Stable,uncleaved HIV-1 envelope glycoprotein gp140 forms a tightly folded trimer with a native-like structure

The HIV-1 envelope spike [trimeric (gp160)₃, cleaved to (gp120/gp41)₃] is the mediator of viral entry and the principal target of humoral immune response to the virus. Production of a recombinant preparation that represents the functional spike poses a challenge for vaccine development, because the (gp120/gp41)₃ complex is prone to dissociation. We have reported previously that stable HIV-1 gp140 trimers, the uncleaved ectodomains of (gp160)₃, have nearly all of the antigenic properties expected for native viral spikes. Because of recent claims that uncleaved gp140 proteins may adopt a nonnative structure with three gp120 moieties “dangling” from a trimeric gp41 ectodomain in its postfusion conformation, we have inserted a long, flexible linker between gp120 and gp41 in our stable gp140 trimers to assess their stability and to analyze their conformation in solution. The modified trimer has biochemical and antigenic properties virtually identical to those of its unmodified counterpart. Both forms bind a single CD4 per trimer, suggesting that the trimeric conformation occludes two of the three CD4 sites even when a flexible linker has relieved the covalent constraint between gp120 and gp41. In contrast, an artificial trimer containing three gp120s flexibly tethered to a trimerization tag binds three CD4s and has antigenicity nearly identical to that of monomeric gp120. Moreover, the gp41 part of both modified and unmodified gp140 trimers has a structure very different from that of postfusion gp41. These results show that uncleaved gp140 trimers from suitable isolates have compact, native-like structures and support their use as candidate vaccine immunogens.The HIV-1 envelope glycoprotein mediates initial steps of virus infection by engaging cellular receptors and facilitating fusion of viral and target-cell membranes (1). Biosynthesis of the virus-encoded envelope glycoprotein yields a precursor, gp160, which following trimerization undergoes cleavage by a furin-like protease into two noncovalently associated fragments: the receptor-binding fragment, gp120, and the fusion fragment, gp41 (1). Three copies each of gp120 and gp41 form the mature envelope spikes (gp120/gp41)₃, the major viral surface antigen. Binding, through a site on gp120, to the host primary receptor, CD4, and then, through a second site, to a coreceptor (e.g., CCR5 or CXCR4) triggers large conformational changes that include reduced interaction between gp120 and gp41 (probably leading to dissociation of the former) and a cascade of ensuing gp41 refolding events (2, 3). Within the precursor gp160, gp41, with its C-terminal transmembrane (TM) segment anchored in the viral membrane, folds into a prefusion conformation. Cleavage of gp160 makes this prefusion conformation metastable with respect to a rearranged, postfusion conformation. Thus, the loss of constraint on gp41 that accompanies coreceptor binding to gp120 triggers a transition in gp41 to an extended, membrane-bridging conformation (sometimes called a “prehairpin” conformation) (4) with a hydrophobic “fusion peptide” at its N terminus inserted into the target-cell membrane and the TM segment in the viral membrane. This relatively long-lived, transient conformation is the target of fusion inhibitors, such as enfuvirtide (5), and of several broadly neutralizing antibodies (bnAbs) (6–8). Folding back of each chain into an α-helical hairpin creates a stable, six-helix bundle—the “postfusion conformation”—placing the fusion peptide and TM segment at the same end of the molecule. This irreversible refolding of gp41 brings the two membranes together, leading to bilayer fusion and viral entry. Thus, during the fusion process, there are at least three distinct conformational states of the envelope protein: the prefusion conformation of (gp120/gp41)₃, the extended intermediate of gp41, and the postfusion conformation of gp41 (with release of free gp120). Moreover, conformational changes of the prefusion form occur upon CD4 and perhaps also coreceptor binding.The envelope glycoprotein is also the primary target of humoral responses in HIV-1–infected individuals. Studies of human monoclonal antibodies (mAbs) have identified a subset members that neutralize a wide range of HIV isolates (see Table S1 for a partial catalog and original references) (9, 10). These bnAbs are of particular interest, because they may guide a search for immunogens to elicit them in vaccinees. Epitopes on gp120 recognized by human bnAbs include the CD4-binding site, a trimer-specific epitope in the relatively invariant parts of the V2 and V3 loops, and a site near the base of the V3 loop involving an N-linked glycan at position 332. A glycan-dependent epitope spans both gp120 and gp41. The membrane-proximal external region (MPER) of gp41 binds a set of bnAbs that were among the earliest broad neutralizers discovered. The domain-swapped, dimeric antibody, 2G12, recognizes only glycans at defined positions, and its reactivity therefore depends on specific glycosylation patterns but not on many other aspects of the gp120 amino acid sequence.Interesting groups of nonneutralizing antibodies, or with a very narrow range of isolates neutralized, include those that bind the so-called CD4-induced (CD4i) epitope, which overlaps the coreceptor site, on the bridging sheet of gp120, when the epitope becomes exposed by the conformational changes that accompany CD4 binding (Table S1). Nonneutralizing antibodies that interact with gp41 fall into two “clusters”: those in cluster I, which recognize the “immunodominant” C-C loop of gp41, and those in cluster II, which bind strongly with a segment just preceding the MPER in the gp41 polypeptide chain, but only when gp41 is in the postfusion conformation. Most of the antibodies listed in Table S1 recognize conformation-dependent epitopes and thus are excellent molecular probes for defining the conformational state of the envelope trimer.A form of gp140, stabilized by a disulfide crosslink between gp120 and gp41 (perhaps related to the disulfide between surface and TM subunits in many oncoretroviruses) was introduced over a decade ago (11) and subsequently modified by introducing an Ile-to-Pro mutation in gp41, to retard formation of the six-helix bundle (12). The product, known as SOSIP (SOS to designate the double cysteine mutations and IP to denote the isoleucine to proline change), is possible only with certain isolates, and the most widely studied has been BG505 SOSIP.664 (13–16). This modified gp140 trimer, which can be cleaved with furin without compromising stability, has greatly facilitated structural analysis, probably by eliminating large-scale conformational fluctuations (13, 14). In this variant, the MPER has been deleted, and the furin site has been replaced with a string of six arginines. Its structure, determined by both electron cryomicroscopy and X-ray crystallography, shows that (as expected from other fusion proteins) the conformation of gp41 in the prefusion state is distinct from the postfusion six-helix bundle (3, 17). The SOSIP.664 structure, which is an extremely important contribution to our understanding of envelope trimer molecular architecture, has some puzzling features. Docking CD4 onto the model suggests that it can bind all three gp120 sites with no clashes. This observation is at odds with a large body of evidence, including a recent biophysical study of the same trimer, showing that CD4 binding induces a substantial structural rearrangement (18–22). Moreover, uncleaved BG505 gp140 without the SOSIP modifications is unstable and heterogeneous. Can one really conclude, as suggested (14), that all HIV-1 uncleaved gp140 trimers are misfolded products resembling three gp120 moieties flexibly linked to a trimer gp41 in the postfusion, six-helix bundle conformation? The answer to this question is of considerable consequence, as it relates directly to design and production of candidate HIV-1 vaccine immunogens.In the work reported here, we have inserted a flexible, 20-residue linker between gp120 and gp41 in the context of a previously characterized, stable gp140. The linker releases the tight covalent constraint between gp120 and gp41, and it should therefore mimic to some extent the furin cleavage (which is effectively a linker of infinite length). If an uncleaved gp140 trimer truly resembles “three balls on a string,” as asserted (14), addition of a linker that can extend as much as 70 Å should exaggerate this property, causing the trimer with the inserted linker to have antigenic properties resembling three monomeric gp120s and a very large hydrodynamic radius. We find, to the contrary, that gp140 with the flexible linker, 20-residue insert (gp140–FL20) has antigenic properties essentially identical to those of the unmodified trimer and very different from those of free gp120 or of a construct with three gp120s held together by a heterologous trimerization tag. Moreover, the linker barely affects the hydrodynamic radius of the trimer, which is even slightly smaller than that of SOSIP. These results show that the stable, uncleaved gp140 trimers we have characterized previously are compact and native-like, and they support our suggestion that they are promising, envelope-based immunogens for clinical testing in vaccine development.     

CD4 mimetics sensitize HIV-1-infected cells to ADCC

HIV-1-infected cells presenting envelope glycoproteins (Env) in the CD4-bound conformation on their surface are preferentially targeted by antibody-dependent cell-mediated cytotoxicity (ADCC). HIV-1 has evolved a sophisticated mechanism to avoid exposure of ADCC-mediating Env epitopes by down-regulating CD4 and by limiting the overall amount of Env at the cell surface. Here we report that small-molecule CD4-mimetic compounds induce the CD4-bound conformation of Env, and thereby sensitize cells infected with primary HIV-1 isolates to ADCC mediated by antibodies present in sera, cervicovaginal lavages, and breast milk from HIV-1-infected individuals. Importantly, we identified one CD4 mimetic with the capacity to sensitize endogenously infected ex vivo-amplified primary CD4 T cells to ADCC killing mediated by autologous sera and effector cells. Thus, CD4 mimetics hold the promise of therapeutic utility in preventing and controlling HIV-1 infection.Worldwide, it is estimated that more than 35 million people are living with HIV. In 2013 alone, around 2.1 million people became newly infected with HIV, and 1.5 million people died from AIDS (1). Measures to prevent HIV-1 transmission are desperately needed. Prevention of HIV-1 transmission and progression likely requires approaches that can specifically target and eliminate HIV-1-infected cells. Interestingly, there is increasing evidence supporting a role of antibody (Ab)-dependent cell-mediated cytotoxicity (ADCC) in controlling HIV-1 transmission and disease progression (2–8). Analysis of the correlates of protection in the RV144 vaccine trial suggested that increased ADCC activity was linked with decreased HIV-1 acquisition (9), and Abs with potent ADCC activity were isolated from some RV144 vaccinees (10). Recent studies reported that the viral accessory proteins Nef and Vpu protect HIV-1-infected cells from anti-HIV-1 envelope (Env)-mediated ADCC responses (11–14). Importantly, we and others reported that Env in the CD4-bound conformation was preferentially targeted by ADCC-mediating Abs and sera from HIV-1-infected individuals (11, 12, 15, 16), which represent a significant proportion of anti-Env Abs elicited during natural HIV infection (11, 17). However, the vast majority of circulating HIV-1 strains worldwide express functional Nef and Vpu proteins, which limit the exposure of CD4-induced (CD4i) Env epitopes at the surface of infected cells, likely preventing ADCC responses.Theoretically, agents promoting the CD4-bound Env conformation should expose CD4i epitopes that are readily recognized by ADCC-mediating Abs and sera from infected individuals (11, 12, 15, 16, 18), resulting in the sensitization of HIV-1-infected cells to ADCC. Importantly, modulating Env conformation at the surface of HIV-1-infected cells has become feasible as a result of the availability of small CD4-mimetic compounds (CD4mc). The prototypes of such compounds, NBD-556 and NBD-557, were discovered in a screen for inhibitors of gp120-CD4 interaction (19). These small-molecule ∼337-Da compounds and recent derivatives (DMJ-I-228, JP-III-48) bind in the Phe-43 cavity (20–22), a highly conserved ∼150-ų pocket in the gp120 glycoprotein located at the interface of the inner domain, outer domain, bridging sheet, and CD4 receptor (23). CD4mc block gp120-CD4 interaction and induce thermodynamic changes in gp120 similar to those observed during CD4 or soluble CD4 (sCD4) binding (24). Accordingly, these small molecules, as well as sCD4, can promote the transition of Env to the CD4-bound conformation, thus sensitizing HIV-1 particles to neutralization by otherwise nonneutralizing CD4i Abs (17, 25). Additional strategies using scaffolded miniproteins targeting critical gp120 elements required for CD4 interaction allowed the identification of CD4 mimetics with nanomolar affinity for gp120 (26). One of these variants, M48U1, displayed remarkably potent neutralization of three HIV-1 isolates (27). Its crystal structure in complex with HIV-1 gp120 was recently solved, showing that M48U1 engages the Phe-43 cavity in a manner similar to that of CD4 (28); thus, M48U1 might induce gp120 to adopt the CD4-bound conformation and expose CD4i epitopes. Previous studies exploring the antiviral properties of CD4mc were performed on viral particles (17, 25, 27). However, whether these compounds are able to engage the large amounts of Env present at the surface of infected cells and modulate Env conformation in a way that allows exposure of ADCC-mediating epitopes is currently not known. In this study, we show that CD4mc strongly sensitize HIV-1-infected primary CD4 T cells to ADCC mediated by sera, cervicovaginal fluids, and breast milk from HIV-1-infected individuals, as well as help eliminate infected, ex vivo-expanded primary CD4 T cells from HIV-1-infected individuals. Therefore, CD4mc possess three valuable complementary antiviral properties: direct inactivation of viral particles, sensitization of viral particles to neutralization by otherwise nonneutralizing Abs, and sensitization of HIV-1-infected cells to ADCC-mediated killing.     

Maturation Pathways of Cross-Reactive HIV-1 Neutralizing Antibodies

Several human monoclonal antibodies (hmAbs) and antibody fragments, including the best characterized in terms of structure-function b12 and Fab X5, exhibit relatively potent and broad HIV-1 neutralizing activity. However, the elicitation of b12 or b12-like antibodies in vivo by vaccine immunogens based on the HIV-1 envelope glycoprotein (Env) has not been successful. B12 is highly divergent from the closest corresponding germline antibody while X5 is less divergent. We have hypothesized that the relatively high degree of specific somatic hypermutations may preclude binding of the HIV-1 envelope glycoprotein (Env) to closest germline antibodies, and that identifying antibodies that are intermediates in the pathways to maturation could help design novel vaccine immunogens to guide the immune system for their enhanced elicitation. In support of this hypothesis we have previously found that a germline-like b12 (monovalent and bivalent scFv as an Fc fusion protein or IgG) lacks measurable binding to an Env as measured by ELISA with a sensitivity in the μM range [1]; here we present evidence confirming and expanding these findings for a panel of Envs. In contrast, a germline-like scFv X5 bound Env with high (nM) affinity. To begin to explore the maturation pathways of these antibodies we identified several possible b12 intermediate antibodies and tested their neutralizing activity. These intermediate antibodies neutralized only some HIV-1 isolates and with relatively weak potency. In contrast, germline-like scFv X5 neutralized a subset of the tested HIV-1 isolates with comparable efficiencies to that of the mature X5. These results could help explain the relatively high immunogenicity of the coreceptor binding site on gp120 and the abundance of CD4-induced (CD4i) antibodies in HIV-1-infected patients (X5 is a CD4i antibody) as well as the maturation pathway of X5. They also can help identify antigens that can bind specifically to b12 germline and intermediate antibodies that together with Envs could be used as a conceptually novel type of candidate vaccines. Such candidate vaccines based on two or more immunogens could help guiding the immune system through complex maturation pathways for elicitation of antibodies that are similar or identical to antibodies with known properties.     

唾液HIV抗体的检测

本文报告唾液标本用明胶颗粒凝集试验和快速免疫印迹试验检测4例HIV感染者的HIV抗体,获得与血清一致的结果。结合文献资料分析,唾液可以代替血液作为群体性艾滋病流行病学监测的理想标本。     

HIV-1 induces cardiomyopathy by cardiomyocyte invasion and gp120, tat, and cytokine apoptotic signaling

We examined heart tissues of AIDS patients with or without HIV cardiomyopathy (HIVCM) by immunohistocheistry, in situ polymerase chain reaction, in situ riboprobe hybridization, and the TUNEL technique for apoptosis. In HIVCM tissues, only inflammatory cells, but not endothelial cells or cardiomyocytes, displayed HIV-1 DNA and RNA. However, macrophages, lymphocytes, and—in a patchy fashion—cardiomyocytes and endothelial cells exhibited virus envelope protein gp 120. Macrophages infiltrated the myocardium in a perivascular fashion and expressed tumor necrosis factor family ligands; adjacent cardiomyocytes suffered apopotosis. in vitro HIV-1 strongly invaded neonatal rat ventricular myocytes (NRVMs) and coronary artery endothelial cells (CAECs) and induced microvilli but did not replicate. HIV-1, gp120, or Tat induced Erk 1/2 phosphorylation, activation of caspase-3, and apoptosis of NRVMs and CAECs; all of these were inhibited by a MAPK/ERK-kinase (MEK) inhibitor U0126. The pathogenesis of HIVCM involves HIV-1 replication in inflammatory cells and induction of cardiomyocyte apoptosis by (1) the extrinsic pathway through apoptotic ligands and (2) the intrinsic pathway through direct virus entry and gp120-and Tat-proapoptotic signaling.     

HIV-1B/C重组毒株gp160假病毒的构建及其活性检测

目的以CD4+T细胞DNA为模板,进行gp160扩增,构建艾滋病病毒Ⅰ型(HIV-1)前病毒假病毒,通过中和试验验证其具有感染活性。方法选择高效抗反转录病毒治疗(HAART)成功的病人3例,分离外周血单核细胞(PBMC),纯化CD4+T细胞,提取DNA,以其为模板扩增gp160全长基因,并克隆到pcDNA3.1(+)表达载体上,酶切验证得到阳性克隆。将此阳性克隆和pNL4-3质粒共转染,获得HIV-1假病毒。用免疫兔血清验证假病毒的感染活性。结果成功地获得了1株假病毒。用免疫后的兔血清测定半数抑制浓度(IC50)的滴度约在1∶90~1∶270之间。病毒的加入量与细胞感染率之间存在良好的线性关系,说明该假病毒感染系统可以通过细胞感染率较好地反映出感染性病毒的含量。结论获得了具有感染活性的HIV-1B/C重组型假病毒。     

Structure and function of broadly reactive antibody PG16 reveal an H3 subdomain that mediates potent neutralization of HIV-1

Development of an effective vaccine against HIV-1 will likely require elicitation of broad and potent neutralizing antibodies against the trimeric surface envelope glycoprotein (Env). Monoclonal antibodies (mAbs) PG9 and PG16 neutralize ~80% of HIV-1 isolates across all clades with extraordinary potency and target novel epitopes preferentially expressed on Env trimers. As these neutralization properties are ideal for a vaccine-elicited antibody response to HIV-1, their structural basis was investigated. The crystal structure of the antigen-binding fragment (Fab) of PG16 at 2.5 Å resolution revealed its unusually long, 28-residue, complementarity determining region (CDR) H3 forms a unique, stable subdomain that towers above the antibody surface. A 7-residue “specificity loop” on the “hammerhead” subdomain was identified that, when transplanted from PG16 to PG9 and vice versa, accounted for differences in the fine specificity and neutralization of these two mAbs. The PG16 electron density maps also revealed that a CDR H3 tyrosine was sulfated, which was confirmed for both PG9 (doubly) and PG16 (singly) by mass spectral analysis. We further showed that tyrosine sulfation plays a role in binding and neutralization. An N-linked glycan modification is observed in the variable light chain, but not required for antigen recognition. Further, the crystal structure of the PG9 light chain at 3.0 Å facilitated homology modeling to support the presence of these unusual features in PG9. Thus, PG9 and PG16 use unique structural features to mediate potent neutralization of HIV-1 that may be of utility in antibody engineering and for high-affinity recognition of a variety of therapeutic targets.     

HIV-1 gp120 induces autophagy in cardiomyocytes via the NMDA receptor

Background

HIV-1 envelope glycoprotein gp120 (gp120) is considered as one of the major virulent proteins responsible for the involvement of the cardiovascular system. Autophagy as a form of self maintenance plays important roles in cell survival and death. HIV-1 gp120 is reported to induce autophagy in a variety of cells. However, the effect of gp120 on autophagy in cardiomyocytes has not been reported. This study aimed to test our hypothesis that gp120 could induce autophagy in cardiomyocytes.

Methods

Rat cardiomyocyte H9c2 cells were treated with gp120 (100 ng/ml) in vitro for 4 h, 1 day and 7 days. The autophagy related proteins were analyzed by Western blot and the autophagosomes were analyzed by confocal microscopy.

Results

The autophagic proteins and autophagosomes were markedly increased in the H9c2 cells after 4 h of gp120 treatment. Furthermore, gp120 induced autophagic proteins and autophagosomes were significantly inhibited by the N-methyl-d-aspartic acid (NMDA) receptor inhibitor MK801, c-Jun N-terminal kinase (JNK) inhibitor SP600125, and the class III phosphoinositide 3-kinase (PI3K) inhibitor 3-methyladenine (3-MA), while there was no change in the cells pretreated with the CXCR4 antagonist AMD3100. In addition, no apparent cell death was observed in the cardiomyocytes treated with gp120 for up to 7 days.

Conclusions

In summary, our research demonstrated for the first time that HIV-1 gp120 could induce autophagy of cardiomyocytes and the NMDA receptor, JNK and class III PI3K were involved in this process. This observation provides a new insight into the mechanisms of in the cardiovascular involvement during HIV-1 infection.     

Production and Characterization of Murine Monoclonal Antibodies to Leishmania Gp63 Antigen

Background : Production of monoclonal antibodies to Leishmania antigens assists the identification and characterization of these organisms. Objective: Production of monoclonal antibodies against epitopes on the gp63. Methods: Two murine monoclonal antibodies to gp63 were produced and characterized. The reactions of both antibodies with soluble leishmanial antigens, purified gp63 and truncated recombinant gp63 molecules were studied by an ELISA assay. These two antibodies reacted with the crude soluble antigens prepared from 4 reference strains of Leishmania, 10 isolates from the patients, purified gp63 and recombinant gp63 molecules. However, no reaction with several non-leishmanial antigens was observed. Reaction of both antibodies with the intact recombinant gp63 and truncated molecules were compared. Results: The results indicated that the two antibodies specifically recognize two different epitopes on the gp63 molecule. Conclusion: Possible applications of such antibodies in searching for immunogenic epitopes are discussed.     

Quaternary Interaction of the HIV-1 Envelope Trimer with CD4 and Neutralizing Antibodies

The entry of HIV-1 into host cells is initiated by the interaction of the viral envelope (Env) spike with the CD4 receptor. During this process, the spike undergoes a series of conformational changes that eventually lead to the exposure of the fusion peptide located at the N-terminus of the transmembrane glycoprotein, gp41. Recent structural and functional studies have provided important insights into the interaction of Env with CD4 at various stages. However, a fine elucidation of the earliest events of CD4 contact and its immediate effect on the Env conformation remains a challenge for investigation. Here, we summarize the discovery of the quaternary nature of the CD4-binding site in the HIV-1 Env and the role of quaternary contact in the functional interaction with the CD4 receptor. We propose two models for this initial contact based on the current knowledge and discuss how a better understanding of the quaternary interaction may lead to improved immunogens and antibodies targeting the CD4-binding site.     

Development of a Monoclonal Antibody Targeting HTLV-1 Envelope gp46 Glycoprotein and Its Application to Near-Infrared Photoimmuno-Antimicrobial Strategy

Human T-cell leukemia virus type 1 (HTLV-1), a retrovirus, causes adult T-cell leukemia-lymphoma, HTLV-1 associated myelopathy/tropical spastic paraparesis, and HTLV-1 uveitis. Currently, no antiretroviral therapies or vaccines are available for HTLV-1 infection. This study aimed to develop an antibody against the HTLV-1 envelope protein (Env) and apply it to a near-infrared photoimmuno-antimicrobial strategy (NIR-PIAS) to eliminate HTLV-1 infected cells. We established mouse monoclonal antibodies (mAbs) against HTLV-1 Env by immunization with a complex of liposome and the recombinant protein. Detailed epitope mapping revealed that one of the mAbs bound to the proline-rich region of gp46 and exhibited no obvious neutralizing activity to inhibit viral infection. Instead, the mAb was rarely internalized intracellularly and remained on the cell surface of HTLV-1-infected cells. The antibody conjugated to the photosensitive dye IRDye700Dx recognized HTLV-1 infected cells and killed them following NIR irradiation. These results suggest that the novel mAb and NIR-PIAS could be developed as a new targeted therapeutic tool against HTLV-1 infected cells.     

Isolation and Characterization of Cross-Reactive Human Monoclonal Antibodies That Potently Neutralize Australian Bat Lyssavirus Variants and Other Phylogroup 1 Lyssaviruses

Australian bat lyssavirus (ABLV) is a rhabdovirus that circulates in four species of pteropid bats (ABLVp) and the yellow-bellied sheath-tailed bat (ABLVs) in mainland Australia. In the three confirmed human cases of ABLV, rabies illness preceded fatality. As with rabies virus (RABV), post-exposure prophylaxis (PEP) for potential ABLV infections consists of wound cleansing, administration of the rabies vaccine and injection of rabies immunoglobulin (RIG) proximal to the wound. Despite the efficacy of PEP, the inaccessibility of human RIG (HRIG) in the developing world and the high immunogenicity of equine RIG (ERIG) has led to consideration of human monoclonal antibodies (hmAbs) as a passive immunization option that offers enhanced safety and specificity. Using a recombinant vesicular stomatitis virus (rVSV) expressing the glycoprotein (G) protein of ABLVs and phage display, we identified two hmAbs, A6 and F11, which completely neutralize ABLVs/ABLVp, and RABV at concentrations ranging from 0.39 and 6.25 µg/mL and 0.19 and 0.39 µg/mL respectively. A6 and F11 recognize overlapping epitopes in the lyssavirus G protein, effectively neutralizing phylogroup 1 lyssaviruses, while having little effect on phylogroup 2 and non-grouped diverse lyssaviruses. These results suggest that A6 and F11 could be effective therapeutic and diagnostic tools for phylogroup 1 lyssavirus infections.     

HIV-1 Envelope Glycoproteins Proteolytic Cleavage Protects Infected Cells from ADCC Mediated by Plasma from Infected Individuals

The HIV-1 envelope glycoprotein (Env) is synthesized in the endoplasmic reticulum as a trimeric gp160 precursor, which requires proteolytic cleavage by a cellular furin protease to mediate virus-cell fusion. Env is conformationally flexible but controls its transition from the unbound “closed” conformation (State 1) to downstream CD4-bound conformations (States 2/3), which are required for fusion. In particular, HIV-1 has evolved several mechanisms that reduce the premature “opening” of Env which exposes highly conserved epitopes recognized by non-neutralizing antibodies (nnAbs) capable of mediating antibody-dependent cellular cytotoxicity (ADCC). Env cleavage decreases its conformational transitions favoring the adoption of the “closed” conformation. Here we altered the gp160 furin cleavage site to impair Env cleavage and to examine its impact on ADCC responses mediated by plasma from HIV-1-infected individuals. We found that infected primary CD4+ T cells expressing uncleaved, but not wildtype, Env are efficiently recognized by nnAbs and become highly susceptible to ADCC responses mediated by plasma from HIV-1-infected individuals. Thus, HIV-1 limits the exposure of uncleaved Env at the surface of HIV-1-infected cells at least in part to escape ADCC responses.     

HIV-1 Envelope Conformation,Allostery, and Dynamics

The HIV-1 envelope glycoprotein (Env) mediates host cell fusion and is the primary target for HIV-1 vaccine design. The Env undergoes a series of functionally important conformational rearrangements upon engagement of its host cell receptor, CD4. As the sole target for broadly neutralizing antibodies, our understanding of these transitions plays a critical role in vaccine immunogen design. Here, we review available experimental data interrogating the HIV-1 Env conformation and detail computational efforts aimed at delineating the series of conformational changes connecting these rearrangements. These studies have provided a structural mapping of prefusion closed, open, and transition intermediate structures, the allosteric elements controlling rearrangements, and state-to-state transition dynamics. The combination of these investigations and innovations in molecular modeling set the stage for advanced studies examining rearrangements at greater spatial and temporal resolution.     

ELISA法检测尿液中HIV-1抗体的研究分析

目的 探讨艾滋病病毒(HIV)感染者晨尿、非晨尿和血液中HIV-1抗体检测结果的一致性，引进尿液HIV-1抗体检测方法。方法 平行采集吸毒人员血液和尿液标本，分别用血液和尿液酶联免疫吸附试验(ELISA)法检测HIV抗体，阳性血清标本送云南省疾病预防控制中心确认。结果 检测483人，血检阳性91人，晨尿检测阳性96人，两种方法一致性98．96％。对应晨尿阳性者采集非晨尿和阴性对照尿液标本各91份(其中血液标本检测HIV抗体阳性者86份)，检出阳性86份，阴性5份，非晨尿标本与血液标本检测结果一致。以血液标本检测结果为准，晨尿标本检测HIV-1抗体的灵敏度100％，特异度98．72％；非晨尿标本检测HIV-1抗体的灵敏度和特异度均为100％。结论 尿液ELISA法检测HIV-1抗体结果可靠，非晨尿可代替晨尿作HIV-1抗体筛查。     

820份蛋白印迹试验HIV-1抗体阳性结果分析

目的 通过研究分析绵阳市1型艾滋病病毒(HIV-1)受试者条带检出情况,提高实验室条带判读能力.方法 对2019年绵阳辖区820份蛋白印迹试验HIV-1抗体检测阳性结果使用描述性分析、卡方检验、Fisher确切概率法和Kappa进行分析.结果 阳性结果中出现全条带的有87.3％(716/820);p51和p66的条带检...     

口腔黏膜渗出液检测HIV1/2型抗体的方法评价

目的 评价口腔黏膜渗出液检测艾滋病病毒Ⅰ/Ⅱ型(HIV-1/2)抗体的敏感性和特异性,及其与血液HIV-1/2抗体检测试剂的一致性.方法 分别取已知HIV感染者及未感染者餐前及餐后10分钟、餐后1小时,饮酒前、饮酒后,牙周疾病患者、健康志愿者的口腔黏膜渗出液标本,检测HIV-1/2抗体.同时平行采集上述人群的血清标本,通过酶联免疫吸附试验(ELISA)检测HIV-1/2抗体.并进一步通过ELISA方法检测标准血清转换盘,同时用此试剂检测该感染者的口腔黏膜渗出液标本中的HIV-1/2抗体.结果 15例已知HIV感染者(餐前、餐后10分钟、餐后1小时,饮酒前、饮酒后)口腔黏膜渗出液标本为阳性,其余口腔黏膜渗出液标本均为阴性;15例已知HIV感染者血清标本均为阳性,1例健康志愿者血清标本呈阳性(后经Western Blot试验确认为阴性),余血清HIV抗体均为阴性.该口腔黏膜渗出液HIV抗体检测试剂的敏感性为100%,特异性为100%,与ELISA检测的一致性为99.85%.血清转换盘实验结果显示,血清标本于感染后的33天检出阳性,口腔黏膜渗出液于感染后的28天检出阳性.结论 利用口腔黏膜渗出液检测HIV-1/2型抗体与现行的血液ELISA检测结果相近.口腔黏膜渗出液标本采集方便而且危险性小,在采血较困难的人群、基层医疗机构、艾滋病自愿咨询检测(VCT)门诊等,可考虑使用口腔黏膜渗出液进行HIV抗体的初筛检测.     

Human Genetic Polymorphisms Affecting HIV-1 Diseases

Human immunodeficiency virus type 1 (HIV-1) infection is generally characterized by a long-term, chronic disease course that gradually progresses to acquired immunodeficiency syndrome (AIDS). However, a small fraction of HIV-1-infected individuals remain both clinically and immunologically healthy for 10 years or more after seroconversion. Conversely, the disease of another significant fraction is characterized by an extremely rapid progression to AIDS within 1 year. There are also individuals not infected with HIV-1 who have had repetitive sexual exposure to HIV-1 in extremely high-risk situations. Determining the host factors involved in these different susceptibility and disease courses would be helpful for better understanding AIDS and its control. Today, a worldwide scientific endeavor called the Human Genome Project has completed the production of a full-length sequence of the 3 billion base pairs that make up the human genome. The Human Genome Project has also revealed the presence of variation in human genomes. Relating these genetic differences to human phenotypes offers a very attractive prospect for a genetic understanding of the different sensitivities to various human diseases, including cancer, brain and heart diseases, allergy, and infectious diseases. In this review, we present examples of human genetic variation that can affect HIV-1 infection and disease progression.