REVIEWS Insights into vaccine development for acquired immune deficiency syndrome from crystal structures of human immunodeficiency virus-1 gp41 and equine infectious anemia virus gp45

Liangwei Duan, Jiansen Du, and Xinqi Liu* State Key Laboratory of Medicinal Chemical Biology, College of Life Sciences, Nankai University, Tianjin 300071, China Received 9 June 2015; Accepted 6 July 2015 DOI: 10.1002/pro.2750 Published online 15 July 2015 proteinscience.org

Abstract: An effective vaccine against acquired immune deficiency syndrome is still unavailable after dozens of years of striving. The glycoprotein gp41 of human immunodeficiency virus is a good candidate as potential immunogen because of its conservation and relatively low glycosylation. As a reference of human immunodeficiency virus gp41, gp45 from equine infectious anemia virus (EIAV) could be used for comparison because both wild-type and vaccine strain of EIAV have been extensively studied. From structural studies of these proteins, the conformational changes during viral invasion could be unveiled, and a more effective acquired immune deficiency syndrome vaccine immunogen might be designed based on this information. Keywords: HIV-1 gp41; EIAV gp45; crystal structure; neutralizing antibodies; vaccine development; MPER; gp140; Env-based immunogen

Introduction Since acquired immune deficiency syndrome (AIDS) was first reported in 1981 in the United States, approximately 78 million people worldwide have been infected by human immunodeficiency virus (HIV), which was identified as the etiologic agent for Grant sponsor: National Key Project on Major Infectious Diseases; Grant number: 2012ZX10001-008; Grant sponsor: National Basic Research Program of China, Ministry of Science and Technology; Grant number: 2010CB911800; Grant sponsor: National Natural Science Foundation of China; Grant number: 31370925. *Correspondence to: Xinqi Liu, College of Life Sciences, Nankai University, Tianjin 300071, China. E-mail: [email protected]

C 2015 The Protein Society Published by Wiley-Blackwell. V

AIDS in 1984.1–4 To date, 39 million patients have died of AIDS (http://www.who.int/gho/hiv/). Advances in medicine have produced therapies that help infected individuals to control the viral load, but HIV still cannot be completely eradicated from the circulation of infected persons, and most infected individuals will eventually lose control of the virus.5,6 A safe and efficacious vaccine is needed to control the worldwide AIDS pandemic. However, despite decades of intensive research and concerted efforts to develop a vaccine against HIV/AIDS, the levels of protection seen in most HIV vaccine trials have been disappointing, except in a recent Thai trial (RV-144), which showed a modest degree of

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short-term protection and ignited new hope in the HIV vaccine development field.7,8 The envelope (Env) glycoproteins of HIV interact with receptors on the target cell surface and mediate virus entry by fusing the viral and cellular membranes.9,10 Mature Env on the viral surface is a heavily glycosylated trimer, which comprises three heterodimers of transmembrane glycoprotein gp41 and surface glycoprotein gp120, both derived from a common gp160 precursor.11 Env interacts sequentially with its primary receptor CD412,13 and a coreceptor CCR5 or CXCR414,15 on the host cell, inducing a cascade of conformational changes in both Env subunits.16,17 In following steps, the gp120 subunits dissociate from the Env glycoprotein, the gp41 ectodomain is refolded into a six-helical bundle structure and drags the virus and cell membrane in close proximity, leading finally to the membrane fusion.18 As the sole virally encoded antigen on the surface of HIV-1 virions, Env is undoubtedly the focus in HIV-1 vaccine development to elicit neutralizing antibodies. A large number of Env-based immunogens have been tested, of which monomeric HIV-1 Env gp120 subunits are used most widely. However, immunization with recombinant gp120 fails to induce potent broadly cross-reactive HIV-1 neutralizing antibodies (bNAbs),19,20 and gp120 did not prove vaccine efficacy in previous clinical trials.21–23 Elsewhere, gp41 is probably a good target for vaccine development because of its high sequence conservation among HIV-1 subtypes and low level of glycosylation. Historically, dozens of broadly neutralizing anti-HIV-1 monoclonal antibodies (mAbs) have been reported, such as b12, 2G12, 2F5, Z13, and 4E10.24–29 Most of these mAbs are directed against gp41 membrane-proximal external region (MPER), which consists of 25 highly conserved residues and plays an important role in viral fusion and infectivity.30–32 Recently, 10E8, which neutralizes up to 98% of HIV-1 isolates tested, has become one of the most potent and broad antibodies that has been described.33 This mAb demonstrates a combination of high potency and minimal autoreactivity, reinvigorating research interest in MPER-based HIV-1 vaccine design.33 Theoretically, the native Env trimer containing the original conformation of viral spikes should be the most ideal immunogen for the elicitation of potent neutralizing antibodies.34,35 However, the inherent conformational lability of the Env trimer has impeded its usage in vaccine trials.36 A variety of approaches have been exploited to generate recombinant, soluble, stable trimers that closely mimic the native HIV-1 spike, for example, engineered so-called “gp140” molecules such as BG505 SOSIP.664 gp140.37 Thanks to spatial information provided by protein crystallography, structure-based immunogen

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design contributes greatly to an effective vaccine, which is consolidated in the case of respiratory syncytial virus.38 Considering the important roles of HIV-1 gp41 in viral infection and inducing antibodies, its crystal structure has been extensively studied. This review summarizes a series of representative crystal structures of HIV-1 gp41, and also its ortholog gp45 from equine infectious anemia virus (EIAV), the first lentivirus for which an effective vaccine was successfully developed. By analyzing the information obtained from these structures, we hope to shed light on the methods of designing further Env-based HIV-1 vaccine immunogens.

Structural Studies of HIV-1 gp41 by X-ray Crystallography The HIV-1 gp41 subunit is composed of 345 amino acids (residues 512–856, HXBc2 gp160 numbering in context), which are organized into three major domains: an N-terminal ectodomain, a transmembrane domain, and a long C-terminal cytoplasmic tail.39 The ectodomain mediates the major functions of gp41 and can be further divided into the following functional regions: the fusion peptide, the Nterminal heptad repeat (NHR or HR1), the Cterminal heptad repeat (CHR or HR2), and the MPER. The NHR and CHR are connected by a disulfide bridge within a structurally conserved hydrophilic loop that is involved in the fusion process.40,41 The first insights into the structure of HIV-1 gp41 were acquired in 1997, when three independent groups of investigators reported the crystal structure of the gp41 core.42–44 Chan et al.42 reported the crystal structure of the HIV-1 gp41 core consisting of N36 (residues 546–581) and C34 (residues 628–661), which were synthetic peptides with blocked termini [Fig. 1(A)]. The X-ray structure determined by Weissenhorn et al.43 was a large trimer designated GCN4/gp41, composed of an Nterminal of gp41 segment (residues 541–590) fused to a 31-residue trimeric coiled coil from GCN4 in place of the N-terminal fusion peptide, plus a Cterminal segment of gp41 (residues 624–665). Tan et al.44 presented the structure of a thermostable subdomain of HIV-1 gp41, in which the N34 (residues 546–579) and C28 peptide fragments (residues 628–655) were connected by a six-residue hydrophilic flexible linker Ser-Gly-Gly-Arg-Gly-Gly instead of the disulfide-bonded “loop” region. All three structures showed almost identical postfusion conformation in spite of varying constructing methods. In this conformation, HIV-1 gp41 folds into a six-helical bundle structure, in which three NHR helices form an interior, parallel coiled-coil trimer with three CHR helices packing in an oblique, antiparallel manner into the highly conserved hydrophobic grooves on the surface of the interior trimer [Fig. 1(A)]. Each groove has a deep

Structural Insights into AIDS Vaccine Development

Figure 1. Representative HIV-1 gp41 structures in the postfusion conformation, and their comparison from different clades. (A, B) Ribbon diagrams of trimeric HIV-1 gp41 core (PDB code 1AIK) and HIV-1 gp41 including both FPPR and MPER (PDB code 2X7R). The Flag sequence, FPPR, NHR, CHR, and MPER are colored in gray, hot pink, deep salmon, lime green, and chartreuse, respectively. (C) Superimposed structures of HIV-1 gp41 core from Clade A (PDB code 3P30, magenta), Clade B (PDB code 1ENV, yellow), and CRF07 B0 /C (PDB code 3WFV, green). The N termini of the NHR helices point toward the top of the page, whereas those of the CHR helices point toward the bottom in all three figures except Figure 3(C).

hydrophobic pocket formed by the cavity-forming sequences (including Leu568, Val570, Trp571, Lys574, and Gln577) located near the C-terminus of the NHR region that interacts with three conserved hydrophobic residues (Trp628, Trp631, and Ile635) of the CHR, which are referred to as the pocketbinding domain.42,45 These interactions seem to contribute largely to the stability of the six-helix bundle and thereby are critical for viral fusion.42,46,47 Based on these gp41 structures and evidence from other study, it is widely accepted that the transition of HIV-1 gp41 into a six-helix bundle, not the bundle configuration, drives membrane fusion.48,49 The six-helix bundle represents only the terminal conformation of a fusogenic Env, which is extremely stable (the melting temperature (Tm) of the gp41 core is in excess of 708C).49 This discovery provides an important insight into why antibodies to the immunodominant six-helix bundle form of gp41 fail to neutralize the virus.49 By the time an antibody is able to react with the six-helix bundle, the fusion events have already taken place. In 2010, Buzon et al.18 reported a more complete crystal structure of HIV-1 gp41, containing residues 528–581 that correspond to the fusion peptide

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proximal region (FPPR) and NHR, and residues 629–683 that correspond to CHR and MPER [Fig. 1(B)]. Both FPPR and MPER form extended helices from the gp41 core structure of six-helix bundle and interact with each other while there is a lack of regular coiled-coil interactions within FPPR and MPER [Fig. 1(B)].18 Inclusion of FPPR and MPER increases the stability of gp41 significantly, as the Tm rises substantially compared with that of the gp41 core; so, there will be more free energy released during the formation of this structure, which can be directly coupled to membrane fusion. This structure indicates that part of MPER can be inserted into the viral membrane within trimeric gp4150 and supports the hypothesis that a number of neutralizing antibodies directed at gp41 recognize MPER in a lipid context.18,51 The emergence of circulating recombinant forms of viruses alerts us the new challenge to handle the complexity of HIV-1 Env proteins by the immune system. Fortunately for gp41, the conformation is quite conserved despite sequence diversity from various clades [Fig. 1(C)]. In understanding the pre-fusion conformation of gp41, the breakthrough come at the end of 2013,

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when Julien et al.52 described the crystal structure of a soluble cleaved HIV-1 envelope trimer termed BG505 SOSIP.664 gp140 in complex with a potent ˚ . This gp140 bNAb, PGT122 at a resolution of 4.7 A contains the whole ectodomain deleting all but several residues of the MPER of gp41. The complex structure reveals the overall organization of a prefusion state of HIV-1 Env, the interaction between gp120 and gp41 subunits, and the complete and complicated epitope of PGT122 in the context of a soluble Env trimer.52 This crystal structure of SOSIP gp140 trimer is in excellent agreement with the accompanying cryo-electron microscopy (cryoEM) structure of the same trimeric Env construct in complex with a CD4 binding site bNAb, PGV04, at ˚ resolution.53 The soluble BG505 SOSIP.664 5.8 A trimer adopts a compact mushroom shape with the gp120 subunits leaning into each other at the top and the gp41 components forming a pedestal at the base, consistent with the previous result determined ˚ .54 by EM at a resolution of 20 A For each gp41, two long, but bent helices could be traced clearly. Part of the first helix (six turns), forming a three-helix bundle structure with the adjacent protomers along the trimer axis and perpendicular to the viral membrane, is ascribed to gp41 NHR.52 The remaining helix (2.5 turns) of NHR, which could not be traced in the accompanying cyro-EM structure, extends from the three-helix bundle but is kinked away from the trimer axis.52,53 However, the exact residues corresponding to the first helix cannot be clearly defined due to the relatively low resolution. The second helix assigned to gp41 CHR is also bent, wrapping around the trimer base and obliquely parallel to viral membrane surface. The residues in the second helix are probably 625–664 because the NAG moieties at glycosylation sites 625 and 637 can be clearly visible in the electron density map and define the location of helical turns.52 This substantially intact recombinant SOSIP gp140 trimer structure provides for the first time the structural insights into the pre-fusion conformation of HIV-1 gp41, probably as it exists in the native envelope glycoprotein spike present on the surface of the viral particle prior to CD4 and coreceptor binding.52 Very recently, Pancera et al.55 presented the crystal structure of BG505 SOSIP.664 gp140 trimer [Fig. 2(A)] captured in a pre-fusion mature closed state by potent bNAbs PGT122 and 35O22 Fabs. By addition of 35O22 that recognizes a novel epitope at the gp120-gp41 interface,56 Pancera et al. improved the resolution of the BG505 SOSIP.664 trimer from ˚ to a moderate 3.5 A ˚ , which a relatively low 4.7 A could trace nonhelical loop regions of HIV-1 gp41 as well as the registry of side chains.52,55 Not surprisingly, besides association of the V1-V3 regions at the

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apex,57 the stabilizing force of gp140 trimers are contributed mainly by gp41.52,53,55 In the pre-fusion state, gp41 encircles extended amino- and carboxy-terminal strands of gp120 with a four-helix membrane-proximal collar [Fig. 2(A)]. The four helices consist of helices a6 (residues 530– 543), a7 (residues 572–595), a8 (residues 619–623), and a9 (residues 628–664), indicating that both NHR and CHR are split up into two smaller helices connected by loops, which is in line with our previously proposed pre-fusion model for gp41 in which both HR1 and HR2 are interspersed by loop segments in the middle, and sequential loop-to-helix transitions will occur when viral fusion begins [Fig. 2(B)].55,58 The gp41 subunit completes a single ring around the N and C termini of gp120 with the side chain of Met530, which is proximal to the fusion peptide, inserted into a triple-tryptophan clasp formed by Trp623, Trp628, and Trp631, derived from a8 and a9 [Fig. 2(A) insert].55 This methionine–tryptophan clasp is further stabilized by the electrostatic complementarities offered by the alignment of neighboring helices a6 and a8.55 As a result, gp41 is kept in the pre-fusion conformation by interaction with gp120, waiting for the triggering force from receptor binding. Notably, when Pancera and colleagues combined glycan shielding and genetic variation, both of which have long been regarded as mechanisms to avoid antibody recognition,59 up to 98% of the BG505 SOSIP.664 gp140 trimer surface is either immunoglobulin-domain inaccessible or shown to have a variability of greater than 10%; much of the rest is located at the membrane-proximal “base” of the spike, which is expected to be protected by steric constrains conferred by the viral membrane.55 Therefore, the recombinant pre-fusion BG505 SOSIP.664 gp140 trimer, with high glycosylation and variability, might not be a good immunogen that can easily induce a potent neutralizing antibody response capable of recognizing highly diverse strains of HIV-1 circulating in the human population. Compared with the pre-fusion closed conformation, the CD4-bound state in which trimeric Env exposes significantly higher levels of glycan-free conserved surface, might be a promising immunogen expected to elicit neutralizing antibodies.55 The gp41 MPER, as a potential region to induce bNAbs, has been well studied by X-ray crystallography in complex with various antibodies, such as 2F5, 4E10, Z13e1, and 10E8. The MPER peptide adopts a largely extended conformation with a central b-turn involving a 664DKW666 core, sometimes followed by a canonical a-helix turn when bound by 2F5,60–62 in comparing with a meandering S-shaped conformation when bound by Z13e1.63 The crystal structure of 4E10 Fab in complex with its gp41 MPER epitope (residues 671–683) reveals an a-

Structural Insights into AIDS Vaccine Development

This figure also includes an iMolecules 3D interactive version that can be accessed via the link at the bottom of this figure’s caption.

Figure 2. Pre-fusion structure of HIV-1 gp41 and comparison with that in the postfusion conformation. (A) Ribbon representation of pre-fusion gp41 within an Env trimer (PDB code 4TVP) in the stereoview. A trimer formed through crystallographic symmetry is shown with removal of associated Fabs. HIV-1 gp120 moiety is shown in light gray. For gp41, monomers 2 and 3 of gp41 moiety are shown in green for clarity, and monomer 1 is colored in yellow. Monomer 1 shows the insertion of a fusion peptide-proximal methionine into a gp41-tryptophan clasp. Zoom insert by stereoview: side chain of Met530 is shown to insert into a tripletryptophan clasp formed by Trp623, Trp628, and Trp631.The side chains of all four residues are shown in stick representation and colored according to the element type (N, blue; O, red; C, cyan; and S, yellow-orange). An interactive view is available in the electronic version of the article. (B) Stereoview of superimposed structures of pre-fusion and postfusion gp41 (PDB code 1ENV). Prefusion gp41 is shown in magenta, and postfusion gp41 is shown as in Figure 1(C).

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helical structure whose N-terminal residues are configured as a short 310-helix, which transitions into a regular a-helix at residue Asp674 and extends to Lys683, the final residue of the MPER.64,65 The Xray structure of an extended MPER peptide (residues 656–683) in complex with 10E8 Fab reveals a helix–kink–helix structure composed of two helices, ˚ in length and oriented 100˚ relative to each 15–20 A 8 each other. This structure is similar to that of an unbound MPER peptide (residues 662–683), which is determined in a lipid environment by a combination of nuclear magnetic resonance and spin-label electron paramagnetic resonance.66 These different structures reveal the conformational flexibility of the MPER region when it is truncated from native Env spikes. High-resolution crystal structures of diverse MPER neutralizing antibodies in complex with the MPER region in the context of native trimeric Env are necessitated to precisely define the detailed and authentic neutralizing epitope(s), which could provide valuable insights into the development of improved antibody-based vaccines for HIV.

X-ray Crystallography Studies of EIAV gp45 EIAV is a member of lentivirus genus belonging to the Retroviridae family67 and has similar genome structure and life cycle as those of HIV, a property qualifying it as a reference for study of other lentiviruses.68 Around 30 years ago, the vaccine against EIAV was successfully developed by Chinese scientists using a live attenuated EIAV strain produced by serial passages on donkey leukocyte cells, which makes EIAV the first effective lentivirus vaccine.69 With the availability of vaccine strain for comparison, EIAV is a good reference for vaccine study of other lentiviruses, including HIV. A number of studies have been carried out to elucidate the mechanism underlying the EIAV vaccine. Val/Ile505 to Thr (V/I505T) mutation in gp45 as well as other mutations in gp90 (analogous to gp120 in HIV) may potentially contribute to the success of the vaccine strain.70–72 Hence, the crystal structures of gp45 and gp90 from both wild-type (WT) and vaccine strains in different conformational states are badly needed to reveal the detailed molecular mechanism of EIAV vaccine-associated mutations within gp45 and gp90. In 2014, Du et al.73 reported the high-resolution X-ray structures of both the WT and V/I505T mutant of EIAV gp45. As expected, both the structures are very similar to that of HIV-1 gp41 corresponding to the postfusion conformation [Fig. 3(A)] and form a stable six-helix bundle, with three NHR forming the inside core and three CHR lying antiparallel to NHR on the outside [Fig. 3(B)].73 However, there are still several differences between the structures of EIAV gp45 and HIV-1 gp41, for

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example, the structure of gp45 is more loosely packed compared with that of gp41, consistent with the observation that WT gp45 has a much lower Tm than HIV-1 gp41 and that there are more coordinated water molecules along the central axis of the six-helix bundle of gp45.42,58,73 The heptad repeat regions in NHR and CHR are composed of several seven-residue repeats designated as a to g. Residues in a and d positions are extremely important for helical bundle stability with their side chains protruding inward. A structure comparison between the gp45 from wild-type and vaccine strains reveals that the V/I505T mutation is located at the d position of the heptad repeat, protruding toward the central axis within the six-helix bundle [Fig. 3(B,C)], where high levels of conservation are observed for other lentiviruses such as HIV and simian immunodeficiency virus. The V/I505T mutation generates a hydrophobic to hydrophilic interaction transition along a threefold symmetry axis and markedly reduces the stability of the helix bundle, which is proven by biochemical studies.73 Surprisingly, even with lower stability, viral replication is not affected by this mutation at normal body temperature, indicating a relative tolerance of this mutation for viral activity. Nevertheless, infection efficiency drops with increasing temperature for viruses harboring this mutation. Therefore, this mutation allows the host to control viral copy numbers and pathogenic load easily by regulation of body temperature, while at the same time maintaining a continuously low level of viral replication asymptomatically. As a consequence, the mutual coevolution of the virus and host is achieved, which might be one of the reasons for the success of vaccine strain in EIAV.73 The pathogen–host coevolution has been extensively studied, and a recent observation in an HIV-1-infected individual strongly supports the mutual adaption of the virus and the host immune system.74 Continuous stimulation may be necessary for an effective immune response and for viral control. On the other hand, the loose packing of WT gp45 itself and the lower stability of gp45 harboring V/I505T mutation might potentially slow down the formation of postfusion conformation and simultaneously stabilize the pre-fusion counterpart, therefore providing a prolonged period for the elicitation of neutralizing antibodies directed at exposed epitopes.73 The study on EIAV gp45 emphasizes the importance of residue 505, which of course prompted investigations into looking for similar mutations in HIV-1 gp41. Subsequent study reveals that HIV-1 gp41 has minor tolerance to the mutations in conserved d positions and most mutants lost their activity completely (our unpublished data). The study in our laboratory also identified several mutations in the Q562 position (analogous to the residue 505

Structural Insights into AIDS Vaccine Development

Figure 3. Structure comparison of EIAV gp45 with HIV-1 gp41, and comparison between wild-type strain (gp45wt) and vaccine strain (gp45vaccine). (A) Superimposition of EIAV gp45wt and HIV-1 gp41 including FPPR and MPER (PDB code 2X7R). HIV-1 gp41 including FPPR and MPER is shown as in Figure 1(B). The TEV sequence, NHR, and CHR of EIAV gp45wt are shown in gray, magenta, and cyan, respectively. (B) Superimposed structures of EIAV gp45wt and gp45vaccine. EIAV gp45wt is shown as in (A). gp45vaccine is colored in yellow for the NHR and green for the CHR. Thr-505 is shown in stick representation and colored according to the element type (N, blue; O, red; and C, cyan). Both the TEV sequences of gp45wt and gp45vaccine are removed for clarity. (C) Thr505 in gp45vaccine. Thr505 is represented as a stick model and viewed down threefold axis.

in EIAV gp45), which retained viral activity. Interestingly, the Q562H mutation acquired even higher activity compared with wild-type virus, a phenomenon needing to be further explored. In summary, all these data support the important roles of d positions in adaptation of virus during evolution, but the translation of EIAV vaccine research into HIV is neither simple nor straightforward. Furthermore, live attenuated HIV vaccines cannot be tested in humans owing to safety concerns.75,76 Nonetheless, structural insights into the successful EIAV vaccine from gp45 in combination with biochemical and virological methods do contribute to the HIV-1 immunogen design for envelope proteins.

Insights into Rational Design of Improved HIV-1 Immunogens The structural study described earlier advances our understanding of the correlations between antigenicity and immunogenicity, which is important for the rational design of an effective Env-based immunogen. As mentioned earlier, the recombinant prefusion BG505 SOSIP.664 gp140 trimer may not be the best immunogen for inducing neutralizing antibodies, as the pre-fusion form of trimeric Env is

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substantially shielded from access of neutralizing antibodies by the dense shield of glycans and highly variable loops. Besides, this gp140 trimer is overly stabilized by an artificially introduced disulfide bond77; so, it is locked in the pre-fusion state even after receptor binding. Therefore, it might be highly antigenic, but may not have the immunogenic potential to induce a neutralizing antibody response. Novel strategies need to be developed to generate recombinant gp140 trimers in which the stabilization and intrinsic flexibility should be balanced. Following fusion, the integrated HIV-1 Env spike probably dissociates into monomeric gp120 subunits and postfusion gp41, both of which are highly immunogenic in that high titers of antibodies specific for these forms of Env are found. These antibodies are not neutralizing as the forms of Env that they target are not functional. It has been shown that a large proportion of the host response to HIV-1 is directed at these nonfunctional Env forms, including shed gp120, which are utilized by the virus to serve as decoys78 to distract the immune system and to elicit ineffective antibody responses in infected individuals.79 We can reasonably speculate that native gp41 in pre-fusion conformation is a promising immunogen,

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rather than the previously defined native Env spike. This form of gp41 contains an exposed and conserved vulnerable site of Env, that is, the MPER region. The MPER-specific or 10E8-like neutralizing antibodies are not rare in healthy HIV-1-infected donors, whose MPER-specific antibodies account for 27% and 10E8-like specificities for 8% generally.33 Another promising immunogen may be the Env trimer in the receptor-bound intermediate states, especially in the CD4-bound conformation, for which no crystal structure is available yet. However, the CD4-bound trimer is just transiently, functionally present in vivo as the fusion-intermediate phase is very short, inhibiting the eliciting and recognizing of neutralizing antibodies. This notion is supported by the observation that there is a substantial increase in immunoreactivity to certain neutralizing antibodies against trimeric Env after its interaction with CD4 on the surface of target cells.56,59,80–82 For instance, the MPER-directed antibodies have been shown to react more efficiently after Env attachment to the CD4 receptor, probably as trimeric Env is ˚ from lipid membrane after its raised or tilted 15 A 56 engagement of CD4. This notion is also supported by the success of the EIAV vaccine. With lower stability of gp45 in vaccine strain, the Env spike might be easier to access for immune system due to prolonged time of conformational transitions from an unliganded state, through a sole receptor-bound intermediate stage, to a postfusion state.73 More focus should be put on the fusion intermediate of Env trimers following CD4 binding, and therefore, a crystal structure of Env in this state would be crucial to direct the design of improved HIV-1 immunogens.

Concluding Remarks and Future Perspective Over past years, some positive news has appeared for the development of an antibody-based HIV-1 vaccine. First, it has been revealed that up to 30% of individuals who are infected with HIV-1 for at least 1 year generate quite broadly cross-reactive neutralizing antibody responses.34,83 Second, large quantities of far more powerful neutralizing mAbs84,85 of substantial breadth have been isolated by use of single-cell antibody cloning techniques.86 Third, passive transfer experiments with one of the recently discovered, more potent bNAbs in rhesus macaques have shown that serum concentrations of as little as 15 lg/mL can provide sterile protection against a single high-dose challenge of chimeric simianhuman immunodeficiency virusSF162P3, which indicates that sterilizing immunity offered by analogous bNAbs may be achievable through vaccination with the development of improved HIV-1 immunogens by a rational strategy.87 Therefore, potent bNAbs are undoubtedly a crucial component of a highly effective HIV-1 vaccine, and designing an immunogen

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that induces such bNAbs is one of the most important goals of HIV vaccine research. Substantial experience has been accumulated following dozens of failed trials on AIDS vaccine development. The strategy of priming with a live vector vaccine and boosting with an improved Env protein to elicit both cellular and humoral immune responses has yielded some of the most effective results thus far, including the ALVAC-prime AIDSVAX-boost RV-144 trial.7 An alternative strategy is to develop a vaccine capable of priming germline B cells with a most promising HIV-1 Env immunogen to express the germline precursors of bNAbs and later boosting with Env proteins from heterologous virus strains and (or) in distinct conformations (polyvalent Env immunogen formulations) to mimic virus diversification, which tries to replicate what happens in infected individuals who develop neutralizing serum activity of considerable breadth and potency.74,88–90 There is still a long way to develop a safe and effective vaccine against HIV/AIDS. However, insights into the structures of HIV-1 trimeric Env in different states, understanding the tug-of-war between virus and host in co-evolution, and advances in technologies for the isolation of more potent neutralizing antibodies, together will bring us closer to the goal of the ultimate success of a safe and effective vaccine.

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