Comparative analysis of retroviral Gag-host cell interactions: focus on the nuclear interactome.

Retroviruses exploit host proteins to assemble and release virions from infected cells. Previously, most studies focused on interacting partners of retroviral Gag proteins that localize to the cytoplasm or plasma membrane. Given that several full-length Gag proteins have been found in the nucleus, identifying the Gag-nuclear interactome has high potential for novel findings involving previously unknown host processes. Here we systematically compared nuclear factors identified in published HIV-1 proteomic studies and performed our own mass spectrometry analysis using affinity-tagged HIV-1 and RSV Gag proteins mixed with nuclear extracts. We identified 57 nuclear proteins in common between HIV-1 and RSV Gag, and a set of nuclear proteins present in our analysis and ≥ 1 of the published HIV-1 datasets. Many proteins were associated with nuclear processes which could have functional consequences for viral replication, including transcription initiation/elongation/termination, RNA processing, splicing, and chromatin remodeling. Examples include facilitating chromatin remodeling to expose the integrated provirus, promoting expression of viral genes, repressing the transcription of antagonistic cellular genes, preventing splicing of viral RNA, altering splicing of cellular RNAs, or influencing viral or host RNA folding or RNA nuclear export. Many proteins in our pulldowns common to RSV and HIV-1 Gag are critical for transcription, including PolR2B, the second largest subunit of RNA polymerase II (RNAPII), and LEO1, a PAF1C complex member that regulates transcriptional elongation, supporting the possibility that Gag influences the host transcription profile to aid the virus. Through the interaction of RSV and HIV-1 Gag with splicing-related proteins CBLL1, HNRNPH3, TRA2B, PTBP1 and U2AF1, we speculate that Gag could enhance unspliced viral RNA production for translation and packaging. To validate one putative hit, we demonstrated an interaction of RSV Gag with Mediator complex member Med26, required for RNA polymerase II-mediated transcription. Although 57 host proteins interacted with both Gag proteins, unique host proteins belonging to each interactome dataset were identified. These results provide a strong premise for future functional studies to investigate roles for these nuclear host factors that may have shared functions in the biology of both retroviruses, as well as functions specific to RSV and HIV-1, given their distinctive hosts and molecular pathology.

Broadly neutralizing antibody epitopes on HIV-1 particles are exposed after virus interaction with host cells.

The envelope (Env) glycoproteins on HIV-1 virions are the sole target of broadly neutralizing antibodies (bNAbs) and the focus of vaccines. However, many cross-reactive conserved epitopes are often occluded on virus particles, contributing to the evasion of humoral immunity. This study aimed to identify the Env epitopes that are exposed/occluded on HIV-1 particles and to investigate the mechanisms contributing to their masking. Using a flow cytometry-based assay, three HIV-1 isolates, and a panel of antibodies, we show that only select epitopes, including V2i, the gp120-g41 interface, and gp41-MPER, are accessible on HIV-1 particles, while V3, V2q, and select CD4bs epitopes are masked. These epitopes become accessible after allosteric conformational changes are induced by the pre-binding of select Abs, prompting us to test if similar conformational changes are required for these Abs to exhibit their neutralization capability. We tested HIV-1 neutralization where the virus-mAb mix was pre-incubated/not pre-incubated for 1 hour prior to adding the target cells. Similar levels of neutralization were observed under both assay conditions, suggesting that the interaction between virus and target cells sensitizes the virions for neutralization via bNAbs. We further show that lectin-glycan interactions can also expose these epitopes. However, this effect is dependent on the lectin specificity. Given that, bNAbs are ideal for providing sterilizing immunity and are the goal of current HIV-1 vaccine efforts, these data offer insight on how HIV-1 may occlude these vulnerable epitopes from the host immune response. In addition, the findings can guide the formulation of effective antibody combinations for therapeutic use. IMPORTANCE The human immunodeficiency virus (HIV-1) envelope (Env) glycoprotein mediates viral entry and is the sole target of neutralizing antibodies. Our data suggest that antibody epitopes including V2q (e.g., PG9, PGT145), CD4bs (e.g., VRC01, 3BNC117), and V3 (2219, 2557) are masked on HIV-1 particles. The PG9 and 2219 epitopes became accessible for binding after conformational unmasking was induced by the pre-binding of select mAbs. Attempts to understand the masking mechanism led to the revelation that interaction between virus and host cells is needed to sensitize the virions for neutralization by broadly neutralizing antibodies (bNAbs). These data provide insight on how bNAbs may gain access to these occluded epitopes to exert their neutralization effects and block HIV-1 infection. These findings have important implications for the way we evaluate the neutralizing efficacy of antibodies and can potentially guide vaccine design.

Commensal Hafnia alvei strain reduces food intake and fat mass in obese mice-a new potential probiotic for appetite and body weight management.

BACKGROUND/OBJECTIVES: Based on the recent identification of E.coli heat shock protein ClpB as a mimetic of the anorexigenic α-melanocyte stimulating hormone (α-MSH), the objective of this study was to preclinically validate Hafnia alvei, a ClpB-producing commensal bacterium as a potential probiotic for appetite and body weight management in overweight and obesity. METHODS: The involvement of enterobacterial ClpB in the putative anti-obesity effects was studied using ClpB-deficient E.coli. A food-grade H. alvei HA4597 strain synthetizing the ClpB protein with an α-MSH-like motif was selected as a candidate probiotic to be tested in ob/ob and high-fat diet (HFD)-fed obese and overweight mice. The relevance of the enterobacterial ClpB gene to human obesity was studied by in silico analysis of fecal metagenomes of 569 healthy individuals from the "MetaHIT" database. RESULTS: Chronic per os administration of native but not ClpB-deficient E.coli strain reduced body weight gain (p < 0.05) and daily meal frequency (p < 0.001) in ob/ob mice. Oral gavage of H.alvei for 18 and 46 days in ob/ob and HFD-fed obese mice, respectively, was well tolerated, reduced body weight gain and fat mass in both obesity models (p < 0.05) and decreased food intake in hyperphagic ob/ob mice (p < 0.001). Elevated fat tissue levels of phosphorylated hormone-sensitive lipase were detected in H.alvei -treated ob/ob mice (p < 0.01). Enterobacterial ClpB gene richness was lower in obese vs. non-obese humans (p < 0.0001) and correlated negatively with BMI in genera of Enterobacter, Klebsiella and Hafnia. CONCLUSIONS: H.alvei HA4597 strain reduces food intake, body weight and fat mass gain in hyperphagic and obese mice. These data combined with low enterobacterial ClpB gene abundance in the microbiota of obese humans provide the rationale for using H.alvei as a probiotic for appetite and body weight management in overweight and obesity.

Insights into the Mechanisms by Which Clostridial Neurotoxins Discriminate between Gangliosides.

Botulinum neurotoxins (BoNTs) and tetanus neurotoxin (TeNT) are the causative agents of the paralytic diseases botulism and tetanus, respectively. Entry of toxins into neurons is mediated through initial interactions with gangliosides, followed by binding to a protein co-receptor. Herein, we aimed to understand the mechanism through which individual neurotoxins recognize the carbohydrate motif of gangliosides. Using cell-based and in vitro binding assays, in conjunction with structure-driven site-directed mutagenesis, a conserved hydrophobic residue within the BoNTs that contributes to both affinity and specificity toward Sia5-containing gangliosides was identified. We demonstrate that targeted mutations within the Sia5 binding pocket result in the generation of neurotoxins that either bind and enter cells more efficiently (BoNT/A1 and BoNT/B) or display altered ganglioside binding specificity (TeNT). These data support a model in which recognition of Sia5 is largely driven by hydrophobic interactions between the sugar and the Sia5 binding site.

An easy to produce and economical three-dimensional brain phantom for stereotactic computed tomographic-guided brain biopsy training in the dog.

OBJECTIVE: To develop and validate a three-dimensional (3D) brain phantom that can be incorporated into existing stereotactic headframes to simulate stereotactic brain biopsy (SBB) and train veterinary surgeons. STUDY DESIGN: Experimental study. SAMPLE POPULATION: Canine brain phantoms were fabricated from osteological skull specimens, agarose brain parenchyma, and cheddar and mozzarella cheese molds (simulating meningiomas and gliomas). METHODS: The neuroradiologic and viscoelastic properties of phantoms were quantified with computed tomography (CT) and oscillatory compression tests, respectively. Phantoms were validated by experienced and novice operators performing SBB on phantoms containing randomly placed, focal targets. Target yield and needle placement error (NPE) were compared between operators. RESULTS: Phantoms were produced in <4 hours, at an average cost of $92. The CT appearances of the phantom skull, agarose, and cheese components approximated the in vivo features of skull, brain parenchyma, and contrast-enhancing tumors of meningeal and glial origin, respectively. The complex moduli of the agarose and cheeses were comparable to the viscoelastic properties of in vivo brain tissues and brain tumors. The overall diagnostic yield of SBB was 88%. Although NPE did not differ between novice (median 3.68 mm; range, 1.46-14.54 mm) and experienced surgeons (median 1.17 mm, range, 0.78-1.58 mm), our results support the relevance of the learning curve associated with the SBB procedure. CONCLUSION: This 3D phantom replicates anatomical, CT, and tactile features of brain tissues and tumors and can be used to develop the technical skills required to perform SBB.

The comparative cytotoxic effects of different local anesthetics on a human neuroblastoma cell line.

BACKGROUND: Although local anesthetics (LAs) are generally accepted as being safe, incidental neuronal damage has been reported for all LAs in humans. Therefore, in this study, we compared the dose corresponding to 50% cell lethality (LD50) of articaine, lidocaine, mepivacaine, bupivacaine, prilocaine, and ropivacaine in human neuroblastoma cells. METHODS: Undifferentiated SH-SY5Y cells were exposed for 20 minutes to different concentrations of each LA. Metabolic activity of viable cells was assessed by a cell viability test with a tetrazolium dye (WST-1) followed by optical density quantification. LD50 was determined by extrapolation of curve response. RESULTS: As expected, all LAs induced cell death in a concentration-dependent manner. The bupivacaine, lidocaine, prilocaine, mepivacaine, articaine, and ropivacaine LD50 were 0.95 ± 0.08, 3.35 ± 0.33, 4.32 ± 0.39, 4.84 ± 1.28, 8.98 ± 2.07, and 13.43 ± 0.61 mM, respectively, after 20 minutes of incubation on SH-SY5Y cells. Ropivacaine LD50 was significantly different from the bupivacaine, lidocaine, mepivacaine, and prilocaine LD50 (all P ≤ 0.009). No significant difference was obtained between ropivacaine and articaine LD50 and between prilocaine, lidocaine, and mepivacaine LD50. Articaine LD50 was significantly different from lidocaine LD50 (P = 0.03). Bupivacaine LD50 was significantly lower compared with all LAs (all P ≤ 0.003). CONCLUSIONS: LA neurotoxicity was tested in a validated in vitro model SH-SY5Y, a human neuroblastoma cell line. Three groups of LAs were identified in terms of toxicity: (1) the less (ropivacaine, articaine); (2) medium (mepivacaine, prilocaine, lidocaine); and (3) the high (bupivacaine). Among dental anesthetics, articaine is the least neurotoxic in SH-SY5Y cells.

Comparative analysis of retroviral Gag-host cell interactions: focus on the nuclear interactome.

Retroviruses exploit a variety of host proteins to assemble and release virions from infected cells. To date, most studies that examined possible interacting partners of retroviral Gag proteins focused on host proteins that localize primarily to the cytoplasm or plasma membrane. Given the recent findings that several full-length Gag proteins localize to the nucleus, identifying the Gag-nuclear interactome has high potential for novel findings that reveal previously unknown host processes. In this study, we systematically compared nuclear factors identified in published HIV-1 proteomic studies which had used a variety of experimental approaches. In addition, to contribute to this body of knowledge, we report results from a mass spectrometry approach using affinity-tagged (His6) HIV-1 and RSV Gag proteins mixed with nuclear extracts. Taken together, the previous studies-as well as our own-identified potential binding partners of HIV-1 and RSV Gag involved in several nuclear processes, including transcription, splicing, RNA modification, and chromatin remodeling. Although a subset of host proteins interacted with both Gag proteins, there were also unique host proteins belonging to each interactome dataset. To validate one of the novel findings, we demonstrated the interaction of RSV Gag with a member of the Mediator complex, Med26, which is required for RNA polymerase II-mediated transcription. These results provide a strong premise for future functional studies to investigate roles for these nuclear host factors that may have shared functions in the biology of both retroviruses, as well as functions specific to RSV and HIV-1, given their distinctive hosts and molecular pathology.

Human-aided dispersal and population bottlenecks facilitate parasitism escape in the most invasive mosquito species.

During biological invasion process, species encounter new environments and partially escape some ecological constraints they faced in their native range, while they face new ones. The Asian tiger mosquito Aedes albopictus is one of the most iconic invasive species introduced in every inhabited continent due to international trade. It has also been shown to be infected by a prevalent yet disregarded microbial entomoparasite Ascogregarina taiwanensis. In this study, we aimed at deciphering the factors that shape the global dynamics of A. taiwanensis infection in natural A. albopictus populations. We showed that A. albopictus populations are highly colonized by several parasite genotypes but recently introduced ones are escaping it. We further performed experiments based on the invasion process to explain such pattern. To that end, we hypothesized that (i) mosquito passive dispersal (i.e. human-aided egg transportation) may affect the parasite infectiveness, (ii) founder effects (i.e. population establishment by a small number of mosquitoes) may influence the parasite dynamics, and (iii) unparasitized mosquitoes are more prompt to found new populations through active flight dispersal. The two first hypotheses were supported as we showed that parasite infection decreases over time when dry eggs are stored and that experimental increase in mosquitoes' density improves the parasite horizontal transmission to larvae. Surprisingly, parasitized mosquitoes tend to be more active than their unparasitized relatives. Finally, this study highlights the importance of global trade as a driver of biological invasion of the most invasive arthropod vector species.

Clinical evaluation of the performance and safety of a new dentine substitute, Biodentine, in the restoration of posterior teeth - a prospective study.

OBJECTIVES: A multicentric randomized, 3-year prospective study was conducted to determine for how long Biodentine, a new biocompatible dentine substitute, can remain as a posterior restoration. MATERIALS AND METHODS: First, Biodentine was compared to the composite Z100®, to evaluate whether and for how long it could be used as a posterior restoration according to selected United States Public Health Service (USPHS)' criteria (mean ± SD). Second, when abrasion occurred, Biodentine was evaluated as a dentine substitute combined with Z100®. RESULTS: A total of 397 cases were included. This interim analysis was conducted on 212 cases that were seen for the 1-year recall. On the day of restoration placement, both materials obtained good scores for material handling, anatomic form (0.12 ± 0.33), marginal adaptation (0.01 ± 0.10) and interproximal contact (0.11 ± 0.39). During the follow-up, both materials scored well in surface roughness (≤1) without secondary decay and post-operative pain. Biodentine kept acceptable surface properties regarding anatomic form score (≤1), marginal adaptation score (≤2) and interproximal contact score (≤1) for up to 6 months after placement. Resistance to marginal discoloration was superior with Biodentine compared to Z100®. When Biodentine was retained as a dentine substitute after pulp vitality control, it was covered systematically with the composite Z100®. This procedure yielded restorations that were clinically sound and symptom free. CONCLUSIONS: Biodentine is able to restore posterior teeth for up to 6 months. When subsequently covered with Z100®, it is a convenient, efficient and well tolerated dentine substitute. CLINICAL RELEVANCE: Biodentine as a dentine substitute can be used under a composite for posterior restorations.

Broadly Neutralizing Antibody Epitopes on HIV-1 Particles are exposed after Virus Interaction with Host Cells.

The envelope glycoproteins (Env) on HIV-1 virions are the sole target of broadly neutralizing antibodies (bNAb) and the focus of vaccines. However, many cross-reactive conserved epitopes are often occluded on virus particles, contributing to the evasion of humoral immunity. This study aimed to identify the Env epitopes that are exposed/occluded on HIV-1 particles and to investigate the mechanisms contributing to their masking. Using a flow cytometry-based assay, three HIV-1 isolates, and a panel of antibodies, we show that only select epitopes including V2i, gp120-g41 interface, and gp41-MPER are accessible on HIV-1 particles, while V3, V2q, and select CD4bs epitopes are masked. These epitopes become accessible after allosteric conformational changes are induced by pre-binding of select Abs, prompting us to test if similar conformational changes are required for these Abs to exhibit their neutralization capability. We tested HIV-1 neutralization where virus-mAb mix was pre-incubated/not pre-incubated for one hour prior to adding the target cells. Similar levels of neutralization were observed under both assay conditions, suggesting that the interaction between virus and target cells sensitizes the virions for neutralization via bNAbs. We further show that lectin-glycan interactions can also expose these epitopes. However, this effect is dependent on the lectin specificity. Given that, bNAbs are the ideal for providing sterilizing immunity and are the goal of current HIV-1 vaccine efforts, these data offer insight on how HIV-1 may occlude these vulnerable epitopes from the host immune response. In addition, the findings can guide the formulation of effective antibody combinations for therapeutic use.

Influence of HIV-1 genomic RNA on the formation of Gag biomolecular condensates.

Biomolecular condensates (BMCs) play an important role in the replication of a growing number of viruses, but many important mechanistic details remain to be elucidated. Previously, we demonstrated that pan-retroviral nucleocapsid (NC) and the HIV-1 pr55 Gag (Gag) proteins phase separate into condensates, and that HIV-1 protease (PR)-mediated maturation of Gag and Gag-Pol precursor proteins yield self-assembling BMCs having HIV-1 core architecture. Using biochemical and imaging techniques, we aimed to further characterize the phase separation of HIV-1 Gag by determining which of its intrinsically disordered regions (IDRs) influence the formation of BMCs and how the HIV-1 viral genomic RNA (gRNA) could influence BMC abundance and size. We found that mutations in the Gag matrix (MA) domain or the NC zinc finger motifs altered condensate number and size in a salt-dependent manner. Gag BMCs were also bimodally influenced by the gRNA, with a condensate-promoting regime at lower protein concentrations and a gel dissolution at higher protein concentrations. Interestingly, incubation of Gag with CD4 + T cell nuclear lysates led to the formation of larger BMCs as compared to much smaller ones observed in the presence of cytoplasmic lysates. These findings suggests that the composition and properties of Gag-containing BMCs may be altered by differential association of host factors in nuclear and cytosolic compartments during virus assembly. This study significantly advances our understanding of HIV-1 Gag BMC formation and provides a foundation for future therapeutic targeting of virion assembly.

Influence of HIV-1 Genomic RNA on the Formation of Gag Biomolecular Condensates.

Biomolecular condensates (BMCs) play an important role in the replication of a growing number of viruses, but many important mechanistic details remain to be elucidated. Previously, we demonstrated that the pan-retroviral nucleocapsid (NC) and HIV-1 pr55Gag (Gag) proteins phase separate into condensates, and that HIV-1 protease (PR)-mediated maturation of Gag and Gag-Pol precursor proteins yields self-assembling BMCs that have HIV-1 core architecture. Using biochemical and imaging techniques, we aimed to further characterize the phase separation of HIV-1 Gag by determining which of its intrinsically disordered regions (IDRs) influence the formation of BMCs, and how the HIV-1 viral genomic RNA (gRNA) could influence BMC abundance and size. We found that mutations in the Gag matrix (MA) domain or the NC zinc finger motifs altered condensate number and size in a salt-dependent manner. Gag BMCs were also bimodally influenced by the gRNA, with a condensate-promoting regime at lower protein concentrations and a gel dissolution at higher protein concentrations. Interestingly, incubation of Gag with CD4+ T cell nuclear lysates led to the formation of larger BMCs compared to much smaller ones observed in the presence of cytoplasmic lysates. These findings suggest that the composition and properties of Gag-containing BMCs may be altered by differential association of host factors in nuclear and cytosolic compartments during virus assembly. This study significantly advances our understanding of HIV-1 Gag BMC formation and provides a foundation for future therapeutic targeting of virion assembly.

The Rous sarcoma virus Gag polyprotein forms biomolecular condensates driven by intrinsically-disordered regions.

Biomolecular condensates (BMCs) play important roles in cellular structures including transcription factories, splicing speckles, and nucleoli. BMCs bring together proteins and other macromolecules, selectively concentrating them so that specific reactions can occur without interference from the surrounding environment. BMCs are often made up of proteins that contain intrinsically disordered regions (IDRs), form phase-separated spherical puncta, form liquid-like droplets that undergo fusion and fission, contain molecules that are mobile, and are disrupted with phase-dissolving drugs such as 1,6-hexanediol. In addition to cellular proteins, many viruses, including influenza A, SARS-CoV-2, and human immunodeficiency virus type 1 (HIV-1) encode proteins that undergo phase separation and rely on BMC formation for replication. In prior studies of the retrovirus Rous sarcoma virus (RSV), we observed that the Gag protein forms discrete spherical puncta in the nucleus, cytoplasm, and at the plasma membrane that co-localize with viral RNA and host factors, raising the possibility that RSV Gag forms BMCs that participate in the virion intracellular assembly pathway. In our current studies, we found that Gag contains IDRs in the N-terminal (MAp2p10) and C-terminal (NC) regions of the protein and fulfills many criteria of BMCs. Although the role of BMC formation in RSV assembly requires further study, our results suggest the biophysical properties of condensates are required for the formation of Gag complexes in the nucleus and the cohesion of these complexes as they traffic through the nuclear pore, into the cytoplasm, and to the plasma membrane, where the final assembly and release of virus particles occurs.

The Rous sarcoma virus Gag Polyprotein Forms Biomolecular Condensates Driven by Intrinsically-disordered Regions.

Biomolecular condensates (BMCs) play important roles incellular structures includingtranscription factories, splicing speckles, and nucleoli. BMCs bring together proteins and other macromolecules, selectively concentrating them so that specific reactions can occur without interference from the surrounding environment. BMCs are often made up of proteins that contain intrinsically disordered regions (IDRs), form phase-separated spherical puncta, form liquid-like droplets that undergo fusion and fission, contain molecules that are mobile, and are disrupted with phase-dissolving drugs such as 1,6-hexanediol. In addition to cellular proteins, many viruses, including influenza A, SARS-CoV-2, and human immunodeficiency virus type 1 (HIV-1) encode proteins that undergo phase separation and rely on BMC formation for replication. In prior studies of the retrovirus Rous sarcoma virus (RSV), we observed that the Gag protein forms discrete spherical puncta in the nucleus, cytoplasm, and at the plasma membrane that co-localize with viral RNA and host factors, raising the possibility that RSV Gag forms BMCs that participate in the intracellular phase of the virion assembly pathway. In our current studies, we found that Gag contains IDRs in the N-terminal (MAp2p10) and C-terminal (NC) regions of the protein and fulfills many criteria of BMCs. Although the role of BMC formation in RSV assembly requires further study, our results suggest the biophysical properties of condensates are required for the formation of Gag complexes in the nucleus and the cohesion of these complexes as they traffic through the nuclear pore, into the cytoplasm, and to the plasma membrane, where the final assembly and release of virus particles occurs.

Immune profiles to distinguish hospitalized versus ambulatory COVID-19 cases in older patients.

A fraction of patients with COVID-19 develops severe disease requiring hospitalization, while the majority, including high-risk individuals, experience mild symptoms. Severe disease has been associated with higher levels of antibodies and inflammatory cytokines but often among patients with diverse demographics and comorbidity status. This study evaluated hospitalized vs. ambulatory patients with COVID-19 with demographic risk factors for severe COVID-19: median age of 63, >80% male, and >85% black and/or Hispanic. Sera were collected four to 243 days after symptom onset and evaluated for binding and functional antibodies as well as 48 cytokines and chemokines. SARS-CoV-2-specific antibody levels and functions were similar in ambulatory and hospitalized patients. However, a strong correlation between anti-S2 antibody levels and the other antibody parameters, along with higher IL-27 levels, was observed in hospitalized but not ambulatory cases. These data indicate that antibodies against the relatively conserved S2 spike subunit and immunoregulatory cytokines such as IL-27 are potential immune determinants of COVID-19.

HIV-1 Envelope Glycosylation and the Signal Peptide.

The RV144 trial represents the only vaccine trial to demonstrate any protective effect against HIV-1 infection. While the reason(s) for this protection are still being evaluated, it serves as justification for widespread efforts aimed at developing new, more effective HIV-1 vaccines. Advances in our knowledge of HIV-1 immunogens and host antibody responses to these immunogens are crucial to informing vaccine design. While the envelope (Env) protein is the only viral protein present on the surface of virions, it exists in a complex trimeric conformation and is decorated with an array of variable N-linked glycans, making it an important but difficult target for vaccine design. Thus far, efforts to elicit a protective humoral immune response using structural mimics of native Env trimers have been unsuccessful. Notably, the aforementioned N-linked glycans serve as a component of many of the epitopes crucial for the induction of potentially protective broadly neutralizing antibodies (bnAbs). Thus, a greater understanding of Env structural determinants, most critically Env glycosylation, will no doubt be of importance in generating effective immunogens. Recent studies have identified the HIV-1 Env signal peptide (SP) as an important contributor to Env glycosylation. Further investigation into the mechanisms by which the SP directs glycosylation will be important, both in the context of understanding HIV-1 biology and in order to inform HIV-1 vaccine design.

The Probiotic Strain H. alvei HA4597® Improves Weight Loss in Overweight Subjects under Moderate Hypocaloric Diet: A Proof-of-Concept, Multicenter Randomized, Double-Blind Placebo-Controlled Study.

Background: Increasing evidence supports the role of the gut microbiota in the control of body weight and feeding behavior. Moreover, recent studies have reported that the probiotic strain Hafnia alvei HA4597® (HA), which produces the satietogenic peptide ClpB mimicking the effect of alpha-MSH, reduced weight gain and adiposity in rodent models of obesity. Methods: To investigate the clinical efficacy of HA, 236 overweight subjects were included, after written informed consent, in a 12-week prospective, double-blind, randomized study. All subjects received standardized counselling for a -20% hypocaloric diet and were asked to maintain their usual physical activity. Subjects of the HA group received two capsules per day providing 100 billion bacteria per day and subjects in the Placebo (P) group received two placebo capsules. The primary endpoint was the percentage of subjects achieving a weight loss of at least 3% after 12 weeks. Intention-to-treat statistical analysis was performed using exact-Fischer, Mann-Whitney and paired-Wilcoxon tests as appropriate. Results: In the HA group, significantly more subjects (+33%) met the primary endpoint than in the P group (54.9 vs. 41.4%, p = 0.048). In the HA group, an increased feeling of fullness (p = 0.009) and a greater loss of hip circumference (p < 0.001) at 12 weeks were also observed. Fasting glycemia at 12 weeks was significantly lower (p < 0.05) in the HA compared to P group. Clinical and biological tolerance was good in both groups. Conclusions: A 12-week treatment with the probiotic strain H. alvei HA4597® significantly improves weight loss, feeling of fullness and reduction of hip circumference in overweight subjects following moderate hypocaloric diet. These data support the use of H. alvei HA4597® in the global management of excess weight.

Effects of Bacterial CLPB Protein Fragments on Food Intake and PYY Secretion.

CLPB (Caseinolytic peptidase B) protein is a conformational mimetic of α-MSH, an anorectic hormone. Previous in vivo studies have already shown the potential effect of CLPB protein on food intake and on the production of peptide YY (PYY) by injection of E. coli wild type (WT) or E. coli ΔClpB. However, until now, no study has shown its direct effect on food intake. Furthermore, this protein can fragment naturally. Therefore, the aim of this study was (i) to evaluate the in vitro effects of CLPB fragments on PYY production; and (ii) to test the in vivo effects of a CLPB fragment sharing molecular mimicry with α-MSH (CLPB25) compared to natural fragments of the CLPB protein (CLPB96). To do that, a primary culture of intestinal mucosal cells from male Sprague-Dawley rats was incubated with proteins extracted from E. coli WT and ΔCLPB after fragmentation with trypsin or after a heat treatment of the CLPB protein. PYY secretion was measured by ELISA. CLPB fragments were analyzed by Western Blot using anti-α-MSH antibodies. In vivo effects of the CLPB protein on food intake were evaluated by intraperitoneal injections in male C57Bl/6 and ob/ob mice using the BioDAQ® system. The natural CLPB96 fragmentation increased PYY production in vitro and significantly decreased cumulative food intake from 2 h in C57Bl/6 and ob/ob mice on the contrary to CLPB25. Therefore, the anorexigenic effect of CLPB is likely the consequence of enhanced PYY secretion.

Detection of Antibody Responses against SARS-CoV-2 in Plasma and Saliva from Vaccinated and Infected Individuals.

Antibodies (Abs) are essential for the host immune response against SARS-CoV-2, and all the vaccines developed so far have been designed to induce Abs targeting the SARS-CoV-2 spike. Many studies have examined Ab responses in the blood from vaccinated and infected individuals. However, since SARS-CoV-2 is a respiratory virus, it is also critical to understand the mucosal Ab responses at the sites of initial virus exposure. Here, we examined plasma versus saliva Ab responses in vaccinated and convalescent patients. Although saliva levels were significantly lower, a strong correlation was observed between plasma and saliva total Ig levels against all SARS-CoV-2 antigens tested. Virus-specific IgG1 responses predominated in both saliva and plasma, while a lower prevalence of IgM and IgA1 Abs was observed in saliva. Antiviral activities of plasma Abs were also studied. Neutralization titers against the initial WA1 (D614G), B.1.1.7 (alpha) and B.1.617.2 (delta) strains were similar but lower against the B.1.351 (beta) strain. Spike-specific antibody-dependent cellular phagocytosis (ADCP) activities were also detected and the levels correlated with spike-binding Ig titers. Interestingly, while neutralization and ADCP potencies of vaccinated and convalescent groups were comparable, enhanced complement deposition to spike-specific Abs was noted in vaccinated versus convalescent groups and corresponded with higher levels of IgG1 plus IgG3 among the vaccinated individuals. Altogether, this study demonstrates the detection of Ab responses after vaccination or infection in plasma and saliva that correlate significantly, although Ig isotypic differences were noted. The induced plasma Abs displayed Fab-mediated and Fc-dependent functions with comparable neutralization and ADCP potencies, but a greater capacity to activate complement was elicited upon vaccination.

Detection of Antibody Responses Against SARS-CoV-2 in Plasma and Saliva From Vaccinated and Infected Individuals.

Antibodies (Abs) are essential for the host immune response against SARS-CoV-2, and all the vaccines developed so far have been designed to induce Abs targeting the SARS-CoV-2 spike. Many studies have examined Ab responses in the blood from vaccinated and infected individuals. However, since SARS-CoV-2 is a respiratory virus, it is also critical to understand the mucosal Ab responses at the sites of initial virus exposure. Here, we examined plasma versus saliva Ab responses in vaccinated and convalescent patients. Although saliva levels were significantly lower, a strong correlation was observed between plasma and saliva total Ig levels against all SARS-CoV-2 antigens tested. Virus-specific IgG1 responses predominated in both saliva and plasma, while a lower prevalence of IgM and IgA1 Abs was observed in saliva. Antiviral activities of plasma Abs were also studied. Neutralization titers against the initial WA1 (D614G), B.1.1.7 (alpha) and B.1.617.2 (delta) strains were similar but lower against the B.1.351 (beta) strain. Spike-specific antibody-dependent cellular phagocytosis (ADCP) activities were also detected and the levels correlated with spike-binding Ig titers. Interestingly, while neutralization and ADCP potencies of vaccinated and convalescent groups were comparable, enhanced complement deposition to spike-specific Abs was noted in vaccinated versus convalescent groups and corresponded with higher levels of IgG1 plus IgG3 among the vaccinated individuals. Altogether, this study demonstrates the detection of Ab responses after vaccination or infection in plasma and saliva that correlate significantly, although Ig isotypic differences were noted. The induced plasma Abs displayed Fab-mediated and Fc-dependent functions with comparable neutralization and ADCP potencies, but a greater capacity to activate complement was elicited upon vaccination.