A Novel Quantitative Multi-Component Serological Assay for SARS-CoV-2 Vaccine Evaluation.

A multi-component microarray, applying a novel analysis algorithm, was developed for quantitative evaluation of the SARS-CoV-2 vaccines' immunogenicity. The array enables simultaneous quantitation of IgG, IgM, and IgA, specific to the SARS-CoV-2 spike, receptor binding domain, and nucleocapsid proteins. The developed methodology is based on calculating an apparent immunoglobulin signal from the linear range of the fluorescent read-outs generated by scanning the microarray slides at different exposure times. A dedicated algorithm, employing a rigorous set of embedded conditions, then generates a normalized signal for each of the unique assays. Qualification of the multi-component array performance (evaluating linearity, extended dynamic-range, specificity, precision, and accuracy) was carried out with an in-house COVID-19, qRT-PCR positive serum, as well as pre-pandemic commercial negative sera. Results were compared to the WHO international standard for anti-SARS-CoV-2 immunoglobulins. Specific IgG, IgM, and IgA signals obtained by this array enabled successful discrimination between SARS-CoV-2 q-RT-PCR positive (seroconverted SARS-CoV-2 patients) and negative (naïve) samples. This array is currently used for evaluation of the humoral response to BriLife, the VSV-based Israeli vaccine during phase I/II clinical trials.

Preliminary nonclinical safety and immunogenicity of an rVSV-ΔG-SARS-CoV-2-S vaccine in mice, hamsters, rabbits and pigs.

rVSV-ΔG-SARS-CoV-2-S is a clinical stage (Phase 2) replication competent recombinant vaccine against SARS-CoV-2. To evaluate the safety profile of the vaccine, a series of non-clinical safety, immunogenicity and efficacy studies were conducted in four animal species, using multiple doses (up to 108 Plaque Forming Units/animal) and dosing regimens. There were no treatment-related mortalities or any noticeable clinical signs in any of the studies. Compared to unvaccinated controls, hematology and biochemistry parameters were unremarkable and no adverse histopathological findings. There was no detectable viral shedding in urine, nor viral RNA detected in whole blood or serum samples seven days post vaccination. The rVSV-ΔG-SARS-CoV-2-S vaccination gave rise to neutralizing antibodies, cellular immune responses, and increased lymphocytic cellularity in the spleen germinal centers and regional lymph nodes. No evidence for neurovirulence was found in C57BL/6 immune competent mice or in highly sensitive type I interferon knock-out mice. Vaccine virus replication and distribution in K18-human Angiotensin-converting enzyme 2-transgenic mice showed a gradual clearance from the vaccination site with no vaccine virus recovered from the lungs. The nonclinical data suggest that the rVSV-ΔG-SARS-CoV-2-S vaccine is safe and immunogenic. These results supported the initiation of clinical trials, currently in Phase 2.

rVSV-ΔG-SARS-CoV-2-S vaccine: repeated intramuscular (IM) toxicity, local tolerance, immunogenicity and biodistribution study in NZW rabbits.

BriLife®, a vector-based vaccine that utilizes the recombinant vesicular stomatitis virus (VSV) platform to express and present the spike antigen of SARS-CoV-2, is undergoing testing in a phase 2 clinical trial in Israel. A nonclinical repeated-dose (GLP) toxicity study in New Zealand white rabbits was performed to evaluate the potential toxicity, local tolerance, immunogenicity and biodistribution of the vaccine. rVSV-ΔG-SARS-CoV-2-S (or vehicle) was administered intramuscularly to two groups of animals (106, 107 PFU/animal, n = 10/sex/group) on three occasions, at 2-week intervals, followed by a 3-week recovery period. Systemic clinical signs, local reactions, body weight, body temperature, food consumption, ophthalmology, urinalysis, clinical pathology, C-reactive protein, viremia and antibody levels were monitored. Gross pathology was performed, followed by organs/tissues collection for biodistribution and histopathological evaluation. Treatment-related changes were restricted to multifocal minimal myofiber necrosis at the injection sites, and increased lymphocytic cellularity in the iliac and mesenteric lymph nodes and in the spleen. These changes were considered related to the inflammatory reaction elicited, and correlated with a trend for recovery. Detection of rVSV-ΔG-SARS-CoV-2-S vaccine RNA was noted in the regional iliac lymph node in animals assigned to the high-dose group, at both termination time points. A significant increase in binding and neutralizing antibody titers was observed following vaccination at both vaccine doses. In view of the findings, it was concluded that the rVSV-ΔG-SARS-CoV-2-S vaccine is safe. These results supported the initiation of clinical trials.

Galectin-3 is a sensor-regulator of toll-like receptor pathways in synovial fibroblasts.

Galectin-3 is a ß-galactoside-binding lectin that plays an important role in the modulation of immune responses. It has been shown to aggravate joint inflammation and destruction in experimental arthritis. We investigated the role of galectin-3 in TLR-induced cell activation in human synovial fibroblasts (SF) in order to better understand the mechanism(s) of the proinflammatory function of galectin-3 in arthritis. Galectin-3 expression in SF obtained from rheumatoid arthritis and osteoarthritis patients was inhibited by siRNA mediated gene-knockdown. Galectin-3 was also inhibited with modified citrus pectin (MCP), a polysaccharide galectin-3 ligand. Galectin-3 knockdown inhibited TLR-2, -3 and -4-induced IL-6 secretion, but not TLR-2, -3 and -4-mediated matrix metalloproteinase-3 or CC chemokine ligand-5 secretion. When the SF were stimulated with phorbol 12-myristate 13-acetate, a protein kinase C activator that bypasses the membranal receptors, galectin-3 knockdown no longer influenced IL-6 secretion. MCP reduced IL-6 levels in a dose-dependent manner. Our results indicate that galectin-3 is a positive sensor-regulator of TLR-induced IL-6 secretion in human synovial fibroblasts, thus adding new insights into the mechanisms by which galectin-3 augments synovial inflammation. These findings corroborate the potential role of glycan inhibitors of galectin-3 as a therapeutic approach for the treatment of inflammatory arthritis.

Predictors of reinfection with pre-Omicron and Omicron variants of concern among individuals who recovered from COVID-19 in the first year of the pandemic.

OBJECTIVES: The predictors of SARS-CoV-2 reinfection are unclear. We examined predictors of reinfection with pre-Omicron and Omicron variants among COVID-19-recovered individuals. METHODS: Randomly selected COVID-19-recovered patients (N = 1004) who donated convalescent plasma during 2020 were interviewed between August 2021 and March 2022 regarding COVID-19 vaccination and laboratory-proven reinfection. The sera from 224 (22.3%) participants were tested for antispike (anti-S) immunoglobulin G and neutralizing antibodies. RESULTS: The participants' median age was 31.1 years (78.6% males). The overall reinfection incidence rate was 12.8%; 2.7% versus 21.6% for the pre-Omicron (mostly Delta) versus Omicron variants. Negative associations were found between fever during the first illness and pre-Omicron reinfection: relative risk 0.29 (95% confidence interval 0.09-0.94), high anti-N level at first illness and Omicron reinfection: 0.53 (0.33-0.85), and overall reinfection: 0.56 (0.37-0.84), as well as between subsequent COVID-19 vaccination with the BNT162b2 vaccine and pre-Omicron 0.15 (0.07-0.32), Omicron 0.48 (0.25-0.45), and overall reinfections 0.38 (0.25-0.58). These variables significantly correlated with immunoglobulin G anti-S follow-up levels. High pre-existing anti-S binding and neutralizing antibody levels against the SARS-CoV-2 Wuhan and Alpha strains predicted protection against Omicron reinfections. CONCLUSION: Strong immune responses after the first COVID-19 infection and subsequent vaccination with the BNT162b2 vaccine provided cross-protection against reinfections with the Delta and Omicron variants.

Neutralization of SARS-CoV-2 Variants by rVSV-ΔG-Spike-Elicited Human Sera.

The emergence of rapidly spreading variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses a major challenge to the ability of vaccines and therapeutic antibodies to provide immunity. These variants contain mutations of specific amino acids that might impede vaccine efficacy. BriLife® (rVSV-ΔG-spike) is a newly developed SARS-CoV-2 vaccine candidate currently in phase II clinical trials. It is based on a replication-competent vesicular stomatitis virus (VSV) platform. The rVSV-ΔG-spike contains several spontaneously acquired spike mutations that correspond to SARS-CoV-2 variants' mutations. We show that human sera from BriLife® vaccinees preserve comparable neutralization titers towards alpha, gamma, and delta variants and show less than a three-fold reduction in the neutralization capacity of beta and omicron compared to the original virus. Taken together, we show that human sera from BriLife® vaccinees overall maintain a neutralizing antibody response against all tested variants. We suggest that BriLife®-acquired mutations may prove advantageous against future SARS-CoV-2 VOCs.

Longterm Efficacy of an Antipneumococcal Polysaccharide Vaccine among Patients with Autoimmune Inflammatory Rheumatic Diseases.

OBJECTIVE: To estimate the longterm humoral response of an antipneumococcal polysaccharide vaccine (PPSV23) in patients with rheumatoid arthritis (RA), psoriatic arthritis (PsA), ankylosing spondylitis (AS), or inflammatory bowel disease (IBD)-associated spondyloarthropathy (SpA), and the effect of demographic and clinical factors and treatment on the longterm efficacy of the vaccine. METHODS: A total of 145 consecutive patients treated with biologics [tumor necrosis factor-α (TNF-α) or interleukin 6 (IL-6) receptor inhibitors] or methotrexate (MTX) participated in this study. Fifteen were excluded because of absent information regarding their vaccination status (n = 9) or because of technical problems in obtaining their serum sample (n = 6). They were diagnosed with RA (n = 63, 48.5%), PsA (n = 29, 22.3%), AS (n = 28, 21.5%), or IBD-associated SpA (n = 3, 2.3%). Their mean age was 54.6 years, and 61.5% were women. Data were collected on the timing of vaccination, demographic and clinical characteristics, and treatment, and patients' serum antipneumococcal antibody levels were tested. RESULTS: Two-thirds of the patients (67.7%) had received PPSV23 45 months (mean) earlier. Treatment included TNF-α inhibitors (73.9%), IL-6 receptor inhibitors (13.1%), or MTX without a biological treatment (13%). The uptake of vaccination was significantly higher in the older population (> 65 yrs). Vaccinated patients had significantly higher antibody levels compared with vaccine-naive patients. The antibody levels had been preserved after 10 years. MTX use, but not biologics, was associated with significantly lower antibody levels. CONCLUSION: The longterm efficacy of the PPSV23 vaccination seems to be preserved among patients with RA, PsA, AS, and IBD-associated SpA for at least 10 years. Efficacy is slightly impaired by MTX, but it is not affected by biologics. These findings suggest that revaccination after 5 years might not be needed for all, and testing the antibody titers should be considered to identify those who may benefit from revaccination.

Prevalence of TNF-α blocker immunogenicity in psoriatic arthritis.

OBJECTIVE: The longterm use of tumor necrosis factor (TNF)-α blockers is limited by the formation of neutralizing antibodies. To the best of our knowledge, immunogenicity in psoriatic arthritis (PsA) has not been investigated in depth. Our objective was to evaluate the prevalence and significance of TNF-α blocker immunogenicity in PsA. METHODS: Consecutive patients with PsA treated with either infliximab (IFX), adalimumab (ADA), or etanercept (ETN) > 3 months participated in our cross-sectional study. Their demographic and clinical characteristics, skin and joint disease activity, and records of use of methotrexate (MTX) and other medications were collected. Drug levels (ELISA) and antidrug antibodies (ADAb; Bridging ELISA) were evaluated before the next injection or infusion. RESULTS: A total of 93 patients with PsA were recruited (48 receiving ADA, 24 IFX, and 21 ETN), with a mean age of 53 years (range 21-83 yrs), composed of 53% women. One-fourth of the patients were concomitantly treated with MTX. Altogether, 77% of the patients demonstrated therapeutic drug levels. High levels of ADAb were found in 29% of patients taking ADA, 21% taking IFX, and 0% taking ETN. ADAb significantly correlated with lower drug levels, higher 28-joint Disease Activity Scores, and higher global assessments. MTX use correlated significantly with a lower prevalence of ADAb. CONCLUSION: Significant levels of ADAb were present in up to 29% of patients with PsA treated with ADA or IFX. ADAb clearly correlated with low therapeutic drug levels and higher disease activity variables. The use of MTX significantly decreased ADAb prevalence, and its use should be strongly considered in combination with TNF-α blocker antibodies in patients with PsA.

In vivo characterization of tumor and tumor vascular network using multi-modal imaging approach.

We present a multi-modal optical diagnostic approach utilizing a combined use of Fluorescence Intravital Microscopy (FIM), Dynamic Light Scattering (DLS) and Spectrally Enhanced Microscopy (SEM) modalities for in vivo imaging of tumor vascular network and blood microcirculation. FIM is used for imaging of tumor surroundings and microenvironment, SEM provides information regarding blood vessels topography, whereas DLS is applied for functional imaging of vascular network and blood microcirculation. This complementary combination of the imaging approaches is extremely useful for functional in vivo imaging of blood vasculature and tumor microenvironment. The technique has also a great potential in vascular biology and can significantly expand the capabilities of tumor angiogenesis studies and notably contribute to the development of cancer treatment.

Permanent occlusion of feeding arteries and draining veins in solid mouse tumors by vascular targeted photodynamic therapy (VTP) with Tookad.

BACKGROUND: Antiangiogenic and anti-vascular therapies present intriguing alternatives to cancer therapy. However, despite promising preclinical results and significant delays in tumor progression, none have demonstrated long-term curative features to date. Here, we show that a single treatment session of Tookad-based vascular targeted photodynamic therapy (VTP) promotes permanent arrest of tumor blood supply by rapid occlusion of the tumor feeding arteries (FA) and draining veins (DV), leading to tumor necrosis and eradication within 24-48 h. METHODOLOGY/PRINCIPAL FINDINGS: A mouse earlobe MADB106 tumor model was subjected to Tookad-VTP and monitored by three complementary, non-invasive online imaging techniques: Fluorescent intravital microscopy, Dynamic Light Scattering Imaging and photosensitized MRI. Tookad-VTP led to prompt tumor FA vasodilatation (a mean volume increase of 70%) with a transient increase (60%) in blood-flow rate. Rapid vasoconstriction, simultaneous blood clotting, vessel permeabilization and a sharp decline in the flow rates then followed, culminating in FA occlusion at 63.2 sec+/-1.5SEM. This blockage was deemed irreversible after 10 minutes of VTP treatment. A decrease in DV blood flow was demonstrated, with a slight lag from FA response, accompanied by frequent changes in flow direction before reaching a complete standstill. In contrast, neighboring, healthy tissue vessels of similar sizes remained intact and functional after Tookad-VTP. CONCLUSION/SIGNIFICANCE: Tookad-VTP selectively targets the tumor feeding and draining vessels. To the best of our knowledge, this is the first mono-therapeutic modality that primarily aims at the larger tumor vessels and leads to high cure rates, both in the preclinical and clinical arenas.