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1.  HIV-1 Therapy with Monoclonal Antibody 3BNC117 Elicits Host Immune Responses against HIV-1 
Science (New York, N.Y.)  2016;352(6288):997-1001.
3BNC117 is a broad and potent anti-HIV-1 neutralizing antibody that targets the CD4 binding site on the viral envelope spike. When administered passively, this antibody can prevent infection in animal models and suppress viremia in HIV-1-infected individuals. Here we report that HIV-1 immunotherapy with a single injection of 3BNC117 impacts host antibody responses in viremic subjects. In comparison to untreated controls that showed little change in their neutralizing activity over a six-month period, 3BNC117 infusion significantly improved neutralizing responses to heterologous tier 2 viruses in nearly all study participants. We conclude that 3BNC117-mediated immunotherapy enhances host humoral immunity to HIV-1.
doi:10.1126/science.aaf0972
PMCID: PMC5151174  PMID: 27199429
2.  HIV-1 antibody 3BNC117 suppresses viral rebound in humans during treatment interruption 
Nature  2016;535(7613):556-560.
Interruption of combination antiretroviral therapy in HIV-1-infected individuals leads to rapid viral rebound. Here we report the results of a phase IIa open label clinical trial evaluating 3BNC117, a broad and potent neutralizing antibody (bNAb) against the CD4 binding site of HIV-1 Env1, in the setting of analytical treatment interruption in 13 HIV-1-infected individuals. Participants with 3BNC117-sensitive virus outgrowth cultures were enrolled. Two or four 30 mg kg−1 infusions of 3BNC117, separated by 3 or 2 weeks, respectively, are generally well tolerated. Infusions are associated with a delay in viral rebound for 5–9 weeks after two infusions, and up to 19 weeks after four infusions, or an average of 6.7 and 9.9 weeks respectively, compared with 2.6 weeks for historical controls (P < 0.00001). Rebound viruses arise predominantly from a single provirus. In most individuals, emerging viruses show increased resistance, indicating escape. However, 30% of participants remained suppressed until antibody concentrations waned below 20 μg ml−1, and the viruses emerging in all but one of these individuals showed no apparent resistance to 3BCN117, suggesting failure to escape over a period of 9–19 weeks. We conclude that administration of 3BNC117 exerts strong selective pressure on HIV-1 emerging from latent reservoirs during analytical treatment interruption in humans.
doi:10.1038/nature18929
PMCID: PMC5034582  PMID: 27338952
3.  Anti-polyQ antibodies recognize a short polyQ stretch in both normal and mutant huntingtin exon 1 
Journal of molecular biology  2015;427(15):2507-2519.
Huntington’s disease (HD) is caused by expansion of a polyglutamine (polyQ) repeat in the huntingtin protein. A structural basis for the apparent transition between normal and disease-causing expanded polyQ repeats of huntingtin is unknown. The ‘linear lattice’ model proposed random-coil structures for both normal and expanded polyQ in the preaggregation state. Consistent with this model, the affinity and stoichiometry of the anti-polyQ antibody MW1 increased with the number of glutamines. An opposing ‘structural toxic threshold’ model proposed a conformational change above the pathogenic polyQ threshold resulting in a specific toxic conformation for expanded polyQ. Support for this model was provided by the anti-polyQ antibody 3B5H10, which was reported to specifically recognize a distinct pathologic conformation of soluble expanded polyQ. To distinguish between these models, we directly compared binding of MW1 and 3B5H10 to normal and expanded polyQ repeats within huntingtin exon 1 fusion proteins. We found similar binding characteristics for both antibodies. First, both antibodies bound to normal, as well as expanded, polyQ in huntingtin exon 1 fusion proteins. Second, an expanded polyQ tract contained multiple epitopes for antigen-binding fragments (Fabs) of both antibodies, demonstrating that 3B5H10 does not recognize a single epitope specific to expanded polyQ. Finally, small angle X-ray scattering and dynamic light scattering revealed similar binding modes for MW1 and 3B5H10 Fab-huntingtin exon 1 complexes. Together, these results support the linear lattice model for polyQ binding proteins, suggesting that the hypothesized pathologic conformation of soluble expanded polyQ is not a valid target for drug design.
Graphical abstract
doi:10.1016/j.jmb.2015.05.023
PMCID: PMC4520773  PMID: 26047735
Equilibrium gel filtration; Huntington’s disease; linear lattice; polyglutamine; small angle X-ray scattering
4.  Structure of an HIV-2 gp120 in Complex with CD4 
Journal of Virology  2016;90(4):2112-2118.
HIV-2 is a nonpandemic form of the virus causing AIDS, and the majority of HIV-2-infected patients exhibit long-term nonprogression. The HIV-1 and HIV-2 envelope glycoproteins, the sole targets of neutralizing antibodies, share 30 to 40% identity. As a first step in understanding the reduced pathogenicity of HIV-2, we solved a 3.0-Å structure of an HIV-2 gp120 bound to the host receptor CD4, which reveals structural similarity to HIV-1 gp120 despite divergence in amino acid sequence.
doi:10.1128/JVI.02678-15
PMCID: PMC4733984  PMID: 26608312
5.  Structural repertoire of HIV-1-neutralizing antibodies targeting the CD4 supersite in 14 donors 
Cell  2015;161(6):1280-1292.
The site on the HIV-1 gp120 glycoprotein that binds the CD4 receptor is recognized by broadly reactive antibodies, several of which neutralize over 90% of HIV-1 strains. To understand how antibodies achieve such neutralization, we isolated CD4-binding-site (CD4bs) antibodies and analyzed 16 co-crystal structures –8 determined here– of CD4bs antibodies from 14 donors. The 16 antibodies segregated by recognition mode and developmental ontogeny into two types: CDR H3-dominated and VH-gene-restricted. Both could achieve greater than 80% neutralization breadth, and both could develop in the same donor. Although paratope chemistries differed, all 16 gp120-CD4bs antibody complexes showed geometric similarity, with antibody-neutralization breadth correlating with antibody-angle of approach relative to the most effective antibody of each type. The repertoire for effective recognition of the CD4 supersite thus comprises antibodies with distinct paratopes arrayed about two optimal geometric orientations, one achieved by CDR H3 ontogenies and the other achieved by VH-gene-restricted ontogenies.
doi:10.1016/j.cell.2015.05.007
PMCID: PMC4683157  PMID: 26004070
6.  3BNC117 a Broadly Neutralizing Antibody Suppresses Viremia in HIV-1-Infected Humans 
Nature  2015;522(7557):487-491.
HIV-1 immunotherapy with a combination of first generation monoclonal antibodies was largely ineffective in pre-clinical and clinical settings and was therefore abandoned1–3. However, recently developed single cell based antibody cloning methods have uncovered a new generation of far more potent broadly neutralizing antibodies (bNAbs) to HIV-14,5. These antibodies can prevent infection and suppress viremia in humanized mice (hu-mice) and nonhuman primates, but their potential for human HIV-1 immunotherapy has not been evaluated6–10. Here we report the results of a first-in-man dose escalation phase 1 clinical trial of 3BNC117, a potent human CD4 binding site antibody11, in uninfected and HIV-1-infected individuals. 3BNC117 infusion was well tolerated and demonstrated favorable pharmacokinetics. A single 30 mg/kg infusion of 3BNC117 reduced the viral load (VL) in HIV-1-infected individuals by 0.8 – 2.5 log10 and viremia remained significantly reduced for 28 days. Emergence of resistant viral strains was variable, with some individuals remaining sensitive to 3BNC117 for a period of 28 days. We conclude that as a single agent 3BNC117 is safe and effective in reducing HIV-1 viremia, and that immunotherapy should be explored as a new modality for HIV-1 prevention, therapy, and cure.
doi:10.1038/nature14411
PMCID: PMC4890714  PMID: 25855300
7.  Antibody engineering for increased potency, breadth and half-life. 
Current opinion in HIV and AIDS  2015;10(3):151-159.
Purpose of review
This review highlights recent developments in HIV-1 antibody engineering and discusses the effects of increased polyreactivity on serum half-lives of engineered antibodies.
Recent Findings
Recent studies have uncovered a wealth of information about the relationship between the sequences and efficacies of anti-HIV-1 antibodies through a combination of bioinformatics, structural characterization, and in vivo studies. This knowledge has stimulated efforts to enhance antibody breadth and potency for therapeutic use. While some engineered antibodies have shown increased polyreactivity and short half-lives, promising efforts are circumventing these problems.
Summary
Antibodies are desirable as therapeutics due to their ability to recognize targets with both specificity and high affinity. Furthermore, the ability of antibodies to stimulate Fc-mediated effector functions can increase their utility. Thus, monoclonal antibodies have become central to strategies for the treatment of various diseases. Using both targeted and library-based approaches, antibodies can be engineered to improve their therapeutic properties. This article will discuss recent antibody engineering efforts to improve the breadth and potency of anti-HIV-1 antibodies. The polyreactivity of engineered HIV-1 bNAbs and the effect on serum half-life will be explored along with strategies to overcome problems introduced by engineering antibodies. Finally, advances in creating bispecific anti-HIV-1 reagents are discussed.
doi:10.1097/COH.0000000000000148
PMCID: PMC4465343  PMID: 25760931
Antibody engineering; HIV-1; potency; breadth; polyreactivity; bispecific reagents
8.  Structural basis for germline antibody recognition of HIV-1 immunogens 
eLife  null;5:e13783.
Efforts to elicit broadly neutralizing antibodies (bNAbs) against HIV-1 require understanding germline bNAb recognition of HIV-1 envelope glycoprotein (Env). The VRC01-class bNAb family derived from the VH1-2*02 germline allele arose in multiple HIV-1–infected donors, yet targets the CD4-binding site on Env with common interactions. Modified forms of the 426c Env that activate germline-reverted B cell receptors are candidate immunogens for eliciting VRC01-class bNAbs. We present structures of germline-reverted VRC01-class bNAbs alone and complexed with 426c-based gp120 immunogens. Germline bNAb–426c gp120 complexes showed preservation of VRC01-class signature residues and gp120 contacts, but detectably different binding modes compared to mature bNAb-gp120 complexes. Unlike typical antibody-antigen interactions, VRC01–class germline antibodies exhibited preformed antigen-binding conformations for recognizing immunogens. Affinity maturation introduced substitutions increasing induced-fit recognition and electropositivity, potentially to accommodate negatively-charged complex-type N-glycans on gp120. These results provide general principles relevant to the unusual evolution of VRC01–class bNAbs and guidelines for structure-based immunogen design.
DOI: http://dx.doi.org/10.7554/eLife.13783.001
eLife digest
When human immunodeficiency virus-1 (HIV-1) infects humans it can cause a serious disease that damages the immune system. Currently there is no cure for this disease and there are no vaccines available to halt the spread of the virus. Researchers are hoping to be able to develop a single vaccine that can protect individuals against every form (or strain) of HIV-1, but this has proved difficult because many different versions of the virus exist.
An effective vaccine triggers long-lasting immunity to a particular virus or microbe by activating the production of proteins called antibodies that identify and help to destroy the threat. Research has shown that most individuals infected with HIV-1 produce antibodies that can only recognize a few HIV strains. However, there are rare individuals who produce “broadly neutralizing antibodies”; that is, antibodies that can recognize and help to kill 90% or more of HIV-1 strains. Understanding how broadly neutralizing antibodies are produced in infected individuals may aid the development of a vaccine that can protect others from the many circulating strains of HIV.
When an individual encounters a virus, immature antibodies are modified to generate mature antibodies that bind more effectively to specific virus proteins. Here, Scharf et al. investigated how a class of broadly neutralizing antibodies called VRC01-class antibodies, which bind to an HIV protein called gp120, are produced. The experiments used a technique called X-ray crystallography to reveal the three-dimensional structures of immature versions of these antibodies when they are bound to gp120.
Scharf et al. discovered that, unlike most antibodies, the overall final structure of VRC01 antibodies is formed before the antibody matures. Instead of making large changes to the structure of these antibodies, the maturation process makes VRC01-class antibodies become more positively charged, which allows them to bind to gp120 proteins on a wider variety of HIV viruses. These findings suggest that it may be possible to use modified gp120 proteins in vaccines to trigger the production of broadly neutralizing antibodies against HIV.
DOI: http://dx.doi.org/10.7554/eLife.13783.002
doi:10.7554/eLife.13783
PMCID: PMC4811768  PMID: 26997349
HIV; broadly neutralizing antibodies; crystallography; Human; Virus
9.  Intra-spike crosslinking overcomes antibody evasion by HIV-1 
Cell  2015;160(3):433-446.
SUMMARY
Antibodies developed during HIV-1 infection lose efficacy as the viral spike mutates. We postulated that anti-HIV-1 antibodies primarily bind monovalently because HIV’s low spike density impedes bivalent binding through inter-spike crosslinking, and the spike structure prohibits bivalent binding through intra-spike crosslinking. Monovalent binding reduces avidity and potency, thus expanding the range of mutations permitting antibody evasion. To test this idea, we engineered antibody-based molecules capable of bivalent binding through intra-spike crosslinking. We used DNA as a “molecular ruler” to measure intra-epitope distances on virion-bound spikes and construct intra-spike crosslinking molecules. Optimal bivalent reagents exhibited up to 2.5 orders-of-magnitude increased potency (>100-fold average increases across virus panels) and identified conformational states of virion-bound spikes. The demonstration that intra-spike crosslinking lowers the concentration of antibodies required for neutralization supports the hypothesis that low spike densities facilitate antibody evasion and the use of molecules capable of intra-spike crosslinking for therapy or passive protection.
doi:10.1016/j.cell.2015.01.016
PMCID: PMC4401576  PMID: 25635457
10.  Spinel–rock salt transformation in LiCoMnO4−δ 
The transformation on heating LiCoMnO4, with a spinel structure, to LiCoMnO3, with a cation-disordered rock salt structure, accompanied by loss of 25% of the oxygen, has been followed using a combination of diffraction, microscopy and spectroscopy techniques. The transformation does not proceed by a topotactic mechanism, even though the spinel and rock salt phases have a similar, cubic close-packed oxygen sublattice. Instead, the transformation passes through two stages involving, first, precipitation of Li2MnO3, leaving behind a Li-deficient, Co-rich non-stoichiometric spinel and, second, rehomogenization of the two-phase assemblage, accompanied by additional oxygen loss, to give the homogeneous rock salt final product; a combination of electron energy loss spectroscopy and X-ray absorption near edge structure analyses showed oxidation states of Co2+ and Mn3+ in LiCoMnO3. Subsolidus phase diagram determination of the Li2O-CoOx-MnOy system has established the compositional extent of spinel solid solutions at approximately 500°C.
doi:10.1098/rspa.2014.0991
PMCID: PMC4786028  PMID: 26997883
LiCoMnO4; Rietveld; electron energy loss spectroscopy; X-ray absorption near edge structure; Raman; oxygen loss
11.  Nitric Oxide Signaling Is Recruited As a Compensatory Mechanism for Sustaining Synaptic Plasticity in Alzheimer's Disease Mice 
The Journal of Neuroscience  2015;35(17):6893-6902.
Synaptic plasticity deficits are increasingly recognized as causing the memory impairments which define Alzheimer's disease (AD). In AD mouse models, evidence of abnormal synaptic function is present before the onset of cognitive deficits, and presents as increased synaptic depression revealed only when synaptic homeostasis is challenged, such as with suppression of ryanodine receptor (RyR)-evoked calcium signaling. Otherwise, at early disease stages, the synaptic physiology phenotype appears normal. This suggests compensatory mechanisms are recruited to maintain a functionally normal net output of the hippocampal circuit. A candidate calcium-regulated synaptic modulator is nitric oxide (NO), which acts presynaptically to boost vesicle release and glutamatergic transmission. Here we tested whether there is a feedforward cycle between the increased RyR calcium release seen in presymptomatic AD mice and aberrant NO signaling which augments synaptic plasticity. Using a combination of electrophysiological approaches, two-photon calcium imaging, and protein biochemistry in hippocampal tissue from presymptomatic 3xTg-AD and NonTg mice, we show that blocking NO synthesis results in markedly augmented synaptic depression mediated through presynaptic mechanisms in 3xTg-AD mice. Additionally, blocking NO reduces the augmented synaptically evoked dendritic calcium release mediated by enhanced RyR calcium release. This is accompanied by increased nNOS levels in the AD mice and is reversed upon normalization of RyR-evoked calcium release with chronic dantrolene treatment. Thus, recruitment of NO is serving a compensatory role to boost synaptic transmission and plasticity during early AD stages. However, NO's dual role in neuroprotection and neurodegeneration may convert to maladaptive functions as the disease progresses.
doi:10.1523/JNEUROSCI.4002-14.2015
PMCID: PMC4412902  PMID: 25926464
calcium; homeostasis; nitric oxide; ryanodine receptor; synaptic depression; synaptic plasticity
12.  Facilitation of Corticostriatal Transmission following Pharmacological Inhibition of Striatal Phosphodiesterase 10A: Role of Nitric Oxide-Soluble Guanylyl Cyclase-cGMP Signaling Pathways 
The Journal of Neuroscience  2015;35(14):5781-5791.
The striatum contains a rich variety of cyclic nucleotide phosphodiesterases (PDEs), which play a critical role in the regulation of cAMP and cGMP signaling. The dual-substrate enzyme PDE10A is the most highly expressed PDE in striatal medium-sized spiny neurons (MSNs) with low micromolar affinity for both cyclic nucleotides. Previously, we have shown that systemic and local administration of the selective PDE10A inhibitor TP-10 potently increased the responsiveness of MSNs to cortical stimulation. However, the signaling mechanisms underlying PDE10A inhibitor-induced changes in corticostriatal transmission are only partially understood. The current studies assessed the respective roles of cAMP and cGMP in the above effects using soluble guanylyl cyclase (sGC) or adenylate cyclase (AC) specific inhibitors. Cortically evoked spike activity was monitored in urethane-anesthetized rats using in vivo extracellular recordings performed proximal to a microdialysis probe during local infusion of vehicle, the selective sGC inhibitor ODQ, or the selective AC inhibitor SQ 22536. Systemic administration of TP-10 (3.2 mg/kg) robustly increased cortically evoked spike activity in a manner that was blocked following intrastriatal infusion of ODQ (50 μm). The effects of TP-10 on evoked activity were due to accumulation of cGMP, rather than cAMP, as the AC inhibitor SQ was without effect. Consistent with these observations, studies in neuronal NO synthase (nNOS) knock-out (KO) mice confirmed that PDE10A operates downstream of nNOS to limit cGMP production and excitatory corticostriatal transmission. Thus, stimulation of PDE10A acts to attenuate corticostriatal transmission in a manner largely dependent on effects directed at the NO-sGC-cGMP signaling cascade.
doi:10.1523/JNEUROSCI.1238-14.2015
PMCID: PMC4388932  PMID: 25855188
cGMP; medium-sized spiny neuron; nitric oxide; nitric oxide synthase; phosphodiesterase 10A; soluble guanylyl cyclase
13.  Antibody Gene Transfer for HIV Immunoprophylaxis 
Nature immunology  2013;14(1):1-5.
Antibody gene transfer, which involves the delivery of genes that encode potent, broadly neutralizing anti-HIV antibodies, is a promising new strategy to prevent HIV infection. A satellite symposium at the AIDS Vaccine 2012 conference brought together many of the groups working in this field.
doi:10.1038/ni.2480
PMCID: PMC4560170  PMID: 23238748
14.  Antibody engineering for increased potency, breadth and half-life 
Current Opinion in HIV and AIDS  2015;10(3):151-159.
Purpose of review
This review highlights recent developments in HIV-1 antibody engineering and discusses the effects of increased polyreactivity on serum half-lives of engineered antibodies.
Recent findings
Recent studies have uncovered a wealth of information about the relationship between the sequences and efficacies of anti-HIV-1 antibodies through a combination of bioinformatics, structural characterization and in vivo studies. This knowledge has stimulated efforts to enhance antibody breadth and potency for therapeutic use. Although some engineered antibodies have shown increased polyreactivity and short half-lives, promising efforts are circumventing these problems.
Summary
Antibodies are desirable as therapeutics due to their ability to recognize targets with both specificity and high affinity. Furthermore, the ability of antibodies to stimulate Fc-mediated effector functions can increase their utility. Thus, mAbs have become central to strategies for the treatment of various diseases. Using both targeted and library-based approaches, antibodies can be engineered to improve their therapeutic properties. This article will discuss recent antibody engineering efforts to improve the breadth and potency of anti-HIV-1 antibodies. The polyreactivity of engineered HIV-1 bNAbs and the effect on serum half-life will be explored along with strategies to overcome problems introduced by engineering antibodies. Finally, advances in creating bispecific anti-HIV-1 reagents are discussed.
doi:10.1097/COH.0000000000000148
PMCID: PMC4465343  PMID: 25760931
antibody engineering; bispecific reagents; breadth; HIV-1; polyreactivity; potency
15.  CATNAP: a tool to compile, analyze and tally neutralizing antibody panels 
Nucleic Acids Research  2015;43(Web Server issue):W213-W219.
CATNAP (Compile, Analyze and Tally NAb Panels) is a new web server at Los Alamos HIV Database, created to respond to the newest advances in HIV neutralizing antibody research. It is a comprehensive platform focusing on neutralizing antibody potencies in conjunction with viral sequences. CATNAP integrates neutralization and sequence data from published studies, and allows users to analyze that data for each HIV Envelope protein sequence position and each antibody. The tool has multiple data retrieval and analysis options. As input, the user can pick specific antibodies and viruses, choose a panel from a published study, or supply their own data. The output superimposes neutralization panel data, virus epidemiological data, and viral protein sequence alignments on one page, and provides further information and analyses. The user can highlight alignment positions, or select antibody contact residues and view position-specific information from the HIV databases. The tool calculates tallies of amino acids and N-linked glycosylation motifs, counts of antibody-sensitive and -resistant viruses in conjunction with each amino acid or N-glycosylation motif, and performs Fisher's exact test to detect potential positive or negative amino acid associations for the selected antibody. Website name: CATNAP (Compile, Analyze and Tally NAb Panels). Website address: http://hiv.lanl.gov/catnap.
doi:10.1093/nar/gkv404
PMCID: PMC4489231  PMID: 26044712
16.  Enhanced HIV-1 immunotherapy by commonly arising antibodies that target virus escape variants 
The Journal of Experimental Medicine  2014;211(12):2361-2372.
Klein et al. find that frequently arising antibodies that normally fail to control HIV-1 infection can synergize with passively transferred bNAbs to prevent the emergence of bNAb viral escape variants.
Antibody-mediated immunotherapy is effective in humanized mice when combinations of broadly neutralizing antibodies (bNAbs) are used that target nonoverlapping sites on the human immunodeficiency virus type 1 (HIV-1) envelope. In contrast, single bNAbs can control simian–human immunodeficiency virus (SHIV) infection in immune-competent macaques, suggesting that the host immune response might also contribute to the control of viremia. Here, we investigate how the autologous antibody response in intact hosts can contribute to the success of immunotherapy. We find that frequently arising antibodies that normally fail to control HIV-1 infection can synergize with passively administered bNAbs by preventing the emergence of bNAb viral escape variants.
doi:10.1084/jem.20141050
PMCID: PMC4235636  PMID: 25385756
17.  Neutralization Properties of Simian Immunodeficiency Viruses Infecting Chimpanzees and Gorillas 
mBio  2015;6(2):e00296-15.
ABSTRACT
Broadly cross-reactive neutralizing antibodies (bNabs) represent powerful tools to combat human immunodeficiency virus type 1 (HIV-1) infection. Here, we examined whether HIV-1-specific bNabs are capable of cross-neutralizing distantly related simian immunodeficiency viruses (SIVs) infecting central (Pan troglodytes troglodytes) (SIVcpzPtt) and eastern (Pan troglodytes schweinfurthii) (SIVcpzPts) chimpanzees (n = 11) as well as western gorillas (Gorilla gorilla gorilla) (SIVgor) (n = 1). We found that bNabs directed against the CD4 binding site (n = 10), peptidoglycans at the base of variable loop 3 (V3) (n = 5), and epitopes at the interface of surface (gp120) and membrane-bound (gp41) envelope glycoproteins (n = 5) failed to neutralize SIVcpz and SIVgor strains. In addition, apex V2-directed bNabs (n = 3) as well as llama-derived (heavy chain only) antibodies (n = 6) recognizing both the CD4 binding site and gp41 epitopes were either completely inactive or neutralized only a fraction of SIVcpzPtt strains. In contrast, one antibody targeting the membrane-proximal external region (MPER) of gp41 (10E8), functional CD4 and CCR5 receptor mimetics (eCD4-Ig, eCD4-Igmim2, CD4-218.3-E51, and CD4-218.3-E51-mim2), as well as mono- and bispecific anti-human CD4 (iMab and LM52) and CCR5 (PRO140, PRO140-10E8) receptor antibodies neutralized >90% of SIVcpz and SIVgor strains with low-nanomolar (0.13 to 8.4 nM) potency. Importantly, the latter antibodies blocked virus entry not only in TZM-bl cells but also in Cf2Th cells expressing chimpanzee CD4 and CCR5 and neutralized SIVcpz in chimpanzee CD4+ T cells, with 50% inhibitory concentrations (IC50s) ranging from 3.6 to 40.5 nM. These findings provide new insight into the protective capacity of anti-HIV-1 bNabs and identify candidates for further development to combat SIVcpz infection.
IMPORTANCE
SIVcpz is widespread in wild-living chimpanzees and can cause AIDS-like immunopathology and clinical disease. HIV-1 infection of humans can be controlled by antiretroviral therapy; however, treatment of wild-living African apes with current drug regimens is not feasible. Nonetheless, it may be possible to curb the spread of SIVcpz in select ape communities using vectored immunoprophylaxis and/or therapy. Here, we show that antibodies and antibody-like inhibitors developed to combat HIV-1 infection in humans are capable of neutralizing genetically diverse SIVcpz and SIVgor strains with considerable breadth and potency, including in primary chimpanzee CD4+ T cells. These reagents provide an important first step toward translating intervention strategies currently developed to treat and prevent AIDS in humans to SIV-infected apes.
doi:10.1128/mBio.00296-15
PMCID: PMC4453581  PMID: 25900654
18.  Structural Insights on the Role of Antibodies in HIV-1 Vaccine and Therapy 
Cell  2014;156(4):633-648.
Despite 30 years of effort, there is no effective vaccine for HIV-1. However, antibodies can prevent HIV-1 infection in humanized mice and macaques when passively transferred. New single-cell-based methods have uncovered many broad and potent donor-derived antibodies, and structural studies have revealed the molecular bases for their activities. The new data suggest why such antibodies are difficult to elicit and inform HIV-1 vaccine development efforts. In addition to protecting against infection, the newly identified antibodies can suppress active infections in mice and macaques, suggesting they could be valuable additions to anti-HIV-1 therapies and to strategies to eradicate HIV-1 infection.
doi:10.1016/j.cell.2014.01.052
PMCID: PMC4041625  PMID: 24529371
19.  Structural basis for enhanced neutralization of HIV-1 by a dimeric IgG form of the glycan-recognizing antibody 2G12 
Cell reports  2013;5(5):1443-1455.
SUMMARY
The human IgG 2G12 recognizes high-mannose carbohydrates on the HIV-1 envelope glycoprotein gp120. Its two antigen-binding fragments (Fabs) are intramolecularly domain exchanged, resulting in a rigid (Fab)2 unit including a third antigen-binding interface not found in antibodies with flexible Fab arms. We determined crystal structures of dimeric 2G12 IgG created by intermolecular domain exchange, which exhibits increased breadth and >50-fold increased neutralization potency compared with monomeric 2G12. The four Fab and two Fc regions of dimeric 2G12 were localized at low resolution in two independent structures, revealing IgG dimers with two (Fab)2 arms analogous to the Fabs of conventional monomeric IgGs. Structures revealed three conformationally-distinct dimers, demonstrating flexibility of the (Fab)2–Fc connections that was confirmed by electron microscopy, small-angle X-ray scattering, and binding studies. We conclude that intermolecular domain exchange, flexibility, and bivalent binding to allow avidity effects are responsible for the increased potency and breadth of dimeric 2G12.
doi:10.1016/j.celrep.2013.11.015
PMCID: PMC3919625  PMID: 24316082
20.  Review: Modulation of striatal neuron activity by cyclic nucleotide signaling and phosphodiesterase inhibition 
Basal ganglia  2013;3(3):137-146.
The cyclic nucleotides cAMP and cGMP are common signaling molecules synthesized in neurons following the activation of adenylyl or guanylyl cyclase. In the striatum, the synthesis and degradation of cAMP and cGMP is highly regulated as these second messengers have potent effects on the activity of striatonigral and striatopallidal neurons. This review will summarize the literature on cyclic nucleotide signaling in the striatum with a particular focus on the impact of cAMP and cGMP on the membrane excitability of striatal medium-sized spiny output neurons (MSNs). The effects of non-selective and selective phosphodiesterase (PDE) inhibitors on membrane activity and synaptic plasticity of MSNs will also be reviewed. Lastly, this review will discuss the implications of the effects PDE modulation on electrophysiological activity of striatal MSNs as it relates to the treatment of neurological disorders such as Huntington’s and Parkinson’s disease.
doi:10.1016/j.baga.2013.08.001
PMCID: PMC3904398  PMID: 24490129
cyclic AMP; cyclic GMP; phosphodiesterase; electrophysiology
21.  Antibody 8ANC195 Reveals a Site of Broad Vulnerability on the HIV-1 Envelope Spike 
Cell reports  2014;7(3):785-795.
Summary
Broadly neutralizing antibodies (bNAbs) to HIV-1 envelope glycoprotein (Env) can prevent infection in animal models. Characterized bNAb targets, although key to vaccine and therapeutic strategies, are currently limited. We defined a new site of vulnerability by solving structures of bNAb 8ANC195 complexed with monomeric gp120 by X-ray crystallography and trimeric Env by electron microscopy. The site includes portions of gp41 and N-linked glycans adjacent to the CD4 binding site on gp120, making 8ANC195 the first donor-derived anti-HIV-1 bNAb with an epitope spanning both Env subunits. Rather than penetrating the glycan shield using a single variable region CDR loop, 8ANC195 inserted its entire heavy chain variable domain into a gap to form a large interface with gp120 glycans and regions of the gp120 inner domain not contacted by other bNAbs. By isolating additional 8ANC195 clonal variants, we identified a more potent variant, which may be valuable for therapeutic approaches using bNAb combinations with non-overlapping epitopes.
doi:10.1016/j.celrep.2014.04.001
PMCID: PMC4109818  PMID: 24767986
22.  Restricting HIV-1 pathways for escape using rationally designed anti–HIV-1 antibodies 
The Journal of Experimental Medicine  2013;210(6):1235-1249.
Mutating anti–HIV-1 broadly neutralizing antibodies increases their breadth and reduces pathways for viral escape through mutation.
Recently identified broadly neutralizing antibodies (bNAbs) that potently neutralize most HIV-1 strains are key to potential antibody-based therapeutic approaches to combat HIV/AIDS in the absence of an effective vaccine. Increasing bNAb potencies and resistance to common routes of HIV-1 escape through mutation would facilitate their use as therapeutics. We previously used structure-based design to create the bNAb NIH45-46G54W, which exhibits superior potency and/or breadth compared with other bNAbs. We report new, more effective NIH45-46G54W variants designed using analyses of the NIH45-46–gp120 complex structure and sequences of NIH45-46G54W–resistant HIV-1 strains. One variant, 45-46m2, neutralizes 96% of HIV-1 strains in a cross-clade panel and viruses isolated from an HIV-infected individual that are resistant to all other known bNAbs, making it the single most broad and potent anti–HIV-1 antibody to date. A description of its mechanism is presented based on a 45-46m2–gp120 crystal structure. A second variant, 45-46m7, designed to thwart HIV-1 resistance to NIH45-46G54W arising from mutations in a gp120 consensus sequence, targets a common route of HIV-1 escape. In combination, 45-46m2 and 45-46m7 reduce the possible routes for the evolution of fit viral escape mutants in HIV-1YU-2–infected humanized mice, with viremic control exhibited when a third antibody, 10–1074, was added to the combination.
doi:10.1084/jem.20130221
PMCID: PMC3674693  PMID: 23712429
23.  Engineering HIV envelope protein to activate germline B cell receptors of broadly neutralizing anti-CD4 binding site antibodies 
Eliminating key glycosylation sites on HIV envelope (Env) restores binding of the germline versions of known broadly neutralizing anti-Env antibodies.
Broadly neutralizing antibodies (bnAbs) against HIV are believed to be a critical component of the protective responses elicited by an effective HIV vaccine. Neutralizing antibodies against the evolutionarily conserved CD4-binding site (CD4-BS) on the HIV envelope glycoprotein (Env) are capable of inhibiting infection of diverse HIV strains, and have been isolated from HIV-infected individuals. Despite the presence of anti–CD4-BS broadly neutralizing antibody (bnAb) epitopes on recombinant Env, Env immunization has so far failed to elicit such antibodies. Here, we show that Env immunogens fail to engage the germline-reverted forms of known bnAbs that target the CD4-BS. However, we found that the elimination of a conserved glycosylation site located in Loop D and two glycosylation sites located in variable region 5 of Env allows Env-binding to, and activation of, B cells expressing the germline-reverted BCRs of two potent broadly neutralizing antibodies, VRC01 and NIH45-46. Our results offer a possible explanation as to why Env immunogens have been ineffective in stimulating the production of such bNAbs. Importantly, they provide key information as to how such immunogens can be engineered to initiate the process of antibody-affinity maturation against one of the most conserved Env regions.
doi:10.1084/jem.20122824
PMCID: PMC3620356  PMID: 23530120
24.  A conditional knockout resource for the genome–wide study of mouse gene function 
Nature  2011;474(7351):337-342.
Gene targeting in embryonic stem cells has become the principal technology for manipulation of the mouse genome, offering unrivalled accuracy in allele design and access to conditional mutagenesis. To bring these advantages to the wider research community, large-scale mouse knockout programmes are producing a permanent resource of targeted mutations in all protein-coding genes. Here we report the establishment of a high-throughput gene-targeting pipeline for the generation of reporter-tagged, conditional alleles. Computational allele design, 96-well modular vector construction and high-efficiency gene-targeting strategies have been combined to mutate genes on an unprecedented scale. So far, more than 12,000 vectors and 9,000 conditional targeted alleles have been produced in highly germline-competent C57BL/6N embryonic stem cells. High-throughput genome engineering highlighted by this study is broadly applicable to rat and human stem cells and provides a foundation for future genome-wide efforts aimed at deciphering the function of all genes encoded by the mammalian genome.
doi:10.1038/nature10163
PMCID: PMC3572410  PMID: 21677750
25.  The mammalian gene function resource: the international knockout mouse consortium 
Bradley, Allan | Anastassiadis, Konstantinos | Ayadi, Abdelkader | Battey, James F. | Bell, Cindy | Birling, Marie-Christine | Bottomley, Joanna | Brown, Steve D. | Bürger, Antje | Bult, Carol J. | Bushell, Wendy | Collins, Francis S. | Desaintes, Christian | Doe, Brendan | Economides, Aris | Eppig, Janan T. | Finnell, Richard H. | Fletcher, Colin | Fray, Martin | Frendewey, David | Friedel, Roland H. | Grosveld, Frank G. | Hansen, Jens | Hérault, Yann | Hicks, Geoffrey | Hörlein, Andreas | Houghton, Richard | Hrabé de Angelis, Martin | Huylebroeck, Danny | Iyer, Vivek | de Jong, Pieter J. | Kadin, James A. | Kaloff, Cornelia | Kennedy, Karen | Koutsourakis, Manousos | Kent Lloyd, K. C. | Marschall, Susan | Mason, Jeremy | McKerlie, Colin | McLeod, Michael P. | von Melchner, Harald | Moore, Mark | Mujica, Alejandro O. | Nagy, Andras | Nefedov, Mikhail | Nutter, Lauryl M. | Pavlovic, Guillaume | Peterson, Jane L. | Pollock, Jonathan | Ramirez-Solis, Ramiro | Rancourt, Derrick E. | Raspa, Marcello | Remacle, Jacques E. | Ringwald, Martin | Rosen, Barry | Rosenthal, Nadia | Rossant, Janet | Ruiz Noppinger, Patricia | Ryder, Ed | Schick, Joel Zupicich | Schnütgen, Frank | Schofield, Paul | Seisenberger, Claudia | Selloum, Mohammed | Simpson, Elizabeth M. | Skarnes, William C. | Smedley, Damian | Stanford, William L. | Francis Stewart, A. | Stone, Kevin | Swan, Kate | Tadepally, Hamsa | Teboul, Lydia | Tocchini-Valentini, Glauco P. | Valenzuela, David | West, Anthony P. | Yamamura, Ken-ichi | Yoshinaga, Yuko | Wurst, Wolfgang
Mammalian Genome  2012;23(9-10):580-586.
In 2007, the International Knockout Mouse Consortium (IKMC) made the ambitious promise to generate mutations in virtually every protein-coding gene of the mouse genome in a concerted worldwide action. Now, 5 years later, the IKMC members have developed high-throughput gene trapping and, in particular, gene-targeting pipelines and generated more than 17,400 mutant murine embryonic stem (ES) cell clones and more than 1,700 mutant mouse strains, most of them conditional. A common IKMC web portal (www.knockoutmouse.org) has been established, allowing easy access to this unparalleled biological resource. The IKMC materials considerably enhance functional gene annotation of the mammalian genome and will have a major impact on future biomedical research.
doi:10.1007/s00335-012-9422-2
PMCID: PMC3463800  PMID: 22968824

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