A gp41 MPER-specific llama VHH requires a hydrophobic CDR3 for neutralization but not for antigen recognition

The membrane proximal external region (MPER) of the HIV-1 glycoprotein gp41 is targeted by the broadly neutralizing antibodies 2F5 and 4E10. To date, no immunization regimen in animals or humans has produced HIV-1 neutralizing MPER-specific antibodies. We immunized llamas with gp41-MPER proteoliposomes and selected a MPER-specific single chain antibody (VHH), 2H10, whose epitope overlaps with that of mAb 2F5. Bi-2H10, a bivalent form of 2H10, which displayed an approximately 20-fold increased affinity compared to the monovalent 2H10, neutralized various sensitive and resistant HIV-1 strains, as well as SHIV strains in TZM-bl cells. X-ray and NMR analyses combined with mutagenesis and modeling revealed that 2H10 recognizes its gp41 epitope in a helical conformation. Notably, tryptophan 100 at the tip of the long CDR3 is not required for gp41 interaction but essential for neutralization. Thus bi-2H10 is an anti-MPER antibody generated by immunization that requires hydrophobic CDR3 determinants in addition to epitope recognition for neutralization similar to the mode of neutralization employed by mAbs 2F5 and 4E10

Analysis of Memory B Cell Responses and Isolation of Novel Monoclonal Antibodies with Neutralizing Breadth from HIV-1-Infected Individuals

BACKGROUND: The isolation of human monoclonal antibodies (mAbs) that neutralize a broad spectrum of primary HIV-1 isolates and the characterization of the human neutralizing antibody B cell response to HIV-1 infection are important goals that are central to the design of an effective antibody-based vaccine. METHODS AND FINDINGS: We immortalized IgG(+) memory B cells from individuals infected with diverse clades of HIV-1 and selected on the basis of plasma neutralization profiles that were cross-clade and relatively potent. Culture supernatants were screened using various recombinant forms of the envelope glycoproteins (Env) in multiple parallel assays. We isolated 58 mAbs that were mapped to different Env surfaces, most of which showed neutralizing activity. One mAb in particular (HJ16) specific for a novel epitope proximal to the CD4 binding site on gp120 selectively neutralized a multi-clade panel of Tier-2 HIV-1 pseudoviruses, and demonstrated reactivity that was comparable in breadth, but distinct in neutralization specificity, to that of the other CD4 binding site-specific neutralizing mAb b12. A second mAb (HGN194) bound a conserved epitope in the V3 crown and neutralized all Tier-1 and a proportion of Tier-2 pseudoviruses tested, irrespective of clade. A third mAb (HK20) with broad neutralizing activity, particularly as a Fab fragment, recognized a highly conserved epitope in the HR-1 region of gp41, but showed striking assay-dependent selectivity in its activity. CONCLUSIONS: This study reveals that by using appropriate screening methods, a large proportion of memory B cells can be isolated that produce mAbs with HIV-1 neutralizing activity. Three of these mAbs show unusual breadth of neutralization and therefore add to the current panel of HIV-1 neutralizing antibodies with potential for passive protection and template-based vaccine design

Analysis of memory B cell responses and isolation of novel monoclonal antibodies with neutralizing breadth from HIV-1-infected individuals

Background The isolation of human monoclonal antibodies (mAbs) that neutralize a broad spectrum of primary HIV-1 isolates and the characterization of the human neutralizing antibody B cell response to HIV-1 infection are important goals that are central to the design of an effective antibody-based vaccine. Methods and Findings We immortalized IgG+ memory B cells from individuals infected with diverse clades of HIV-1 and selected on the basis of plasma neutralization profiles that were cross-clade and relatively potent. Culture supernatants were screened using various recombinant forms of the envelope glycoproteins (Env) in multiple parallel assays. We isolated 58 mAbs that were mapped to different Env surfaces, most of which showed neutralizing activity. One mAb in particular (HJ16) specific for a novel epitope proximal to the CD4 binding site on gp120 selectively neutralized a multi-clade panel of Tier-2 HIV-1 pseudoviruses, and demonstrated reactivity that was comparable in breadth, but distinct in neutralization specificity, to that of the other CD4 binding site-specific neutralizing mAb b12. A second mAb (HGN194) bound a conserved epitope in the V3 crown and neutralized all Tier-1 and a proportion of Tier-2 pseudoviruses tested, irrespective of clade. A third mAb (HK20) with broad neutralizing activity, particularly as a Fab fragment, recognized a highly conserved epitope in the HR-1 region of gp41, but showed striking assay-dependent selectivity in its activity. Conclusions This study reveals that by using appropriate screening methods, a large proportion of memory B cells can be isolated that produce mAbs with HIV-1 neutralizing activity. Three of these mAbs show unusual breadth of neutralization and therefore add to the current panel of HIV-1 neutralizing antibodies with potential for passive protection and template-based vaccine design

Novel Strategy for Inhibiting Viral Entry by Use of a Cellular Receptor-Plant Virus Chimera

The plant virus cowpea mosaic virus (CPMV) has recently been developed as a biomolecular platform to display heterologous peptide sequences. Such CPMV-peptide chimeras can be easily and inexpensively produced in large quantities from experimentally infected plants. This study utilized the CPMV chimera platform to create an antiviral against measles virus (MV) by displaying a peptide known to inhibit MV infection. This peptide sequence corresponds to a portion of the MV binding site on the human MV receptor CD46. The CPMV-CD46 chimera efficiently inhibited MV infection of HeLa cells in vitro, while wild-type CPMV did not. Furthermore, CPMV-CD46 protected mice from mortality induced by an intracranial challenge with MV. Our results indicate that the inhibitory CD46 peptide expressed on the surface of CPMV retains virus-binding activity and is capable of inhibiting viral entry both in vitro and in vivo. The CD46 peptide presented in the context of CPMV is also up to 100-fold more effective than the soluble CD46 peptide at inhibiting MV infection in vitro. To our knowledge, this study represents the first utilization of a plant virus chimera as an antiviral agent

Measles Virus (MV) Hemagglutinin: Evidence that Attachment Sites for MV Receptors SLAM and CD46 Overlap on the Globular Head

Measles virus hemagglutinin (MVH) residues potentially responsible for attachment to the wild-type (wt) MV receptor SLAM (CD150) have been identified and localized on the MVH globular head by reference to a revised hypothetical structural model for MVH (www.pepscan.nl/downloads/measlesH.pdb). We show that the mutation of five charged MVH residues which are conserved among morbillivirus H proteins has major effects on both SLAM downregulation and SLAM-dependent fusion. In the three-dimensional surface representation of the structural model, three of these residues (D505, D507, and R533) align the rim on one side of the cavity on the top surface of the MVH globular head and form the basis of a single continuous site that overlaps with the 546-548-549 CD46 binding site. We show that the overlapping sites fall within the footprint of an anti-MVH monoclonal antibody that neutralizes both wt and laboratory-vaccine MV strains and whose epitope contains R533. Our study does not exclude the possibility that Y481 binds CD46 directly but suggests that the N481Y mutation of wt MVH could influence, at a distance, the conformation of the overlapping sites so that affinity to CD46 increases. The relevance of these results to present concepts of MV receptor usage is discussed, and an explanation is proposed as to why morbillivirus attachment proteins are H, whereas those from the other paramyxoviruses are HN (hemagglutinin-neuraminidase)

Evidence of a Potential Receptor-Binding Site on the Nipah Virus G Protein (NiV-G): Identification of Globular Head Residues with a Role in Fusion Promotion and Their Localization on an NiV-G Structural Model

As a preliminary to the localization of the receptor-binding site(s) on the Nipah virus (NiV) glycoprotein (NiV-G), we have undertaken the identification of NiV-G residues that play a role in fusion promotion. To achieve this, we have used two strategies. First, as NiV and Hendra virus (HeV) share a common receptor and their cellular tropism is similar, we hypothesized that residues functioning in receptor attachment could be conserved between their respective G proteins. Our initial strategy was to target charged residues (which can be expected to be at the surface of the protein) conserved between the NiV-G and HeV-G globular heads. Second, we generated NiV variants that escaped neutralization by anti-NiV-G monoclonal antibodies (MAbs) that neutralize NiV both in vitro and in vivo, likely by blocking receptor attachment. The sequencing of such “escape mutants” identified NiV-G residues present in the epitopes to which the neutralizing MAbs are directed. Residues identified via these two strategies whose mutation had an effect on fusion promotion were localized on a new structural model for the NiV-G protein. Our results suggest that seven NiV-G residues, including one (E533) that was identified using both strategies, form a contiguous site on the top of the globular head that is implicated in ephrinB2 binding. This site commences near the shallow depression in the center of the top surface of the globular head and extends to the rim of the barrel-like structure on the top loops of β-sheet 5. The topology of this site is strikingly similar to that proposed to form the SLAM receptor site on another paramyxovirus attachment protein, that of the measles virus hemagglutinin

HIV-1 anchor inhibitors and membrane fusion inhibitors target distinct but overlapping steps in virus entry

HIV-1 entry into cells is mediated by the envelope glycoprotein (Env) and represents an attractive target for therapeutic intervention. Two drugs that inhibit HIV entry are approved for clinical use: the membrane fusion-inhibitor T20 (Fuzeon, enfuvirtide) and the C-C chemokine receptor type 5 (CCR5) blocker maraviroc (Selzentry). Another class of entry inhibitors supposedly target the fusion peptide (FP) and are termed anchor inhibitors. These include the VIRIP peptide and VIRIP derivatives such as VIR165, VIR353, and VIR576. Here, we investigated the mechanism of inhibition by VIR165. We show that substitutions within the FP modulate sensitivity to VIR165, consistent with the FP being the drug target. Our results also revealed that VIR165 acts during an intermediate post-CD4 – binding entry step that is overlapping but not identical to the step inhibited by fusion inhibitors such as T20. We found that some but not all resistance mutations to heptad repeat 2 (HR2)-targeting fusion inhibitors can provide cross-resistance to VIR165. In contrast, resistance mutations in the HR1-binding site for the fusion inhibitors did not cause cross-resistance to VIR165. However, Env with mutations located outside this binding site and thought to affect fusion kinetics, exhibited decreased sensitivity to VIR165. Although we found a strong correlation between Env stability and resistance to HR2-based fusion inhibitors, such correlation was not observed for Env stability and VIR165 resistance. We conclude that VIRIP analogs target the FP during an intermediate, post-CD4 – binding entry step that overlaps with but is distinct from the step(s) inhibited by HR2-based fusion inhibitors

Selection of T1249-Resistant Human Immunodeficiency Virus Type 1 Variants▿

Human immunodeficiency virus type 1 (HIV-1) entry is an attractive target for therapeutic intervention. Two drugs that inhibit this process have been approved: the fusion inhibitor T20 (enfuvirtide [Fuzeon]) and, more recently, the CCR5 blocker maraviroc (Selzentry). T1249 is a second-generation fusion inhibitor with improved antiviral potency compared to the first-generation peptide T20. We selected T1249-resistant HIV-1 variants in vitro by serial virus passage in the presence of increasing T1249 doses after passage with wild-type and T20-resistant variants. Sequence analysis revealed the acquisition of substitutions within the HR1 region of the gp41 ectodomain. The virus acquired mutations of residue V38 to either E or R in 10 of 19 cultures. Both E and R at position 38 were confirmed to cause resistance to T1249, as well as cross-resistance to T20 and C34, but not to the third-generation fusion inhibitor T2635. We also observed substitutions at residues 79 and 90 (Q79E and K90E), which provide modest resistance to T1249 and, interestingly, T2635. Thus, the gp41 amino acid position implicated in T20 resistance (V38 replaced by A, G, or W) is also responsible for T1249 resistance (V38 replaced by E, R, or K). These results indicate that T20 and T1249 exhibit very similar inhibition modes that call for similar but not identical resistance mutations. All T1249-resistant viruses with changes at position 38 are cross resistant to T20, but not vice versa. Furthermore, substitutions at position 38 do not provide resistance to the third-generation inhibitor T2635, while substitution at positions 79 and 90 do, suggesting different resistance mechanisms