Mucosal Targeting of a BoNT/A Subunit Vaccine Adjuvanted with a Mast Cell Activator Enhances Induction of BoNT/A Neutralizing Antibodies in Rabbits

We previously reported that the immunogenicity of Hcβtre, a botulinum neurotoxin A (BoNT/A) immunogen, was enhanced by fusion to an epithelial cell binding domain, Ad2F, when nasally delivered to mice with cholera toxin (CT). This study was performed to determine if Ad2F would enhance the nasal immunogenicity of Hcβtre in rabbits, an animal model with a nasal cavity anatomy similar to humans. Since CT is not safe for human use, we also tested the adjuvant activity of compound 48/80 (C48/80), a mast cell activating compound previously determined to safely exhibit nasal adjuvant activity in mice.New Zealand White or Dutch Belted rabbits were nasally immunized with Hcβtre or Hcβtre-Ad2F alone or combined with CT or C48/80, and serum samples were tested for the presence of Hcβtre-specific binding (ELISA) or BoNT/A neutralizing antibodies.Hcβtre-Ad2F nasally administered with CT induced serum anti-Hcβtre IgG ELISA and BoNT/A neutralizing antibody titers greater than those induced by Hcβtre + CT. C48/80 provided significant nasal adjuvant activity and induced BoNT/A-neutralizing antibodies similar to those induced by CT.Ad2F enhanced the nasal immunogenicity of Hcβtre, and the mast cell activator C48/80 was an effective adjuvant for nasal immunization in rabbits, an animal model with a nasal cavity anatomy similar to that in humans

Symptom-based stratification of patients with primary Sjögren's syndrome: multi-dimensional characterisation of international observational cohorts and reanalyses of randomised clinical trials

Background Heterogeneity is a major obstacle to developing effective treatments for patients with primary Sjögren's syndrome. We aimed to develop a robust method for stratification, exploiting heterogeneity in patient-reported symptoms, and to relate these differences to pathobiology and therapeutic response. Methods We did hierarchical cluster analysis using five common symptoms associated with primary Sjögren's syndrome (pain, fatigue, dryness, anxiety, and depression), followed by multinomial logistic regression to identify subgroups in the UK Primary Sjögren's Syndrome Registry (UKPSSR). We assessed clinical and biological differences between these subgroups, including transcriptional differences in peripheral blood. Patients from two independent validation cohorts in Norway and France were used to confirm patient stratification. Data from two phase 3 clinical trials were similarly stratified to assess the differences between subgroups in treatment response to hydroxychloroquine and rituximab. Findings In the UKPSSR cohort (n=608), we identified four subgroups: Low symptom burden (LSB), high symptom burden (HSB), dryness dominant with fatigue (DDF), and pain dominant with fatigue (PDF). Significant differences in peripheral blood lymphocyte counts, anti-SSA and anti-SSB antibody positivity, as well as serum IgG, κ-free light chain, β2-microglobulin, and CXCL13 concentrations were observed between these subgroups, along with differentially expressed transcriptomic modules in peripheral blood. Similar findings were observed in the independent validation cohorts (n=396). Reanalysis of trial data stratifying patients into these subgroups suggested a treatment effect with hydroxychloroquine in the HSB subgroup and with rituximab in the DDF subgroup compared with placebo. Interpretation Stratification on the basis of patient-reported symptoms of patients with primary Sjögren's syndrome revealed distinct pathobiological endotypes with distinct responses to immunomodulatory treatments. Our data have important implications for clinical management, trial design, and therapeutic development. Similar stratification approaches might be useful for patients with other chronic immune-mediated diseases. Funding UK Medical Research Council, British Sjogren's Syndrome Association, French Ministry of Health, Arthritis Research UK, Foundation for Research in Rheumatology

Multiorgan MRI findings after hospitalisation with COVID-19 in the UK (C-MORE): a prospective, multicentre, observational cohort study

Introduction: The multiorgan impact of moderate to severe coronavirus infections in the post-acute phase is still poorly understood. We aimed to evaluate the excess burden of multiorgan abnormalities after hospitalisation with COVID-19, evaluate their determinants, and explore associations with patient-related outcome measures. Methods: In a prospective, UK-wide, multicentre MRI follow-up study (C-MORE), adults (aged ≥18 years) discharged from hospital following COVID-19 who were included in Tier 2 of the Post-hospitalisation COVID-19 study (PHOSP-COVID) and contemporary controls with no evidence of previous COVID-19 (SARS-CoV-2 nucleocapsid antibody negative) underwent multiorgan MRI (lungs, heart, brain, liver, and kidneys) with quantitative and qualitative assessment of images and clinical adjudication when relevant. Individuals with end-stage renal failure or contraindications to MRI were excluded. Participants also underwent detailed recording of symptoms, and physiological and biochemical tests. The primary outcome was the excess burden of multiorgan abnormalities (two or more organs) relative to controls, with further adjustments for potential confounders. The C-MORE study is ongoing and is registered with ClinicalTrials.gov, NCT04510025. Findings: Of 2710 participants in Tier 2 of PHOSP-COVID, 531 were recruited across 13 UK-wide C-MORE sites. After exclusions, 259 C-MORE patients (mean age 57 years [SD 12]; 158 [61%] male and 101 [39%] female) who were discharged from hospital with PCR-confirmed or clinically diagnosed COVID-19 between March 1, 2020, and Nov 1, 2021, and 52 non-COVID-19 controls from the community (mean age 49 years [SD 14]; 30 [58%] male and 22 [42%] female) were included in the analysis. Patients were assessed at a median of 5·0 months (IQR 4·2–6·3) after hospital discharge. Compared with non-COVID-19 controls, patients were older, living with more obesity, and had more comorbidities. Multiorgan abnormalities on MRI were more frequent in patients than in controls (157 [61%] of 259 vs 14 [27%] of 52; p<0·0001) and independently associated with COVID-19 status (odds ratio [OR] 2·9 [95% CI 1·5–5·8]; padjusted=0·0023) after adjusting for relevant confounders. Compared with controls, patients were more likely to have MRI evidence of lung abnormalities (p=0·0001; parenchymal abnormalities), brain abnormalities (p<0·0001; more white matter hyperintensities and regional brain volume reduction), and kidney abnormalities (p=0·014; lower medullary T1 and loss of corticomedullary differentiation), whereas cardiac and liver MRI abnormalities were similar between patients and controls. Patients with multiorgan abnormalities were older (difference in mean age 7 years [95% CI 4–10]; mean age of 59·8 years [SD 11·7] with multiorgan abnormalities vs mean age of 52·8 years [11·9] without multiorgan abnormalities; p<0·0001), more likely to have three or more comorbidities (OR 2·47 [1·32–4·82]; padjusted=0·0059), and more likely to have a more severe acute infection (acute CRP >5mg/L, OR 3·55 [1·23–11·88]; padjusted=0·025) than those without multiorgan abnormalities. Presence of lung MRI abnormalities was associated with a two-fold higher risk of chest tightness, and multiorgan MRI abnormalities were associated with severe and very severe persistent physical and mental health impairment (PHOSP-COVID symptom clusters) after hospitalisation. Interpretation: After hospitalisation for COVID-19, people are at risk of multiorgan abnormalities in the medium term. Our findings emphasise the need for proactive multidisciplinary care pathways, with the potential for imaging to guide surveillance frequency and therapeutic stratification

Ad2 fiber protein enhances the nasal immunogenicity of BoNT/A β-trefoil in NZW rabbits.

<p>Female NZW rabbits were immunized on days 0, 14 and 28 with the indicated vaccine formulation. Intramuscular immunization with 10 µg of BoNT/A toxoid combined with alum (n = 4) served as a control. BoNT/A Hcβtre (10 µg) was nasally delivered in the absence of adjuvant (n = 5) or combined with CT (2 µg; n = 6) or C48/80 (120 µg; n = 6). BoNT/A Hcβtre-Ad2F (20 µg) was delivered nasally in the absence of adjuvant (n = 5) or combined with CT (2 µg; n = 6) or C48/80 (120 µg; n = 6). BoNT/A Hc (20 µg) was delivered nasally combined with CT (2 µg; n = 3) or C48/80 (120 µg; n = 3). Serum samples collected on day 27 and day 40 were tested for the presence of anti-BoNT/A β-trefoil IgG by ELISA. Serum antibody titers were compared between groups by ANOVA followed by Tukey's multiple comparison test (GraphPad, Prism). <b>a</b>: serum anti-BoNT/A β-trefoil IgG titers significantly greater than those induced by intramuscular immunization with toxoid, nasal immunization with Hcβtre, nasal immunization with Hcβtre + CT, nasal immunization with Hcβtre + C48/80, nasal immunization with Hcβtre-Ad2F, nasal immunization with Hc + CT and nasal immunization with Hc + C48/80. <b>b</b>: serum anti-BoNT/A β-trefoil IgG titers significantly greater than those induced by nasal immunization with Hcβtre + CT and nasal immunization with Hcβtre + C48/80. <b>c</b>: serum anti-BoNT/A β-trefoil IgG titers significantly greater than those induced by intramuscular immunization with toxoid, nasal immunization with Hcβtre, nasal immunization with Hcβtre-Ad2F, nasal immunization with Hcβtre + CT, nasal immunization with Hcβtre + C48/80, nasal immunization with Hc + CT and nasal immunization with Hc + C48/80. <b>d</b>: serum anti-BoNT/A β-trefoil IgG titers significantly greater than those induced by intramuscular immunization with toxoid, nasal immunization with Hcβtre, nasal immunization with Hcβtre + CT, immunization with Hcβtre + C48/80, nasal immunization with Hcβtre-Ad2F and nasal immunization with Hc + C48/80.</p

Ad2 fiber protein enhances the induction of BoNT/A-neutralizing antibodies in nasally immunized rabbits.

<p>Female Dutch Belted rabbits (4 per group) were nasally immunized on days 0, 14, 28 and 91 with the indicated vaccine formulation. <b>A</b>. Serum collected on day 105 was tested for the presence of BoNT/A-neutralizing antibodies using a mouse neutralization assay. <b>a</b>: serum BoNT/A-neutralizing activity significantly greater than the neutralizing activity measured in rabbits nasally immunized with Hcβtre, Hcβtre + CT, Hcβtre + C48/80 or Hcβtre-Ad2F; ANOVA and Tukey's multiple comparison, p<0.05. <b>b</b>: serum BoNT/A-neutralizing activity significantly greater than the neutralizing activity measured in rabbits nasally immunized with Hcβtre, Hcβtre + CT, Hcβtre + C48/80 or Hcβtre-Ad2F; ANOVA and Tukey's multiple comparison, p<0.05. <b>B</b>. Hcβtre-Ad2F is superior to Hcβtre for the induction of BoNT/A neutralizing antibodies when delivered with CT or C48/80 as adjuvants. <b>a</b>: p = 0.0006, Mann-Whitney test.</p

BoNT/A Hcβtre immunogens induce antibodies that recognize epitopes distinct from those induced by BoNT/A toxoid.

<p>Day 40 sera from a subset of rabbits included in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016532#pone-0016532-g001" target="_blank">Figure 1</a> were tested for the presence of antibodies specific for BoNT/A Hc or BoNT/A toxoid by ELISA. <b>a</b>: serum anti-BoNT/A toxoid IgG titers significantly greater than those induced by all other groups. There were no other significant differences between groups.</p

Schematic representation of Hcβtre-Ad2F fusion protein.

<p><b>A</b>. schematic representation of botulinum neurotoxin type A (heavy and light chains). <b>B</b>. Schematic of β-trefoil domain of BoNT/A heavy chain (Hcβtre). <b>C</b>. Schematic of fusion protein containing β-trefoil domain of BoNT/A heavy chain (Hcβtre) and the adenovirus type 2 fiber protein (Hcβtre-Ad2F).</p

Ad2 fiber protein enhances the nasal immunogenicity of BoNT/A β-trefoil in Dutch Belted rabbits.

<p>Female Dutch Belted rabbits (4 per group) were nasally immunized on days 0, 14, 28 and 91 with the indicated vaccine formulation. Serum collected on days 27, 41 and 105 was tested for the presence of anti-BoNT/A β-trefoil IgG by ELISA. <b>a</b>: serum anti-BoNT/A β-trefoil IgG titers significantly greater than those induced by nasal immunization with Hcβtre or nasal immunization with Hcβtre + C48/80. <b>b</b>: serum anti-BoNT/A β-trefoil IgG titers significantly greater than those induced by nasal immunization with Hcβtre, nasal immunization with Hcβtre + C48/80 or nasal immunization with Hcβtre-Ad2F. <b>c</b>: serum anti-BoNT/A β-trefoil IgG titers significantly greater than those induced by nasal immunization with Hcβtre, nasal immunization with Hcβtre + CT, nasal immunization with Hcβtre + C48/80. <b>d</b>: serum anti-BoNT/A β-trefoil IgG titers significantly greater than those induced by nasal immunization with Hcβtre or nasal immunization with Hcβtre + C48/80. <b>e</b>: serum anti-BoNT/A β-trefoil IgG titers significantly greater than those induced by nasal immunization with Hcβtre.</p

Adjuvants enhance the avidity of anti-BoNT/A β-trefoil IgG antibodies.

<p>The relative avidity of anti-BoNT/A IgG serum antibodies was measured using day 162 serum using a modified ELISA as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0016532#s2" target="_blank">Materials and Methods</a>. <b>A</b>. The percent antibody bound in the ELISA in the presence of 3 M ammonium thiocyanate is indicated for individual serum samples that had measurable anti-β-trefoil IgG. A higher percent antibody bound represents a greater relative avidity for vaccine-induced, antigen-specific antibody. •: represents serum anti-β-trefoil IgG with no detectable BoNT/A neutralizing activity. ○: represents serum anti-β-trefoil IgG with detectable BoNT/A neutralizing activity. <b>B</b>. Average percent antibody bound when samples were organized by the neutralization capacity of the serum. Vaccine-induced BoNT/A neutralizing antibody has significantly greater avidity than non-neutralizing antibody. <b>a</b>: % antibody bound significantly greater than in the no neutralization group. Two-tailed Mann Whitney, p = 0.0239.</p

Harvard HIV and Aging Workshop: Perspectives and Priorities from Claude D. Pepper Centers and Centers for AIDS Research

People aging with HIV (PAWH) infection experience greater impairments in physical and cognitive function, in addition to higher rates of peripheral comorbid conditions (e.g., renal failure, diabetes, bone fracture, hypertension, cardiovascular disease, polypharmacy, and multimorbidity). While multifactorial drivers, including HIV infection itself, antiretroviral therapy-related toxicities, disparities in care, and biobehavioral factors, likely contribute, there remains an overarching question as to what are the relevant age-related mechanisms and models that could inform interventions that promote health span and life span in PAWH? This workshop was convened to hear from experts on the biology of aging and HIV researchers studying PAWH to focus on advancing investigations at the interface of HIV and Aging. In this study, we summarize the discussions from the Harvard Center for AIDS Research and Boston Claude D. Pepper cosponsored workshop on HIV and Aging, which took place in October 2018