Single-Domain Antibody-Based Protein Degrader for Synucleinopathies.

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 ligand 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 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.

Transmembrane domain-driven PD-1 dimers mediate T cell inhibition.

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.

Human CD4-Binding Site Antibody Elicited by Polyvalent DNA Prime-Protein Boost Vaccine Neutralizes Cross-Clade Tier-2-HIV Strains.

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.

Single-domain antibody-based noninvasive in vivo imaging of α-synuclein or tau pathology.

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.

An anatomical study of the tibial nerve branches innervating the posterior tibial artery.

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.

Single domain antibodies targeting pathological tau protein: Influence of four IgG subclasses on efficacy and toxicity.

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).

Differential V2-directed antibody responses in non-human primates infected with SHIVs or immunized with diverse HIV vaccines.

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.

VSV-Displayed HIV-1 Envelope Identifies Broadly Neutralizing Antibodies Class-Switched to IgG and IgA.

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.

The structural features that distinguish PD-L2 from PD-L1 emerged in placental mammals.

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.

Author Correction: The wide utility of rabbits as models of human diseases.

This article was originally published under a CC BY-NC-SA License, but has now been made available under a CC BY 4.0 License.

Select gp120 V2 domain specific antibodies derived from HIV and SIV infection and vaccination inhibit gp120 binding to α4ß7.

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.

Structural Comparison of Human Anti-HIV-1 gp120 V3 Monoclonal Antibodies of the Same Gene Usage Induced by Vaccination and Chronic Infection.

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.

The wide utility of rabbits as models of human diseases.

Studies using the European rabbit Oryctolagus cuniculus contributed to elucidating numerous fundamental aspects of antibody structure and diversification mechanisms and continue to be valuable for the development and testing of therapeutic humanized polyclonal and monoclonal antibodies. Additionally, during the last two decades, the use of the European rabbit as an animal model has been increasingly extended to many human diseases. This review documents the continuing wide utility of the rabbit as a reliable disease model for development of therapeutics and vaccines and studies of the cellular and molecular mechanisms underlying many human diseases. Examples include syphilis, tuberculosis, HIV-AIDS, acute hepatic failure and diseases caused by noroviruses, ocular herpes, and papillomaviruses. The use of rabbits for vaccine development studies, which began with Louis Pasteur's rabies vaccine in 1881, continues today with targets that include the potentially blinding HSV-1 virus infection and HIV-AIDS. Additionally, two highly fatal viral diseases, rabbit hemorrhagic disease and myxomatosis, affect the European rabbit and provide unique models to understand co-evolution between a vertebrate host and viral pathogens.

Increased Epitope Complexity Correlated with Antibody Affinity Maturation and a Novel Binding Mode Revealed by Structures of Rabbit Antibodies against the Third Variable Loop (V3) of HIV-1 gp120.

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.

Contrasting antibody responses to intrasubtype superinfection with CRF02_AG.

HIV superinfection describes the sequential infection of an individual with two or more unrelated HIV strains. Intersubtype superinfection has been shown to cause a broader and more potent heterologous neutralizing antibody response when compared to singly infected controls, yet the effects of intrasubtype superinfection remain controversial. Longitudinal samples were analyzed phylogenetically for pol and env regions using Next-Generation Sequencing and envelope cloning. The impact of CRF02_AG intrasubtype superinfection was assessed for heterologous neutralization and antibody binding responses. We compared two cases of CRF02_AG intrasubtype superinfection that revealed complete replacement of the initial virus by superinfecting CRF02_AG variants with signs of recombination. NYU6564, who became superinfected at an early time point, exhibited greater changes in antibody binding profiles and generated a more potent neutralizing antibody response post-superinfection compared to NYU6501. In contrast, superinfection occurred at a later time point in NYU6501 with strains harboring significantly longer V1V2 regions with no observable changes in neutralization patterns. Here we show that CRF02_AG intrasubtype superinfection can induce a cross-subtype neutralizing antibody response, and our data suggest timing and/or superinfecting viral envelope characteristics as contributing factors. These results highlight differential outcomes in intrasubtype superinfection and provide the first insight into cases with CRF02_AG, the fourth most prevalent HIV-1 strain worldwide.

Rationally Targeted Mutations at the V1V2 Domain of the HIV-1 Envelope to Augment Virus Neutralization by Anti-V1V2 Monoclonal Antibodies.

HIV-1 envelope glycoproteins (Env) are the only viral antigens present on the virus surface and serve as the key targets for virus-neutralizing antibodies. However, HIV-1 deploys multiple strategies to shield the vulnerable sites on its Env from neutralizing antibodies. The V1V2 domain located at the apex of the HIV-1 Env spike is known to encompass highly variable loops, but V1V2 also contains immunogenic conserved elements recognized by cross-reactive antibodies. This study evaluates human monoclonal antibodies (mAbs) against V2 epitopes which overlap with the conserved integrin α4ß7-binding LDV/I motif, designated as the V2i (integrin) epitopes. We postulate that the V2i Abs have weak or no neutralizing activities because the V2i epitopes are often occluded from antibody recognition. To gain insights into the mechanisms of the V2i occlusion, we evaluated three elements at the distal end of the V1V2 domain shown in the structure of V2i epitope complexed with mAb 830A to be important for antibody recognition of the V2i epitope. Amino-acid substitutions at position 179 that restore the LDV/I motif had minimal effects on virus sensitivity to neutralization by most V2i mAbs. However, a charge change at position 153 in the V1 region significantly increased sensitivity of subtype C virus ZM109 to most V2i mAbs. Separately, a disulfide bond introduced to stabilize the hypervariable region of V2 loop also enhanced virus neutralization by some V2i mAbs, but the effects varied depending on the virus. These data demonstrate that multiple elements within the V1V2 domain act independently and in a virus-dependent fashion to govern the antibody recognition and accessibility of V2i epitopes, suggesting the need for multi-pronged strategies to counter the escape and the shielding mechanisms obstructing the V2i Abs from neutralizing HIV-1.

Structural analysis of a novel rabbit monoclonal antibody R53 targeting an epitope in HIV-1 gp120 C4 region critical for receptor and co-receptor binding.

The fourth conserved region (C4) in the HIV-1 envelope glycoprotein (Env) gp120 is a structural element that is important for its function, as it binds to both the receptor CD4 and the co-receptor CCR5/CXCR4. It has long been known that this region is highly immunogenic and that it harbors B-cell as well as T-cell epitopes. It is the target of a number of antibodies in animal studies, which are called CD4-blockers. However, the mechanism by which the virus shields itself from such antibody responses is not known. Here, we determined the crystal structure of R53 in complex with its epitope peptide using a novel anti-C4 rabbit monoclonal antibody R53. Our data show that although the epitope of R53 covers a highly conserved sequence (433)AMYAPPI(439), it is not available in the gp120 trimer and in the CD4-bound conformation. Our results suggest a masking mechanism to explain how HIV-1 protects this critical region from the human immune system.

Functional and Structural Characterization of Human V3-Specific Monoclonal Antibody 2424 with Neutralizing Activity against HIV-1 JRFL.

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.

The V1V2 Region of HIV-1 gp120 Forms a Five-Stranded Beta Barrel.

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.

Imbalanced expression of Tif1γ inhibits pancreatic ductal epithelial cell growth.

Transcriptional intermediary factor 1 gamma (Tif1γ) (Ectodermin/PTC7/RFG7/TRIM33) is a transcriptional cofactor with an important role in the regulation of the TGFß pathway. It has been suggested that it competes with Smad2/Smad3 for binding to Smad4, or alternatively that it may target Smad4 for degradation, although its role in carcinogenesis is unclear. In this study, we showed that Tif1γ interacts with Smad1/Smad4 complex in vivo, using both yeast two-hybrid and coimmunoprecipitation assays. We demonstrated that Tif1γ inhibits transcriptional activity of the Smad1/Smad4 complex through its PHD domain or bromo-domainin pancreatic cells by luciferase assay. Additionally, there is a dynamic inverse relationship between the levels of Tif1γ and Smad4 in benign and malignant pancreatic cell lines. Overexpression of Tif1γ resulted in decreased level of Smad4. Both overexpression and knockdown of Tif1γ resulted in growth inhibition in both benign and cancerous pancreatic cell lines, attributable to a G2-phase cell cycle arrest, but only knockdown of Tif1γ reduces tumor cell invasiveness in vitro. Our study demonstrated that imbalanced expression of Tif1γ results in inhibition of pancreatic ductal epithelial cell growth. In addition, knockdown of Tif1γ may inhibit tumor invasion. These data suggest that Tif1γ might serve as a potential therapeutic target for pancreatic cancer.