the preventive potential of eCD4-Ig in the rhesus macaque model, and it is the outcome of this part of the study that has particularly spurred the publicity.
To deliver the eCD4-Ig (which was modified to match the rhesus macaque form of the protein), the researchers chose to incorporate the gene encoding eCD4-Ig into an AAV vector. AAV vectors are taken up by muscle tissue and act as a factory for producing the encoded protein; for this reason they have been widely used in gene therapy studies (to deliver factor IX to hemophiliacs, for example) and are also under evaluation as a method to deliver neutralizing antibodies against HIV. One small twist with the eCD4-Ig macaque study is that the researchers delivered an 80/20 mix of two AAV vectors: one encoding eCD4-Ig, the other encoding the gene for tyrosylprotein sulfotransferase 2 (TPST2). The role of the TPST2 is to alter the eCD4-Ig protein via a process called sulfation, and this is necessary to make the eCD4-Ig active. It’s not evident from the paper whether preliminary experiments indicated the addition of TPST2 was needed, or if the decision was based solely on biological considerations.
Four macaques were administered the AAV vector mix and four served as controls. All animals were subsequently challenged intravenously with escalating doses of a hybrid SIV/HIV virus (SHIV-AD8). The four controls all eventually became infected, but the AAV recipients resisted infection despite multiple challenges over a 34-week period. Serum levels of eCD4-Ig were stable at the end of the 40-week study, and sera from the macaques neutralized HIV in vitro just as effectively as eCD4-Ig produced in the laboratory. Although antibody responses against the eCD4-Ig could be detected in the macaques, the levels were very low (much lower than were seen against the broadly neutralizing monoclonal antibodies 3BNC117, NIH45-45, 10-1074 and PGT121).
The researchers cite several features of the approach that may make it suitable for advancing into human trials.
- Protection was achieved at eCD4-Ig concentrations that are likely sustainable in humans, and the challenge dose of virus was higher than is the case in most human transmission events.
- Previous macaque studies using AAV vectors to encode bNAbs have shown that protective titers can be sustained for at least several years.
- A related protein that only targeted the CD4 binding site of HIV, CD4-Ig (also known as PRO 542), has been tested in human trials and found to be safe.
- eCD4-Ig does not appear likely to provoke significant immune responses against itself in humans.
But there are also some potential hurdles and caveats:
- The macaque experiment involved intravenous challenges and efficacy against a mucosal challenge has yet to be assessed.
- Whether eCD4-Ig can safely be administered to humans remains to be established.
- Advancing what is essentially a gene therapy approach into healthy individuals raises safety concerns that regulatory agencies will want to scrutinize very carefully (notably, the first ever phase I human clinical trial of an AAV vector encoding a bNAb against HIV began in the UK last year).
- Further research is needed to establish if HIV can become resistant to eCD4-Ig, although the researchers believe that if the virus did develop the ability to bind CCR5 and CD4 in the presence of eCD4-Ig, the binding efficiency would be extremely impaired.
- The safety of delivering the adjunctive TPST2 protein will need to be established. Rather than use a separate AAV vector to deliver TPST2, as was the case in the macaque study, the aim is to develop a single AAV vector that encodes both eCD4-Ig and TPST2. Human TPST2 is naturally expressed in multiple tissues, including muscle, giving some reason to hope that delivery via AAV vector would not be problematic.
- There is evidence that the presence of AAV-specific memory CD8 T cell responses in humans can limit the ability of AAV vectors to deliver their protein cargo (a problem seen in hemophilia gene therapy trials). Whether the problem might affect AAV-based approaches for HIV is not yet known. Strategies to limit CD8 T cell recognition of AAV vectors are actively being pursued.
The paper does not explicitly discuss whether eCD4-Ig might have therapeutic potential (focusing instead on prevention) but notes that it is a potent inducer of antibody-dependent cell-mediated cytotoxicity (ADCC); it has been postulated that induction of ADCC against HIV-infected cells could be a component of a combination strategy to eliminate HIV reservoirs. A press release from the National Institute of Allergy and Infectious Diseases (NIAID) addresses the issue more directly and refers to eCD4-Ig as a “potential long-acting HIV therapeutic,” with a quote from Michael Farzan stating: “if one could inject either eCD4-Ig or our gene therapy tool into people with HIV infection, it might control HIV for extended periods in the absence of antiretroviral drugs.” The release also notes that therapeutic studies in macaques are getting underway, funded under NIAID’s recent Beyond HAART: Innovative Therapies to Control HIV-1 RFA.
According to an article in the New York Times, the researchers are considering a stepwise development plan that would first test infusions of the eCD4-Ig protein in healthy volunteers, then evaluate AAV-delivered eCD4-Ig (and, presumably, TPST2) in HIV-positive individuals, then finally move the AAV-delivered version into trials in HIV-negative populations at high risk of HIV infection. In the Wall Street Journal, Farzan articulates the hope that “human trials could begin within a year” but this may be overly optimistic; given the additional work that needs to be done, two or three years might be more realistic.
Links to several news articles about the research are below.
Stopping HIV with an artificial protein - Jon Cohen, Science Magazine, February 18, 2015
New Approach to Blocking H.I.V. Raises Hopes for an AIDS Vaccine - Donald G. McNeil Jr., New York Times, February 18, 2015
Molecule Shows Ability to Block AIDS Virus - Betsy McKay, Wall Street Journal, Feb. 18, 2015
Update 3/6/15: A webcast of Michael Farzan's presentation at CROI 2015 is now available. In the talk Farzan reveals that, since the publication of the Nature paper, the eCD4-Ig recipients have been challenged with two higher doses of SHIV-AD8 and remain uninfected.
Nature (2015) doi:10.1038/nature14264
Received 29 June 2013 Accepted 27 January 2015 Published online 18 February 2015
Matthew R. Gardner, Lisa M. Kattenhorn, Hema R. Kondur, Markus von Schaewen, Tatyana Dorfman, Jessica J. Chiang, Kevin G. Haworth, Julie M. Decker, Michael D. Alpert, Charles C. Bailey, Ernest S. Neale, Christoph H. Fellinger, Vinita R. Joshi, Sebastian P. Fuchs, Jose M. Martinez-Navio, Brian D. Quinlan, Annie Y. Yao, Hugo Mouquet, Jason Gorman, Baoshan Zhang, Pascal Poignard, Michel C. Nussenzweig, Dennis R. Burton, Peter D. Kwong, Michael Piatak Jeffrey D. Lifson, Guangping Gao, Ronald C. Desrosiers, David T. Evans, Beatrice H. Hahn, Alexander Ploss, Paula M. Cannon, Michael S. Seaman & Michael Farzan
Long-term in vivo expression of a broad and potent entry inhibitor could circumvent the need for a conventional vaccine for HIV-1. Adeno-associated virus (AAV) vectors can stably express HIV-1 broadly neutralizing antibodies (bNAbs)1, 2. However, even the best bNAbs neutralize 10–50% of HIV-1 isolates inefficiently (80% inhibitory concentration (IC80) > 5 μg ml−1), suggesting that high concentrations of these antibodies would be necessary to achieve general protection3, 4, 5, 6. Here we show that eCD4-Ig, a fusion of CD4-Ig with a small CCR5-mimetic sulfopeptide, binds avidly and cooperatively to the HIV-1 envelope glycoprotein (Env) and is more potent than the best bNAbs (geometric mean half-maximum inhibitory concentration (IC50) < 0.05 μg ml−1). Because eCD4-Ig binds only conserved regions of Env, it is also much broader than any bNAb. For example, eCD4-Ig efficiently neutralized 100% of a diverse panel of neutralization-resistant HIV-1, HIV-2 and simian immunodeficiency virus isolates, including a comprehensive set of isolates resistant to the CD4-binding site bNAbs VRC01, NIH45-46 and 3BNC117. Rhesus macaques inoculated with an AAV vector stably expressed 17–77 μg ml−1 of fully functional rhesus eCD4-Ig for more than 40 weeks, and these macaques were protected from several infectious challenges with SHIV-AD8. Rhesus eCD4-Ig was also markedly less immunogenic than rhesus forms of four well-characterized bNAbs. Our data suggest that AAV-delivered eCD4-Ig can function like an effective HIV-1 vaccine.
Nature (2015) doi:10.1038/nature14205
Published online 18 February 2015
Nancy L. Haigwood