Antibody-directed evolution reveals a mechanism for enhanced neutralization at the HIV-1 fusion peptide site.

The HIV-1 fusion peptide (FP) represents a promising vaccine target, but global FP sequence diversity among circulating strains has limited anti-FP antibodies to ~60% neutralization breadth. Here we evolve the FP-targeting antibody VRC34.01 in vitro to enhance FP-neutralization using site saturation mutagenesis and yeast display. Successive rounds of directed evolution by iterative selection of antibodies for binding to resistant HIV-1 strains establish a variant, VRC34.01_mm28, as a best-in-class antibody with 10-fold enhanced potency compared to the template antibody and ~80% breadth on a cross-clade 208-strain neutralization panel. Structural analyses demonstrate that the improved paratope expands the FP binding groove to accommodate diverse FP sequences of different lengths while also recognizing the HIV-1 Env backbone. These data reveal critical antibody features for enhanced neutralization breadth and potency against the FP site of vulnerability and accelerate clinical development of broad HIV-1 FP-targeting vaccines and therapeutics.

The light chain of the L9 antibody is critical for binding circumsporozoite protein minor repeats and preventing malaria.

L9 is a potent human monoclonal antibody (mAb) that preferentially binds two adjacent NVDP minor repeats and cross-reacts with NANP major repeats of the Plasmodium falciparum circumsporozoite protein (PfCSP) on malaria-infective sporozoites. Understanding this mAb's ontogeny and mechanisms of binding PfCSP will facilitate vaccine development. Here, we isolate mAbs clonally related to L9 and show that this B cell lineage has baseline NVDP affinity and evolves to acquire NANP reactivity. Pairing the L9 kappa light chain (L9κ) with clonally related heavy chains results in chimeric mAbs that cross-link two NVDPs, cross-react with NANP, and more potently neutralize sporozoites in vivo compared with their original light chain. Structural analyses reveal that the chimeric mAbs bound minor repeats in a type-1 ß-turn seen in other repeat-specific antibodies. These data highlight the importance of L9κ in binding NVDP on PfCSP to neutralize sporozoites and suggest that PfCSP-based immunogens might be improved by presenting ≥2 NVDPs.

Protective effects of combining monoclonal antibodies and vaccines against the Plasmodium falciparum circumsporozoite protein.

Combinations of monoclonal antibodies (mAbs) against different epitopes on the same antigen synergistically neutralize many viruses. However, there are limited studies assessing whether combining human mAbs against distinct regions of the Plasmodium falciparum (Pf) circumsporozoite protein (CSP) enhances in vivo protection against malaria compared to each mAb alone or whether passive transfer of PfCSP mAbs would improve protection following vaccination against PfCSP. Here, we isolated a panel of human mAbs against the subdominant C-terminal domain of PfCSP (C-CSP) from a volunteer immunized with radiation-attenuated Pf sporozoites. These C-CSP-specific mAbs had limited binding to sporozoites in vitro that was increased by combination with neutralizing human "repeat" mAbs against the NPDP/NVDP/NANP tetrapeptides in the central repeat region of PfCSP. Nevertheless, passive transfer of repeat- and C-CSP-specific mAb combinations did not provide enhanced protection against in vivo sporozoite challenge compared to repeat mAbs alone. Furthermore, combining potent repeat-specific mAbs (CIS43, L9, and 317) that respectively target the three tetrapeptides (NPDP/NVDP/NANP) did not provide additional protection against in vivo sporozoite challenge. However, administration of either CIS43, L9, or 317 (but not C-CSP-specific mAbs) to mice that had been immunized with R21, a PfCSP-based virus-like particle vaccine that induces polyclonal antibodies against the repeat region and C-CSP, provided enhanced protection against sporozoite challenge when compared to vaccine or mAbs alone. Collectively, this study shows that while combining mAbs against the repeat and C-terminal regions of PfCSP provide no additional protection in vivo, repeat mAbs do provide increased protection when combined with vaccine-induced polyclonal antibodies. These data should inform the implementation of PfCSP human mAbs alone or following vaccination to prevent malaria infection.

Vaccination in a humanized mouse model elicits highly protective PfCSP-targeting anti-malarial antibodies.

Repeat antigens, such as the Plasmodium falciparum circumsporozoite protein (PfCSP), use both sequence degeneracy and structural diversity to evade the immune response. A few PfCSP-directed antibodies have been identified that are effective at preventing malaria infection, including CIS43, but how these repeat-targeting antibodies might be improved has been unclear. Here, we engineered a humanized mouse model in which B cells expressed inferred human germline CIS43 (iGL-CIS43) B cell receptors and used both vaccination and bioinformatic analysis to obtain variant CIS43 antibodies with improved protective capacity. One such antibody, iGL-CIS43.D3, was significantly more potent than the current best-in-class PfCSP-directed antibody. We found that vaccination with a junctional epitope peptide was more effective than full-length PfCSP at recruiting iGL-CIS43 B cells to germinal centers. Structure-function analysis revealed multiple somatic hypermutations that combinatorically improved protection. This mouse model can thus be used to understand vaccine immunogens and to develop highly potent anti-malarial antibodies.

Enhancing durability of CIS43 monoclonal antibody by Fc mutation or AAV delivery for malaria prevention.

CIS43 is a potent neutralizing human mAb that targets a highly conserved "junctional" epitope in the Plasmodium falciparum (Pf) circumsporozoite protein (PfCSP). Enhancing the durability of CIS43 in vivo will be important for clinical translation. Here, 2 approaches were used to improve the durability of CIS43 in vivo while maintaining potent neutralization. First, the Fc domain was modified with the LS mutations (CIS43LS) to increase CIS43 binding affinity for the neonatal Fc receptor (FcRn). CIS43LS and CIS43 showed comparable in vivo protective efficacy. CIS43LS had 9- to 13-fold increased binding affinity for human (6.2 nM versus 54.2 nM) and rhesus (25.1 nM versus 325.8 nM) FcRn at endosomal pH 6.0 compared with CIS43. Importantly, the half-life of CIS43LS in rhesus macaques increased from 22 days to 39 days compared with CIS43. The second approach for sustaining antibody levels of CIS43 in vivo is through adeno-associated virus (AAV) expression. Mice administered once with AAV-expressing CIS43 had sustained antibody levels of approximately 300 µg/mL and mediated protection against sequential malaria challenges up to 36 weeks. Based on these data, CIS43LS has advanced to phase I clinical trials, and AAV delivery provides a potential next-generation approach for malaria prevention.

A Potent Anti-Malarial Human Monoclonal Antibody Targets Circumsporozoite Protein Minor Repeats and Neutralizes Sporozoites in the Liver.

Discovering potent human monoclonal antibodies (mAbs) targeting the Plasmodium falciparum circumsporozoite protein (PfCSP) on sporozoites (SPZ) and elucidating their mechanisms of neutralization will facilitate translation for passive prophylaxis and aid next-generation vaccine development. Here, we isolated a neutralizing human mAb, L9 that preferentially bound NVDP minor repeats of PfCSP with high affinity while cross-reacting with NANP major repeats. L9 was more potent than six published neutralizing human PfCSP mAbs at mediating protection against mosquito bite challenge in mice. Isothermal titration calorimetry and multiphoton microscopy showed that L9 and the other most protective mAbs bound PfCSP with two binding events and mediated protection by killing SPZ in the liver and by preventing their egress from sinusoids and traversal of hepatocytes. This study defines the subdominant PfCSP minor repeats as neutralizing epitopes, identifies an in vitro biophysical correlate of SPZ neutralization, and demonstrates that the liver is an important site for antibodies to prevent malaria.

Garcinoic Acid Is a Natural and Selective Agonist of Pregnane X Receptor.

Pregnane X receptor (PXR) is a master xenobiotic-sensing transcription factor and a validated target for immune and inflammatory diseases. The identification of chemical probes to investigate the therapeutic relevance of the receptor is still highly desired. In fact, currently available PXR ligands are not highly selective and can exhibit toxicity and/or potential off-target effects. In this study, we have identified garcinoic acid as a selective and efficient PXR agonist. The properties of this natural molecule as a specific PXR agonist were demonstrated by the screening on a panel of nuclear receptors, the assessment of the physical and thermodynamic binding affinity, and the determination of the PXR-garcinoic acid complex crystal structure. Cytotoxicity, transcriptional, and functional properties were investigated in human liver cells, and compound activity and target engagement were confirmed in vivo in mouse liver and gut tissue. In conclusion, garcinoic acid is a selective natural agonist of PXR and a promising lead compound toward the development of new PXR-regulating modulators.

Preclinical Development of a Fusion Peptide Conjugate as an HIV Vaccine Immunogen.

The vaccine elicitation of broadly neutralizing antibodies against HIV-1 is a long-sought goal. We previously reported the amino-terminal eight residues of the HIV-1-fusion peptide (FP8) - when conjugated to the carrier protein, keyhole limpet hemocyanin (KLH) - to be capable of inducing broadly neutralizing responses against HIV-1 in animal models. However, KLH is a multi-subunit particle derived from a natural source, and its manufacture as a clinical product remains a challenge. Here we report the preclinical development of recombinant tetanus toxoid heavy chain fragment (rTTHC) linked to FP8 (FP8-rTTHC) as a suitable FP-conjugate vaccine immunogen. We assessed 16 conjugates, made by coupling the 4 most prevalent FP8 sequences with 4 carrier proteins: the aforementioned KLH and rTTHC; the H. influenzae protein D (HiD); and the cross-reactive material from diphtheria toxin (CRM197). While each of the 16 FP8-carrier conjugates could elicit HIV-1-neutralizing responses, rTTHC conjugates induced higher FP-directed responses overall. A Sulfo-SIAB linker yielded superior results over an SM(PEG)2 linker but combinations of carriers, conjugation ratio of peptide to carrier, or choice of adjuvant (Adjuplex or Alum) did not significantly impact elicited FP-directed neutralizing responses in mice. Overall, SIAB-linked FP8-rTTHC appears to be a promising vaccine candidate for advancing to clinical assessment.

Antibody Lineages with Vaccine-Induced Antigen-Binding Hotspots Develop Broad HIV Neutralization.

The vaccine-mediated elicitation of antibodies (Abs) capable of neutralizing diverse HIV-1 strains has been a long-standing goal. To understand how broadly neutralizing antibodies (bNAbs) can be elicited, we identified, characterized, and tracked five neutralizing Ab lineages targeting the HIV-1-fusion peptide (FP) in vaccinated macaques over time. Genetic and structural analyses revealed two of these lineages to belong to a reproducible class capable of neutralizing up to 59% of 208 diverse viral strains. B cell analysis indicated each of the five lineages to have been initiated and expanded by FP-carrier priming, with envelope (Env)-trimer boosts inducing cross-reactive neutralization. These Abs had binding-energy hotspots focused on FP, whereas several FP-directed Abs induced by immunization with Env trimer-only were less FP-focused and less broadly neutralizing. Priming with a conserved subregion, such as FP, can thus induce Abs with binding-energy hotspots coincident with the target subregion and capable of broad neutralization.

Bioinspired supramolecular engineering of self-assembling immunofibers for high affinity binding of immunoglobulin G.

Many one-dimensional (1D) nanostructures are constructed by self-assembly of peptides or peptide conjugates containing a short ß-sheet sequence as the core building motif essential for the intermolecular hydrogen bonding that promotes directional, anisotropic growth of the resultant assemblies. While this molecular engineering strategy has led to the successful production of a plethora of bioactive filamentous ß-sheet assemblies for interfacing with biomolecules and cells, concerns associated with effective presentation of α-helical epitopes and their function preservation have yet to be resolved. In this context, we report on the direct conjugation of the protein A mimicking peptide Z33, a motif containing two α-helices, to linear hydrocarbons to create self-assembling immuno-amphiphiles (IAs). Our results suggest that the resulting amphiphilic peptides can, despite lacking the essential ß-sheet segment, effectively associate under physiological conditions into supramolecular immunofibers (IFs) while preserving their native α-helical conformation. Isothermal titration calorimetry (ITC) measurements confirmed that these self-assembling immunofibers can bind to the human immunoglobulin G class 1 (IgG1) with high specificity at pH 7.4, but with significantly weakened binding at pH 2.8. We further demonstrated the accessibility of Z33 ligand in the immunofibers using transmission electron microscopy (TEM) and confocal imaging. We believe these results shed important light into the supramolecular engineering of α-helical peptides into filamentous assemblies that may possess an important potential for antibody isolation.

Denatured state aggregation parameters derived from concentration dependence of protein stability.

Protein aggregation is a major issue affecting the long-term stability of protein preparations. Proteins exist in equilibrium between the native and denatured or partially denatured conformations. Often denatured or partially denatured conformations are prone to aggregate because they expose to solvent the hydrophobic core of the protein. The aggregation of denatured protein gradually shifts the protein equilibrium toward increasing amounts of denatured and ultimately aggregated protein. Recognizing and quantitating the presence of denatured protein and its aggregation at the earliest possible time will bring enormous benefits to the identification and selection of optimal solvent conditions or the engineering of proteins with the best stability/aggregation profile. In this article, a new approach that allows simultaneous determination of structural stability and the amount of denatured and aggregated protein is presented. This approach is based on the analysis of the concentration dependence of the Gibbs energy (ΔG) of protein stability. It is shown that three important quantities can be evaluated simultaneously: (i) the population of denatured protein, (ii) the population of aggregated protein, and (iii) the fraction of denatured protein that is aggregated.

HIV-1 Vif N-terminal motif is required for recruitment of Cul5 to suppress APOBEC3.

BACKGROUND: HIV-1 Vif promotes the degradation of host anti-retroviral factor family, APOBEC3 proteins via the recruitment of a multi-subunit E3 ubiquitin ligase complex. The complex is composed of a scaffold protein, Cullin 5 (Cul5), RING-box protein (Rbx), a SOCS box binding protein complex, Elongins B/C (Elo B/C), as well as newly identified host co-factor, core binding factor beta (CBF-ß). Cul5 has previously been shown to bind amino acids within an HCCH domain as well as a PPLP motif at the C-terminus of Vif; however, it is unclear whether Cul5 binding requires additional regions of the Vif polypeptide. RESULTS: Here, we provide evidence that an amino terminal region of full length Vif is necessary for the Vif-Cul5 interaction. Single alanine replacement of select amino acids spanning residues 25-30 (25VXHXMY30) reduced the ability for Vif to bind Cul5, but not CBF-ß or Elo B/C in pull-down experiments. In addition, recombinant Vif mutants had a reduced binding affinity for Cul5 compared to wild-type as measured by isothermal titration calorimetry. N-terminal mutants that demonstrated reduced Cul5 binding were also unable to degrade APOBEC3G as well as APOBEC3F and were unable to restore HIV infectivity, in the presence of APOBEC3G. Although the Vif N-terminal amino acids were necessary for Cul5 interaction, the mutation of each residue to alanine induced a change in the secondary structure of the Vif-CBF-ß-Elo B/C complex as suggested by results from circular dichroism spectroscopy and size-exclusion chromatography experiments. Surprisingly, the replacement of His108 to alanine also contributed to the Vif structure. Thus, it is unclear whether the amino acids contribute to a direct interaction with Cul5 or whether the amino acids are responsible for the structural organization of the Vif protein that promotes Cul5 binding. CONCLUSIONS: Taken together, we propose a novel Vif N-terminal motif that is responsible for Vif recruitment of Cul5. Motifs in Vif that are absent from cellular proteins represent attractive targets for future HIV pharmaceutical design.

Development of potent dipeptide-type SARS-CoV 3CL protease inhibitors with novel P3 scaffolds: design, synthesis, biological evaluation, and docking studies.

We report the design and synthesis of a series of dipeptide-type inhibitors with novel P3 scaffolds that display potent inhibitory activity against SARS-CoV 3CLpro. A docking study involving binding between the dipeptidic lead compound 4 and 3CLpro suggested the modification of a structurally flexible P3 N-(3-methoxyphenyl)glycine with various rigid P3 moieties in 4. The modifications led to the identification of several potent derivatives, including 5c-k and 5n with the inhibitory activities (Ki or IC50) in the submicromolar to nanomolar range. Compound 5h, in particular, displayed the most potent inhibitory activity, with a Ki value of 0.006 µM. This potency was 65-fold higher than the potency of the lead compound 4 (Ki=0.39 µM). In addition, the Ki value of 5h was in very good agreement with the binding affinity (16 nM) observed in isothermal titration calorimetry (ITC). A SAR study around the P3 group in the lead 4 led to the identification of a rigid indole-2-carbonyl unit as one of the best P3 moieties (5c). Further optimization showed that a methoxy substitution at the 4-position on the indole unit was highly favorable for enhancing the inhibitory potency.

Nudel/NudE and Lis1 promote dynein and dynactin interaction in the context of spindle morphogenesis.

Lis1, Nudel/NudE, and dynactin are regulators of cytoplasmic dynein, a minus end-directed, microtubule (MT)-based motor required for proper spindle assembly and orientation. In vitro studies have shown that dynactin promotes processive movement of dynein on MTs, whereas Lis1 causes dynein to enter a persistent force-generating state (referred to here as dynein stall). Yet how the activities of Lis1, Nudel/NudE, and dynactin are coordinated to regulate dynein remains poorly understood in vivo. Working in Xenopus egg extracts, we show that Nudel/NudE facilitates the binding of Lis1 to dynein, which enhances the recruitment of dynactin to dynein. We further report a novel Lis1-dependent dynein-dynactin interaction that is essential for the organization of mitotic spindle poles. Finally, using assays for MT gliding and spindle assembly, we demonstrate an antagonistic relationship between Lis1 and dynactin that allows dynactin to relieve Lis1-induced dynein stall on MTs. Our findings suggest the interesting possibility that Lis1 and dynactin could alternately engage with dynein to allow the motor to promote spindle assembly.

Design, synthesis, and biological evaluation of novel dipeptide-type SARS-CoV 3CL protease inhibitors: structure-activity relationship study.

This work describes the design, synthesis, and evaluation of low-molecular weight peptidic SARS-CoV 3CL protease inhibitors. The inhibitors were designed based on the potent tripeptidic Z-Val-Leu-Ala(pyrrolidone-3-yl)-2-benzothiazole (8; Ki = 4.1 nM), in which the P3 valine unit was substituted with a variety of distinct moieties. The resulting series of dipeptide-type inhibitors displayed moderate to good inhibitory activities against 3CL(pro). In particular, compounds 26m and 26n exhibited good inhibitory activities with Ki values of 0.39 and 0.33 µM, respectively. These low-molecular weight compounds are attractive leads for the further development of potent peptidomimetic inhibitors with pharmaceutical profiles. Docking studies were performed to model the binding interaction of the compound 26m with the SARS-CoV 3CL protease. The preliminary SAR study of the peptidomimetic compounds with potent inhibitory activities revealed several structural features that boosted the inhibitory activity: (i) a benzothiazole warhead at the S1' position, (ii) a γ-lactam unit at the S1-position, (iii) an appropriately hydrophobic leucine moiety at the S2-position, and (iv) a hydrogen bond between the N-arylglycine unit and a backbone hydrogen bond donor at the S3-position.

Non-natural peptide triazole antagonists of HIV-1 envelope gp120.

We investigated the derivation of non-natural peptide triazole dual receptor site antagonists of HIV-1 Env gp120 to establish a pathway for developing peptidomimetic antiviral agents. Previously we found that the peptide triazole HNG-156 [R-I-N-N-I-X-W-S-E-A-M-M-CONH(2), in which X=ferrocenyltriazole-Pro (FtP)] has nanomolar binding affinity to gp120, inhibits gp120 binding to CD4 and the co-receptor surrogate mAb 17b, and has potent antiviral activity in cell infection assays. Furthermore, truncated variants of HNG-156, typified by UM-24 (Cit-N-N-I-X-W-S-CONH(2)) and containing the critical central stereospecific (L)X-(L)W cluster, retain the functional characteristics of the parent peptide triazole. In the current work, we examined the possibility of replacing natural with unnatural residue components in UM-24 to the greatest extent possible. The analogue with the critical "hot spot" residue Trp 6 replaced with L-3-benzothienylalanine (Bta) (KR-41), as well as a completely non-natural analogue containing D-amino acid substitutions outside the central cluster (KR-42, (D)Cit-(D)N-(D)N-(D)I-X-Bta-(D)S-CONH(2)), retained the dual receptor site antagonism/antiviral activity signature. The results define differential functional roles of subdomains within the peptide triazole and provide a structural basis for the design of metabolically stable peptidomimetic inhibitors of HIV-1 Env gp120.

Some binding-related drug properties are dependent on thermodynamic signature.

The binding affinity is determined by the Gibbs energy of binding (ΔG) which is the sum of enthalpic (ΔH) and entropic (-TΔS) contributions. Because the enthalpy and entropy contribute in an additive way to the binding energy, the same binding affinity can be achieved by many different combinations of enthalpic and entropic contributions; however, do compounds with similar binding affinities but different thermodynamic signatures (i.e., different ΔH, -TΔS combinations) exhibit the same functional effects? Are there characteristics of compounds that can be modulated by modifying their thermodynamic signatures? In this paper, we consider the minimization of unwanted conformational effects arising during the development of CD4/gp120 inhibitors, a new class of HIV-1 cell entry inhibitors. Competitive inhibitors of protein/protein interactions run the risk of triggering the very same signals that they are supposed to inhibit. Here, we show that for CD4/gp120 inhibitors, the magnitude of those unwanted effects is related to the proportion in which the enthalpy and entropy changes contribute to the binding affinity. The thermodynamic optimization plot (TOP) previously proposed to optimize binding affinity can also be used to obtain appropriate enthalpy/entropy combinations for drug candidates.

The active core in a triazole peptide dual-site antagonist of HIV-1 gp120.

In an effort to identify broadly active inhibitors of HIV-1 entry into host cells, we previously reported a family of dodecamer triazole-peptide conjugates with nanomolar affinity for the viral surface protein gp120. This peptide class exhibits potent antiviral activity and the capacity to simultaneously inhibit interaction of the viral envelope protein with both CD4 and co-receptor. In this investigation, we minimized the structural complexity of the lead triazole inhibitor HNG-156 (peptide 1) to explore the limits of the pharmacophore that enables dual antagonism and to improve opportunities for peptidomimetic design. Truncations of both carboxy- and amino-terminal residues from the parent 12-residue peptide 1 were found to have minimal effects on both affinity and antiviral activity. In contrast, the central triazole(Pro)-Trp cluster at residues 6 and 7 with ferrocenyl-triazole(Pro) (Ftp) was found to be critical for bioactivity. Amino-terminal residues distal to the central triazole(Pro)-Trp sequence tolerated decreasing degrees of side chain variation upon approaching the central cluster. A peptide fragment containing residues 3-7 (Asn-Asn-Ile-Ftp-Trp) exhibited substantial direct binding affinity, antiviral potency, dual receptor site antagonism, and induction of gp120 structuring, all properties that define the functional signature of the parent compound 1. This active core contains a stereochemically specific hydrophobic triazole(Pro)-Trp cluster, with a short N-terminal peptide extension providing groups for potential main chain and side chain hydrogen bonding. The results of this work argue that the pharmacophore for dual antagonism is structurally limited, thereby enhancing the potential to develop minimized peptidomimetic HIV-1 entry inhibitors that simultaneously suppress binding of envelope protein to both of its host cell receptors. The results also argue that the target epitope on gp120 is relatively small, pointing to a localized allosteric inhibition site in the HIV-1 envelope that could be targeted for small-molecule inhibitor discovery.

Structural determinants for affinity enhancement of a dual antagonist peptide entry inhibitor of human immunodeficiency virus type-1.

Structure-activity correlations were investigated for substituted peptide conjugates that function as dual receptor site antagonists of HIV-1 gp120. A series of peptide conjugates were constructed via click reaction of both aryl and alkyl acetylenes with an internally incorporated azidoproline 6 derived from the parent peptide 1 (12p1, RINNIPWSEAMM). Compared to 1, many of these conjugates were found to exhibit several orders of magnitude increase in both affinity for HIV-1 gp120 and inhibition potencies at both the CD4 and coreceptor binding sites of gp120. We sought to determine structural factors in the added triazole grouping responsible for the increased binding affinity and antiviral activity of the dual inhibitor conjugates. We measured peptide conjugate potencies in both kinetic and cell infection assays. High affinity was sterically specific, being exhibited by the cis- but not the trans-triazole. The results demonstrate that aromatic, hydrophobic, and steric features in the residue 6 side-chain are important for increased affinity and inhibition. Optimizing these features provides a basis for developing gp120 dual inhibitors into peptidomimetic and increasingly smaller molecular weight entry antagonist leads.