Profound structural conservation of chemically cross-linked HIV-1 envelope glycoprotein experimental vaccine antigens.

Chemical cross-linking is used to stabilize protein structures with additional benefits of pathogen and toxin inactivation for vaccine use, but its use has been restricted by the potential for local or global structural distortion. This is of particular importance when the protein in question requires a high degree of structural conservation for inducing a biological outcome such as the elicitation of antibodies to conformationally sensitive epitopes. The HIV-1 envelope glycoprotein (Env) trimer is metastable and shifts between different conformational states, complicating its use as a vaccine antigen. Here we have used the hetero-bifunctional zero-length reagent 1-Ethyl-3-(3-Dimethylaminopropyl)-Carbodiimide (EDC) to cross-link two soluble Env trimers, selected well-folded trimer species using antibody affinity, and transferred this process to good manufacturing practice (GMP) for experimental medicine use. Cross-linking enhanced trimer stability to biophysical and enzyme attack. Cryo-EM analysis revealed that cross-linking retained the overall structure with root-mean-square deviations (RMSDs) between unmodified and cross-linked Env trimers of 0.4-0.5 Å. Despite this negligible distortion of global trimer structure, we identified individual inter-subunit, intra-subunit, and intra-protomer cross-links. Antigenicity and immunogenicity of the trimers were selectively modified by cross-linking, with cross-linked ConS retaining bnAb binding more consistently than ConM. Thus, the EDC cross-linking process improves trimer stability whilst maintaining protein folding, and is readily transferred to GMP, consistent with the more general use of this approach in protein-based vaccine design

Immunogenicity of protein aldehydes

Protein carbonylation introduces diverse non-enzymatic post-translational modifications (PTMs) containing aldehydes and ketones (reactive carbonyls) during oxidative or glycative stress, and is a hallmark of unhealthy ageing and various neurological, cardiovascular, respiratory, musculoskeletal and metabolic diseases. The activation of innate and adaptive immune responses to carbonylated proteins is thought to trigger or exacerbate these pathologies, although how specific PTMs activate the immune system is not well understood. In this thesis, the protein hen egg lysozyme (HEL) was modified with glycolaldehyde (GA) as a model to investigate the immunogenicity of protein aldehydes. Mice immunised with HEL GA in the absence of adjuvant produced higher IgG titres to native HEL protein. Chemical reduction of aldehydes to non-reactive alcohol groups maintained equivalent levels of protein cross-linking and mostly abrogated immunogenic responses. Moreover, mice immunised with GA-modified self-homologue mouse lysozyme (ML) produced anti-ML IgG responses to break self-tolerance. T and B cell adaptive immune responses were investigated to determine their role in GA-mediated immunogenicity. Antigen-specific CD4+ T cell recall responses to native HEL protein were not elevated in mice immunised with HEL GA compared with controls. In contrast, the number of HEL-specific antibody secreting cells (ASCs) was increased in mice after HEL GA immunisation. Innate immune responses were then considered as potential factors mediating enhanced GA-protein immunogenicity. Binding assays showed that HEL GA had increased mouse C3 degradation product opsonisation compared with controls. Mice depleted of complement C3 using cobra venom factor then immunised with HEL GA suggested greater early anti-HEL IgG responses compared with non-depleted mice. C3 knockout mice immunised with HEL GA also appeared to show increased anti-HEL IgG responses, suggesting an immunoregulatory rather than immunoenhancing role for complement in GA-mediated immunogenicity. Pull-down and co-immunoprecipitation experiments to determine whether GA-modified proteins could engage innate immune factors confirmed increased complement activation and immunoglobulin binding. In summary, protein aldehydation induced by GA treatment increases IgG anti-body responses to native protein and can break self-tolerance in the absence of adjuvant. Protein aldehydes are confirmed to be the immunogenic moiety underlying this immunogenicity because their chemical reduction abrogates most of the ensuing antibody response. Protein aldehydes appear to induce greater B cell activation and differentiation to ASCs, although the GA-enhanced B cell response does not seem to be mediated by CD4+ T helper cells. The mechanism underlying GA-enhanced immunogenicity remains to be determined, although protein aldehydes have altered interactions with various soluble factors including complement and immunoglobulin. This thesis adds to existing knowledge about how protein aldehyde PTMs may activate innate and adaptive immune responses to ultimately lead to unhealthy ageing and associated pathologies.</p

Web-based integrated bipolar parenting intervention (IBPI) for parents with bipolar disorder:intervention development and evaluation in a randomised controlled pilot trial

Background: People with bipolar disorder (BD) experience parenting challenges associated with mood fluctuations. This talk describes the development and evaluation of a novel web- based self- management approach (Integrated Bipolar Parenting Intervention; IBPI) to support parents with BD developed with individuals with lived experience of bipolar disorder. Methods: IBPI content and structure was developed in collaboration with service users coordinated by a service user grant holder. Parents with BD with children aged 3- 10 years randomised to IBPI plus treatment as usual (TAU) or waitlist control (WL). IBPI offered 16 weeks access to interactive self- management information concerning BD and parenting issues. The trial was designed to access feasibility and acceptability as well as provide initial indications of potential benefits of IBPI. Feasibility was through recruitment, retention and web usage. Clinical outcomes were assessed at baseline, 16, 24, 36 and 48 weeks. A nested qualitative study was conducted focusing on participant experiences of the IBPI intervention. Results: The intervention and trial design were both feasible and acceptable. 97 participants were recruited with 90% retention to final follow- up. 77% of IBPI participants accessed the website. Child behaviour, parenting sense of competence and parenting stress improved significantly in IBPI compared to WL to end of intervention, sustained to 48 weeks. Qualitative feedback indicated participants’ valued this approach and made suggestions for further improvements. Conclusions: Developing an online self- management support for parents with BD is feasible, with promising improvements in parenting and child behaviour outcomes. A definitive clinical and cost- effectiveness trial is required

Cohort profile: the ESC EURObservational Research Programme Non-ST-segment elevation myocardial infraction (NSTEMI) Registry

Aims The European Society of Cardiology (ESC) EURObservational Research Programme (EORP) Non-ST-segment elevation myocardial infarction (NSTEMI) Registry aims to identify international patterns in NSTEMI management in clinical practice and outcomes against the 2015 ESC Guidelines for the management of acute coronary syndromes in patients presenting without ST-segment-elevation. Methods and results Consecutively hospitalised adult NSTEMI patients (n = 3620) were enrolled between 11 March 2019 and 6 March 2021, and individual patient data prospectively collected at 287 centres in 59 participating countries during a two-week enrolment period per centre. The registry collected data relating to baseline characteristics, major outcomes (inhospital death, acute heart failure, cardiogenic shock, bleeding, stroke/transient ischaemic attack, and 30-day mortality) and guideline-recommended NSTEMI care interventions: electrocardiogram pre- or in-hospital, prehospitalization receipt of aspirin, echocardiography, coronary angiography, referral to cardiac rehabilitation, smoking cessation advice, dietary advice, and prescription on discharge of aspirin, P2Y12 inhibition, angiotensin converting enzyme inhibitor (ACEi)/angiotensin receptor blocker (ARB), beta-blocker, and statin. Conclusion The EORP NSTEMI Registry is an international, prospective registry of care and outcomes of patients treated for NSTEMI, which will provide unique insights into the contemporary management of hospitalised NSTEMI patients, compliance with ESC 2015 NSTEMI Guidelines, and identify potential barriers to optimal management of this common clinical presentation associated with significant morbidity and mortality