ASK1 inhibitors are potential pan-antiviral drugs, which dampen replication of diverse viruses including SARS-CoV2.

Apoptosis signal-regulating kinase 1 (ASK1)/MAP3K5 is a stress response kinase that is activated by various stimuli. It is known as an upstream activator of p38- Mitogen-activated protein kinase (p38MAPK) and c-Jun N-terminal kinase (JNK) that are reactive oxygen species (ROS)-induced kinases. Accumulating evidence show that ROS accumulate in virus-infected cells. Here, we investigated the relationship between viruses and ASK1/p38MAPK or ASK1/JNK pathways. Our findings suggest that virus infection activates ASK1 related pathways. In parallel, ASK1 inhibition led to a remarkable reduction in the replication of a broad range of viruses including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), vaccinia virus (VV), vesicular stomatitis virus (VSV), Herpes Simplex Virus (HSV), and Human Immunodeficiency virus (HIV) in different human cell lines. Our work demonstrates the potential therapeutic use of Selonsertib, an ASK1 inhibitor, as a pan-antiviral drug in humans. Surprisingly, we observed differential effects of Selonsertib in in vitro and in vivo hamster models, suggesting caution in using rodent models to predict clinical and therapeutic outcomes in humans.

Sensitivity to Neutralizing Antibodies and Resistance to Type I Interferons in SARS-CoV-2 R.1 Lineage Variants, Canada.

Isolating and characterizing emerging SARS-CoV-2 variants is key to understanding virus pathogenesis. In this study, we isolated samples of the SARS-CoV-2 R.1 lineage, categorized as a variant under monitoring by the World Health Organization, and evaluated their sensitivity to neutralizing antibodies and type I interferons. We used convalescent serum samples from persons in Canada infected either with ancestral virus (wave 1) or the B.1.1.7 (Alpha) variant of concern (wave 3) for testing neutralization sensitivity. The R.1 isolates were potently neutralized by both the wave 1 and wave 3 convalescent serum samples, unlike the B.1.351 (Beta) variant of concern. Of note, the R.1 variant was significantly more resistant to type I interferons (IFN-α/ß) than was the ancestral isolate. Our study demonstrates that the R.1 variant retained sensitivity to neutralizing antibodies but evolved resistance to type I interferons. This critical driving force will influence the trajectory of the pandemic.

Altered microRNA Transcriptome in Cultured Human Airway Cells upon Infection with SARS-CoV-2.

Numerous proteomic and transcriptomic studies have been carried out to better understand the current multi-variant SARS-CoV-2 virus mechanisms of action and effects. However, they are mostly centered on mRNAs and proteins. The effect of the virus on human post-transcriptional regulatory agents such as microRNAs (miRNAs), which are involved in the regulation of 60% of human gene activity, remains poorly explored. Similar to research we have previously undertaken with other viruses such as Ebola and HIV, in this study we investigated the miRNA profile of lung epithelial cells following infection with SARS-CoV-2. At the 24 and 72 h post-infection time points, SARS-CoV-2 did not drastically alter the miRNome. About 90% of the miRNAs remained non-differentially expressed. The results revealed that miR-1246, miR-1290 and miR-4728-5p were the most upregulated over time. miR-196b-5p and miR-196a-5p were the most downregulated at 24 h, whereas at 72 h, miR-3924, miR-30e-5p and miR-145-3p showed the highest level of downregulation. In the top significantly enriched KEGG pathways of genes targeted by differentially expressed miRNAs we found, among others, MAPK, RAS, P13K-Akt and renin secretion signaling pathways. Using RT-qPCR, we also showed that SARS-CoV-2 may regulate several predicted host mRNA targets involved in the entry of the virus into host cells (ACE2, TMPRSS2, ADAM17, FURIN), renin-angiotensin system (RAS) (Renin, Angiotensinogen, ACE), innate immune response (IL-6, IFN1ß, CXCL10, SOCS4) and fundamental cellular processes (AKT, NOTCH, WNT). Finally, we demonstrated by dual-luciferase assay a direct interaction between miR-1246 and ACE-2 mRNA. This study highlights the modulatory role of miRNAs in the pathogenesis of SARS-CoV-2.

Discovery and Use of Long dsRNA Mediated RNA Interference to Stimulate Antiviral Protection in Interferon Competent Mammalian Cells.

In invertebrate cells, RNA interference (RNAi) acts as a powerful immune defense that stimulates viral gene knockdown thereby preventing infection. With this pathway, virally produced long dsRNA (dsRNA) is cleaved into short interfering RNA (siRNA) by Dicer and loaded into the RNA-induced silencing complex (RISC) which can then destroy/disrupt complementary viral mRNA sequences. Comparatively, in mammalian cells it is believed that the type I interferon (IFN) pathway is the cornerstone of the innate antiviral response. In these cells, dsRNA acts as a potent inducer of the IFN system, which is dependent on dsRNA length, but not sequence, to stimulate an antiviral state. Although the cellular machinery for RNAi is intact and functioning in mammalian cells, its role to trigger an antiviral response using long dsRNA (dsRNAi) remains controversial. Here we show that dsRNAi is not only functional but has a significant antiviral effect in IFN competent mammalian cells. We found that pre-soaking mammalian cells with concentrations of sequence specific dsRNA too low to induce IFN production could significantly inhibit vesicular stomatitis virus expressing green fluorescent protein (VSV-GFP), and the human coronaviruses (CoV) HCoV-229E and SARS-CoV-2 replication. This phenomenon was shown to be dependent on dsRNA length, was comparable in effect to transfected siRNAs, and could knockdown multiple sequences at once. Additionally, knockout cell lines revealed that functional Dicer was required for viral inhibition, revealing that the RNAi pathway was indeed responsible. These results provide the first evidence that soaking with gene-specific long dsRNA can generate viral knockdown in mammalian cells. We believe that this novel discovery provides an explanation as to why the mammalian lineage retained its RNAi machinery and why vertebrate viruses have evolved methods to suppress RNAi. Furthermore, demonstrating RNAi below the threshold of IFN induction has uses as a novel therapeutic platform, both antiviral and gene targeting in nature.

The HIV-1 accessory protein Nef increases surface expression of the checkpoint receptor Tim-3 in infected CD4+ T cells.

Prolonged immune activation drives the upregulation of multiple checkpoint receptors on the surface of virus-specific T cells, inducing their exhaustion. Reversing HIV-1-induced T cell exhaustion is imperative for efficient virus clearance; however, viral mediators of checkpoint receptor upregulation remain largely unknown. The enrichment of checkpoint receptors on T cells upon HIV-1 infection severely constrains the generation of an efficient immune response. Herein, we examined the role of HIV-1 Nef in mediating the upregulation of checkpoint receptors on peripheral blood mononuclear cells. We demonstrate that the HIV-1 accessory protein Nef upregulates cell surface levels of the checkpoint receptor T-cell immunoglobulin mucin domain-3 (Tim-3) and that this is dependent on Nef's dileucine motif LL164/165. Furthermore, we used a bimolecular fluorescence complementation assay to demonstrate that Nef and Tim-3 form a complex within cells that is abrogated upon mutation of the Nef dileucine motif. We also provide evidence that Nef moderately promotes Tim-3 shedding from the cell surface in a dileucine motif-dependent manner. Treating HIV-1-infected CD4+ T cells with a matrix metalloprotease inhibitor enhanced cell surface Tim-3 levels and reduced Tim-3 shedding. Finally, Tim-3-expressing CD4+ T cells displayed a higher propensity to release the proinflammatory cytokine interferon-gamma. Collectively, our findings uncover a novel mechanism by which HIV-1 directly increases the levels of a checkpoint receptor on the surface of infected CD4+ T cells.

Experimental and natural evidence of SARS-CoV-2-infection-induced activation of type I interferon responses.

Type I interferons (IFNs) are our first line of defense against virus infection. Recent studies have suggested the ability of SARS-CoV-2 proteins to inhibit IFN responses. Emerging data also suggest that timing and extent of IFN production is associated with manifestation of COVID-19 severity. In spite of progress in understanding how SARS-CoV-2 activates antiviral responses, mechanistic studies into wild-type SARS-CoV-2-mediated induction and inhibition of human type I IFN responses are scarce. Here we demonstrate that SARS-CoV-2 infection induces a type I IFN response in vitro and in moderate cases of COVID-19. In vitro stimulation of type I IFN expression and signaling in human airway epithelial cells is associated with activation of canonical transcriptions factors, and SARS-CoV-2 is unable to inhibit exogenous induction of these responses. Furthermore, we show that physiological levels of IFNα detected in patients with moderate COVID-19 is sufficient to suppress SARS-CoV-2 replication in human airway cells.

Efferocytic Defects in Early Atherosclerosis Are Driven by GATA2 Overexpression in Macrophages.

The loss of efferocytosis-the phagocytic clearance of apoptotic cells-is an initiating event in atherosclerotic plaque formation. While the loss of macrophage efferocytosis is a prerequisite for advanced plaque formation, the transcriptional and cellular events in the pre-lesion site that drive these defects are poorly defined. Transcriptomic analysis of macrophages recovered from early-stage human atherosclerotic lesions identified a 50-fold increase in the expression of GATA2, a transcription factor whose expression is normally restricted to the hematopoietic compartment. GATA2 overexpression in vitro recapitulated many of the functional defects reported in patient macrophages, including deficits at multiple stages in the efferocytic process. These findings included defects in the uptake of apoptotic cells, efferosome maturation, and in phagolysosome function. These efferocytic defects were a product of GATA2-driven alterations in the expression of key regulatory proteins, including Src-family kinases, Rab7 and components of both the vacuolar ATPase and NADPH oxidase complexes. In summary, these data identify a mechanism by which efferocytic capacity is lost in the early stages of plaque formation, thus setting the stage for the accumulation of uncleared apoptotic cells that comprise the bulk of atherosclerotic plaques.

HIV-1 Vpu Downregulates Tim-3 from the Surface of Infected CD4+ T Cells.

Along with other immune checkpoints, T cell immunoglobulin and mucin domain-containing protein 3 (Tim-3) is expressed on exhausted CD4+ and CD8+ T cells and is upregulated on the surface of these cells upon infection by human immunodeficiency virus type 1 (HIV-1). Recent reports have suggested an antiviral role for Tim-3. However, the molecular determinants of HIV-1 which modulate cell surface Tim-3 levels have yet to be determined. Here, we demonstrate that HIV-1 Vpu downregulates Tim-3 from the surface of infected primary CD4+ T cells, thus attenuating HIV-1-induced upregulation of Tim-3. We also provide evidence that the transmembrane domain of Vpu is required for Tim-3 downregulation. Using immunofluorescence microscopy, we determined that Vpu is in close proximity to Tim-3 and alters its subcellular localization by directing it to Rab 5-positive (Rab 5+) vesicles and targeting it for sequestration within the trans- Golgi network (TGN). Intriguingly, Tim-3 knockdown and Tim-3 blockade increased HIV-1 replication in primary CD4+ T cells, thereby suggesting that Tim-3 expression might represent a natural immune mechanism limiting viral spread.IMPORTANCE HIV infection modulates the surface expression of Tim-3, but the molecular determinants remain poorly understood. Here, we show that HIV-1 Vpu downregulates Tim-3 from the surface of infected primary CD4+ T cells through its transmembrane domain and alters its subcellular localization. Tim-3 blockade increases HIV-1 replication, suggesting a potential negative role of this protein in viral spread that is counteracted by Vpu.

PACS-1 and adaptor protein-1 mediate ACTH trafficking to the regulated secretory pathway.

The regulated secretory pathway is a specialized form of protein secretion found in endocrine and neuroendocrine cell types. Pro-opiomelanocortin (POMC) is a pro-hormone that utilizes this pathway to be trafficked to dense core secretory granules (DCSGs). Within this organelle, POMC is processed to multiple bioactive hormones that play key roles in cellular physiology. However, the complete set of cellular membrane trafficking proteins that mediate the correct sorting of POMC to DCSGs remain unknown. Here, we report the roles of the phosphofurin acidic cluster sorting protein - 1 (PACS-1) and the clathrin adaptor protein 1 (AP-1) in the targeting of POMC to DCSGs. Upon knockdown of PACS-1 and AP-1, POMC is readily secreted into the extracellular milieu and fails to be targeted to DCSGs.

Identification of Novel Subcellular Localization and Trafficking of HIV-1 Nef Variants from Reference Strains G (F1.93.HH8793) and H (BE.93.VI997).

The human immunodeficiency virus type 1 (HIV-1) accessory protein Nef, plays an essential role in disease progression and pathogenesis via hijacking the host cellular membrane-trafficking machinery. Interestingly, HIV-1 group-M subtypes display differences in the rate of disease progression. However, few reports investigated how the cellular behaviors and activities of Nef isolates from reference strains may differ between HIV-1 group-M subtypes. Here, we characterize how differing cellular distributions of Nef proteins across group-M subtypes may impact protein function using immunofluorescence microscopy and flow cytometric analysis. We demonstrate that Nef variants isolated from HIV-1 group-M subtypes display differences in expression, with low expressing Nef proteins from reference strains of subtypes G (F1.93.HH8793) and H (BE.93.VI997) also displaying decreased functionality. Additionally, we demonstrate variations in the subcellular distribution and localization of these Nef proteins. Nef from subtype G (F1.93.HH8793) and H (BE.93.VI997) reference strains also failed to colocalize with the trans-Golgi network, and were not differentially localized to cellular markers of multivesicular bodies or lysosomes. Strikingly, our results demonstrate that HIV-1 Nef proteins from reference strains G (F1.93.HH8793) and H (BE.93.VI997) highly colocalize with labeled mitochondrial compartments.

Molecular Basis of Unusually High Neutralization Resistance in Tier 3 HIV-1 Strain 253-11.

Understanding the mechanisms used by HIV-1 to evade antibody neutralization may contribute to the design of a high-coverage vaccine. The tier 3 virus 253-11 is poorly neutralized by subtype-matched and subtype C sera, even compared to other tier 3 viruses, and is also recognized poorly by V3/glycan-targeting monoclonal antibodies (MAbs). We found that sequence polymorphisms in the V3 loop and N-linked glycosylation sites contribute only minimally to the high neutralization resistance of 253-11. Interestingly, the 253-11 membrane-proximal external region (MPER) is rarely recognized by sera in the context of the wild-type virus but is commonly recognized in the context of an HIV-2 chimera, suggesting steric or kinetic hindrance of binding to MPER in the native envelope (Env). Mutations in the 253-11 MPER, which were previously reported to increase the lifetime of the prefusion Env conformation, affected the resistance of 253-11 to antibodies targeting various epitopes on HIV-1 Env, presumably destabilizing its otherwise stable, closed trimer structure. To gain insight into the structure of 253-11, we constructed and crystallized a recombinant 253-11 SOSIP trimer. The resulting structure revealed that the heptad repeat helices in gp41 are drawn in close proximity to the trimer axis and that gp120 protomers also showed a relatively compact disposition around the trimer axis. These observations give substantial insight into the molecular features of an envelope spike from a tier 3 virus and into possible mechanisms that may contribute to its unusually high neutralization resistance.IMPORTANCE HIV-1 isolates that are highly resistant to broadly neutralizing antibodies could limit the efficacy of an antibody-based vaccine. We studied 253-11, which is highly resistant to commonly elicited neutralizing antibodies. To further understand its resistance, we made mutations that are known to delay fusion and thus increase the time that the virus spends in the open conformation following CD4 binding. Interestingly, we found that these mutations affect the 253-11 envelope (Env) spike before CD4 binding, presumably by destabilizing the trimer structure. To gain further information about the structure of the 253-11 Env trimer, we generated a recombinant 253-11 SOSIP trimer. The crystal structure of the SOSIP trimer revealed that the gp41 helices and the gp120 protomers were drawn in toward the center of the molecule compared to most solved HIV-1 Env structures. These observations provide insight into the distinct molecular features of a tier 3 envelope spike.

The HIV-1 accessory proteins Nef and Vpu downregulate total and cell surface CD28 in CD4+ T cells.

BACKGROUND: The HIV-1 accessory proteins Nef and Vpu alter cell surface levels of multiple host proteins to modify the immune response and increase viral persistence. Nef and Vpu can downregulate cell surface levels of the co-stimulatory molecule CD28, however the mechanism of this function has not been completely elucidated. RESULTS: Here, we provide evidence that Nef and Vpu decrease cell surface and total cellular levels of CD28. Moreover, using inhibitors we implicate the cellular degradation machinery in the downregulation of CD28. We shed light on the mechanisms of CD28 downregulation by implicating the Nef LL165 and DD175 motifs in decreasing cell surface CD28 and Nef DD175 in decreasing total cellular CD28. Moreover, the Vpu LV64 and S52/56 motifs were required for cell surface CD28 downregulation, while, unlike for CD4 downregulation, Vpu W22 was dispensable. The Vpu S52/56 motif was also critical for Vpu-mediated decreases in total CD28 protein level. Finally, the ability of Vpu to downregulate CD28 is conserved between multiple group M Vpu proteins and infection with viruses encoding or lacking Nef and Vpu have differential effects on activation upon stimulation. CONCLUSIONS: We report that Nef and Vpu downregulate cell surface and total cellular CD28 levels. We identified inhibitors and mutations within Nef and Vpu that disrupt downregulation, shedding light on the mechanisms utilized to downregulate CD28. The conservation and redundancy between the abilities of two HIV-1 proteins to downregulate CD28 highlight the importance of this function, which may contribute to the development of latently infected cells.

The interaction between HIV-1 Nef and adaptor protein-2 reduces Nef-mediated CD4+ T cell apoptosis.

Acquired Immune Deficiency Syndrome is characterized by a decline in CD4+ T cells. Here, we elucidated the mechanism underlying apoptosis in Human Immunodeficiency Virus-1 (HIV-1) infection by examining host apoptotic pathways hijacked by the HIV-1 Nef protein in the CD4+ T-cell line Sup-T1. Using a panel of Nef mutants unable to bind specific host proteins we uncovered that Nef generates pro- and anti-apoptotic signals. Apoptosis increased upon mutating the motifs involved in the interaction of Nef:AP-1 (NefM20A or NefEEEE62-65AAAA) or Nef:AP-2 (NefLL164/165AA), implying these interactions limit Nef-mediated apoptosis. In contrast, disrupting the Nef:PAK2 interaction motifs (NefH89A or NefF191A) reduced apoptosis. To validate further, apoptosis was measured after short-hairpin RNA knock-down of AP-1, AP-2 and PAK2. AP-2α depletion enhanced apoptosis, demonstrating that disrupting the Nef:AP-2α interaction limits Nef-mediated apoptosis. Collectively, we describe a mechanism by which HIV-1 regulates cell survival and demonstrate the consequence of interfering with Nef:host protein interactions.

HIV-1 Nef sequesters MHC-I intracellularly by targeting early stages of endocytosis and recycling.

A defining characteristic of HIV-1 infection is the ability of the virus to persist within the host. Specifically, MHC-I downregulation by the HIV-1 accessory protein Nef is of critical importance in preventing infected cells from cytotoxic T-cell mediated killing. Nef downregulates MHC-I by modulating the host membrane trafficking machinery, resulting in the endocytosis and eventual sequestration of MHC-I within the cell. In the current report, we utilized the intracellular protein-protein interaction reporter system, bimolecular fluorescence complementation (BiFC), in combination with super-resolution microscopy, to track the Nef/MHC-I interaction and determine its subcellular localization in cells. We demonstrate that this interaction occurs upon Nef binding the MHC-I cytoplasmic tail early during endocytosis in a Rab5-positive endosome. Disruption of early endosome regulation inhibited Nef-dependent MHC-I downregulation, demonstrating that Nef hijacks the early endosome to sequester MHC-I within the cell. Furthermore, super-resolution imaging identified that the Nef:MHC-I BiFC complex transits through both early and late endosomes before ultimately residing at the trans-Golgi network. Together we demonstrate the importance of the early stages of the endocytic network in the removal of MHC-I from the cell surface and its re-localization within the cell, which allows HIV-1 to optimally evade host immune responses.

The 23-Hour Observation Unit Admissions Within the Emergency Service at a National Tertiary Psychiatric Hospital: Clarifying Clinical Profiles, Outcomes, and Predictors of Subsequent Hospitalization.

OBJECTIVE: We examined health care utilization, clinical profiles (such as sociodemographic features, clinical severity), and outcomes (inpatient admission, revisit within 24 hours of discharge) of patients who were admitted to a 23-hour observation unit within the emergency service of a tertiary psychiatric hospital and hypothesized that a specific clinical profile (greater clinical severity, lower psychosocial functioning) predicted subsequent inpatient hospitalization. METHOD: The medical records of all patients admitted to the observation unit from February 5, 2007, to February 4, 2012 (N = 2,158) were assessed for relevant data. Clinical severity and level of psychosocial functioning were assessed using Clinical Global Impressions-Severity (CGI-S) and Global Assessment of Functioning (GAF) scales, respectively. RESULTS: Overall, the patients seen were predominantly Chinese males > 36 years old who had diagnoses including stress-related, anxiety, affective spectrum, and psychotic disorders. The clinical severity score (CGI-S) improved significantly following discharge from the observation unit (t 1,1848 = 23.316; P < .001). Logistic regression analyses revealed that self-referred (P = .001), older patients (P = .007) with past psychiatric history (P = .019), lower GAF scores (P = .025), and less improvement of CGI-S scores (P = .001) were associated with inpatient admission after a 23-hour stay in the observation unit. CONCLUSIONS: Our study findings affirmed our hypothesis and supported the utility of the observation unit in monitoring the overall clinical status of patients, which was linked with subsequent inpatient admissions. Better management of these patients at the outpatient level can potentially decrease unnecessary hospitalization and reduce health care cost as well as illness burden on patients and caregivers.

Viral bimolecular fluorescence complementation: a novel tool to study intracellular vesicular trafficking pathways.

The Human Immunodeficiency Virus type 1 (HIV-1) accessory protein Nef interacts with a multitude of cellular proteins, manipulating the host membrane trafficking machinery to evade immune surveillance. Nef interactions have been analyzed using various in vitro assays, co-immunoprecipitation studies, and more recently mass spectrometry. However, these methods do not evaluate Nef interactions in the context of viral infection nor do they define the sub-cellular location of these interactions. In this report, we describe a novel bimolecular fluorescence complementation (BiFC) lentiviral expression tool, termed viral BiFC, to study Nef interactions with host cellular proteins in the context of viral infection. Using the F2A cleavage site from the foot and mouth disease virus we generated a viral BiFC expression vector capable of concurrent expression of Nef and host cellular proteins; PACS-1, MHC-I and SNX18. Our studies confirmed the interaction between Nef and PACS-1, a host membrane trafficking protein involved in Nef-mediated immune evasion, and demonstrated co-localization of this complex with LAMP-1 positive endolysosomal vesicles. Furthermore, we utilized viral BiFC to localize the Nef/MHC-I interaction to an AP-1 positive endosomal compartment. Finally, viral BiFC was observed between Nef and the membrane trafficking regulator SNX18. This novel demonstration of an association between Nef and SNX18 was localized to AP-1 positive vesicles. In summary, viral BiFC is a unique tool designed to analyze the interaction between Nef and host cellular proteins by mapping the sub-cellular locations of their interactions during viral infection.

Anti-V3/Glycan and Anti-MPER Neutralizing Antibodies, but Not Anti-V2/Glycan Site Antibodies, Are Strongly Associated with Greater Anti-HIV-1 Neutralization Breadth and Potency.

UNLABELLED: The membrane-proximal external region (MPER), the V2/glycan site (initially defined by PG9 and PG16 antibodies), and the V3/glycans (initially defined by PGT121-128 antibodies) are targets of broadly neutralizing antibodies and potential targets for anti-HIV-1 antibody-based vaccines. Recent evidence shows that antibodies with moderate neutralization breadth are frequently attainable, with 50% of sera from chronically infected individuals neutralizing ≥ 50% of a large, diverse set of viruses. Nonetheless, there is little systematic information addressing which specificities are preferentially targeted among such commonly found, moderately broadly neutralizing sera. We explored associations between neutralization breadth and potency and the presence of neutralizing antibodies targeting the MPER, V2/glycan site, and V3/glycans in sera from 177 antiretroviral-naive HIV-1-infected (>1 year) individuals. Recognition of both MPER and V3/glycans was associated with increased breadth and potency. MPER-recognizing sera neutralized 4.62 more panel viruses than MPER-negative sera (95% prediction interval [95% PI], 4.41 to 5.20), and V3/glycan-recognizing sera neutralized 3.24 more panel viruses than V3/glycan-negative sera (95% PI, 3.15 to 3.52). In contrast, V2/glycan site-recognizing sera neutralized only 0.38 more panel viruses (95% PI, 0.20 to 0.45) than V2/glycan site-negative sera and no association between V2/glycan site recognition and breadth or potency was observed. Despite autoreactivity of many neutralizing antibodies recognizing MPER and V3/glycans, antibodies to these sites are major contributors to neutralization breadth and potency in this cohort. It may therefore be appropriate to focus on developing immunogens based upon the MPER and V3/glycans. IMPORTANCE: Previous candidate HIV vaccines have failed either to induce wide-coverage neutralizing antibodies or to substantially protect vaccinees. Therefore, current efforts focus on novel approaches never before successfully used in vaccine design, including modeling epitopes. Candidate immunogen models identified by broadly neutralizing antibodies include the membrane-proximal external region (MPER), V3/glycans, and the V2/glycan site. Autoreactivity and polyreactivity of anti-MPER and anti-V3/glycan antibodies are thought to pose both direct and indirect barriers to achieving neutralization breadth. We found that antibodies to the MPER and the V3/glycans contribute substantially to neutralization breadth and potency. In contrast, antibodies to the V2/glycan site were not associated with neutralization breadth/potency. This suggests that the autoreactivity effect is not critical and that the MPER and the V3/glycans should remain high-priority vaccine candidates. The V2/glycan site result is surprising because broadly neutralizing antibodies to this site have been repeatedly observed. Vaccine design priorities should shift toward the MPER and V3/glycans.

Jasmine flower extract lowers prolactin.

BACKGROUND: Antipsychotic drugs frequently cause amenorrhoea and galactorrhoea. Jasmine flowers used topically were as effective as oral Bromocriptine in suppressing puerperal lactation. This study aims to evaluate the efficacy and safety of intranasal jasmine flower extract (JFE) to reduce prolactin levels of patients on stable doses of antipsychotic drugs. METHOD: This is a randomized, double blind, crossover clinical trial. An aqueous-ethanol extract of jasmine flowers was prepared and used as nasal drops. A decrease in serum prolactin of ≥25 ng/mL was considered a significant response. RESULTS: Ten out of 35 women had a significant drop in the serum prolactin while on the JFE. The non-responders to JFE were on higher doses of antipsychotic drugs. The main side effect was a transient and mild burning sensation in the nose. A cost analysis favoured JFE over dopamine agonists. CONCLUSION: JFE contains a prolactin-lowering substance which needs further characterisation.

The grafting of universal T-helper epitopes enhances immunogenicity of HIV-1 Tat concurrently improving its safety profile.

Extracellular Tat (eTat) plays an important role in HIV-1 pathogenesis. The presence of anti-Tat antibodies is negatively correlated with disease progression, hence making Tat a potential vaccine candidate. The cytotoxicity and moderate immunogenicity of Tat however remain impediments for developing Tat-based vaccines. Here, we report a novel strategy to concurrently enhance the immunogenicity and safety profile of Tat. The grafting of universal helper T-lymphocyte (HTL) epitopes, Pan DR Epitope (PADRE) and Pol711 into the cysteine rich domain (CRD) and the basic domain (BD) abolished the transactivation potential of the Tat protein. The HTL-Tat proteins elicited a significantly higher titer of antibodies as compared to the wild-type Tat in BALB/c mice. While the N-terminal epitope remained immunodominant in HTL-Tat immunizations, an additional epitope in exon-2 was recognized with comparable magnitude suggesting a broader immune recognition. Additionally, the HTL-Tat proteins induced cross-reactive antibodies of high avidity that efficiently neutralized exogenous Tat, thus blocking the activation of a Tat-defective provirus. With advantages such as presentation of multiple B-cell epitopes, enhanced antibody response and importantly, transactivation-deficient Tat protein, this approach has potential application for the generation of Tat-based HIV/AIDS vaccines.