New HIV Vaccine, gene therapy -"preventative drug and as a treatment" - NIH Study - Potential Long-Acting HIV Therapeutic eCD4-Ig"
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NIH-Funded Scientists Create Potential Long-Acting HIV Therapeutic
New Molecule also Might Prevent HIV Infection
Scientists have created a new molecule that shows promise for controlling HIV without daily antiretroviral drugs. The molecule foils a wider range of HIV strains in the laboratory than any known broadly neutralizing HIV antibody and is more powerful than some of the most potent of these antibodies. In addition, the molecule safely protected monkeys from infection with an HIV-like virus during a 40-week study period. Together, the data suggest that the molecule could, with further research, be used to subdue HIV in humans. The authors note that the molecule potentially could be used as both a preventative drug and as a treatment. The new findings appear in the February 18 issue of the journal Nature.
"This innovative research holds promise for moving us toward two important goals: achieving long-term protection from HIV infection, and putting HIV into sustained remission in chronically infected people," said Anthony S. Fauci, M.D., director of the National Institute of Allergy and Infectious Diseases (NIAID), part of the National Institutes of Health.
The research was led by Michael Farzan, Ph.D., a professor in the department of infectious diseases at The Scripps Research Institute in Jupiter, Florida. The work was funded primarily by NIAID.
The new molecule is called eCD4-Ig and works by tightly binding to two unchanging sites on the surface of HIV that the virus uses to attach to receptors on cells called CD4 and CCR5. Typically, when HIV attaches to these receptors, it unlocks a door to the cell and gets inside. However, when eCD4-Ig binds to HIV, it effectively takes away the virus's key, locking it out of the cell and preventing it from multiplying.
To make eCD4-Ig, the scientists took an antibody-like molecule that latches onto the CD4 binding site but does not neutralize HIV on its own, and fused it with a short protein fragment that attaches tightly to the CCR5 binding site. Together, these two arms of the molecule are much more effective at stopping HIV than either one is alone.
To test whether eCD4-Ig would protect monkeys from an HIV-like virus (simian immunodeficiency virus, or SIV), the scientists synthesized genetic instructions for making the molecule and placed them inside a harmless carrier virus called adeno-associated virus, or AAV. This gene-therapy tool was designed such that once the AAV-modified virus infected a cell, it would cause the cell to make eCD4-Ig indefinitely. The researchers injected the genetically modified AAV into four monkeys. Then they exposed both the treated monkeys and four untreated monkeys to SIV six times at increasingly higher doses over a 34-week period. None of the treated monkeys became infected with SIV, while all of the untreated monkeys did. The eCD4-Ig molecule made in the monkey's cells persisted in the animals' blood in a fully functional form and at protective concentrations for the entire 40-week study period.
In addition, the scientists found that while monkeys' immune systems view both eCD4-Ig and broadly neutralizing antibodies to HIV-like viruses as foreign molecules to some degree, the undesirable immune response generated by eCD4-Ig is milder than that generated by infusions of broadly neutralizing HIV antibodies. Scientists have been investigating these antibodies as another promising approach to long-acting treatment for HIV.
"Our molecule appears to be the most potent and broadest inhibitor of HIV entry so far described in a preclinical study," said Dr. Farzan. "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. Further research will help illuminate the promise of these approaches."
To build on their findings, the scientists are studying both the therapeutic potential of eCD4-Ig in monkeys infected with HIV-like viruses and the ability of eCD4-Ig to prevent infection against a wider range of HIV and HIV-like strains.
NIAID has funded the newly published and ongoing research through an initiative called Beyond HAART: Innovative Therapies to Control HIV-1, which supports the development of strategies for suppressing HIV in the absence of antiretroviral drugs. Dr. Farzan's principal partners in this endeavor are Ronald C.
Desrosiers, Ph.D., of the University of Miami Miller School of Medicine; Guangping Gao, Ph.D., of the University of Massachusetts Medical School; and Tajib A. Mirzabekov, Ph.D., of Biomirex Inc.
This research was supported by NIAID grants numbers R01 AI091476, R01 AI080324, P01 AI100263, and R01 AI058715. It was also supported by grant number P51 RR000168 from the National Center for Research Resources at NIH.
"research suggesting that protection against HIV infection may be achievable through a gene-therapy approach, rather than by relying on eliciting protective immune responses by vaccination......Gardner et al. went on to show that the eCD4-Ig construct imparted resistance to HIV-1 when infused into mice that model human HIV infections."
The study raises several questions and a few caveats. First, the modified protein is not natural and required the co-expression of an enzyme to perform the efficient addition of the sulfate moiety onto tyrosine residues. Second, the sample size of the monkey studies was quite small, and larger experiments in non-human primates are warranted. Furthermore, the intravenous challenge route, although rigorous, is not representative of the vast number of HIV-1 exposures worldwide, and it remains to be seen how expression of eCD4-Ig would affect virus challenges at mucosal sites, which better mimic natural routes of infection. It is also not yet clear whether the construct needs to be expressed close to the challenge sites. This, too, could be tested in non-human primate models.
Another major question rests in understanding the safety of eCD4-Ig in humans. Immune responses against the protein were elicited in the monkeys, albeit less strongly than against human NmAbs, and such responses could undermine its efficacy. But perhaps the greatest caveat to clinical application of the construct is how it, or future derivatives, will be used in humans. Such a complex molecule is unlikely to be administered repeatedly to those at risk of HIV infection, although that might be considered if it could be applied topically. The risks of expressing the construct as a transgene, in a similar manner to Gardner and colleagues' monkey experiments, are not known, and this approach would require careful and stepwise clinical safety testing. However, in the absence of a vaccine that can elicit broadly protective immunity and prevent infection, and given the lack of major breakthroughs on the horizon to provide one, the idea of conferring potent, sustained vaccine-like protection against HIV infection through gene therapy is certainly worth strong consideration.
HIV: Tied down by its own receptor (commentary) (original article below follows)
18 February 2015
Nancy L. Haigwood
the Division of Pathobiology & Immunology, Oregon National Primate Research Center, Oregon
Health & Science University, Beaverton, Oregon 97006, USA.
An engineered protein that binds to the envelope of HIV viruses protects monkeys against infection with a simian-human virus that causes AIDS. This gene-therapy approach might provide an alternative to elusive HIV vaccines.
The past 30 years have been marked by a long and discouraging search for an effective HIV vaccine. In 2009, the 'Thai trial' of the candidate vaccine RV144 was the first to demonstrate any success, measuring a 31.2% reduction in the rate of infection, although efficacy decreased over the first year after vaccination1. The difficulty in developing a more effective vaccine has forced investigators to explore problems that are posed by other intractable pathogens, including persistence in the host, a high degree of variability of certain regions, masking of common regions, and pathogen-induced inhibition of host immunity. But in a paper published on Nature's website today, Gardner et al.2 describe research suggesting that protection against HIV infection may be achievable through a gene-therapy approach, rather than by relying on eliciting protective immune responses by vaccination.
The trimeric envelope protein that is found on the surface of the viral particle of nearly all HIV strains binds directly to the CD4 receptor protein on the surface of many human immune cells, such as T cells and macrophages. This binding event causes a major shift in the envelope conformation, allowing the virus to bind to other co-receptors and enter the cell. It has been known since 1984 that CD4 is the receptor for HIV3, 4, and various forms of stabilized CD4 tethered to human immunoglobulin molecules (CD4-Ig) have been proposed and tested as potential therapeutics - the idea was that viral binding to these constructs would 'neutralize' the virus by preventing it binding to and entering cells. But this approach failed. Gardner and colleagues' findings provide the first logical explanation for this failure, and suggest an elegant way of using human CD4 derivatives to prevent infection.
The researchers engineered CD4 by fusing it with a mimetic of the amino terminus of CCR5, the host-cell co-receptor used by most HIV-1 strains during infection and disease progression. The CCR5 terminus has two sulfated tyrosine amino-acid residues that bind to the HIV envelope and facilitate viral entry5, so the peptide mimetic is a sulfopeptide. The mimetic was based on an antibody that binds to the CCR5 binding site of the viral envelope; the authors modified and positioned it in the CD4-Ig construct for maximum activity and fit.
This synthetic compound, named eCD4-Ig, has potent and broad neutralizing activity against all HIV isolates tested, including viral strains that are typically thought of as highly resistant to neutralization. It achieved these effects at lower concentrations than required when using the neutralizing monoclonal antibodies (NmAbs) that arise during the immune responses of some patients to HIV, and which are currently a major focus of attempts to develop HIV vaccines that prevent infection, rather than modulate viraemia once infection has occurred6. Furthermore, the construct was more effective than previous CD4-Ig constructs or the NmAb b12 at inducing immune killing of HIV-infected cells - a process known as antibody-dependent cellular cytotoxicity, which functions in concert with viral neutralization.
Gardner et al. went on to show that the eCD4-Ig construct imparted resistance to HIV-1 when infused into mice that model human HIV infections. As a further test of in vivo activity, the authors treated monkeys with an adenovirus-associated virus (AAV) that expressed the gene encoding a rhesus macaque version of the eCD4-Ig construct and with a separate AAV vector expressing a rhesus macaque enzyme to promote efficient sulfation. This gene-therapy vector allows continuous expression of the desired proteins in host cells by integrating into the host genome.
The animals expressed the transgene stably, although at different levels, and all were fully protected against repeated challenge with increasing doses of SHIV (a virus combining parts of the simian immunodeficiency virus (SIV) and HIV genomes). This protection was sustained for as long as 34 weeks after AAV transduction, and was achieved despite the monkeys receiving the virus intravenously, which is considered the infection route that provides the most stringent test of protection. These findings improve on an earlier test of the AAV transduction system to express a NmAb specific for SIV in monkeys7, in which only a subset of monkeys that expressed the transgene were protected from SIV challenge.
Why did Gardner and colleagues' construct work? In a nutshell, it all comes down to the way that eCD4-Ig binds to the virus (Fig. 1). Human NmAbs that are able to neutralize a broad range of HIV-1 strains do so by binding with very high affinity to shared viral structures (epitopes) that have precise but relatively small footprints. However, HIV has a variety of tricks to shield these shared epitopes from the immune system, although some infected individuals - referred to as elite neutralizers - do produce NmAbs of this sort. By contrast, CD4 binds to the envelope of all HIV-1 strains, albeit at lower affinity than these 'super potent' NmAbs. However, CD4 binding leads to a conformational change in the envelope that exposes the CCR5 binding site, thus potentially promoting HIV-1 infection in CCR5-expressing cells8. The modifications introduced by Gardner et al. into their eCD4-Ig construct seem to overcome this problem by preventing the engagement of envelope proteins with CCR5, while at the same time engaging multiple parts of the viral envelope, thereby increasing the binding power of their construct.
AAV-expressed eCD4-Ig provides durable protection from multiple SHIV challenges
18 February 2015
Matthew R. Gardner1*, Lisa M. Kattenhorn2*, Hema R. Kondur1, Markus von Schaewen3, Tatyana Dorfman1, Jessica J. Chiang2,
Kevin G. Haworth4, Julie M. Decker5, Michael D. Alpert2,6, Charles C. Bailey1, Ernest S. Neale Jr2, Christoph H. Fellinger1,
Vinita R. Joshi1, Sebastian P. Fuchs7, Jose M.Martinez-Navio7, Brian D.Quinlan1,Annie Y.Yao2,HugoMouquet8,9, Jason Gorman10,
Baoshan Zhang10, Pascal Poignard11, Michel C. Nussenzweig8,12, Dennis R. Burton11,13, Peter D. Kwong10, Michael Piatak Jr14,
Jeffrey D. Lifson14, Guangping Gao15, Ronald C. Desrosiers2,7, David T. Evans16, Beatrice H. Hahn5, Alexander Ploss3,
Paula M. Cannon4, Michael S. Seaman17 & Michael Farzan1
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.
Rhesus macaques inoculated with an AAV-based gene-therapy vector express antibody-like immunoadhesins for years, and these immunoadhesins afforded partial protection from a neutralization-sensitive simian immunodeficiency virus (SIV)2, suggesting that long-term sterilizing protection from HIV-1 might be achievable without a conventional vaccine. Full-length AAV-expressed bNAbs also protected humanized mice from an HIV-1 challenge1, 7. However, a large fraction of HIV-1 isolates remain partially or wholly resistant to even the best bNAbs, with IC80 values greater than 5 μg ml-1 measured under optimal in vitro conditions3, 4, 5, 6 (Extended Data Table 1). Higher concentrations will probably be necessary for broad-based protection in vivo, but primate studies suggest that these concentrations will be difficult to establish in humans2, 8. An effective AAV-based vaccine may therefore require broader and more potent inhibitors of HIV-1 entry.
The breadth of an antibody depends on the conservation of its epitope. The two most conserved epitopes of HIV-1 Env are its CD4- and coreceptor-binding sites9, 10, 11. The immunoadhesin form of CD4, CD4-Ig, has been extensively studied as a therapeutic. It neutralizes most isolates, irreversibly inactivates Env, and is demonstrated safe for use in humans12, 13, 14, 15. However, its affinity for Env is lower than those of bNAbs16, and its potency is further compromised by its parallel ability to promote infection17. Mimetics of the primary HIV-1 coreceptor CCR5, in particular peptides based on its tyrosine-sulfated amino terminus, have also been characterized18, 19. These sulfopeptides bind Env specifically but with low affinity in the absence of CD4, in part because they include hydrophobic residues and O-linked glycosylation that impede their association with Env18, 20. CCR5mim1, a 15-amino-acid sulfopeptide derived from the HIV-1 neutralizing antibody E51 (ref. 21), lacks these interfering elements (Fig. 1a) and binds Env with higher affinity than CCR5-based peptides20, 22. Reflecting the conservation of the sulfotyrosine-binding pockets of Env9, 10, CCR5mim1 binds both CCR5- and CXCR4-dependent Env proteins from all HIV-1 clades20, 22.
We reasoned that a fusion of CD4-Ig and CCR5mim1 would bind Env cooperatively and with higher avidity than either molecule alone. Accordingly, three fusion proteins were generated (sequences in Extended Data Fig. 1). CCR5mim1 was inserted at either the CD4-Ig amino terminus (fusion 1), between the CD4 and Fc domain (fusion 2), or at the CD4-Ig carboxy terminus (fusion 3, renamed eCD4-Ig). All three CD4-Ig variants neutralized CCR5- and CXCR4-dependent isolates more efficiently than did CD4-Ig, with eCD4-Ig consistently the most potent (Extended Data Fig. 2a, b). eCD4-Ig neutralized a wider panel of HIV-1 isolates and SIVmac316 with 10- to 100-fold lower IC50 values than CD4-Ig (Fig. 1b). Improved neutralization of SIVmac316 is consistent with conservation of the sulfotyrosine-binding pockets of Env9, 10, and a first indication of the exceptional breadth of eCD4-Ig.
To understand better the markedly greater potency of eCD4-Ig relative to CD4-Ig, we compared their abilities to bind cell-surface-expressed Env trimers (Fig. 1c). At low concentrations, eCD4-Ig bound these trimers more efficiently than did CD4-Ig. Surprisingly, eCD4-Ig saturated trimer-expressing cells with approximately one-third less bound protein than CD4-Ig, suggesting that the sulfopeptides of eCD4-Ig made some CD4-binding sites inaccessible. eCD4-Ig also less efficiently promoted HIV-1 infection of CCR5-positive, CD4-negative cells than CD4-Ig (Fig. 1d), presumably because its sulfopeptides blocked virion access to cell-surface CCR5. Heterodimers of CD4-Ig and eCD4-Ig23 neutralized less potently than eCD4-Ig (Fig. 1e and Extended Data Fig. 2c-e), indicating that both eCD4-Ig sulfopeptides engage the Env trimer, consistent with a model of eCD4-Ig bound to Env (Extended Data Fig. 3) and previous studies of CCR5mim1 (ref. 24). Thus, the markedly greater potency of eCD4-Ig relative to CD4-Ig is due in part to the higher avidity with which it binds Env and to its decreased ability to promote infection.
We next assessed eCD4-Ig under more physiological conditions. We observed that eCD4-Ig, but not CD4-Ig, halted replication of infectious viruses in human peripheral blood mononuclear cells (PBMC) at concentrations as low as 125 ng ml-1 (Extended Data Fig. 1f, g). We administered sufficient eCD4-Ig to humanized NOD/SCID/Il2rg-/- (NSG) mice to maintain serum concentrations of 2-4 μg ml-1 at the time of challenge. Five eCD4-Ig-treated mice and six control mice were challenged intravenously with 5 x 104 infectious units of HIV-1NL4-3. Five out of six control mice, but no eCD4-Ig-inoculated mice, were infected (Fig. 1f and Extended Data Fig. 2h). Five weeks later, three eCD4-Ig-treated mice and the uninfected control mouse were rechallenged. Again, no eCD4-Ig-treated mouse was infected, whereas the control mouse became infected.
We then characterized the ability of eCD4-Ig to neutralize a diverse panel of neutralization-resistant tier 2 and 3 viruses25 (Extended Data Figs 4a and 5a). In parallel, we assayed three additional eCD4-Ig variants. In the first, eCD4-Igmim2, CCR5mim1 was replaced by CCR5mim2, which differs from CCR5mim1 by a single Ala to Tyr substitution22. We also introduced a previously characterized Gln 40 to Ala mutation into the CD4 domain 1 of eCD4-Ig (eCD4-IgQ40A)16. Both mutations were combined in a final variant (eCD4-IgQ40A,mim2). eCD4-Ig and these variants substantially outperformed CD4-Ig for every virus in the panel, typically improving neutralization potency by 20- to >200-fold. Underscoring its breadth, eCD4-Ig neutralized SIVmac251 33 times more efficiently than CD4-Ig. In general, the more neutralization-resistant a virus, the better eCD4-Ig and its variants performed relative to CD4-Ig. In most cases, replacement of CCR5mim1 with CCR5mim2 modestly improved neutralization. Similarly, the Gln40Ala mutation also improved neutralization of most HIV-1 isolates, but not of SIVmac251.
We compared eCD4-Ig, eCD4-Igmim2 and eCD4-IgQ40A,mim2 with a panel of 12 antibodies and inhibitors using three additional HIV-1 isolates (Fig. 2a and Extended Data Fig. 6a, b). eCD4-Ig and its variants neutralized the SG3 and YU2 isolates more efficiently than any of these inhibitors. Five bNAbs neutralized JR-CSF more efficiently than any eCD4-Ig variant, but four of these could not neutralize SG3. All eCD4-Ig variants neutralized these isolates with IC50 values less than 0.3 μg ml-1, which is more efficiently than CD4-Ig, the tetrameric CD4-Ig variant PRO-542 (refs 12, 14), or the antibodies 2G12, 4E10 and VRC01. eCD4-Ig and its variants, but not three CD4-binding site bNAbs, neutralized the neutralization-resistant SIVmac239 as well as HIV-2 strain ST (Fig. 2b and Extended Data Fig. 6c). As observed with SIVmac251, the Gln40Ala variant was less efficient at neutralizing SIVmac239 and HIV-2. The potency of these eCD4-Ig variants was also reflected in their abilities to mediate antibody-dependent cell-mediated cytotoxicity (ADCC). eCD4-Ig, eCD4-Igmim2 and eCD4-IgQ40A,mim2 each facilitated 30-40 times more killing of infected cells by CD16+ natural killer cells26 than did CD4-Ig or the antibody IgGb12 (Fig. 2c). Thus the C-terminal modification of eCD4-Ig did not interfere with the ADCC effector function of its Fc domain.
We further evaluated eCD4-Ig, eCD4-Igmim2, eCD4-IgQ40A,mim2 and the bNAb NIH45-46 using nearly every isolate reported to be resistant to either of the CD4bs antibodies NIH45-46 or 3BNC117 (Extended Data Figs 4b and 5b). Both eCD4-Ig variants efficiently neutralized all 38 resistant isolates assayed with IC50 values ranging from <0.001 to 1.453 μg ml-1. By contrast, 26 isolates in this panel were confirmed to be resistant to NIH45-46. Previous reports found 29 and 18 isolates to be resistant to 3BCN117 and VRC01, respectively4, 6. Figure 3 and Extended Data Fig. 7 summarize the neutralization studies compiled from the experiments in Figs 1 and 2 and Extended Data Figs 4, 5, 6, and from previous studies of VRC01 and 3BNC117 against the same isolates4. They show that the geometric mean IC50 and IC80 values of eCD4-Ig and its variants are less than 0.05 μg ml-1 (500 pM) and 0.2 μg ml-1 (2 nM), respectively, roughly 3-4 times lower than those of VRC01, NIH45-46 or 3BNC117. Importantly, our lead eCD4-Ig variant, eCD4-Igmim2, neutralized 100% of the isolates assayed at concentrations (IC50 < 1.5 μg ml-1; IC80 < 5.2 μg ml-1) that are probably sustainable in humans.
Finally, using a rhesus macaque form of eCD4-Igmim2, we investigated whether AAV-delivered eCD4-Ig could function like an HIV-1 vaccine. To minimize potential adverse reactions, the Fc domain of rhesus IgG2, which binds Fc receptors and complement less efficiently than IgG1, was used. We also introduced an Ile39Asn mutation into the CD4 domain27 to correct partially the lower affinity of rhesus CD4 for most HIV-1 isolates (Extended Data Fig. 8a, b). The gene for the resulting construct, rh-eCD4-IgG2I39N,mim2 (described hereafter as rh-eCD4-Ig), was cloned into a single-stranded AAV2 vector (AAV-rh-eCD4-Ig; Extended Data Fig. 8c). A total of 2.5 x 1013 AAV1-encapsidated particles delivering this vector were administered into the quadriceps of four four-year-old male Indian-origin rhesus macaques. To promote rh-eCD4-Ig sulfation, a separate single-stranded AAV vector expressing rhesus tyrosine-protein sulfotransferase 2 (AAV-rh-TPST2; Extended Data Fig. 8c) was co-administered with AAV-rh-eCD4-Ig at a 1:4 ratio. No adverse reactions were observed in any of the AAV-rh-eCD4-Ig-inoculated macaques. These macaques and four age- and gender-matched controls were challenged intravenously with increasing doses of SHIV-AD8 (Fig. 4a, b). Sixteen weeks after AAV inoculation, two control macaques became infected following challenge with 200 pg p27. A subsequent 400 pg challenge infected a third control animal, and, after resisting an additional 400 pg challenge, the final control was infected with 800 pg, 34 weeks from the date of AAV inoculation. None of these challenges infected AAV-rh-eCD4-Ig-inoculated macaques, indicating that eCD4-Ig protected them from four doses capable of infecting control animals.
Measured rh-eCD4-Ig titres in the serum stabilized to between 17 and 77 μg ml-1 over the last 10 weeks of the 40-week study period (Fig. 4c). Two macaques expressed less than 20 μg ml-1 at the time of the final 800 pg challenge, suggesting that this concentration could prevent many otherwise infectious exposures in humans. Sera from inoculated macaques neutralized HIV-1 as efficiently as laboratory-prepared rh-eCD4-Ig mixed with pre-inoculation sera (Fig. 4d and Extended Data Fig. 8d), indicating that the eCD4-Ig was efficiently sulfated and fully active in vivo. We also compared macaque humoral responses to expressed rh-eCD4-Ig and to four AAV-expressed bNAbs inoculated for a separate study. 3BNC117, NIH45-45, 10-1074 and PGT121, each bearing rhesus IgG2 and light-chain constant domains, elicited markedly higher endogenous antibody responses than did rh-eCD4-Ig, consistent with their high levels of somatic hypermutation (Fig. 4e). To investigate the target of the anti-rh-eCD4-Ig responses, we increased the sensitivity of our assay and compared longitudinally the reactivity of inoculated rhesus sera to a series of antigens. rh-eCD4-Ig (Fig. 4f) and rh-CD4-Ig (without the CCR5mim2 sulfopeptide; Fig. 4g) were recognized by rhesus sera with nearly the same reactivity, whereas CCR5mim2 fused to a human IgG1 Fc domain was not (Fig. 4h), indicating that the sulfopeptide was not immunogenic. Rhesus CD4 domains 1 and 2 fused to a human IgG1 Fc was much less reactive than the same CD4 domains fused to the rhesus IgG2 Fc, without or with the Ile39Asn mutation (Extended Data Fig. 8e, f), whereas an unrelated construct bearing the rhesus IgG2 Fc domain showed no reactivity (Extended Data Fig. 8g), suggesting that a neo-epitope formed by the rhesus CD4 and Fc domains was recognized by most anti-rh-eCD4-Ig antibodies. Thus eCD4-Ig is less immunogenic than bNAbs, and can be expressed for at least 40 weeks at concentrations that are well tolerated and protective against several robust SHIV-AD8 challenges.
A key question is whether eCD4-Ig or a similar construct could be used to prevent new HIV-1 infections in a population, and whether it might do so more effectively than a bNAb. We show that AAV-delivered rhesus eCD4-Ig protected all inoculated macaques from multiple infectious doses that are probably higher than those present in most human transmission events, although we have not yet tested protection from mucosal challenges. Protection lasted at least 34 weeks after inoculation (Fig. 4b), and other studies indicate that these protective titres can be sustained for several years2. Previous studies of CD4-Ig indicate that it is safe when passively administered12, 14, and in particular it does not engage MHC II or otherwise interfere with immune function13, although further safety studies of eCD4-Ig are warranted. eCD4-Ig has fewer non-self B- and T-cell epitopes than heavily hypermutated bNAbs, and thus elicits fewer endogenous antibodies that can impair its expression and activity (Fig. 4e). Its most prominent non-self element is its sulfopeptide, which did not elicit any measurable antibody responses (Fig. 4f-h). However, the clearest advantage of eCD4-Ig over bNAbs is its potency and its unmatched breadth (Fig. 3 and Extended Data Figs 4, 5, 6, 7). The breadth of eCD4-Ig arises from the necessary conservation of its binding sites on Env, suggesting that emergence of eCD4-Ig escape variants in a population is less likely than with bNAbs. Moreover, any virus that does bypass prophylaxis is likely to bind CD4 and CCR5 less efficiently in the continued presence of eCD4-Ig, and may therefore be less efficiently retransmitted. Its potency suggests that relatively lower concentrations of eCD4-Ig will be sufficient to protect against most circulating viruses, a feature that may be critical to its use with AAV in humans. Although there are remaining challenges, these observations suggest that AAV-expressed eCD4-Ig could provide effective, long-term and near universal protection from HIV-1.