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1.
Clin Anat ; 36(6): 875-880, 2023 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-36527146

RESUMO

The arteries of the lower limbs are innervated by vascular branches (VBs) originating from the lumbar sympathetic trunk and branches of the spinal nerve. Although lumbar sympathectomy is used to treat nonreconstructive critical lower limb ischemia (CLLI), it has limited long-term effects. In addition, the anatomical structure of tibial nerve (TN) VBs remain incompletely understood. This study aimed to clarify their anatomy and better inform the surgical approach for nonreconstructive CLLI. Thirty-six adult cadavers were dissected under surgical microscopy to observe the patterns and origin points of VBs under direct vision. The calves were anatomically divided into five equal segments, and the number of VB origin points found in each was expressed as a proportion of the total found in the whole calf. Immunofluorescence staining was used to identify the sympathetic nerve fibers of the VBs. Our results showed that the TN gave off 3-4 VBs to innervate the posterior tibial artery (PTA), and the distances between VBs origin points and the medial tibial condyle were: 24.7 ± 16.3 mm, 91.7 ± 66.1 mm, 199.6 ± 52.0 mm, 231.7 ± 38.5 mm, respectively. They were mainly located in the first (40.46%) and fourth (31.68%) calf segments, and immunofluorescence staining showed that they contained tyrosine hydroxylase-positive sympathetic nerve fibers. These findings indicate that the TN gives off VBs to innervate the PTA and that these contain sympathetic nerve fibers. Therefore, these VBs may need to be cut to surgically treat nonreconstructable CLLI.


Assuntos
Artérias da Tíbia , Nervo Tibial , Adulto , Humanos , Perna (Membro)/irrigação sanguínea , Perna (Membro)/inervação , Fibras Nervosas , Doenças Vasculares Periféricas/cirurgia , Tíbia , Artérias da Tíbia/inervação , Nervo Tibial/anatomia & histologia , Cadáver
2.
J Biol Chem ; 295(14): 4372-4380, 2020 04 03.
Artigo em Inglês | MEDLINE | ID: mdl-31882544

RESUMO

Programmed cell death protein 1 (PD-1) is an inhibitory receptor on T lymphocytes that is critical for modulating adaptive immunity. As such, it has been successfully exploited for cancer immunotherapy. Programmed death ligand 1 (PD-L1) and PD-L2 are ligands for PD-1; the former is ubiquitously expressed in inflamed tissues, whereas the latter is restricted to antigen-presenting cells. PD-L2 binds to PD-1 with 3-fold stronger affinity compared with PD-L1. To date, this affinity discrepancy has been attributed to a tryptophan (W110PD-L2) that is unique to PD-L2 and has been assumed to fit snuggly into a pocket on the PD-1 surface. Contrary to this model, using surface plasmon resonance to monitor real-time binding of recombinantly-expressed and -purified proteins, we found that W110PD-L2 acts as an "elbow" that helps shorten PD-L2 engagement with PD-1 and therefore lower affinity. Furthermore, we identified a "latch" between the C and D ß-strands of the binding face as the source of the PD-L2 affinity advantage. We show that the 3-fold affinity advantage of PD-L2 is the consequence of these two opposing features, the W110PD-L2 "elbow" and a C-D region "latch." Interestingly, using phylogenetic analysis, we found that these features evolved simultaneously upon the emergence of placental mammals, suggesting that PD-L2-affinity tuning was part of the alterations to the adaptive immune system required for placental gestation.


Assuntos
Antígeno B7-H1/química , Placenta/metabolismo , Proteína 2 Ligante de Morte Celular Programada 1/química , Sequência de Aminoácidos , Animais , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Linfócitos T CD4-Positivos/citologia , Linfócitos T CD4-Positivos/metabolismo , Proliferação de Células , Feminino , Humanos , Ligantes , Ativação Linfocitária , Camundongos , Mutagênese Sítio-Dirigida , Filogenia , Gravidez , Proteína 2 Ligante de Morte Celular Programada 1/classificação , Proteína 2 Ligante de Morte Celular Programada 1/genética , Proteína 2 Ligante de Morte Celular Programada 1/metabolismo , Ligação Proteica , Domínios Proteicos , Estrutura Terciária de Proteína , Alinhamento de Sequência , Eletricidade Estática
3.
PLoS Pathog ; 14(8): e1007278, 2018 08.
Artigo em Inglês | MEDLINE | ID: mdl-30153309

RESUMO

The GI tract is preferentially targeted during acute/early HIV-1 infection. Consequent damage to the gut plays a central role in HIV pathogenesis. The basis for preferential targeting of gut tissues is not well defined. Recombinant proteins and synthetic peptides derived from HIV and SIV gp120 bind directly to integrin α4ß7, a gut-homing receptor. Using both cell-surface expressed α4ß7 and a soluble α4ß7 heterodimer we demonstrate that its specific affinity for gp120 is similar to its affinity for MAdCAM (its natural ligand). The gp120 V2 domain preferentially engages extended forms of α4ß7 in a cation -sensitive manner and is inhibited by soluble MAdCAM. Thus, V2 mimics MAdCAM in the way that it binds to α4ß7, providing HIV a potential mechanism to discriminate between functionally distinct subsets of lymphocytes, including those with gut-homing potential. Furthermore, α4ß7 antagonists developed for the treatment of inflammatory bowel diseases, block V2 binding to α4ß7. A 15-amino acid V2 -derived peptide is sufficient to mediate binding to α4ß7. It includes the canonical LDV/I α4ß7 binding site, a cryptic epitope that lies 7-9 amino acids amino terminal to the LDV/I, and residues K169 and I181. These two residues were identified in a sieve analysis of the RV144 vaccine trial as sites of vaccine -mediated immune pressure. HIV and SIV V2 mAbs elicited by both vaccination and infection that recognize this peptide block V2-α4ß7 interactions. These mAbs recognize conformations absent from the ß- barrel presented in a stabilized HIV SOSIP gp120/41 trimer. The mimicry of MAdCAM-α4ß7 interactions by V2 may influence early events in HIV infection, particularly the rapid seeding of gut tissues, and supports the view that HIV replication in gut tissue is a central feature of HIV pathogenesis.


Assuntos
Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/imunologia , Proteína gp120 do Envelope de HIV/metabolismo , Infecções por HIV/prevenção & controle , Integrinas/metabolismo , Síndrome de Imunodeficiência Adquirida dos Símios/prevenção & controle , Vacinas contra a AIDS/química , Vacinas contra a AIDS/imunologia , Vacinas contra a AIDS/metabolismo , Animais , Anticorpos Monoclonais , Sítios de Ligação/imunologia , Linhagem Celular Tumoral , Epitopos/química , Epitopos/imunologia , Anticorpos Anti-HIV/química , Anticorpos Anti-HIV/imunologia , Anticorpos Anti-HIV/metabolismo , Infecções por HIV/imunologia , HIV-1/imunologia , Macaca , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas/imunologia , Vacinas contra a SAIDS/química , Vacinas contra a SAIDS/imunologia , Vacinas contra a SAIDS/metabolismo , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Vírus da Imunodeficiência Símia/imunologia , Vacinação/métodos
4.
J Virol ; 92(18)2018 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-29997214

RESUMO

Elucidating the structural basis of antibody (Ab) gene usage and affinity maturation of vaccine-induced Abs can inform the design of immunogens for inducing desired Ab responses in HIV vaccine development. Analyses of monoclonal Abs (MAbs) encoded by the same immunoglobulin genes at different stages of maturation can help to elucidate the maturation process. We have analyzed four human anti-V3 MAbs with the same VH1-3*01 and VL3-10*01 gene usage. Two MAbs, TA6 and TA7, were developed from a vaccinee in the HIV vaccine phase I trial DP6-001 with a polyvalent DNA prime/protein boost regimen, and two others, 311-11D and 1334, were developed from HIV-infected patients. The somatic hypermutation (SHM) rates in VH of vaccine-induced MAbs are lower than in chronic HIV infection-induced MAbs, while those in VL are comparable. Crystal structures of the antigen-binding fragments (Fabs) in complex with V3 peptides show that these MAbs bind the V3 epitope with a new cradle-binding mode and that the V3 ß-hairpin lies along the antigen-binding groove, which consists of residues from both heavy and light chains. Residues conserved from the germ line sequences form specific binding pockets accommodating conserved structural elements of the V3 crown hairpin, predetermining the Ab gene selection, while somatically mutated residues create additional hydrogen bonds, electrostatic interactions, and van der Waals contacts, correlating with an increased binding affinity. Our data provide a unique example of germ line sequences determining the primordial antigen-binding sites and SHMs correlating with affinity maturation of Abs induced by vaccine and natural HIV infection.IMPORTANCE Understanding the structural basis of gene usage and affinity maturation for anti-HIV-1 antibodies may help vaccine design and development. Antibodies targeting the highly immunogenic third variable loop (V3) of HIV-1 gp120 provide a unique opportunity for detailed structural investigations. By comparing the sequences and structures of four anti-V3 MAbs at different stages of affinity maturation but of the same V gene usage, two induced by vaccination and another two by chronic infection, we provide a fine example of how germ line sequence determines the essential elements for epitope recognition and how affinity maturation improves the antibody's recognition of its epitope.


Assuntos
Vacinas contra a AIDS/imunologia , Anticorpos Monoclonais/química , Anticorpos Anti-HIV/química , Proteína gp120 do Envelope de HIV/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Sequência de Aminoácidos , Anticorpos Monoclonais/genética , Anticorpos Monoclonais/imunologia , Formação de Anticorpos , Especificidade de Anticorpos , Cristalização , Genes de Imunoglobulinas , Anticorpos Anti-HIV/genética , Anticorpos Anti-HIV/imunologia , Proteína gp120 do Envelope de HIV/genética , Humanos , Ligação de Hidrogênio , Alinhamento de Sequência , Hipermutação Somática de Imunoglobulina , Vacinação
5.
J Virol ; 92(7)2018 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-29343576

RESUMO

The third variable (V3) loop of HIV-1 gp120 is an immunodominant region targeted by neutralizing antibodies (nAbs). Despite limited breadth, better characterization of the structural details of the interactions between these nAbs and their target epitopes would enhance our understanding of the mechanism of neutralization and facilitate designing better immunogens to induce nAbs with greater breadth. Recently, we isolated two anti-V3 neutralizing monoclonal antibodies (MAbs), 10A3 and 10A37, from a rabbit immunized with gp120 of the M group consensus sequence. In this study, crystal structures of these MAbs bound to target epitopes were determined. 10A3 binds to the V3 crown (303TRKSIHIGPGRAF317) using the cradle binding mode, similar to human V3 MAbs encoded by IGHV5-51 germ line genes, and its epitope structure resembles that bound to the human antibodies. In contrast, 10A37, which exhibits greater breadth and potency than 10A3, binds the V3 crown and the succeeding stem region (308HIGPGRAFYTTGEI323). Unexpectedly, the 315RAFYTT320 portion of the epitope existed as helical turns, a V3 structure that has not been observed previously. Its main chain-dominated antigen-antibody interactions not only explain the broad neutralization of 10A37 but also show that its epitope is a potential vaccine target to be further evaluated. In conclusion, our study provides novel insights about neutralization-susceptible epitope structures of the V3 loop of HIV-1 gp120 and demonstrates that, despite low amino acid sequence similarity to human antibody germ line genes, rabbits can serve as a useful animal model to evaluate human vaccine candidates.IMPORTANCE The apex crown of V3 of HIV-1 gp120 is the most immunogenic region of the surface glycoprotein, and many MAbs targeting this region have been developed. Structural understanding of V3 crown MAbs not only can help understand how antibody responses target this unique region but also contribute to immunogen design for vaccine development. We present here crystal structures of two neutralizing V3 MAbs, 10A3 and 10A37, developed from a rabbit immunized with gp120. Our analysis of 10A3 in complex with V3 provided a detailed example of how epitope complexity can evolve with affinity maturation, while that of 10A37 revealed a novel V3 binding mode targeting the C-terminal side of the V3 crown and showed that this region can form a helical structure. Our study provides novel insights about neutralization-susceptible V3 epitope structures and demonstrates that rabbits can serve as a useful animal model to evaluate human vaccine candidates.


Assuntos
Anticorpos Monoclonais/imunologia , Epitopos/imunologia , Anticorpos Anti-HIV/química , Anticorpos Anti-HIV/imunologia , Proteína gp120 do Envelope de HIV/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/isolamento & purificação , Anticorpos Neutralizantes/química , Anticorpos Neutralizantes/imunologia , Anticorpos Neutralizantes/isolamento & purificação , Afinidade de Anticorpos , Formação de Anticorpos , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/metabolismo , Infecções por HIV/virologia , Humanos , Testes de Neutralização , Conformação Proteica , Coelhos
6.
J Virol ; 89(15): 8003-10, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26018158

RESUMO

UNLABELLED: The region consisting of the first and second variable regions (V1V2) of gp120 plays vital roles in the functioning of the HIV-1 envelope (Env). V1V2, which harbors multiple glycans and is highly sequence diverse, is located at the Env apex and stabilizes the trimeric gp120 spike on the virion surface. It shields V3 and the coreceptor binding sites in the prefusion state and exposes them upon CD4 binding. Data from the RV144 human HIV-1 vaccine trial suggested that antibody responses targeting the V1V2 region inversely correlated with the risk of infection; thus, understanding the antigenic structure of V1V2 can contribute to vaccine design. We have determined a crystal structure of a V1V2 scaffold molecule (V1V2ZM109-1FD6) in complex with 830A, a human monoclonal antibody that recognizes a V1V2 epitope overlapping the integrin-binding motif in V2. The structure revealed that V1V2 assumes a five-stranded beta barrel structure with the region of the integrin-binding site (amino acids [aa] 179 to 181) included in a "kink" followed by an extra beta strand. The complete barrel structure naturally presents the glycans on its outer surface and packs into its core conserved hydrophobic residues, including the Ile at position 181 which was highly correlated with vaccine efficacy in RV144. The epitope of monoclonal antibody 830A is discontinuous and composed of three segments: (i) Thr175, Tyr177, Leu179, and Asp180 at the kink overlapping the integrin-binding site; (ii) Arg153 and Val154 in V1; and (iii) Ile194 at the C terminus of V2. This report thus provides the atomic details of the immunogenic "V2i epitope." IMPORTANCE: Data from the RV144 phase III clinical trial suggested that the presence of antibodies to the first and second variable regions (V1V2) of gp120 was associated with the modest protection afforded by the vaccine. V1V2 is a highly variable and immunogenic region of HIV-1 surface glycoprotein gp120, and structural information about this region and its antigenic landscape will be crucial in the design of an effective HIV-1 vaccine. We have determined a crystal structure of V1V2 in complex with human MAb 830A and have shown that MAb 830A recognizes a region overlapping the α4ß7 integrin-binding site. We also showed that V1V2 forms a 5-stranded beta barrel, an elegant structure allowing sequence variations in the strand-connecting loops while preserving a conserved core.


Assuntos
Proteína gp120 do Envelope de HIV/química , Infecções por HIV/virologia , HIV-1/química , Motivos de Aminoácidos , Sequência de Aminoácidos , Proteína gp120 do Envelope de HIV/genética , Proteína gp120 do Envelope de HIV/metabolismo , HIV-1/genética , HIV-1/metabolismo , Humanos , Dados de Sequência Molecular , Estrutura Secundária de Proteína
7.
J Virol ; 89(17): 9090-102, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26109728

RESUMO

UNLABELLED: The V3 region of HIV-1 gp120 is important for virus-coreceptor interaction and highly immunogenic. Although most anti-V3 antibodies neutralize only the sensitive tier 1 viruses, anti-V3 antibodies effective against the more resistant viruses exist, and a better understanding of these antibodies and their epitopes would be beneficial for the development of novel vaccine immunogens against HIV. The HIV-1 isolate JRFL with its cryptic V3 is resistant to most V3-specific monoclonal antibodies (MAbs). However, the V3 MAb 2424 achieves 100% neutralization against JRFL. 2424 is encoded by IGHV3-53 and IGLV2-28 genes, a pairing rarely used by the other V3 MAbs. 2424 also has distinct binding and neutralization profiles. Studies of 2424-mediated neutralization of JRFL produced with a mannosidase inhibitor further revealed that its neutralizing activity is unaffected by the glycan composition of the virus envelope. To understand the distinct activity of 2424, we determined the crystal structure of 2424 Fab in complex with a JRFL V3 peptide and showed that the 2424 epitope is located at the tip of the V3 crown ((307)IHIGPGRAFYT(319)), dominated by interactions with His(P308), Pro(P313), and Arg(P315). The binding mode of 2424 is similar to that of the well-characterized MAb 447-52D, although 2424 is more side chain dependent. The 2424 epitope is focused on the very apex of V3, away from nearby glycans, facilitating antibody access. This feature distinguishes the 2424 epitope from the other V3 crown epitopes and indicates that the tip of V3 is a potential site to target and incorporate into HIV vaccine immunogens. IMPORTANCE: HIV/AIDS vaccines are crucial for controlling the HIV epidemics that continue to afflict millions of people worldwide. However, HIV vaccine development has been hampered by significant scientific challenges, one of which is the inability of HIV vaccine candidates evaluated thus far to elicit production of potent and broadly neutralizing antibodies. The V3 loop is one of the few immunogenic targets on the virus envelope glycoprotein that can induce neutralizing antibodies, but in many viruses, parts of V3 are inaccessible for antibody recognition. This study examined a V3-specific monoclonal antibody that can completely neutralize HIV-1 JRFL, a virus isolate resistant to most V3 antibodies. Our data reveal that this antibody recognizes the most distal tip of V3, which is not as occluded as other parts of V3. Hence, the epitope of 2424 is in one of the vulnerable sites on the virus that may be exploited in designing HIV vaccine immunogens.


Assuntos
Anticorpos Monoclonais/imunologia , Anticorpos Neutralizantes/imunologia , Anticorpos Anti-HIV/imunologia , Proteína gp120 do Envelope de HIV/imunologia , HIV-1/imunologia , Vacinas contra a AIDS/imunologia , Anticorpos Monoclonais/ultraestrutura , Especificidade de Anticorpos/imunologia , Linhagem Celular , Cristalografia por Raios X , Epitopos/imunologia , Células HEK293 , Antígenos HIV/imunologia , Humanos , Fragmentos Fab das Imunoglobulinas/imunologia , Fragmentos Fab das Imunoglobulinas/ultraestrutura , Manosidases/antagonistas & inibidores , Dados de Sequência Molecular , Polissacarídeos/imunologia , Estrutura Terciária de Proteína
8.
PLoS Pathog ; 9(2): e1003173, 2013 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-23468623

RESUMO

Antibodies that neutralize (nAbs) genetically diverse HIV-1 strains have been recovered from a subset of HIV-1 infected subjects during chronic infection. Exact mechanisms that expand the otherwise narrow neutralization capacity observed during early infection are, however, currently undefined. Here we characterized the earliest nAb responses in a subtype A HIV-1 infected Rwandan seroconverter who later developed moderate cross-clade nAb breadth, using (i) envelope (Env) glycoproteins from the transmitted/founder virus and twenty longitudinal nAb escape variants, (ii) longitudinal autologous plasma, and (iii) autologous monoclonal antibodies (mAbs). Initially, nAbs targeted a single region of gp120, which flanked the V3 domain and involved the alpha2 helix. A single amino acid change at one of three positions in this region conferred early escape. One immunoglobulin heavy chain and two light chains recovered from autologous B cells comprised two mAbs, 19.3H-L1 and 19.3H-L3, which neutralized the founder Env along with one or three of the early escape variants carrying these mutations, respectively. Neither mAb neutralized later nAb escape or heterologous Envs. Crystal structures of the antigen-binding fragments (Fabs) revealed flat epitope contact surfaces, where minimal light chain mutation in 19.3H-L3 allowed for additional antigenic interactions. Resistance to mAb neutralization arose in later Envs through alteration of two glycans spatially adjacent to the initial escape signatures. The cross-neutralizing nAbs that ultimately developed failed to target any of the defined V3-proximal changes generated during the first year of infection in this subject. Our data demonstrate that this subject's first recognized nAb epitope elicited strain-specific mAbs, which incrementally acquired autologous breadth, and directed later B cell responses to target distinct portions of Env. This immune re-focusing could have triggered the evolution of cross-clade antibodies and suggests that exposure to a specific sequence of immune escape variants might promote broad humoral responses during HIV-1 infection.


Assuntos
Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Infecções por HIV/imunologia , HIV-1/imunologia , Evasão da Resposta Imune/imunologia , Sequência de Aminoácidos , Anticorpos Neutralizantes/química , Reações Cruzadas/imunologia , Feminino , Infecções por HIV/virologia , Soropositividade para HIV/imunologia , Soropositividade para HIV/virologia , Humanos , Cadeias Pesadas de Imunoglobulinas/imunologia , Cadeias Leves de Imunoglobulina/imunologia , Masculino , Dados de Sequência Molecular , Estrutura Terciária de Proteína , Alinhamento de Sequência
9.
J Virol ; 87(18): 10221-31, 2013 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-23864637

RESUMO

The rabbit is a commonly used animal model in studying antibody responses in HIV/AIDS vaccine development. However, no rabbit monoclonal antibodies (MAbs) have been developed previously to study the epitope-specific antibody responses against HIV-1 envelope (Env) glycoproteins, and little is known about how the rabbit immune system can mimic the human immune system in eliciting such antibodies. Here we present structural analyses of two rabbit MAbs, R56 and R20, against the third variable region (V3) of HIV-1 gp120. R56 recognizes the well-studied immunogenic region in the V3 crown, while R20 targets a less-studied region at the C terminus of V3. By comparison of the Fab/epitope complex structures of these two antibodies raised by immunization with that of the corresponding human antibodies derived from patients chronically infected with HIV-1, we found that rabbit antibodies can recognize immunogenic regions of gp120 and mimic the binding modes of human antibodies. This result can provide new insight into the use of the rabbit as an animal model in AIDS vaccine development.


Assuntos
Anticorpos Monoclonais/metabolismo , Anticorpos Anti-HIV/metabolismo , Proteína gp120 do Envelope de HIV/metabolismo , HIV-1/imunologia , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Cristalografia por Raios X , Epitopos/química , Epitopos/imunologia , Epitopos/metabolismo , Anticorpos Anti-HIV/química , Anticorpos Anti-HIV/imunologia , Proteína gp120 do Envelope de HIV/química , Proteína gp120 do Envelope de HIV/imunologia , HIV-1/química , Humanos , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Coelhos
10.
Mol Neurodegener ; 19(1): 44, 2024 May 31.
Artigo em Inglês | MEDLINE | ID: mdl-38816762

RESUMO

Synucleinopathies are a group of neurodegenerative diseases characterized by the accumulation of α-synuclein (α-syn) in the brain, leading to motor and neuropsychiatric symptoms. Currently, there are no known cures for synucleinopathies, and treatments mainly focus on symptom management. In this study, we developed a single-domain antibody (sdAb)-based protein degrader with features designed to enhance proteasomal degradation of α-syn. This sdAb derivative targets both α-syn and Cereblon (CRBN), a substrate-receptor for the E3-ubiquitin ligase CRL4CRBN, and thereby induces α-syn ubiquitination and proteasomal degradation. Our results indicate that this therapeutic candidate enhances proteasomal degradation of α-syn, in addition to the endogenous lysosomal degradation machinery. By promoting proteasomal degradation of α-syn, we improved clearance of α-syn in primary culture and mouse models of synucleinopathy. These findings indicate that our sdAb-based protein degrader is a promising therapeutic candidate for synucleinopathies. Considering that only a small percentage of antibodies enter the brain, more potent sdAbs with greater brain entry than whole antibodies could enhance clinical benefits of antibody-based therapies.


Assuntos
Sinucleinopatias , alfa-Sinucleína , Animais , Sinucleinopatias/metabolismo , alfa-Sinucleína/metabolismo , alfa-Sinucleína/imunologia , Camundongos , Humanos , Anticorpos de Domínio Único , Modelos Animais de Doenças , Encéfalo/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo
11.
bioRxiv ; 2024 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-38558982

RESUMO

Synucleinopathies are a group of neurodegenerative diseases characterized by the accumulation of α-synuclein (α-syn) in the brain, leading to motor and neuropsychiatric symptoms. Currently, there are no known cures for synucleinopathies, and treatments mainly focus on symptom management. In this study, we developed a single-domain antibody (sdAb)-based protein degrader with features designed to enhance proteasomal degradation of α-syn. This sdAb derivative targets both α-syn and Cereblon (CRBN), a substrate-receptor for the E3-ubiquitin ligase CRL4CRBN, and thereby induces α-syn ubiquitination and proteasomal degradation. Our results indicate that this therapeutic candidate enhances proteasomal degradation of α-syn, in addition to the endogenous lysosomal degradation machinery. By promoting proteasomal degradation of α-syn, we improved clearance of α-syn in primary culture and mouse models of synucleinopathy. These findings indicate that our sdAb-based protein degrader is a promising therapeutic candidate for synucleinopathies. Considering that only a small percentage of antibodies enter the brain, more potent sdAbs with greater brain entry than whole antibodies could enhance clinical benefits of antibody-based therapies.

12.
Sci Immunol ; 9(93): eade6256, 2024 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-38457513

RESUMO

Programmed cell death-1 (PD-1) is a potent immune checkpoint receptor on T lymphocytes. Upon engagement by its ligands, PD-L1 or PD-L2, PD-1 inhibits T cell activation and can promote immune tolerance. Antagonism of PD-1 signaling has proven effective in cancer immunotherapy, and conversely, agonists of the receptor may have a role in treating autoimmune disease. Some immune receptors function as dimers, but PD-1 has been considered monomeric. Here, we show that PD-1 and its ligands form dimers as a consequence of transmembrane domain interactions and that propensity for dimerization correlates with the ability of PD-1 to inhibit immune responses, antitumor immunity, cytotoxic T cell function, and autoimmune tissue destruction. These observations contribute to our understanding of the PD-1 axis and how it can potentially be manipulated for improved treatment of cancer and autoimmune diseases.


Assuntos
Doenças Autoimunes , Neoplasias , Humanos , Receptor de Morte Celular Programada 1 , Tolerância Imunológica , Ativação Linfocitária , Domínios Proteicos
13.
Nat Commun ; 15(1): 4301, 2024 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-38773089

RESUMO

The vaccine elicitation of HIV tier-2-neutralization antibodies has been a challenge. Here, we report the isolation and characterization of a CD4-binding site (CD4bs) specific monoclonal antibody, HmAb64, from a human volunteer immunized with a polyvalent DNA prime-protein boost HIV vaccine. HmAb64 is derived from heavy chain variable germline gene IGHV1-18 and light chain germline gene IGKV1-39. It has a third heavy chain complementarity-determining region (CDR H3) of 15 amino acids. On a cross-clade panel of 208 HIV-1 pseudo-virus strains, HmAb64 neutralized 20 (10%), including tier-2 strains from clades B, BC, C, and G. The cryo-EM structure of the antigen-binding fragment of HmAb64 in complex with a CNE40 SOSIP trimer revealed details of its recognition; HmAb64 uses both heavy and light CDR3s to recognize the CD4-binding loop, a critical component of the CD4bs. This study demonstrates that a gp120-based vaccine can elicit antibodies capable of tier 2-HIV neutralization.


Assuntos
Vacinas contra a AIDS , Anticorpos Neutralizantes , Antígenos CD4 , Anticorpos Anti-HIV , HIV-1 , Humanos , Vacinas contra a AIDS/imunologia , HIV-1/imunologia , Anticorpos Anti-HIV/imunologia , Anticorpos Neutralizantes/imunologia , Antígenos CD4/imunologia , Antígenos CD4/metabolismo , Vacinas de DNA/imunologia , Anticorpos Monoclonais/imunologia , Infecções por HIV/prevenção & controle , Infecções por HIV/imunologia , Infecções por HIV/virologia , Microscopia Crioeletrônica , Proteína gp120 do Envelope de HIV/imunologia , Proteína gp120 do Envelope de HIV/química , Sítios de Ligação , Regiões Determinantes de Complementaridade/imunologia , Regiões Determinantes de Complementaridade/química
14.
Sci Adv ; 9(19): eadf3775, 2023 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-37163602

RESUMO

Intracellular deposition of α-synuclein and tau are hallmarks of synucleinopathies and tauopathies, respectively. Recently, several dye-based imaging probes with selectivity for tau aggregates have been developed, but suitable imaging biomarkers for synucleinopathies are still unavailable. Detection of both of these aggregates early in the disease process may allow for prophylactic therapies before functional impairments have manifested, highlighting the importance of developing specific imaging probes for these lesions. In contrast to the ß sheet dyes, single-domain antibodies, found in camelids and a few other species, are highly specific, and their small size allows better brain entry and distribution than whole antibodies. Here, we have developed such imaging ligands via phage display libraries derived from llamas immunized with α-synuclein and tau preparations, respectively. These probes allow noninvasive and specific in vivo imaging of α-synuclein versus tau pathology in mice, with the brain signal correlating strongly with lesion burden. These small antibody derivatives have great potential for in vivo diagnosis of these diseases.


Assuntos
Anticorpos de Domínio Único , Sinucleinopatias , Tauopatias , Camundongos , Animais , alfa-Sinucleína , Proteínas tau , Anticorpos , Corantes
15.
Res Sq ; 2023 Oct 20.
Artigo em Inglês | MEDLINE | ID: mdl-37886518

RESUMO

The vaccine elicitation of HIV-neutralizing antibodies with tier-2-neutralization breadth has been a challenge. Here, we report the isolation and characteristics of a CD4-binding site specific monoclonal antibody, HmAb64, from a human volunteer immunized with a polyvalent gp120 DNA prime-protein boost vaccine. HmAb64 derived from heavy chain variable germline gene IGHV1-18, light chain germline gene IGKV1-39, and had a 3rd heavy chain complementarity determining region (CDR H3) of 15 amino acids. On a cross-clade panel of 208 HIV-1 pseudo-virus strains, HmAb64 neutralized 21 (10%), including tier-2 neutralization resistant strains from clades B, BC, C, and G. The cryo-EM structure of the antigen-binding fragment of HmAb64 bound to a conformation between prefusion closed and occluded open forms of envelope trimer, using both heavy and light CDR3s to recognize the CD4-binding loop, a critical component of the CD4-binding site. A gp120 subunit-based vaccine can thus elicit an antibody capable of tier 2-HIV neutralization.

16.
Nat Commun ; 13(1): 903, 2022 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-35173151

RESUMO

V2p and V2i antibodies (Abs) that are specific for epitopes in the V1V2 region of the HIV gp120 envelope (Env) do not effectively neutralize HIV but mediate Fc-dependent anti-viral activities that have been correlated with protection from, or control of HIV, SIV and SHIV infections. Here, we describe a novel molecular toolbox that allows the discrimination of antigenically and functionally distinct polyclonal V2 Ab responses. We identify different patterns of V2 Ab induction by SHIV infection and three separate vaccine regimens that aid in fine-tuning an optimized immunization protocol for inducing V2p and V2i Abs. We observe no, or weak and sporadic V2p and V2i Abs in non-vaccinated SHIV-infected NHPs, but strong V2p and/or V2i Ab responses after immunization with a V2-targeting vaccine protocol. The V2-focused vaccination is superior to both natural infection and to immunization with whole Env constructs for inducing functional V2p- and V2i-specific responses. Strikingly, levels of V2-directed Abs correlate inversely with Abs specific for peptides of V3 and C5. These data demonstrate that a V1V2-targeting vaccine has advantages over the imprecise targeting of SIV/SHIV infections and of whole Env-based immunization regimens for inducing a more focused functional V2p- and V2i-specific Ab response.


Assuntos
Vacinas contra a AIDS/imunologia , Anticorpos Anti-HIV/sangue , Proteína gp120 do Envelope de HIV/imunologia , HIV-1/imunologia , Vírus da Imunodeficiência Símia/imunologia , Animais , Anticorpos Monoclonais/imunologia , Epitopos/imunologia , Feminino , Macaca mulatta , Masculino , Síndrome de Imunodeficiência Adquirida dos Símios/imunologia , Síndrome de Imunodeficiência Adquirida dos Símios/prevenção & controle , Vacinação
17.
EBioMedicine ; 84: 104249, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-36099813

RESUMO

BACKGROUND: Eleven tau immunoglobulin G (IgG) antibodies have entered clinical trials to treat tauopathies, including Alzheimer's disease, but it is unclear which IgG subclass/subtype has the ideal efficacy and safety profile. Only two subtypes, with or without effector function, have been examined in the clinic and not for the same tau antibody. The few preclinical studies on this topic have only compared two subtypes of one antibody each and have yielded conflicting results. METHODS: We selected two single domain antibodies (sdAbs) derived from a llama immunized with tau proteins and utilized them to generate an array of Fc-(sdAb)2 subclasses containing identical tau binding domains but differing Fc region. Unmodified sdAbs and their IgG subclasses were tested for efficacy in primary cultures and in vivo microdialysis using JNPL3 tauopathy mice. FINDINGS: Unmodified sdAbs were non-toxic, blocked tau toxicity and promoted tau clearance. However, the efficacy/safety profile of their Fc-(sdAb)2 subclasses varied greatly within and between sdAbs. For one of them, all its subtypes were non-toxic, only those with effector function cleared tau, and were more effective in vivo than unmodified sdAb. For the other sdAb, all its subtypes were toxic in tauopathy cultures but not in wild-type cells, suggesting that bivalent binding of its tau epitope stabilizes a toxic conformation of tau, with major implications for tau pathogenesis. Likewise, its subclasses were less effective than the unmodified sdAb in clearing tau in vivo. INTERPRETATION: These findings indicate that tau antibodies with effector function are safe and better at clearing pathological tau than effectorless antibodies, Furthermore, tau antibodies can provide a valuable insight into tau pathogenesis, and some may aggravate it. FUNDING: Funding for these studies was provided by the National Institute of Health (R01 AG032611, R01 NS077239, RF1 NS120488, R21 AG 069475, R21 AG 058282, T32AG052909), and the NYU Alzheimer's Disease Center Pilot Grant Program (via P30 AG008051).


Assuntos
Doença de Alzheimer , Anticorpos de Domínio Único , Tauopatias , Doença de Alzheimer/metabolismo , Animais , Epitopos , Imunoglobulina G , Camundongos , Tauopatias/metabolismo , Proteínas tau/metabolismo
18.
Blood ; 113(24): 6051-60, 2009 Jun 11.
Artigo em Inglês | MEDLINE | ID: mdl-19218546

RESUMO

Anti-platelet integrin GPIIIa49-66 antibody (Ab) induces complement-independent platelet oxidative fragmentation and death by generation of platelet peroxide following NADPH oxidase activation. A C-terminal 385-amino acid fragment of ADAMTS-18 (a disintegrin metalloproteinase with thrombospondin motifs produced in endothelial cells) induces oxidative platelet fragmentation in an identical kinetic fashion as anti-GPIIIa49-66 Ab. Endothelial cell ADAMTS-18 secretion is enhanced by thrombin and activated by thrombin cleavage to fragment platelets. Platelet aggregates produced ex vivo with ADP or collagen and fibrinogen are destroyed by the C-terminal ADAMTS-18 fragment. Anti-ADAMTS-18 Ab shortens the tail vein bleeding time. The C-terminal fragment protects against FeCI3-induced carotid artery thrombosis as well as cerebral infarction in a postischemic stroke model. Thus, a new mechanism is proposed for platelet thrombus clearance, via platelet oxidative fragmentation induced by thrombin cleavage of ADAMTS-18.


Assuntos
Proteínas ADAM/farmacologia , Plaquetas/metabolismo , Estenose das Carótidas/prevenção & controle , Fragmentos de Peptídeos/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Acidente Vascular Cerebral/prevenção & controle , Proteínas ADAM/metabolismo , Proteínas ADAMTS , Animais , Tempo de Sangramento , Plaquetas/patologia , Estenose das Carótidas/metabolismo , Estenose das Carótidas/patologia , AMP Cíclico/farmacologia , Endotélio Vascular/metabolismo , Humanos , Técnicas Imunoenzimáticas , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Biblioteca de Peptídeos , Complexo Glicoproteico GPIIb-IIIa de Plaquetas/farmacologia , Reação em Cadeia da Polimerase , Acidente Vascular Cerebral/metabolismo , Acidente Vascular Cerebral/patologia , Trombina/farmacologia , Veias Umbilicais/citologia
19.
J Cell Mol Med ; 14(12): 2780-9, 2010 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19840198

RESUMO

Hormones and their receptors play an important role in the development and progression of breast carcinoma. Although the primary focus has been on oestrogen and oestrogen receptor (ER), androgen, androgen receptor (AR) and its coactivator(s) have been implicated in tumorigenesis of breast carcinoma and warrant further investigation. AR coactivator p44/Mep50 is identified as a subunit of methylosome complex and lately characterized as an AR coactivator that enhances AR mediated transcription activity in a ligand dependent manner. In prostate cancer, p44 is expressed in the nucleus of benign epithelia and translocated into the cytoplasm in cancer cells. Furthermore, nuclear expression of p44 inhibits prostate cancer growth. In this report, we examined the expression and function of p44 in breast cancer. In addition to being an AR coactivator, p44 also functions as an ER coactivator. In contrast to findings in prostate cancer, the expression of p44 shows strong cytoplasmic expression in morphologically normal terminal ductal lobular units, while nuclear p44 is observed in both ductal carcinoma in situ and invasive carcinoma. Further, overexpression of nuclear-localized p44 stimulates proliferation and invasion in MCF7 breast cancer cells in the presence of oestrogen and the process is ERα dependent. These findings strongly suggest that p44 plays a role in mediating the effects of hormones during tumorigenesis in breast.


Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Carcinoma Ductal de Mama/metabolismo , Carcinoma Ductal de Mama/patologia , Fatores de Transcrição/metabolismo , Neoplasias da Mama/genética , Carcinoma Ductal de Mama/genética , Linhagem Celular Tumoral , Proliferação de Células , Estrogênios/metabolismo , Feminino , Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Glândulas Mamárias Humanas/metabolismo , Glândulas Mamárias Humanas/patologia , Invasividade Neoplásica , Receptores de Estrogênio/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Transdução de Sinais , Fatores de Transcrição/genética
20.
Cell Host Microbe ; 27(6): 963-975.e5, 2020 06 10.
Artigo em Inglês | MEDLINE | ID: mdl-32315598

RESUMO

The HIV-1 envelope (Env) undergoes conformational changes during infection. Broadly neutralizing antibodies (bNAbs) are typically isolated by using soluble Env trimers, which do not capture all Env states. To address these limitations, we devised a vesicular stomatitis virus (VSV)-based probe to display membrane-embedded Env trimers and isolated five bNAbs from two chronically infected donors, M4008 and M1214. Donor B cell receptor (BCR) repertoires identified two bNAb lineages, M4008_N1 and M1214_N1, that class-switched to immunoglobulin G (IgG) and IgA. Variants of these bNAbs reconstituted as IgA demonstrated broadly neutralizing activity, and the IgA fraction of M1214 plasma conferred neutralization. M4008_N1 epitope mapping revealed a glycan-independent V3 epitope conferring tier 2 virus neutralization. A 4.86-Å-resolution cryogenic electron microscopy (cryo-EM) structure of M1214_N1 complexed with CH505 SOSIP revealed another elongated epitope, the V2V5 corridor, extending from V2 to V5. Overall, the VSVENV probe identified bNAb lineages with neutralizing IgG and IgA members targeting distinct sites of HIV-1 Env vulnerability.


Assuntos
Anticorpos Amplamente Neutralizantes/imunologia , Anticorpos Anti-HIV/imunologia , Imunoglobulina A/imunologia , Imunoglobulina G/imunologia , Envelope Viral/imunologia , Produtos do Gene env do Vírus da Imunodeficiência Humana/imunologia , Vacinas contra a AIDS , Anticorpos Neutralizantes/imunologia , Microscopia Crioeletrônica , Mapeamento de Epitopos , Epitopos/imunologia , Feminino , Células HEK293 , Anticorpos Anti-HIV/química , Anticorpos Anti-HIV/genética , Anticorpos Anti-HIV/metabolismo , Infecções por HIV/virologia , HIV-1/imunologia , Células HeLa , Humanos , Modelos Moleculares , Conformação Proteica , Alinhamento de Sequência , Vesiculovirus , Produtos do Gene env do Vírus da Imunodeficiência Humana/química
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