Antibodies to HIV-1: aiming at the right target (original) (raw)
Related papers
Virology, 2006
HIV envelope glycoprotein (Env) is the target for inducing neutralizing antibodies. Env is present on the virus surface as a trimer, and, upon binding to CD4, a cascade of events leads to structural rearrangement exposing the co-receptor binding site and entry into the CD4+ host target cells. We have designed monomeric and trimeric Env constructs with and without deletion of the variable loop 2 (ΔV2) from SF162, a subtype B primary isolate, and performed biophysical, biochemical and immunological studies to establish a potential structure-functional relationship. We expressed these Envs in CHO cells, purified the proteins to homogeneity and performed biophysical studies to define the binding properties to CD4, structural characteristics and exposure of epitopes recognized by b12 and CD4i mAb (17B) on both full-length and mutant HIV Env proteins. Parameters evaluated include oligomerization state, number and affinity of CD4 binding sites, enthalpy and entropy of the Env-CD4 interaction and affinity for b12 and 17b mAbs. We observed one CD4 binding site per monomer and three active CD4 binding sites per trimer. A 40-fold difference in affinity of the gp120 monomer vs. the o-gp140 trimer towards CD4 was observed (Kd = 58 nM and 1.5 nM, respectively), whereas only a 2-fold difference was observed for the V2 deleted Envs (Kd of gp120ΔV2 = 19 nM, Kd of o-gp140DV2 = 9.3 nM). Monomers had 3-fold higher affinity to the mAb 17b and at least 3-fold weaker affinity to b12 compared to trimers, with gp120DV2 having the weakest affinity for b12 (Kd = 446 nM). Affinity of CD4 binding correlated with proportion of the antibodies induced against the conformational epitopes by the corresponding Envs, and changes in mAb binding correlated with the induction of antibodies directed against linear epitopes. Furthermore, biophysical analysis reveals that the V2 deletion has broad structural implications in the monomer not shared by the trimer, and these changes are reflected in the quality of the immune responses induced in rabbits. These data suggest that biophysical characteristics of HIV Env, such as affinity for CD4, and exposure of important neutralizing epitopes, such as those recognized by b12 mAb, may be important predictors of its in vivo efficacy and may serve as important surrogate markers for screening Env structures as potential vaccine candidates.
Evolution of an HIV glycan–dependent broadly neutralizing antibody epitope through immune escape
Nature Medicine, 2012
l e t t e r s nature medicine advance online publication Neutralizing antibodies are likely to play a crucial part in a preventative HIV-1 vaccine. Although efforts to elicit broadly cross-neutralizing (BCN) antibodies by vaccination have been unsuccessful 1-3 , a minority of individuals naturally develop these antibodies after many years of infection 4-7 . How such antibodies arise, and the role of viral evolution in shaping these responses, is unknown. Here we show, in two HIV-1-infected individuals who developed BCN antibodies targeting the glycan at Asn332 on the gp120 envelope, that this glycan was absent on the initial infecting virus. However, this BCN epitope evolved within 6 months, through immune escape from earlier strainspecific antibodies that resulted in a shift of a glycan to position 332. Both viruses that lacked the glycan at amino acid 332 were resistant to the Asn332-dependent BCN monoclonal antibody PGT128 (ref. 8), whereas escaped variants that acquired this glycan were sensitive. Analysis of large sequence and neutralization data sets showed the 332 glycan to be significantly underrepresented in transmitted subtype C viruses compared to chronic viruses, with the absence of this glycan corresponding with resistance to PGT128. These findings highlight the dynamic interplay between early antibodies and viral escape in driving the evolution of conserved BCN antibody epitopes.
A potent and broad neutralizing antibody recognizes and penetrates the HIV glycan shield
Science (New York, N.Y.), 2011
The HIV envelope (Env) protein gp120 is protected from antibody recognition by a dense glycan shield. However, several of the recently identified PGT broadly neutralizing antibodies appear to interact directly with the HIV glycan coat. Crystal structures of antigen-binding fragments (Fabs) PGT 127 and 128 with Man(9) at 1.65 and 1.29 angstrom resolution, respectively, and glycan binding data delineate a specific high mannose-binding site. Fab PGT 128 complexed with a fully glycosylated gp120 outer domain at 3.25 angstroms reveals that the antibody penetrates the glycan shield and recognizes two conserved glycans as well as a short β-strand segment of the gp120 V3 loop, accounting for its high binding affinity and broad specificity. Furthermore, our data suggest that the high neutralization potency of PGT 127 and 128 immunoglobulin Gs may be mediated by cross-linking Env trimers on the viral surface.
Neutralization function affected by single amino acid replacement in the HIV-1 antibody targets
Bioinformation, 2015
The viral envelope glycoproteins are essential for entry into their host cells and studied extensively for designing vaccines. We hypothesize that the glycosylation on the HIV-1 viral envelope glycoprotein 41(gp41) at critical residues offers viral escape from the specific immune surveillant neutralizing antibodies Z13, 4E10 and 10E8 targeted to their linear epitopes in the Membrane Proximal External Region (MPER). The glycosylation occurring on the 50th residue (Asparagine) contained in the target (NWFNIT) can mask itself to be inaccessible for these neutralizing antibodies. The glycosylation rate of the epitopes which are shared by the Z13, 4E10 and 10E8 neutralizing antibodies of HIV-1 were predicited in silico. We analyzed the reliable frequency of glycosylation on the HIV-1 envelope gp41 using prediction tools to unravel the plausibility of the glycosylation by a mannose at 50th residue in the 59 amino acid long HIV-gp41 trimer (PDBID: 2M7W and 2LP7). It is evident that the glycosylation by a mannose that masks these targets is possible only when the 50th amino-acid is N (Asparagine, Asn) which is not possible when N is mutated to D (Aspartatic acid, Asp). The additive advantage for the retrovirus is its error-prone reverse transcriptase which can choose to copy these survivable mutants with Asn N-50 that can be glycosylated as explained by the Copy-choice model. So the glycan shields varying in their intensity and patterns have to be essentially studied to understand the viral escape strategies that will give a way forward towards a successful vaccine that can elicit a neutralizing antibody response to confer protection.
Nature structural & molecular biology, 2016
HIV-1 vaccine design is informed by structural studies elucidating mechanisms by which broadly neutralizing antibodies (bNAbs) recognize and/or accommodate N-glycans on the trimeric envelope glycoprotein (Env). Variability in high-mannose and complex-type Env glycoforms leads to heterogeneity that usually precludes visualization of the native glycan shield. We present 3.5-Å- and 3.9-Å-resolution crystal structures of the HIV-1 Env trimer with fully processed and native glycosylation, revealing a glycan shield of high-mannose and complex-type N-glycans, which we used to define complete epitopes of two bNAbs. Env trimer was complexed with 10-1074 (against the V3-loop) and IOMA, a new CD4-binding site (CD4bs) antibody. Although IOMA derives from VH1-2*02, the germline gene of CD4bs-targeting VRC01-class bNAbs, its light chain lacks the short CDRL3 that defines VRC01-class bNAbs. Thus IOMA resembles 8ANC131-class/VH1-46-derived CD4bs bNAbs, which have normal-length CDRL3s. The existence...
Scientific Reports
The HIV-1 envelope (Env) glycoprotein is the primary target of the humoral immune response and a critical vaccine candidate. However, Env is densely glycosylated and thereby substantially protected from neutralisation. Importantly, glycan N301 shields V3 loop and CD4 binding site epitopes from neutralising antibodies. Here, we use molecular dynamics techniques to evaluate the structural rearrangements that maintain the protective qualities of the glycan shield after the loss of glycan N301. We examined a naturally occurring subtype C isolate and its N301A mutant; the mutant not only remained protected against neutralising antibodies targeting underlying epitopes, but also exhibited an increased resistance to the VRC01 class of broadly neutralising antibodies. Analysis of this mutant revealed several glycans that were responsible, independently or through synergy, for the neutralisation resistance of the mutant. These data provide detailed insight into the glycan shield's ability to compensate for the loss of a glycan, as well as the cascade of glycan movements on a protomer, starting at the point mutation, that affects the integrity of an antibody epitope located at the edge of the diminishing effect. These results present key, previously overlooked, considerations for HIV-1 Env glycan research and related vaccine studies. A key scientific challenge in the field of HIV-1 vaccine development is the design of immunogens that elicit antibodies capable of neutralising the wide range of HIV-1 isolates in circulation, despite its immense global diversity 1. Due to immune-mediated selection pressure, the majority of this diversity is in the viral envelope gene that encodes the Env proteins on the surface of a virion 2. The Env proteins facilitate viral entry to target cells and are formed by gp120/gp41 heterodimers that non-covalently associate, forming a trimer of heterodimers 3,4. Even though the majority of HIV-infected individuals mount an immune response targeting these Env trimers, within-host diversity ensures that certain strains continue to evade recognition and neutralisation 5-8. Large sections of the Env trimers are covered by dense glycosylation and roughly half of its molecular mass is made up by glycans 9,10 , which have been suggested to protect the virus from antibody binding and neutralisation 11-15. Changes in these glycosylation patterns can therefore have a large impact on its ability to escape from immune attack. Once HIV infiltrates the host cells, it takes advantage of host cellular biosynthetic pathways for its own benefit, which includes protein glycosylation as one of the main post-translational modifications 16. N-linked glycosylation occurs in the endoplasmic reticulum and Golgi apparatus, where glycans are attached to asparagine residues within an Asn-X-Ser/Thr motif (X is any amino acid except proline 16). The attached glycans, initially assumed to be immunologically inert "self " molecules, were until recently considered a largely insurmountable challenge for antibody recognition; hence termed, the "glycan shield" 8,17 .
Broad and Potent Neutralizing Antibodies Recognize the Silent Face of the HIV Envelope
Immunity, 2019
Broadly neutralizing antibodies (bNAbs) against HIV-1 envelope (Env) inform vaccine design and are potential therapeutic agents. We identified SF12 and related bNAbs with up to 62% neutralization breadth from an HIV-infected donor. SF12 recognized a glycan-dominated epitope on Env's silent face and was potent against clade AE viruses, which are poorly covered by V3-glycan bNAbs. A 3.3Å cryo-EM structure of a SF12-Env trimer complex showed additional contacts to Env protein residues by SF12 compared with VRC-PG05, the only other known donor-derived silentface antibody, explaining SF12's increased neutralization breadth, potency, and resistance to Env mutation routes. Asymmetric binding of SF12 was associated with distinct N-glycan conformations across Env protomers, demonstrating intra-Env glycan heterogeneity. Administrating SF12 to HIV-1-infected humanized mice suppressed viremia and selected for viruses lacking the N448 gp120 glycan. Effective bNAbs can therefore be raised against HIV-1 Env's silent face, suggesting their potential for HIV-1 prevention, therapy, and vaccine development.
Viruses, 2015
The mechanism of antibody-mediated protection is a major focus of HIV-1 vaccine development and a significant issue in the control of viremia. Virus neutralization, Fc-mediated effector function, or both, are major mechanisms of antibody-mediated protection against HIV-1, although other mechanisms, such as virus aggregation, are known. The interplay between virus neutralization and Fc-mediated effector function in protection against HIV-1 is complex and only partially understood. Passive immunization studies using potent broadly neutralizing antibodies (bnAbs) show that both neutralization and Fc-mediated effector function provides the widest dynamic range of protection; however, a vaccine to elicit these responses remains elusive. By contrast, active immunization studies in both humans and non-human primates using HIV-1 vaccine candidates suggest that weakly neutralizing or non-neutralizing antibodies can protect by Fc-mediated effector function, albeit with a much lower dynamic ra...
The Journal of …, 2007
Neutralizing Abs constitute a pivotal mechanism of the adaptive immune response against HIV-1 infection. Yet, most of the Abs that appear in the circulation during HIV infection are nonneutralizing. In this study, we report a dramatic change of the neutralizing properties of a human Ab reactive with the nonneutralizing epitope termed cluster I on the HIV-1 transmembrane protein gp41 when the Ab was produced in Chinese hamster ovary (CHO)-K1 cells. Our laboratory has previously reported that the Ab F240, when produced in a hybridoma, is nonneutralizing as assessed by standard neutralization assays. The F240 IgG1 Ab expressed in CHO cells acquired a strong neutralization activity against a broad range of HIV isolates without a change in immunoreactivity. Sequencing of the F240 mRNAs produced in the parental hybridoma and CHO cells revealed identical sequences, suggesting that acquired neutralization resulted from cell-specific posttranslational modifications. We found that the Ab produced by CHO cells is glycosylated to a greater extent than the parental Ab produced by the hybridoma. Moreover, treatment with peptide N-glycosidase F abrogated F240 neutralization, in an isolate-specific manner, but not Ab b12 neutralization. Interestingly, the F240 isotype-switched variants IgG3 and IgG4, also expressed in CHO cells, exhibited identical immunoreactivity to IgG1 isotypes but had clear differences in viral neutralization. These results suggest that structural features of the Ig molecule other than the primary sequence of the variable regions play a more prominent role in HIV neutralization than anticipated.
Molecular structures of trimeric HIV-1 Env in complex with small antibody derivatives
Proceedings of the National Academy of Sciences, 2013
The extensive carbohydrate coat, the variability of protein structural features on HIV-1 envelope glycoproteins (Env), and the steric constraints of the virus-cell interface during infection, present challenges to the elicitation of effective full-length (∼150 kDa), neutralizing antibodies against HIV. These hurdles have motivated the engineering of smaller antibody derivatives that can bind Env and neutralize the virus. To further understand the mechanisms by which these proteins neutralize HIV-1, we carried out cryoelectron tomography of native HIV-1 BaL virions complexed separately to two small (∼15 kDa) HIV-neutralizing proteins: A12, which binds the CD4-binding site on Env, and m36, whose binding to Env is enhanced by CD4 binding. We show that despite their small size, the presence of these proteins and their effects on the quaternary conformation of trimeric Env can be visualized in molecular structures derived by cryoelectron tomography combined with subvolume averaging. Binding of Env to A12 results in a conformational change that is comparable to changes observed upon its binding to the CD4-binding site antibody, b12. In contrast, binding of Env to m36 results in an "open" quaternary conformation similar to that seen with binding of soluble CD4 or the CD4i antibody, 17b. Because these small neutralizing proteins are less sterically hindered than full-length antibodies at zones of virus-cell contact, the finding that their binding has the same structural consequences as that of other broadly neutralizing antibodies highlights their potential for use in therapeutic applications. gp120 | gp41 | cryoelectron microscopy | AIDS vaccine | virus entry T he HIV-1 envelope glycoprotein (Env) is anchored in the viral membrane and facilitates infection through its interaction with T-cell membrane proteins. Env is a trimer of dimers composed of gp120 and gp41 polypeptides, which associate noncovalently on the surface of the virus. Three copies of this heterodimer assemble to form a functional Env trimer "spike" that is visible on the viral surface in electron micrographs of purified virus particles. HIV entry into the cell is initiated when gp120 makes contact with the cell surface receptor CD4. The quaternary molecular structures of Env and the associated conformational changes that result from its binding to CD4 and numerous monoclonal antibodies have been analyzed by cryoelectron tomography (1-5). These studies have identified three distinct quaternary conformations of trimeric Env. A "closed" conformation, defined by the close positioning of adjacent gp120 V1/V2 loops at the apex of the spike, is observed when trimeric Env is unliganded and when it is bound to the CD4-binding sitedirected neutralizing antibodies VRC01, VRC02, or VRC03 (2, 3). A second, "partially open" conformation is found when Env is bound by the CD4-binding site antibody, b12, and is characterized by a slight outward and rotational displacement of the gp120 monomers with respect to the central axis of the spike (2). Finally, a third, "open" Env structure featuring a quaternary conformation with large rearrangements of gp120 and gp41 is observed upon binding of soluble CD4 or the CD4-induced (CD4i) antibody, 17b (