Membrane-proximal External Region Epitopes Research Articles

Stable expression of HIV-1 MPER extended epitope on the surface of the recombinant probiotic bacteria Escherichia Coli Nissle 1917 using CRISPR/Cas9

BackgroundMucosal vaccines have the potential to induce protective immune responses at the sites of infection. Applying CRISPR/Cas9 editing, we aimed to develop a probiotic-based vaccine candidate expressing the HIV-1 envelope membrane-proximal external region (MPER) on the surface of E. coli Nissle 1917.ResultsThe HIV-1 MPER epitope was successfully introduced in the porin OmpF of the E. coli Nissle 1917 (EcN-MPER) and the modification was stable over 30 passages of the recombinant bacteria on the DNA and protein level. Furthermore, the introduced epitope was recognized by a human anti-HIV-1 gp41 (2F5) antibody using both live and heat-killed EcN-MPER, and this antigenicity was also retained over 30 passages. Whole-cell dot blot suggested a stronger binding of anti-HIV-1 gp41 (2F5) to heat-killed EcN-MPER than their live counterpart. An outer membrane vesicle (OMV) – rich extract from EcN-MPER culture supernatant was equally antigenic to anti-HIV-1 gp41 antibody which suggests that the MPER antigen could be harboured in EcN-MPER OMVs. Using quantitative ELISA, we determined the amount of MPER produced by the modified EcN to be 14.3 µg/108 cfu.ConclusionsThe CRISPR/Cas9 technology was an effective method for establishment of recombinant EcN-MPER bacteria that was stable over many passages. The developed EcN-MPER clone was devoid of extraneous plasmids and antibiotic resistance genes which eliminates the risk of plasmid transfer to animal hosts, should this clone be used as a vaccine. Also, the EcN-MPER clone was recognised by anti-HIV-1 gp41 (2F5) both as live and heat-killed bacteria making it suitable for pre-clinical evaluation. Expression of OmpF on bacterial surfaces and released OMVs identifies it as a compelling candidate for recombinant epitope modification, enabling surface epitope presentation on both bacteria and OMVs. By applying the methods described in this study, we present a potential platform for cost-effective and rational vaccine antigen expression and administration, offering promising prospects for further research in the field of vaccine development.

Dependence on a variable residue limits the breadth of an HIV MPER neutralizing antibody, despite convergent evolution with broadly neutralizing antibodies

Broadly neutralizing antibodies (bNAbs) that target the membrane-proximal external region (MPER) of HIV gp41 envelope, such as 4E10, VRC42.01 and PGZL1, can neutralize >80% of viruses. These three MPER-directed monoclonal antibodies share germline antibody genes (IGHV1-69 and IGKV3-20) and form a bNAb epitope class. Furthermore, convergent evolution within these two lineages towards a 111.2GW111.3 motif in the CDRH3 is known to enhance neutralization potency. We have previously isolated an MPER neutralizing antibody, CAP206-CH12, that uses these same germline heavy and light chain genes but lacks breadth (neutralizing only 6% of heterologous viruses). Longitudinal sequencing of the CAP206-CH12 lineage over three years revealed similar convergent evolution towards 111.2GW111.3 among some lineage members. Mutagenesis of CAP206-CH12 from 111.2GL111.3 to 111.2GW111.3 and the introduction of the double GWGW motif into CAP206-CH12 modestly improved neutralization potency (2.5–3-fold) but did not reach the levels of potency of VRC42.01, 4E10 or PGZL1. To explore the lack of potency/breadth, viral mutagenesis was performed to map the CAP206-CH12 epitope. This indicated that CAP206-CH12 is dependent on D674, a highly variable residue at the solvent-exposed elbow of MPER. In contrast, VRC42.01, PGZL1 and 4E10 were dependent on highly conserved residues (W672, F673, T676, and W680) facing the hydrophobic patch of the MPER. Therefore, while CAP206-CH12, VRC42.01, PGZL1 and 4E10 share germline genes and show some evidence of convergent evolution, their dependence on different amino acids, which impacts orientation of binding to the MPER, result in differences in breadth and potency. These data have implications for the design of HIV vaccines directed at the MPER epitope.

Immunogenicity of a germline-targeting nanoparticle in knock-in mice expressing human B cell receptors of the HIV gp41 neutralizing antibody, DH511

Abstract The Membrane Proximal External Region (MPER) of HIV Envelope represents a key target for vaccine development due to high neutralization breadth and potency of MPER-specific broadly neutralizing antibodies (bnAbs). However, neutralizing antibody responses to MPER epitopes are restricted by tolerance control and the MPER epitope is absent from many HIV immunogens under clinical investigation. Using computational design and yeast display, a candidate germline-targeting (GT5) immunogen was developed that bound strongly to the inferred human unmutated common ancestor (UCA) of the distal MPER bnAb DH511, as well as to several human DH511-like potential precursor antibodies. We studied the immunogenicity of a multimeric nanoparticle of the GT5 immunogen in a knock-in mouse line expressing human DH511.UCA B cell receptors (BCRs). Naïve DH511.UCA knock-in mice exhibited a reduction in overall B cell numbers, and DH511.UCA-bearing B cells expressed low levels of surface IgM and IgD, suggesting that DH511.UCA expression is subject to immune tolerance control. Nonetheless, following immunization with GT5 nanoparticles mixed with a saponin/monophosphoryl lipid A adjuvant, knock-in mice mounted robust anti-GT5 humoral responses, including anti-GT5 IgG in serum and GT5-specific germinal center B cells and T follicular helper cells in lymphoid tissues. Sequencing analysis of IgG+ GT5-specific B cells revealed improbable mutations in knock-in immunoglobulin genes of DH511.UCA. These studies will guide further optimization of immunogens with potential to select for development of bnAbs against MPER epitopes. Supported by a grant from NIH (P01-AI138211)

N-terminal Transmembrane-Helix Epitope Tag for X-ray Crystallography and Electron Microscopy of Small Membrane Proteins

Structural studies of membrane proteins, especially small membrane proteins, are associated with well-known experimental challenges. Complexation with monoclonal antibody fragments is a common strategy to augment such proteins; however, generating antibody fragments that specifically bind a target protein is not trivial. Here we identify a helical epitope, from the membrane-proximal external region (MPER) of the gp41-transmembrane subunit of the HIV envelope protein, that is recognized by several well-characterized antibodies and that can be fused as a contiguous extension of the N-terminal transmembrane helix of a broad range of membrane proteins. To analyze whether this MPER-epitope tag might aid structural studies of small membrane proteins, we determined an X-ray crystal structure of a membrane protein target that does not crystallize without the aid of crystallization chaperones, the Fluc fluoride channel, fused to the MPER epitope and in complex with antibody. We also demonstrate the utility of this approach for single particle electron microscopy with Fluc and two additional small membrane proteins that represent different membrane protein folds, AdiC and GlpF. These studies show that the MPER epitope provides a structurally defined, rigid docking site for antibody fragments that is transferable among diverse membrane proteins and can be engineered without prior structural information. Antibodies that bind to the MPER epitope serve as effective crystallization chaperones and electron microscopy fiducial markers, enabling structural studies of challenging small membrane proteins.

Oral administration of a self-assembling nanofiber-based vaccine generates neutralizing antibodies against HIV and expands CD8+ memory T cell populations

Abstract Human immunodeficiency virus (HIV) is the world’s sixth leading cause of death. Antiretroviral therapy (ART) is the current standard of treatment, but issues of accessibility and compliance pose additional hurdles. In 2018, there were 37.9 million people living with HIV, but only 62% were on ART. The development of a successful vaccine is imperative to combat the transmission of HIV, of which one of the major goals is to generate broadly neutralizing antibodies (bnAb). The membrane-proximal external region (MPER) of gp41 in the envelope glycoprotein (Env) is a largely conserved motif, a key player in fusion, and the target of bnAb in a subset of patients with long-term HIV infections. We previously demonstrated the efficacy of a nanofiber vaccine platform as evidenced by the generation of both strong humoral and cell-mediated responses against viral targets, and enhanced protection against infection. In recent studies, we observed that serial oral dosing of nanofibers (NF) bearing the MPER epitope (NF-MPER) in mice correlated with a significant increase in serum and mucosal neutralizing antibodies against HIV. The NF-MPER vaccine also augmented CD8+ memory T cell populations in the colon, which were further characterized as effector memory T cells (TEM), using surface marker phenotypes (CD69+/− CD103+ CD44+ CD62Llo). Additionally, we observed significant expansion of CD8+ TEM pools in the spleen, a reservoir for HIV, a subset of which were similar to tissue resident memory T cells (TRM) seen in human elite controllers. These results suggest that oral immunization with a NF-based vaccine elicits the production of bnAb and enhanced CD8+ TEM populations, in non-lymphoid and lymphoid tissues, that are necessary for a successful HIV vaccine.

Structural Basis for Broad HIV-1 Neutralization by a Novel MPER-Specific Human Broadly Neutralizing Antibody

Potent and broadly neutralizing antibodies (bnAbs) are the hallmark of HIV-1 protection by vaccination. The membrane-proximal external region (MPER) of the HIV-1 gp41 fusion protein is targeted by the most broadly reactive HIV-1 neutralizing antibodies. Here, we examine the structural and molecular mechansims of neutralization by anti-MPER bnAb, LN01, which was isolated from lymph-node-derived germinal center B cells of an elite controller and exhibits broad neutralization breadth. LN01 engages both MPER and the transmembrane (TM) region, which together form a continuous helix in complex with LN01. The tilted TM orientation allows LN01 to interact simultaneously with the peptidic component of the MPER epitope and membrane via two specific lipid binding sites of the antibody paratope. Although LN01 carries a high load of somatic mutations, most key residues interacting with the MPER epitope and lipids are germline encoded, lending support for the LN01 epitope as a candidate for lineage-based vaccine development.

The Bilayer Collective Properties Govern the Interaction of an HIV-1 Antibody with the Viral Membrane

Efficient engagement with the envelope glycoprotein membrane-proximal external region (MPER) results in robust blocking of viral infection by a class of broadly neutralizing antibodies (bnAbs) against human immunodeficiency virus (HIV). Developing an accommodation surface that engages with the viral lipid envelope appears to correlate with the neutralizing potency displayed by these bnAbs. The nature of the interactions established between the antibody and the lipid is nonetheless a matter of debate, with some authors arguing that anti-MPER specificity arises only under pathological conditions in autoantibodies endowed with stereospecific binding sites for phospholipids. However, bnAb-lipid interactions are often studied in systems that do not fully preserve the biophysical properties of lipid bilayers, and therefore, questions on binding specificity and the effect of collective membrane properties on the interaction are still open. Here, to evaluate the specificity of lipid interactions of an anti-MPER bnAb (4E10) in an intact membrane context, we determine quantitatively its association with lipid bilayers by means of scanning fluorescence correlation spectroscopy and all-atom molecular dynamic simulations. Our data support that 4E10 establishes electrostatic and hydrophobic interactions with the viral membrane surface and that the collective physical properties of the lipid bilayer influence 4E10 dynamics therein. We conclude that establishment of peripheral, nonspecific electrostatic interactions with the viral membrane through accommodation surfaces may assist high-affinity binding of HIV-1 MPER epitope at membrane interfaces. These findings highlight the importance of considering antibody-lipid interactions in the design of antibody-based anti-HIV strategies.

Structural Basis for Broad HIV-1 Neutralization by the MPER-Specific Human Broadly Neutralizing Antibody LN01

SummaryPotent and broadly neutralizing antibodies (bnAbs) are the hallmark of HIV-1 protection by vaccination. The membrane-proximal external region (MPER) of the HIV-1 gp41 fusion protein is targeted by the most broadly reactive HIV-1 neutralizing antibodies. Here, we examine the structural and molecular mechansims of neutralization by anti-MPER bnAb, LN01, which was isolated from lymph-node-derived germinal center B cells of an elite controller and exhibits broad neutralization breadth. LN01 engages both MPER and the transmembrane (TM) region, which together form a continuous helix in complex with LN01. The tilted TM orientation allows LN01 to interact simultaneously with the peptidic component of the MPER epitope and membrane via two specific lipid binding sites of the antibody paratope. Although LN01 carries a high load of somatic mutations, most key residues interacting with the MPER epitope and lipids are germline encoded, lending support for the LN01 epitope as a candidate for lineage-based vaccine development.

HIV-1 Membrane-Proximal External Region Fused to Diphtheria Toxin Domain-A Elicits 4E10-Like Antibodies in Mice

The production of broadly neutralizing antibodies (bNAbs) is a major goal in the development of an HIV-1 vaccine. The membrane-proximal external region (MPER) of gp41, which plays a critical role in the virus membrane fusion process, is highly conserved and targeted by bNAbs 2F5, 4E10, and 10E8. As such, MPER could be a promising epitope for vaccine design. In this study, diphtheria toxin domain A (CRM197, amino acids 1–191) was used as a scaffold to display the 2F5 and 4E10 epitopes of MPER, named CRM197-A-2F5 and CRM197-A-4E10. Modest neutralizing activities were detected against HIV-1 clade B and D viruses in the sera from mice immunized with CRM197-A-4E10. Monoclonal antibodies raised from CRM197-A-4E10 could neutralize several HIV-1 strains, and epitope-mapping analysis indicated that some antibodies recognized the same amino acids as 4E10. Collectively, we show that 4E10-like antibodies can be induced by displaying MPER epitopes using an appropriate scaffold. These results provide insights for HIV-1 MPER-based immunogens design.

Molecular recognition of the native HIV-1 MPER revealed by STED microscopy of single virions

Antibodies against the Membrane-Proximal External Region (MPER) of the Env gp41 subunit neutralize HIV-1 with exceptional breadth and potency. Due to the lack of knowledge on the MPER native structure and accessibility, different and exclusive models have been proposed for the molecular mechanism of MPER recognition by broadly neutralizing antibodies. Here, accessibility of antibodies to the native Env MPER on single virions has been addressed through STED microscopy. STED imaging of fluorescently labeled Fabs reveals a common pattern of native Env recognition for HIV-1 antibodies targeting MPER or the surface subunit gp120. In the case of anti-MPER antibodies, the process evolves with extra contribution of interactions with the viral lipid membrane to binding specificity. Our data provide biophysical insights into the recognition of the potent and broadly neutralizing MPER epitope on HIV virions, and as such is of importance for the design of therapeutic interventions.

Structure of the membrane proximal external region of HIV-1 envelope glycoprotein

The membrane-proximal external region (MPER) of the HIV-1 envelope glycoprotein (Env) bears epitopes of broadly neutralizing antibodies (bnAbs) from infected individuals; it is thus a potential vaccine target. We report an NMR structure of the MPER and its adjacent transmembrane domain in bicelles that mimic a lipid-bilayer membrane. The MPER lies largely outside the lipid bilayer. It folds into a threefold cluster, stabilized mainly by conserved hydrophobic residues and potentially by interaction with phospholipid headgroups. Antigenic analysis and comparison with published images from electron cryotomography of HIV-1 Env on the virion surface suggest that the structure may represent a prefusion conformation of the MPER, distinct from the fusion-intermediate state targeted by several well-studied bnAbs. Very slow bnAb binding indicates that infrequent fluctuations of the MPER structure give these antibodies occasional access to alternative conformations of MPER epitopes. Mutations in the MPER not only impede membrane fusion but also influence presentation of bnAb epitopes in other regions. These results suggest strategies for developing MPER-based vaccine candidates.

Function of the RNA Coliphage Qβ Proteins in Medical In Vitro Evolution.

Qβ is a positive (+) single-stranded RNA bacteriophage covered by a 25 nm icosahedral shell. Qβ belongs to the family of Leviviridae and is found throughout the world (bacterial isolates and sewage). The genome of Qβ is about 4.2 kb, coding for four proteins. This genome is surrounded by 180 copies of coat proteins (capsomers) each comprised of 132 residues of amino acids. The other proteins, the subunit II (β) of a replicase, the maturation protein (A2) and the read-through or minor coat protein (A1), play a key role in phage infection. With the replicase protein, which lacks proofreading activity, as well as its short replication time, and high population size, Qβ phage has attractive features for in vitro evolution. The A1 protein gene shares the same initiation codon with the coat protein gene and is produced during translation when the coat protein’s UGA stop codon triplet (about 400 nucleotides from the initiation) is suppressed by a low level of ribosome misincorporation of tryptophan. Thus, A1 is termed the read-through protein. This RNA phage platform technology not only serves to display foreign peptides but is also exceptionally suited to address questions about in vitro evolution. The C-terminus of A1 protein confers to this RNA phage platform an exceptional feature of not only a linker for foreign peptide to be displayed also a model for evolution. This platform was used to present a peptide library of the G-H loop of the capsid region P1 of the foot-and-mouth disease virus (FMDV) called VP1 protein. The library was exposed on the exterior surface of Qβ phages, evolved and selected with the monoclonal antibodies (mAbs) SD6 of the FMDV. These hybrid phages could principally be good candidates for FMDV vaccine development. Separately, the membrane proximal external region (MPER) of human immunodeficiency virus type 1 (HIV-1) epitopes was fused with the A1 proteins and exposed on the Qβ phage exterior surface. The engineered phages with MPER epitopes were recognized by anti-MPER specific antibodies. This system could be used to overcome the challenge of effective presentation of MPER to the immune system. A key portion of this linear epitope could be randomized and evolved with the Qβ system. Overall, antigens and epitopes of RNA viruses relevant to public health can be randomized, evolved and selected in pools using the proposed Qβ model to overcome their plasticity and the challenge of vaccine development. Major epitopes of a particular virus can be engineered or displayed on the Qβ phage surface and used for vaccine efficacy evaluation, thus avoiding the use of live viruses.

Functional Optimization of Broadly Neutralizing HIV-1 Antibody 10E8 by Promotion of Membrane Interactions.

The 10E8 antibody targets a helical epitope in the membrane-proximal external region (MPER) and transmembrane domain (TMD) of the envelope glycoprotein (Env) subunit gp41 and is among the broadest known neutralizing antibodies against HIV-1. Accordingly, this antibody and its mechanism of action valuably inform the design of effective vaccines and immunotherapies. 10E8 exhibits unusual adaptations to attain specific, high-affinity binding to the MPER at the viral membrane interface. Reversing the charge of the basic paratope surface (from net positive to net negative) reportedly lowered its neutralization potency. Here, we hypothesized that by increasing the net positive charge in similar polar surface patches, the neutralization potency of the antibody may be enhanced. We found that an increased positive charge at this paratope surface strengthened an electrostatic interaction between the antibody and lipid bilayers, enabling 10E8 to interact spontaneously with membranes. Notably, the modified 10E8 antibody did not gain any apparent polyreactivity and neutralized virus with a significantly greater potency. Binding analyses indicated that the optimized 10E8 antibody bound with a higher affinity to the epitope peptide anchored in lipid bilayers and to Env spikes on virions. Overall, our data provide a proof of principle for the rational optimization of 10E8 via manipulation of its interaction with the membrane element of its epitope. However, the observation that a similar mutation strategy did not affect the potency of the first-generation anti-MPER antibody 4E10 shows possible limitations of this principle. Altogether, our results emphasize the crucial role played by the viral membrane in the antigenicity of the MPER-TMD of HIV-1.IMPORTANCE The broadly neutralizing antibody 10E8 blocks infection by nearly all HIV-1 isolates, a capacity which vaccine design seeks to reproduce. Engineered versions of this antibody also represent a promising treatment for HIV infection by passive immunization. Understanding its mechanism of action is therefore important to help in developing effective vaccines and biologics to combat HIV/AIDS. 10E8 engages its helical MPER epitope where the base of the envelope spike submerges into the viral membrane. To enable this interaction, this antibody evolved an unusual property: the ability to interact with the membrane surface. Here, we provide evidence that 10E8 can be made more effective by enhancing its interactions with membranes. Our findings strengthen the idea that to elicit antibodies similar to 10E8, vaccines must reproduce the membrane environment where these antibodies perform their function.

Exposure of the HIV-1 broadly neutralizing antibody 10E8 MPER epitope on the membrane surface by gp41 transmembrane domain scaffolds

The 10E8 antibody achieves near-pan neutralization of HIV-1 by targeting the remarkably conserved gp41 membrane-proximal external region (MPER) and the connected transmembrane domain (TMD) of the HIV-1 envelope glycoprotein (Env). Thus, recreating the structure that generates 10E8-like antibodies is a major goal of the rational design of anti-HIV vaccines. Unfortunately, high-resolution information of this segment in the native Env is lacking, limiting our understanding of the behavior of the crucial 10E8 epitope residues.In this report, two sequences, namely, MPER-TMD1 (gp41 residues 671–700) and MPER-TMD2 (gp41 residues 671–709) were compared both experimentally and computationally, to assess the TMD as a potential membrane integral scaffold for the 10E8 epitope. These sequences were selected to represent a minimal (MPER-TMD1) or full-length (MPER-TMD2) TMD membrane anchor according to mutagenesis results reported by Yue et al. (2009) J. Virol. 83, 11,588. Immunochemical assays revealed that MPER-TMD1, but not MPER-TMD2, effectively exposed the MPER C-terminal stretch, harboring the 10E8 epitope on the surface of phospholipid bilayers containing a cholesterol concentration equivalent to that of the viral envelope. Molecular dynamics simulations, using the recently resolved TMD trimer structure combined with the MPER in a cholesterol-enriched model membrane confirmed these results and provided an atomistic mechanism of epitope exposure which revealed that TMD truncation at position A700 combined with N-terminal addition of lysine residues positively impacts epitope exposure. Overall, these results provide crucial insights into the design of effective MPER-TMD derived immunogens.

Surface Engineering of Recombinant RNA Coliphage Qβ to Display gp41 MembraneProximal External-Region Epitopes from HIV-1

Introduction: The membrane proximal external region (MPER) of HIV-1 envelope glycoprotein-41 (gp41) is targeted by several broadly neutralizing antibodies whose conserved linear epitopes are promising targets for vaccine design. However, a formidable challenge has remained the difficulty to design and deliver MPER based immunogens for the efficient induction of such broadly neutralizing HIV-1 specific antibodies (bnAb). This is mainly because the linear bnAb MPER epitopes are poorly accessible to the immune system. The overall objective of this study therefore was the development of a novel RNA Qβ phage display system not only for monitoring anti-MPER specific antibody responses but equally as potential immunogens in future HIV-1 vaccine designs. Method: To overcome the challenge of effective presentation of MPER to the immune system we have selectively engineered the surface of the RNA coliphage Qβ to expose all MPER bnAb epitopes. Briefly, DNA representing a 50 amino acids consensus region within the HIV-1 gp41 MPER was fused in frame with the minor coat protein A1of the Qβ phage. Three variant MPER expression cassettes were obtained with the MPER cDNA in frame with the minor coat protein A1 gene, including pQβMPER, pQβMPERHis and pQβMPERN. The expression cassettes were used for the production of QβMPER recombinant phages after transformation of E. coli HB101 strain. Antigencity of the phages was assessed with plasma from long standing anti-retroviral naïve HIV-1 infected people from the CIRCB AFRODEC cohort while immunogenicity studies were done in female Balb/c mice. Results: The initial titers of all recombinant phages including QβMPER, QβMPERHis and QβMPERN were 104 plaque forming units/ml (pfu/ml). This was significantly lower (P<0.001) relative to the 108 pfu/ml of wild type phage, but was scaled up to 1014pfu/ml. The fusion of MPER and Qβ genes was confirmed by RT-PCR followed by gel electrophoresis and sequencing. Specific recognition of some reported bnAb epitopes within MPER were confirmed in ELISA using the three recombinant QβMPER phages together with an MPER restrictive peptide as antigens and the bnAb 4E10, Z13e1, 2F5 and 10E8 as antibodies. Next the prevalence of MPER-specific antibodies was determined in plasma from long standing antiretroviral naïve HIV-1 infected participants of the CIRCB AFRODEC cohort. The greater majority (84%) of participants’ plasma showed MPER peptide specific reactivity with anti-MPER specific IgG antibody titers ranging from 200 to 409600 comparative to background IgG antibody titer with the Qβ phage alone as antigen or plasma from seronegative participants. In immunogenicity studies in Balb/c mice the recombinant phages QβMPERN and QβMPERHis induced significantly high Anti-MPER-specific IgG antibody responses (P<0.04) in at least 60 % of mice following three inoculations of each recombinant phage. Conclusion: Thus, these novel recombinantQβMPER phages can be used to monitor MPER-specific immune responses in HIV-1 exposed or infected people. In addition the recombinant QβMPER phages could be used as immunogens either alone as demonstrated here in mice or in combination with other strategies for the induction of MPER specific immunity against HIV-1.

Immunologic Insights on the Membrane Proximal External Region: A Major Human Immunodeficiency Virus Type-1 Vaccine Target.

Broadly neutralizing antibodies (bNAbs) targeting conserved regions within the human immunodeficiency virus type-1 (HIV-1) envelope glycoprotein (Env) can be generated by the human immune system and their elicitation by vaccination will be a key point to protect against the wide range of viral diversity. The membrane proximal external region (MPER) is a highly conserved region within the Env gp41 subunit, plays a major role in membrane fusion and is targeted by naturally induced bNAbs. Therefore, the MPER is considered as an attractive vaccine target. However, despite many attempts to design MPER-based immunogens, further study is still needed to understand its structural complexity, its amphiphilic feature, and its limited accessibility by steric hindrance. These particular features compromise the development of MPER-specific neutralizing responses during natural infection and limit the number of bNAbs isolated against this region, as compared with other HIV-1 vulnerability sites, and represent additional hurdles for immunogen development. Nevertheless, the analysis of MPER humoral responses elicited during natural infection as well as the MPER bNAbs isolated to date highlight that the human immune system is capable of generating MPER protective antibodies. Here, we discuss the recent advances describing the immunologic and biochemical features that make the MPER a unique HIV-1 vulnerability site, the different strategies to generate MPER-neutralizing antibodies in immunization protocols and point the importance of extending our knowledge toward new MPER epitopes by the isolation of novel monoclonal antibodies. This will be crucial for the redesign of immunogens able to skip non-neutralizing MPER determinants.

Differential Antibody Responses to Conserved HIV-1 Neutralizing Epitopes in the Context of Multivalent Scaffolds and Native-Like gp140 Trimers.

ABSTRACTBroadly neutralizing antibodies (bNAbs) have provided valuable insights into the humoral immune response to HIV-1. While rationally designed epitope scaffolds and well-folded gp140 trimers have been proposed as vaccine antigens, a comparative understanding of their antibody responses has not yet been established. In this study, we probed antibody responses to the N332 supersite and the membrane-proximal external region (MPER) in the context of heterologous protein scaffolds and native-like gp140 trimers. Ferritin nanoparticles and fragment crystallizable (Fc) regions were utilized as multivalent carriers to display scaffold antigens with grafted N332 and MPER epitopes, respectively. Trimeric scaffolds were also identified to stabilize the MPER-containing BG505 gp140.681 trimer in a native-like conformation. Following structural and antigenic evaluation, a subset of scaffold and trimer antigens was selected for immunization in BALB/c mice. Serum binding revealed distinct patterns of antibody responses to these two bNAb targets presented in different structural contexts. For example, the N332 nanoparticles elicited glycan epitope-specific antibody responses that could also recognize the native trimer, while a scaffolded BG505 gp140.681 trimer generated a stronger and more rapid antibody response to the trimer apex than its parent gp140.664 trimer. Furthermore, next-generation sequencing (NGS) of mouse splenic B cells revealed expansion of antibody lineages with long heavy-chain complementarity-determining region 3 (HCDR3) loops upon activation by MPER scaffolds, in contrast to the steady repertoires primed by N332 nanoparticles and a soluble gp140.664 trimer. These findings will facilitate the future development of a coherent vaccination strategy that combines both epitope-focused and trimer-based approaches.

Enhanced antibody-mediated neutralization of HIV-1 variants that are resistant to fusion inhibitors.

BackgroundHIV-1 typically develops resistance to any single antiretroviral agent. Combined anti-retroviral therapy to reduce drug-resistance development is necessary to control HIV-1 infection. Here, to assess the utility of a combination of antibody and fusion inhibitor treatments, we investigated the potency of monoclonal antibodies at neutralizing HIV-1 variants that are resistant to fusion inhibitors.ResultsMutations that confer resistance to four fusion inhibitors, enfuvirtide, C34, SC34, and SC34EK, were introduced into the envelope of HIV-1JR-FL, a CCR5-tropic tier 2 strain. Pseudoviruses with these mutations were prepared and used for the assessment of neutralization sensitivity to an array of antibodies. The resulting neutralization data indicate that the potencies of some antibodies, especially of those against the CD4 binding site, V3 loop, and membrane-proximal external region epitopes, were increased by the mutations in gp41 that conferred resistance to the fusion inhibitors. C34-, SC34-, and SC34EK-resistant mutants showed more sensitivity to monoclonal antibodies than enfuvirtide-resistant mutants. An analysis of C34-resistant mutations revealed that the I37K mutation in gp41 HR1 is a key mutation for C34 resistance, low infectivity, neutralization sensitivity, epitope exposure, and slow fusion kinetics. The N126K mutation in the gp41 HR2 domain contributed to C34 resistance and neutralization sensitivity to anti-CD4 binding site antibodies. In the absence of L204I, the effect of N126K was antagonistic to that of I37K. The results of a molecular dynamic simulation of the envelope trimer confirmation suggest that an I37K mutation induces the augmentation of structural fluctuations prominently in the interface between gp41 and gp120. Our observations indicate that the “conformational unmasking” of envelope glycoprotein by an I37K mutation is one of the mechanisms of neutralization sensitivity enhancement. Furthermore, the enhanced neutralization of C34-resistant mutants in vivo was shown by its high rate of neutralization by IgG from HIV patient samples.ConclusionsMutations in gp41 that confer fusion inhibitor resistance exert enhanced sensitivity to broad neutralizing antibodies (e.g., VRC01 and 10E8) and other conventional antibodies developed in HIV-1 infected patients. Therefore, next-generation fusion inhibitors and monoclonal antibodies could be a potential combination for future regimens of combined antiretroviral therapy.Electronic supplementary materialThe online version of this article (doi:10.1186/s12977-016-0304-7) contains supplementary material, which is available to authorized users.

N-terminal residues of an HIV-1 gp41 membrane-proximal external region antigen influence broadly neutralizing 2F5-like antibodies.

The Human immunodeficiency virus type 1 (HIV-1) gp41 membrane proximal external region (MPER) is targeted by broadly neutralizing antibodies (e.g. 2F5, 4E10, Z13e and m66.6), which makes this region a promising target for vaccine design. One strategy to elicit neutralizing antibodies against the MPER epitope is to design peptide immunogens mimicking neutralization structures. To probe 2F5-like neutralizing antibodies, two yeast-displayed antibody libraries from peripheral blood mononuclear cells from a HIV-1 patient were screened against the 2F5 epitope peptide SP62. Two 2F5-like antibodies were identified that specifically recognized SP62. However, these antibodies only weakly neutralized HIV-1 primary isolates. The epitopes recognized by these two 2F5-like antibodies include not only the 2F5 epitope (amino acids (aa) 662-667 in the MPER) but also several other residues (aa 652-655) locating at the N-terminus in SP62. Experimental results suggest that residues of SP62 adjacent to the 2F5 epitope influence the response of broadly neutralizing 2F5-like antibodies in vaccination. Our findings may aid the design of vaccine immunogens and development of therapeutics against HIV-1 infection.

Thermodynamic Analysis of the Binding of 2F5 (Fab and Immunoglobulin G Forms) to Its gp41 Epitope Reveals a Strong Influence of the Immunoglobulin Fc Region on Affinity

Immunotherapies and vaccines based on the induction of broadly neutralizing monoclonal antibodies (bNAbs) have become outstanding strategies against HIV-1. Diverse bNAbs recognizing different regions of the HIV-1 envelope have been identified and extensively studied. However, there is little information about the thermodynamics of binding of these bNAbs and their epitopes. We used isothermal titration calorimetry to characterize thermodynamically the interactions between bNAb2F5 (in both the IgG and Fab forms) and its functional and core epitope peptides. We found that these interactions are enthalpically driven and opposed by a negative entropy change. The highest affinity was found for 2F5 IgG for its functional epitope, indicating that additional interactions involving residues flanking the core epitope contribute strongly to higher affinity. In addition, the strong influence of the Fc region on the binding affinity suggests long-range allosteric effects within IgG. Our results provide useful information for developing new therapeutics against HIV-1 and, in a broader scope, contribute to a better understanding of antigen-antibody recognition.