A Theileria Annulata sporozoite surface antigen as a potential vaccine for tropical Theileriosis

The aim of this study was to identify potentially protective antigens of the infective, sporozoite, stage of Theileria annulata and to clone the relevant gene(s) to obtain one or more of these antigens as a recombinant protein with which to perform immunisation trials.Theileria annulata and the disease it causes, tropical theileriosis, were described with particular reference to literature concerning characteristics of the sporozoite stage, immunity and immunisation. The application of biotechnology to the production of recombinant DNA vaccines was reviewed and relevant examples were detailed.To this end, antisporozoite monoclonal antibodies (Mabs) were raised and those giving positive fluorescence of formalin fixed sporozoites by the indirect fluorescent antibody test (IFAT) were selected. Nineteen such Mabs were screened for the ability to neutralise sporozoite infectivity for bovine peripheral blood mono¬ nuclear cells in vitro. Two antisporozoite Mabs, 1A7 and 4B11, which demonstrated surface immunofluorescence of live sporozoites, exhibited a significant degree of sporozoite inhibition. These were chosen for further investigation. The in vitro assay was also used to detect sporozoite neutralising activity in hyperimmune bovine sera, sera from calves exposed to irradiated sporozoites and serum from rabbits on which infected ticks had fed.Using SDS-PAGE Western blotting, Mabs 1A7 and 4B11 were shown to identify different epitopes on the sporozoite surface. Mab 1A7 specifically recognised an epitope present on several sporozoite V proteins of approximate molecular weights 85, 72, 63 and 54 kilodaltons (kdal) in Western blots. This was believed to reflect pro¬ cessing of a high molecular weight precursor. Mab 4B11 recognised a low molecular weight protein of 17-20 kdal, also located on the sporozoite surface. Some immune bovine sera and antisporozoite rabbit serum also detected these two sporozoite epitopes.Large amounts of one of the epitopes were made available as a recombinant protein by cloning and expressing the relevant theilerial gene fragment in Escherichia coli (E. coli) . To achieve this a λgtl 1 expression library, constructed using genomic DNA from T. annulata piroplasm DNA, was screened with Mabs 1A7 and 4B11. Two recombinant clones, λ gtl1-SR1 and λgtll-SR2, were obtained, both of which contained the gene sequence coding for the epitope recognised by Mab 1A7. The theilerial DNA insert of clone Agtl1-SRl was 330 base pairs in size and hybridised to three DNA bands in EcoRl digested genomic DNA from an uncloned parasite stock. These bands were segregated to single copies of the gene sequence in the DNA from cloned parasite material. Northern blot analysis using RNA from infected tick salivary glands showed expression of the λgtll-SRl insert to be stage specific, occurring only in sporoblast and sporozoite stages and not in macroschizonts or piroplasms, nor in uninfected tick salivary glands.The recombinant DNA cloned in λgtll-SRl and λgtll-SR2 was expressed in E. coli as B-galactosidase fusion proteins of 135 and 147 kdal respectively. These proteins reacted specifically with Mab 1A7 and also with antisporozoite rabbit serum and certain sera from calves exposed to live or irradiated sporozoites. Immunisation trials using the purified 135 kdal protein from λgtl1-SRl, were performed in mice, rabbits and calves. Inoculation of rabbits and calves with this fusion protein combined with Freund's adjuvant elicited a strong and specific antibody response. These antibodies alpo recognised the native sporozoite epitope when assessed by IFAT and Western blotting, the latter revealing exactly the same multiple reactivity of sporo¬ zoite proteins with anti λgtll-SRl sera as observed with Mab 1A7. Significantly, the same sera also neutralised sporozoite infectivity in vitro very effectively, while control sera failed to do so. On challenge with live virulent sporozoites, the calves immunised with theλgtll-SRl protein became infected and underwent clinical reactions which were not significantly different from those observed in control B-galactosidase immunised or unimmunised calves. Discussion of these results concentrated on whether this failure to stimulate protective immunity reflected an inadequate method of antigen presentation, or whether antisporozoite immunity alone is insufficient to protect otherwise fully susceptible calves

Core outcome measurement instruments for use in clinical and research settings for adults with post-COVID-19 condition: an international Delphi consensus study.

Post-COVID-19 condition (also known as long COVID) is a new, complex, and poorly understood disorder. A core outcome set (COS) for post-COVID-19 condition in adults has been developed and agreement is now required on the most appropriate measurement instruments for these core outcomes. We conducted an international consensus study involving multidisciplinary experts and people with lived experience of long COVID. The study comprised a literature review to identify measurement instruments for the core outcomes, a three-round online modified Delphi process, and an online consensus meeting to generate a core outcome measurement set (COMS). 594 individuals from 58 countries participated. The number of potential instruments for the 12 core outcomes was reduced from 319 to 19. Consensus was reached for inclusion of the modified Medical Research Council Dyspnoea Scale for respiratory outcomes. Measures for two relevant outcomes from a previously published COS for acute COVID-19 were also included: time until death, for survival, and the Recovery Scale for COVID-19, for recovery. Instruments were suggested for consideration for the remaining nine core outcomes: fatigue or exhaustion, pain, post-exertion symptoms, work or occupational and study changes, and cardiovascular, nervous system, cognitive, mental health, and physical outcomes; however, consensus was not achieved for instruments for these outcomes. The recommended COMS and instruments for consideration provide a foundation for the evaluation of post-COVID-19 condition in adults, which should help to optimise clinical care and accelerate research worldwide. Further assessment of this COMS is warranted as new data emerge on existing and novel measurement instruments

SARS-CoV-2 Omicron is an immune escape variant with an altered cell entry pathway

Vaccines based on the spike protein of SARS-CoV-2 are a cornerstone of the public health response to COVID-19. The emergence of hypermutated, increasingly transmissible variants of concern (VOCs) threaten this strategy. Omicron (B.1.1.529), the fifth VOC to be described, harbours multiple amino acid mutations in spike, half of which lie within the receptor-binding domain. Here we demonstrate substantial evasion of neutralization by Omicron BA.1 and BA.2 variants in vitro using sera from individuals vaccinated with ChAdOx1, BNT162b2 and mRNA-1273. These data were mirrored by a substantial reduction in real-world vaccine effectiveness that was partially restored by booster vaccination. The Omicron variants BA.1 and BA.2 did not induce cell syncytia in vitro and favoured a TMPRSS2-independent endosomal entry pathway, these phenotypes mapping to distinct regions of the spike protein. Impaired cell fusion was determined by the receptor-binding domain, while endosomal entry mapped to the S2 domain. Such marked changes in antigenicity and replicative biology may underlie the rapid global spread and altered pathogenicity of the Omicron variant

Synthesis, Antibacterial Activity, and Nephrotoxicity of Polymyxin B Analogues Modified at Leu-7, d -Phe-6, and the N-Terminus Enabled by S-Lipidation

With the post-antibiotic era rapidly approaching, many have turned their attention to developing new treatments, often by structural modification of existing antibiotics. Polymyxins, a family of lipopeptide antibiotics that are used as a last line of defense in the clinic, have recently developed resistance and exhibit significant nephrotoxicity issues. Using thiol-ene chemistry, the facile preparation of six unique S-lipidated building blocks was demonstrated and used to generate lipopeptide mimetics upon incorporation into solid-phase peptide synthesis (SPPS). We then designed and synthesized 38 polymyxin analogues, incorporating these unique building blocks at the N-terminus, or to replace hydrophobic residues at positions 6 and 7 of the native lipopeptides. Several polymyxin analogues bearing one or more S-linked lipids were found to be equipotent to polymyxin, showed minimal kidney nephrotoxicity, and demonstrated activity against several World Health Organisation (WHO) priority pathogens. The S-lipidation strategy has demonstrated potential as a novel approach to prepare innovative new lipopeptide antibiotics.</p

Synthesis, Antibacterial Activity, and Nephrotoxicity of Polymyxin B Analogues Modified at Leu-7, d‑Phe-6, and the N‑Terminus Enabled by S‑Lipidation

With the post-antibiotic era rapidly approaching, many have turned their attention to developing new treatments, often by structural modification of existing antibiotics. Polymyxins, a family of lipopeptide antibiotics that are used as a last line of defense in the clinic, have recently developed resistance and exhibit significant nephrotoxicity issues. Using thiol–ene chemistry, the facile preparation of six unique S-lipidated building blocks was demonstrated and used to generate lipopeptide mimetics upon incorporation into solid-phase peptide synthesis (SPPS). We then designed and synthesized 38 polymyxin analogues, incorporating these unique building blocks at the N-terminus, or to replace hydrophobic residues at positions 6 and 7 of the native lipopeptides. Several polymyxin analogues bearing one or more S-linked lipids were found to be equipotent to polymyxin, showed minimal kidney nephrotoxicity, and demonstrated activity against several World Health Organisation (WHO) priority pathogens. The S-lipidation strategy has demonstrated potential as a novel approach to prepare innovative new lipopeptide antibiotics

New insights into the genetic etiology of Alzheimer’s disease and related dementias

Characterization of the genetic landscape of Alzheimer’s disease (AD) and related dementias (ADD) provides a unique opportunity for a better understanding of the associated pathophysiological processes. We performed a two-stage genome-wide association study totaling 111,326 clinically diagnosed/‘proxy’ AD cases and 677,663 controls. We found 75 risk loci, of which 42 were new at the time of analysis. Pathway enrichment analyses confirmed the involvement of amyloid/tau pathways and highlighted microglia implication. Gene prioritization in the new loci identified 31 genes that were suggestive of new genetically associated processes, including the tumor necrosis factor alpha pathway through the linear ubiquitin chain assembly complex. We also built a new genetic risk score associated with the risk of future AD/dementia or progression from mild cognitive impairment to AD/dementia. The improvement in prediction led to a 1.6- to 1.9-fold increase in AD risk from the lowest to the highest decile, in addition to effects of age and the APOE ε4 allele

New insights into the genetic etiology of Alzheimer’s disease and related dementias

Characterization of the genetic landscape of Alzheimer’s disease (AD) and related dementias (ADD) provides a unique opportunity for a better understanding of the associated pathophysiological processes. We performed a two-stage genome-wide association study totaling 111,326 clinically diagnosed/‘proxy’ AD cases and 677,663 controls. We found 75 risk loci, of which 42 were new at the time of analysis. Pathway enrichment analyses confirmed the involvement of amyloid/tau pathways and highlighted microglia implication. Gene prioritization in the new loci identified 31 genes that were suggestive of new genetically associated processes, including the tumor necrosis factor alpha pathway through the linear ubiquitin chain assembly complex. We also built a new genetic risk score associated with the risk of future AD/dementia or progression from mild cognitive impairment to AD/dementia. The improvement in prediction led to a 1.6- to 1.9-fold increase in AD risk from the lowest to the highest decile, in addition to effects of age and the APOE ε4 allele