The membrane-proximal external region of HIV-1 gp41: a vaccine target worth exploring (2005)

HIV-1 vaccine design and the membrane-proximal external region (MPER) of gp41 Neutralizing antibody titers are correlated to protection for most major vaccines [1–3], but eliciting robust neutralizing antibody responses against HIV remains a major stumbling block for vaccine development [4–7]. Even during HIV-1 infection, strong neutralizing antibody titers against heterologous primary isolates are rare, and quite slow to develop [8–10]. Nevertheless, passive transfer of neutralizing monoclonal antibodies can completely protect against lentiviral challenge, at least in animal models, demonstrating a potential for vaccine-elicited antibodies to prevent an established infection with HIV-1 [11–14]. As only antibodies against conserved epitopes on HIV-1 will neutralize a broad range of different viral isolates, eliciting such antibodies should be a priority for vaccine design. Fortunately, a few human monoclonal antibodies have been described that can neutralize primary HIV-1 with exceptional breadth and potency. These antibodies target conserved regions on the envelope glycoproteins, gp120 and gp41, which putatively assemble as a trimer of gp120–gp41 heterodimers on the surface of the virus. Among the broadly neutralizing antibodies, IgG1b12 [15,16] and 2G12 [17] cross react with monomeric gp120, and strategies to elicit anti-gp120 antibodies are discussed elsewhere [18–23]. The major focus of this review will be the target on the transmembrane protein, gp41, of the broadly neutralizing antibodies 2F5, 4E10 and Z13 [24–27]. The finding that these three antibodies recognize neighboring sites on the conserved membrane-proximal external region (MPER) on gp41, defined here roughly as residues 660 to 683 of HIV-1 gp160, has brought attention to this whole region for vaccine design [26]. Subsequently, interest in the MPER of gp41 as a vaccine target has risen, as numerous studies have broadened our understanding of the structure and function of the MPER of gp41, as well as of the antibodies that bind to it. It should be made clear, though, that a vaccine designed to elicit neutralizing antibodies against HIV will most likely integrate conserved targets on both gp120 and gp41. It should also be established that non-neutralizing antibodies appear to dominate in the antibody response against gp41, both during natural infection, and through immunization [28–30]. These antibodies typically target membrane-distal regions, which tend to be occluded by quaternary interactions within the HIV-1 envelope glycoprotein (Env) trimer [31–33]; the MPER appears to be an exception. This review will cover what is known about the structure and function of the MPER of gp41, compare it with other vaccine targets, and address some of the challenges of exploiting membrane-proximal epitopes on HIV-1 in vaccine design. In-vivo and in-vitro anti-HIV-1 activity of 2F5, 4E10 and Z13 The main justification for targeting the MPER of gp41 for vaccine design is the great breadth with which the human monoclonal antibodies 2F5 and 4E10 neutralize primary isolates of HIV-1 (see below). Importantly, however, these antibodies, often in various cocktails, can contribute some level of protection against infection in animal models, although their individual contributions are sometimes difficult to assess [11,13,14,34–36]. In a recent clinical study, a high-dose cocktail of 2F5, 4E10 and 2G12 could delay viral rebound after cessation of antiretroviral therapy (ART) in some individuals [37]. In that study, the average serum levels were higher for 2G12 than 2F5 and 4E10, and viral escape mutants to 2G12, but not 2F5 and 4E10 were observed. Many questions about the clinical efficacy of 2F5 and 4E10 have now been raised that will require future studies, perhaps using each antibody at higher doses. Note that neutralizing antibodies are not expected to have a profound effect once an infection with HIV-1 has been established, but may be more effective at lower levels in a vaccination setting, in which neutralization of free virus may have a greater impact [34]. In the most comprehensive study of cross-clade neutralization to date, 2F5 and 4E10 neutralized 67 and 100%, respectively, of the diverse primary isolates tested (n = 90) [38]. Similarly, 2F5 and 4E10 neutralized 80 and 100%, respectively, of viruses (n = 91, presumably clade B) that were cloned directly from newly infected patients that had a negative or indeterminate serology [39]. In both of these examples, a relatively sensitive pseudovirus assay was used. Some experiments involving a classical peripheral blood mononuclear cell (PBMC)-based assay have shown that these antibodies can be less potent in this format, although still remarkably broad in their activity. For example, at 50 μg/ml, 2F5 and 4E10 neutralized (IC90) 60 and 8%, respectively, of primary isolates (n = 25) in a PBMC assay [38]. In other studies using the PBMC assay, 4E10 neutralized 71% (n = 28), 82% (n = 22) or 98% (n = 58) of the primary isolates tested [26,27,40]. In the latter study, an increased susceptibility to 4E10 and 2F5 of viruses taken during acute, relative to chronic infection was observed [40]. The molecular bases of these observed differences, and of some of the discrepancies in neutralization between pseudovirus and PBMC-derived virus, are not yet clear but may be instructive for vaccine design. A clear understanding of the mechanism of neutralization of HIV-1 by antibodies against the MPER is essential for rational approaches to elicit such antibodies. In general, neutralization of HIV-1 by antibody has been attributed to the ability of the antibody to bind to the native Env trimer, largely irrespective of epitope specificity [32,41–45]. Accordingly, studies have suggested that this explanation also extends to 2F5 [46,47]. To complicate the matter slightly, however, 2F5 and 4E10 also appear to neutralize subsequent to receptor engagement, a mechanism of inhibition that has been well established for the peptide drug, T20 (also named DP178, fuzeon and enfuvirtide) [48,49] and for other polypeptides corresponding to the N-heptad repeat (NHR) and C-heptad repeat (CHR) regions of gp41 [50–52] (Fig. 1). For example, a mutant HIV-1 pseudovirion, in which infectivity is controlled by an inducible disulfide bond in Env, is neutralizable by 2F5 and 4E10 post-attachment, albeit at a diminished potency relative to the neutralization observed under standard conditions [53]. Other studies have suggested that the 2F5 epitope is present on both native and a fusion-intermediate form of gp41 [54–56]. Moreover, substitutions that appear to affect Env stability and/or fusion kinetics have been shown to increase the sensitivity of HIV-1 to 2F5 and 4E10 [57–59]. Thus, there are many potential factors, both structural and kinetic, that can affect the access of antibody to the MPER (Table 1). It is also noteworthy that 2F5 and 4E10 can neutralize with several-fold greater potency as whole IgGs as compared to Fabs [60,61]. These results suggest that increased bulk does not impair access of bivalent antibodies to the MPER, in contrast to what has been observed with whole IgGs against CD4-induced epitopes that overlap the coreceptor binding site on gp120 [62]. However, access to the MPER may still be limited, as an IgM version of 4E10 appears to have little neutralizing activity [63].Fig. 1: Model of HIV-1 envelope-mediated fusion and its inhibition by targeting gp41. (a) A cartoon showing that, prior to attachment of gp120 to CD4 and co-receptor (CoR), gp41 is mostly occluded within the trimeric Env spike, leaving the MPER of gp41 (pink) partially exposed. Antibody binding to the MPER on the trimer is inferred from the neutralizing activity of antibodies 2F5, Z13 and 4E10. (b) Following receptor activation, gp41 assumes a transient fusion-intermediate state in which the N-terminal fusion peptide (orange) inserts into the host cell membrane. Access to this ‘pre-hairpin’ structure is brief (several minutes) and is somewhat restricted by the close proximity of the viral and host membranes and by other host and viral proteins. (c) Membrane fusion occurs upon formation of a stable six-helix bundle structure. (d) Fusion may be blocked by ‘class 1’ (e.g. T20, C34) or ‘class 2’ (e.g. 5-helix, IZN17, N35-NCCG-N13) inhibitors, against NHR or CHR regions, respectively [33,51,52,145]; or by 2F5, Z13 or 4E10, which may neutralize by binding to the native Env trimer (as in Panel (a)), but may also have activity post-receptor engagement (Panel (d); see text).Table 1: Potential factors affecting exposure of gp41 MPER to antibodies.Fab Z13 is less well characterized than 2F5 and 4E10, and whereas the latter two antibodies were obtained using combined polyethylene glycol-electrofusion of PBMCs and heteromyeloma cells [25], Z13 was isolated from an antibody phage display library prepared from the bone marrow RNA of a donor whose sera had broad neutralizing antibody titers [26]. Z13 binds to a determinant that overlaps the 4E10 epitope, has cross-clade neutralizing activity, but apparently neutralizes primary isolates with less breadth and potency than 4E10 [26]. In addition, Fab Z13 appears to bind with poorer affinity than 4E10 to the MPER, and its activity is dependent on a somewhat variable residue on gp41, D674, by which 4E10 recognition is less affected [26]. Future structural studies with Z13, and comparison of its epitope with that of 4E10 may shed light on how best to present the most conserved elements of this region to the immune system. HIV-1 gp41 MPER structure The first biophysical insights into the MPER of gp41 with the of the structure of a residues to 683 of HIV-1 This peptide an in in which the and residues are the (Fig. in have more and The defined structure of this MPER peptide a with regions of the gp41 that had been shown to into using Thus, the NHR and the CHR (Fig. the latter of which the N-terminal of the MPER of gp41, can to such that the NHR polypeptides form a trimeric which the CHR at a The is stable and the of its formation is to the fusion of the virus and host cell membranes during infection Importantly, prior to or receptor engagement, gp41 is to be in a different and less is known of the structure of this native to the MPER is the which is most likely as are most stable as in membranes the membrane-proximal external region (MPER) of HIV-1 gp41. A of HIV-1 gp41 the fusion peptide N-terminal and repeat regions and MPER here as residues of the transmembrane and the The clade of the MPER is which is an of the residues that and as using the The residues for 2F5 and 4E10 neutralization are by A of the CHR region the N-terminal of the 2F5 epitope, shown in The structure of the peptide corresponding to residues of gp160, as in using is shown with the residues of the 2F5 and 4E10 epitopes in and These residues are in the of the and and as by The structural of and were obtained from the and using be best for vaccine design to the structure of gp41 in the of the virus. However, there are in this structure that to the and of HIV-1 The antibodies 2F5, Z13 and 4E10 are as structural bind to the Env trimer (as of their neutralizing the structure was for a of 2F5 with a with a These studies that this of the MPER is not in a but an with a at the in the of the peptide epitope (Fig. The also that the residues are the most in the that these residues are on the Env trimer prior to a structure was of a of 4E10 and a peptide the the peptide is shown to 4E10 in a (Fig. the corresponding region on the structure of the MPER peptide in the peptide has a of the and N-terminal to the residues and which are in the of 4E10 Thus, which most into 4E10, be well at some prior to that most of the MPER is at some in on the Env trimer, this an or of the on the of the trimer, and the or be occluded within the Env trimer in the as some have Note that the of the to prior to 4E10 recognition be by interactions made elsewhere by and other have also been to the structural of in although the of this to vaccine design has been For example, a peptide has been shown to a in In addition, 2F5 binds to mutants of the peptide the In a into a peptide to a recognition of the peptide by 2F5 The of 2F5 may these as may bind to 2F5 using different binding It was suggested that the has structural that its corresponding structure within the HIV-1 Env spike, at only structure from the virus In of the 2F5 epitope was into the MPER of the protein, the the were shown to be neutralized by 2F5 It be however, that it can be to the binding affinity of a neutralizing antibody for an to the in between that and the corresponding viral in the MPER of gp41 on virus that the only to neutralization to 2F5 in residue or of the 4E10 by three two and were in the epitope, and was residues to these two residues on the virus that are not for the neutralizing activity of 2F5, can the affinity of to 2F5 in the in apparently the should not other of the MPER of gp41, to which as yet neutralizing antibodies may the that between the 2F5 and 4E10 epitopes an or and is it to neutralizing its proximity to the the MPER of gp41 may relatively molecular that is of in in this may not be a of a designed on the MPER of gp41, antibodies may to a more variable This may be in is to different target cell and MPER as a for vaccine the MPER of gp41 should be in a that the structure that is 2F5 and/or 4E10 bind the virus. Note that the virus may not be the best with the of 2F5 and antibodies that are during natural infection, and that and non-neutralizing epitopes are present in HIV-1 viral is that, in comparison to the neutralizing epitopes on gp120, by antibodies and 2G12, the 2F5 and 4E10 epitopes appear to be less on virus and on infected cells HIV-1 gp41 MPER function studies have shown the of the MPER of gp41 in viral or cell to cell of residues for However, of the conserved residues a in which Env does not but does between host and target a in with this mutant In a recent study to the in the fusion mechanism with the mutant it was that the mutant Env, on the host cell was to form six-helix bundle by The in the MPER of gp41 was suggested to the in the of the such that the fusion peptide is to into the target cell and that of the fusion peptide to and the that was observed. In this the MPER of gp41 as a which the fusion in a with fusion at the of the MPER of gp41 also contribute to the fusion and/or viral is not The MPER peptide that was shown to be an in using can also bind and Thus, it has been suggested that the MPER directly with the during fusion The of an between the MPER and has been in structural studies with the 2F5 epitope and in studies showing that the affinity of 2F5 and 4E10 to that have been on For virus the stability of the bundle on N-terminal to the and on the MPER Thus, for at the MPER may directly contribute the fusion bundle the MPER of gp41 may affect Env or For example, to the MPER of gp41 (e.g. can the of Env into and virus although appears to be and on the cell and appears to be with these mutants The studies on the function of the MPER of gp41 to its However, many substitutions to the MPER of gp41 have little or effect on virus in the natural that occurs in the MPER of gp41 is an that some of this region of gp41 are not directly for a mutant of with three to substitutions in the MPER was in and a was that to the was a N-terminal of the MPER and is in the MPER of gp41, are some residues still a major for the that is observed in the MPER of gp41 is that these are the least in of the virus, and the most in the viral Thus, there are at least two that the MPER of antibody as a viral and of function that be the of a for the fusion the of fusion an or by Env trimer stability (Table of the HIV-1 gp41 membrane-proximal external region residues in Many other viruses also have of residues in the MPER region of their transmembrane virus virus or or virus virus, virus and to For it has been shown that within a of the MPER prevent viral infection affecting virus attachment to cells for of three conserved in the MPER of viral fusion an corresponding to this region on can block viral in an activity that is by a peptide from the N-terminal of the The of the from and human viruses are for viral fusion as well For however, only but not or within the MPER this has been attributed to a effect of the and region between the NHR and CHR regions of the Even the and which are in and have residues in their The of in the of these can fusion some viruses not appear to require their membrane-proximal residues for viral or into target In the of for example, of the conserved membrane-proximal residues has effect on viral and little effect on fusion activity, although of the MPER the virus completely on many transmembrane of diverse the as well as have shown that residues are typically at the that between the and the of appear to in the as well As corresponding to the MPER of gp41 have been shown to bind and Moreover, gp41 that overlap with the MPER can in an on the surface or have activity In addition, studies have the ability of the in the MPER of gp41 The latter studies have the activity to the ability of HIV-1 Env to into or not this activity has a effect on the antibody response to the MPER of gp41 is the strong of it is not that residues may into the although this be residues in the MPER of the protein, may an for the that is suggested to be to the such that residues can with the The MPER of as a for subsequent that is by formation in the studies have that, the by bundle formation is to the membranes this may be the MPER is not for membrane-proximal epitopes In to the peptide a of have been to display the 2F5 epitope using such as the surface virus binding gp120 virus and eliciting antibody titers against the epitope these have not neutralizing antibody against primary some neutralizing antibodies have been with using gp41 and with display However, these approaches will require and the studies how difficult it is to elicit antibodies, and how or that overlap the of the on these elicit non-neutralizing antibodies. It may be that some non-neutralizing antibodies to of the MPER are during natural infection, although there is to a binding assay was to the of titers of antibodies against the fusion NHR and CHR regions, and the MPER of gp41 that region of the gp41 is The study between neutralizing antibody and the antibody against of the gp41 In study, a of HIV-1 sera was using and an site was in which the residues were most for recognition However, other serum using not the MPER as an at least in comparison with some other on Env such as on gp120 or the on gp41 Note that antibodies against epitopes may be using peptide their non-neutralizing antibodies in serum the or of the MPER of gp41 may to elicit neutralizing antibodies to the MPER, their be antibodies are as prior to their as by by the viral could the or at least the of by the to the MPER of gp41. The of that are to on a is for example, at but at and this has been attributed to the of the to the it is close to the an antibody the MPER from the in which it antibody against the membrane-proximal of the Env of virus appears to this The monoclonal was with in contrast to other antibodies to binds to than to virus or infected the to its epitope on the virus is by the In example, with a peptide corresponding to the MPER of the of antibodies that were to bind to and were non-neutralizing In this however, it is the MPER was a that the corresponding epitope on with a form of HIV-1 gp41 that the CHR the MPER and the recognition by both 2F5 and 4E10, this was to elicit HIV-1 neutralizing antibodies in In this a was but of the native trimer was and the of N-terminal to the MPER may have antibodies, which have been to this region The of into which the MPER is may also affect its This effect has been in a study involving the protein, which was on of different the of the at the of the the exposure of membrane-proximal epitopes on this The of surface has also been to be dependent upon the both a negative and at least in the 2F5 and 4E10 were shown to cross react with a few It was suggested that the conserved MPER may be a molecular of such as such that antibodies against it may be from the or upon immunization using MPER In diverse are in both and the latter of which is to a by the that with both and are and are often an against In that react more with which can and less with which may should the in a immune or not the MPER of gp41 is a molecular of will In addition, the molecular of 4E10 and 2F5 to bind to can be using to the and neutralizing activity are Note that antibodies a different to recognition of a it is that antibodies with close to of the described broadly neutralizing antibodies will be Thus, may to neutralizing antibodies against the MPER of gp41 that not with such antibodies be for vaccine design. Fab Z13 can be as a whole and tested for may be other of the broadly neutralizing antibodies that could but antibodies difficult to studies have suggested that the of 2F5 is for its neutralizing activity, which may be a molecular for other neutralizing antibodies against its epitope [60,61]. Other HIV-1 neutralizing antibodies have that are or as 4E10 and Fab is by in cells in the bone in the of the to and to and and the of at the contribute to the and/or of there is described for the to a antibody with a be for the of antibodies with by are the described broadly neutralizing and are are to which can to or from and can the the activity of which will tend to the Thus, in at receptor and activity may however, this is only and the of antibody is Moreover, are non-neutralizing antibodies against non-neutralizing than neutralizing antibodies To this more immunization approaches the may be perhaps more is that typically not such and may be for vaccination experiments using MPER Nevertheless, as the and of antibody and are to the human antibody response to the MPER of HIV-1 gp41, by other than may and can be to the of antibody and antibody although the is and on the MPER to or it into different or may also be in its the the of the structure of the MPER be For it be that the to elicit neutralizing antibodies to HIV-1 by designed has typically been attributed to but in epitope exposure and/or between the in and the Env trimer Future The of eliciting broadly neutralizing antibodies against the MPER of gp41 elsewhere on HIV-1 will at the in design. As the MPER is not on viral future on on it may require approaches (Table a or vaccine is the MPER will most likely to be The epitopes of 2F5, Z13 and 4E10, as well as neutralizing epitopes on the MPER, also be as to be by the perhaps on a surface or into a protein, but in such a as to the of of the antibodies to that which is with to the epitope on the virus. understanding of the most MPER the of the molecular the MPER, the or will be in the design of structural neutralizing and non-neutralizing antibodies against the MPER, and to on gp41, may in the exposure of neutralizing immunization or may be to the of the MPER A to and should the MPER of gp41 will be a of a HIV-1 strategies to elicit neutralizing antibodies against the MPER of has to light this have shown that sera from that were by immunization with of and the HIV-1 gp41 MPER, and with the gp41 MPER to the were to neutralize at serum in a pseudovirus assay This on some experiments with virus and a in which the has been as an to elicit antibodies against the MPER of the The and other in eliciting neutralizing antibodies against the gp41 MPER will now require development to neutralizing antibody titers against primary HIV-1 at levels with protection to for of the and for many for with to and for with the and the for