Rapid detection of vaccinia virus using biofunctionalized fiber-optic ball-tip biosensors.

In this work, we present the development and biofunctionalization of a fiber-optic ball-resonator biosensor for the real-time detection of vaccinia poxvirus. We fabricated several ball-tip resonators, functionalized through a silanization process to immobilize two bioreceptors: the monoclonal anti-L1R antibody targeting the L1R protein, and the polyclonal rabbit serum antibodies targeting the whole vaccinia virus (VV) pathogen. Experimental measurements were carried out to detect VV in concentrations from 103 to 108 plaque-forming units (PFU), with a limit of detection of around 1.7-4.3 × 103 PFU and a log-quadratic pattern, with a response up to 5 × 10-4 RIU (refractive index units). The specificity was assessed against herpes simplex virus, used as a non-specific control, with the best results obtained with anti-L1R monoclonal antibodies, and through the detection of vaccinia virus/herpes simplex-1 combination. The obtained results provide a real-time viral recognition with a label-free sensing platform, having rapid response and ease of manufacturing, and paving the road to the seamless detection of poxviruses affecting different human and animal species using optical fibers.

Rational Engineering of a Sub-Picomolar HIV-1 Blocker.

With the aim of rationally devising a refined and potent HIV-1 blocker, the cDNA of CCL5 5p12 5m, an extremely potent CCR5 antagonist, was fused to that of C37, a gp41-targeted fusion inhibitor. The resulting CCL5 5p12 5m-C37 fusion protein was expressed in E. coli and proved to be capable of inhibiting R5 HIV-1 strains with low to sub-picomolar IC50, maintaining its antagonism toward CCR5. In addition, CCL5 5p12 5m-C37 inhibits R5/X4 and X4 HIV-1 strains in the picomolar concentration range. The combination of CCL5 5p12 5m-C37 with tenofovir (TDF) exhibited a synergic effect, promoting this antiviral cocktail. Interestingly, a CCR5-targeted combination of maraviroc (MVC) with CCL5 5p12 5m-C37 led to a synergic effect that could be explained by an extensive engagement of different CCR5 conformational populations. Within the mechanism of HIV-1 entry, the CCL5 5p12 5m-C37 chimera may fit as a powerful blocker in several instances. In its possible consideration for systemic therapy or pre-exposure prophylaxis, this protein design represents an interesting lead in the combat of HIV-1 infection.

Role of periostin in inflammatory bowel disease development and synergistic effects mediated by the CCL5-CCR5 axis.

Inflammatory bowel disease (IBD), comprising mainly Crohn's disease (CD) and ulcerative colitis (UC), is a chronic inflammatory disease of the gastrointestinal tract. In recent years, a wealth of data has been accumulated demonstrating the complex interplay of many different factors in the pathogenesis of IBD. Among these are factors impacting the epithelial barrier function, including vessel and extracellular matrix (ECM) formation, the gut microbiome (e.g., bacterial antigens), and, most importantly, the production of cytokines (pro- and anti-inflammatory) directly shaping the immune response. Patients failing to resolve the acute intestinal inflammation develop chronic inflammation. It has been shown that the expression of the matricellular protein periostin is enhanced during IBD and is one of the drivers of this disease. The C-C chemokine receptor 5 (CCR5) is engaged by the chemotactic mediators CCL3/MIP-1α, CCL4/MIP-1ß, and CCL5/RANTES. CCR5 blockade has been reported to ameliorate inflammation in a murine IBD model. Thus, both periostin and CCR5 are involved in the development of IBD. In this study, we investigated the potential crosstalk between the two signaling systems and tested a highly potent CCL5 derivative acting as a CCR5 antagonist in a murine model of IBD. We observed that the absence of periostin influences the CCR5-expressing cell population of the gut. Our data further support the notion that targeted modulation of the periostin and CCR5 signaling systems bears therapeutic potential for IBD.

Filling the Gaps in Antagonist CCR5 Binding, a Retrospective and Perspective Analysis.

The large number of pathologies that position CCR5 as a central molecular determinant substantiates the studies aimed at understanding receptor-ligand interactions, as well as the development of compounds that efficiently block this receptor. This perspective focuses on CCR5 antagonism as the preferred landscape for therapeutic intervention, thus the receptor active site occupancy by known antagonists of different origins is overviewed. CCL5 is a natural agonist ligand for CCR5 and an extensively studied scaffold for CCR5 antagonists production through chemokine N-terminus modification. A retrospective 3D modeling analysis on recently developed CCL5 mutants and their contribution to enhanced anti-HIV-1 activity is reported here. These results allow us to prospect the development of conceptually novel amino acid substitutions outside the CCL5 N-terminus hotspot. CCR5 interaction improvement in regions distal to the chemokine N-terminus, as well as the stabilization of the chemokine hydrophobic core are strategies that influence binding affinity and stability beyond the agonist/antagonist dualism. Furthermore, the development of allosteric antagonists topologically remote from the orthosteric site (e.g., intracellular or membrane-embedded) is an intriguing new avenue in GPCR druggability and thus a conceivable novel direction for CCR5 blockade. Ultimately, the three-dimensional structure elucidation of the interaction between various ligands and CCR5 helps illuminate the active site occupancy and mechanism of action.

Ultralow Limit Detection of Soluble HER2 Biomarker in Serum with a Fiber-Optic Ball-Tip Resonator Assisted by a Tilted FBG.

An optical-fiber biosensor has been developed for the detection of the breast cancer biomarker soluble human epidermal growth factor receptor-2 (sHER2). The sensor was fabricated by combining a tilted fiber Bragg grating (TFBG) with a ball resonator, allowing us to achieve an excellent sensitivity compared to other optical-fiber-based sensors. The sensor exhibits a resonance comb excited by the TFBG and the spectral profile of the ball resonator. The detection of sHER2 at extremely low concentrations was carried out by tracking the amplitude change of selected resonances. The therapeutic anti-HER2 monoclonal antibody Trastuzumab has been used to functionalize the biosensor with silane surface chemistry. The sensor features a sensitivity of 4034 dB/RIU with a limit of detection (LoD) in buffer and in a 1/10 diluted serum of 151.5 ag/mL and 3.7 pg/mL, respectively. At relatively high protein concentrations (64 ng/mL) binding to sHER (7.36 dB) as compared to control proteins (below 0.7 dB) attested the high specificity of sHER2 detection.

Perspectives on Assembling Coronavirus Spikes on Fiber Optics to Reveal Broadly Recognizing Antibodies and Generate a Universal Coronavirus Detector.

In time of COVID-19 biological detection technologies are of crucial relevance. We propose here the use of state of the art optical fiber biosensors to address two aspects of the fight against SARS-CoV-2 and other pandemic human coronaviruses (HCoVs). Fiber optic biosensors functionalized with HCoV spikes could be used to discover broadly neutralizing antibodies (bnAbs) effective against known HCoVs (SARS-CoV, MERS-CoV and SARS-CoV-2) and likely future ones. In turn, identified bnAbs, once immobilized onto fiber optic biosensors, should be capable to detect HCoVs as diagnostic and environmental sensing devices. The therapeutic and preventative value of bnAbs is immense as they can be used for passive immunization and for the educated development of a universal vaccine (active immunization). Hence, HCoV bnAbs represent an extremely important resource for future preparedness against coronavirus-borne pandemics. Furthermore, the assembly of bnAb-based biosensors constitutes an innovative approach to counteract public health threats, as it bears diagnostic competence additional to environmental detection of a range of pandemic strains. This concept can be extended to different pandemic viruses, as well as bio-warfare threats that entail existing, emerging and extinct viruses (e.g., the smallpox-causing Variola virus). We report here the forefront fiber optic biosensor technology that could be implemented to achieve these aims.

Effects of omalizumab on basophils: Potential biomarkers in asthma and chronic spontaneous urticaria.

Omalizumab is an anti-IgE humanized monoclonal antibody approved for the treatment of severe asthma and chronic spontaneous urticaria. Omalizumab binds free serum IgE and antagonizes its interaction with FcεRI, which is considered the main pharmacodynamic mechanism responsible for the clinical response to the treatment. The reduction of IgE serum concentration down-regulates the cellular expression of FcεRI on basophils. However, the biological events occurring on basophils during the therapy with omalizumab are multiple and complex. Here we review the current evidence regarding the specific biological effects of omalizumab on basophils in patients with asthma and chronic spontaneous urticaria. In addition to the modulation of IgE receptors, omalizumab may affect basophils homeostasis, intra-cellular signaling, cellular responsiveness/activation and cytokine release. These effects may be partially responsible for the clinical success of omalizumab and potentially provide useful biological markers for future assessment of the clinical response to the treatment. However, further investigation is required to better elucidate the role of basophils during the treatment with omalizumab.

Staphylococcus aureus Infection and Persistence in Chronic Rhinosinusitis: Focus on Leukocidin ED.

Chronic rhinosinusitis (CRS) is thought to be a multifactorial disease that includes a direct involvement of bacteria that trigger inflammation and contribute to CRS pathogenesis. Staphylococcus aureus infection and persistence is associated with chronic rhinosinusitis (CRS), and it may be particularly relevant in the form with nasal polyps (CRSwNP). The large array of exotoxins deployed by S. aureus is instrumental for the bacterium to warrant its infection and dissemination in different human body districts. Here, we analyze the common Th2 environment in CRSwNP and prospect a possible dynamic role played by S. aureus leukocidins in promoting this chronic inflammation, considering leukocidin ED (LukED) as a strong prototype candidate worth of therapeutic investigation. CCR5 is an essential target for LukED to exert its cytotoxicity towards T cells, macrophages and dendritic cells. Therefore, CCR5 blockade might be an interesting therapeutic option for CRS and, more specifically, persistent and relapsing CRSwNP. In this perspective, the arsenal of CCR5 antagonists being developed to inhibit HIV-1 entry (CCR5 being the major HIV-1 co-receptor) could be easily repurposed for CRS therapeutic investigation. Finally, direct targeting of LukED by neutralizing antibodies could represent an important additional solution to S. aureus infection.

Prepare for the Future: Dissecting the Spike to Seek Broadly Neutralizing Antibodies and Universal Vaccine for Pandemic Coronaviruses.

Learning from the lengthy fight against HIV-1, influenza, and Ebola virus infection, broadly neutralizing antibodies (bnAbs), directed at conserved regions of surface proteins crucial to virus entry (Env, hemagglutinin, and GP, respectively), are an essential resource for passive as well as active immunization. Rare in their emergence and antigen recognition mode, bnAbs are active toward a large set of different viral strains. Isolation, characterization and production of bnAbs lead to their possible use in passive immunotherapy and form the basis for an educated effort in the development of vaccines for universal coverage. SARS-CoV-2-specific antibodies targeting the spike receptor binding domain (RBD) may lead to antibody dependent enhancement (ADE) of infection, possibly hampering the field of vaccine development. This perspective points to the identification of conserved regions in the spike of SARS-CoV-2, SARS-CoV, and MERS-CoV through investigation, dissection and recombinant production of isolated moieties. These spike moieties should be capable of independent folding and allow the detection as well as the elicitation of bnAbs, thus setting the basis for an effective passive immunotherapy and the development of a universal vaccine against human epidemic coronaviruses (HCoVs). SARS, MERS and, most of all, COVID-19 demonstrate that humanity is the target of HCoV, preparedness for future hits is thus no longer an option.

Functionalized etched tilted fiber Bragg grating aptasensor for label-free protein detection.

An aptasensor based on etched tilted fiber Bragg grating (eTFBG) is developed on a single-mode optical fiber targeting biomolecule detection. TFBGs were chemically etched using hydrofluoric acid (HF) to partially remove the fiber cladding. The sensor response was coarsely interrogated, resulting on a sensitivity increase from 1.25 nm/RIU (refractive index unit) at the beginning of the process, up to 23.38 nm/RIU at the end of the etching, for a RI range from 1.3418 to 1.4419 RIU. The proposed aptasensor showed improved RI sensitivity as compared to the unetched TFBG, without requiring metal depositions on the fiber surface or polarization control during the measurements. The proposed sensor was tested for the detection of thrombin-aptamer interactions based on silane-coupling surface chemistry, with thrombin concentrations ranging from 2.5 to 40 nM. Functionalized eTFBGs provided a competitive platform for biochemical interaction measurements, showing sensitivity values ranging from 2.3 to 3.3 p.m./nM for the particular case of thrombin detection.

AllergoOncology: Microbiota in allergy and cancer-A European Academy for Allergy and Clinical Immunology position paper.

The microbiota can play important roles in the development of human immunity and the establishment of immune homeostasis. Lifestyle factors including diet, hygiene, and exposure to viruses or bacteria, and medical interventions with antibiotics or anti-ulcer medications, regulate phylogenetic variability and the quality of cross talk between innate and adaptive immune cells via mucosal and skin epithelia. More recently, microbiota and their composition have been linked to protective effects for health. Imbalance, however, has been linked to immune-related diseases such as allergy and cancer, characterized by impaired, or exaggerated immune tolerance, respectively. In this AllergoOncology position paper, we focus on the increasing evidence defining the microbiota composition as a key determinant of immunity and immune tolerance, linked to the risk for the development of allergic and malignant diseases. We discuss novel insights into the role of microbiota in disease and patient responses to treatments in cancer and in allergy. These may highlight opportunities to improve patient outcomes with medical interventions supported through a restored microbiome.

Rational CCL5 mutagenesis integration in a lactobacilli platform generates extremely potent HIV-1 blockers.

Efforts to improve existing anti-HIV-1 therapies or develop preventatives have identified CCR5 as an important target and CCL5 as an ideal scaffold to sculpt potent HIV-1 entry inhibitors. We created novel human CCL5 variants that exhibit exceptional anti-HIV-1 features using recombinant lactobacilli (exploited for live microbicide development) as a screening platform. Protein design, expression and anti-HIV-1 activity flowed in iterative cycles, with a stepwise integration of successful mutations and refinement of an initial CCL5 mutant battery towards the generation of two ultimate CCL5 derivatives, a CCR5 agonist and a CCR5 antagonist with similar anti-HIV-1 potency. The CCR5 antagonist was tested in human macrophages and against primary R5 HIV-1 strains, exhibiting cross-clade low picomolar IC50 activity. Moreover, its successful combination with several HIV-1 inhibitors provided the ground for conceiving therapeutic and preventative anti-HIV-1 cocktails. Beyond HIV-1 infection, these CCL5 derivatives may now be tested against several inflammation-related pathologies where the CCL5:CCR5 axis plays a relevant role.

The Expanding Therapeutic Perspective of CCR5 Blockade.

CCR5 and its interaction with chemokine ligands have been crucial for understanding and tackling HIV-1 entry into target cells. However, over time, CCR5 has witnessed an impressive transition from being considered rather unimportant in physiology and pathology to becoming central in a growing number of pathophysiological conditions. It now turns out that the massive efforts devoted to combat HIV-1 entry by interfering with CCR5, and the subsequent production of chemokine ligand variants, small chemical compounds, and other molecular entities and strategies, may set the therapeutic standards for a wealth of different pathologies. Expressed on various cell types, CCR5 plays a vital role in the inflammatory response by directing cells to sites of inflammation. Aside HIV-1, CCR5 has been implicated in other infectious diseases and non-infectious diseases such as cancer, atherosclerosis, and inflammatory bowel disease. Individuals carrying the CCR5Δ32 mutation live a normal life and are warranted a natural barrier to HIV-1 infection. Therefore, CCR5 antagonism and gene-edited knockout of the receptor gained growing interest for the therapeutic role that CCR5 blockade may play in the attenuation of the severity or progression of numerous diseases.

Cutting Edge: IgE Plays an Active Role in Tumor Immunosurveillance in Mice.

Exogenous IgE acts as an adjuvant in tumor vaccination in mice, and therefore a direct role of endogenous IgE in tumor immunosurveillance was investigated. By using genetically engineered mice, we found that IgE ablation rendered mice more susceptible to the growth of transplantable tumors. Conversely, a strengthened IgE response provided mice with partial or complete resistance to tumor growth, depending on the tumor type. By genetic crosses, we showed that IgE-mediated tumor protection was mostly lost in mice lacking FcεRI. Tumor protection was also lost after depletion of CD8(+) T cells, highlighting a cross-talk between IgE and T cell-mediated tumor immunosurveillance. Our findings provide the rationale for clinical observations that relate atopy with a lower risk for developing cancer and open new avenues for the design of immunotherapeutics relevant for clinical oncology.

Combination of the CCL5-derived peptide R4.0 with different HIV-1 blockers reveals wide target compatibility and synergic cobinding to CCR5.

R4.0, a synthetic CCL5/RANTES-derived peptide, exerts potent anti-HIV-1 activity via its nonactivating interaction with CCR5, the major HIV-1 coreceptor. CCR5 chronic activation may promote undesirable inflammatory effects and enhance viral infection; thus, receptor antagonism is a necessary requisite. HIV-1 gp120, CCL5, and maraviroc dock on CCR5 by sharing two receptor sites: the N terminus and the second extracellular loop. In combination studies, R4.0, CCL5, and maraviroc exhibited concomitant interactions with CCR5 and promoted synergic inhibition of HIV-1 in acute-infection assays. Furthermore, various degrees of additive/synergic HIV-1 inhibition were observed when R4.0 was tested in combination with drugs and lead compounds directed toward different viral targets (gp120, gp41, reverse transcriptase, and protease). In combination with tenofovir, R4.0 provides cross-clade synergic inhibition of primary HIV-1 isolates. Remarkably, an in vitro-generated maraviroc-resistant R5 HIV-1 strain was inhibited by R4.0 comparably to the wild-type strain, suggesting the presence of viral resistance barriers similar to those reported for CCL5. Overall, R4.0 appears to be a promising lead peptide with potential for combination in anti-HIV-1 therapy and in microbicide development to prevent sexual HIV-1 transmission.

Enhancement of anti-HIV-1 activity by hot spot evolution of RANTES-derived peptides.

CCR5, the major HIV-1 coreceptor, is a primary target for HIV-1 entry inhibition strategies. CCL5/RANTES, a natural CCR5 ligand, is one of the most potent HIV-1 entry inhibitors and, therefore, an ideal candidate to derive HIV-1 blockers. Peptides spanning the RANTES N-loop/ß1-strand region act as specific CCR5 antagonists, with their hydrophobic N- and C termini playing a crucial role in virus blockade. Here, hydrophobic surfaces were enhanced by tryptophan substitution of aromatic residues, highlighting position 27 as a critical hot spot for HIV-1 blockade. In a further molecular evolution step, C-terminal engraftment of RANTES 40' loop produced a peptide with the highest solubility and anti-HIV-1 activity. These modified peptides represent leads for the development of effective HIV-1 inhibitors and microbicides.

In search of a vaccine for mouse allergy: significant reduction of Mus m 1 allergenicity by structure-guided single-point mutations.

BACKGROUND: Mouse urinary proteins are relevant allergens from mice urine. We used the recombinant protein Mus m 1 as an allergen model to identify if, by altering Mus m 1 architecture via single-point mutations, we could effectively modify its allergenicity. METHODS: Based on structural considerations, we synthesized two single-point mutants, Mus m 1-Y120A and Mus m 1-Y120F, which were expected to harbor large structural alterations. Circular dichroism and fluorescence analysis showed significant conformational rearrangements of the aromatic side chains in the internal cavity of Mus m 1-Y120A when compared to Mus m 1-Y120F and Mus m 1. Evaluation of the allergenic potential of the recombinant molecules was performed in vitro with both immunochemical approaches and assays based on the measurement of basophil degranulation. Moreover, to assess the integrity of the T cell epitopes and as an in vitro measure of immunogenicity, we tested the reactivity of T lymphocytes from subjects allergic to mouse urine against proteins and synthetic peptides encompassing the immunodominant linear epitope containing the mutation. RESULTS: We found that the selected point mutation was able to modulate the protein allergenicity, and to severely impair the recognition of Mus m 1 by IgE, while T cell reactivity was fully maintained. CONCLUSIONS: In silico predicted, minimum selected structural modifications allowed to design one protein with reduced allergenicity and preserved immunogenicity. Structurally guided mutations can direct the design of proteins with reduced allergenicity which can be used as vaccines for a safer and more effective immunotherapy of allergic disorders.

An antitumor cellular vaccine based on a mini-membrane IgE.

The IgE-mediated immune system activation can be redirected to combat tumors. Mouse and human IgE have been shown to provide a potent adjuvant effect in antitumor vaccination, with a crucial role played by FcεRI. This effect results from T cell-mediated adaptive immune response. Modified vaccinia virus Ankara (MVA) has been used to infect IgE-loaded tumor cells. These results led to a shift toward a highly safe protocol employing membrane IgE (mIgE), thus eliminating any possible anaphylactogenicity caused by circulating IgE. Evidence that human mIgE and a truncated version lacking IgE Fabs (tmIgE) bind and activate FcεRI has been fundamental and forms the core of this report. Human tmIgE has been engineered into a recombinant MVA (rMVA-tmIgE), and the expression of tmIgE and its transport to the surface of rMVA-tmIgE-infected cells has been detected by Western blot and cytofluorimetry, respectively. FcεRI activation by tmIgE has been confirmed by the release of ß-hexosaminidase in a cell-to-cell contact assay using human FcεRI-transfected RBL-SX38 cells. The rMVA-tmIgE antitumor vaccination strategy has been investigated in FcεRIα(-/-) human FcεRIα(+) mice, with results indicating a level of protection comparable to that obtained using soluble human IgE tumor cell loading. The rMVA-tmIgE vector represents a device that suits safe IgE-based antitumor vaccines, harboring the possibility to couple tmIgE with other gene insertions that might enhance the antitumor effect, thus bringing the field closer to the clinics.

Molecular engineering of RANTES peptide mimetics with potent anti-HIV-1 activity.

The chemokine receptor CCR5 is utilized as a critical coreceptor by most primary HIV-1 strains. While the lack of structural information on CCR5 has hampered the rational design of specific inhibitors, mimetics of the chemokines that naturally bind CCR5 can be molecularly engineered. We used a structure-guided approach to design peptide mimetics of the N-loop and ß1-strand regions of regulated on activation normal T-cell-expressed and secreted (RANTES)/CCL5, which contain the primary molecular determinants of HIV-1 blockade. Rational modifications were sequentially introduced into the N-loop/ß1-strand sequence, leading to the generation of mimetics with potent activity against a broad spectrum of CCR5-specific HIV-1 isolates (IC(50) range: 104-640 nM) but lacking activity against CXCR4-specific HIV-1 isolates. Functional enhancement was initially achieved with the stabilization of the N loop in the ß-extended conformation adopted in full-length RANTES, as confirmed by nuclear magnetic resonance (NMR) analysis. However, the most dramatic increase in antiviral potency resulted from the engraftment of an in silico-optimized linker segment designed using de novo structure-prediction algorithms to stabilize the C-terminal α-helix and experimentally validated by NMR. Our mimetics exerted CCR5-antagonistic effects, demonstrating that the antiviral and proinflammatory functions of RANTES can be uncoupled. RANTES peptide mimetics provide new leads for the development of safe and effective HIV-1 entry inhibitors.