Yper Hall

Inhibition of Release of Neurotransmitters from Rat Dorsal Root Ganglia by a Novel Conjugate of a Clostridium botulinum Toxin A Endopeptidase Fragment and Erythrina cristagalli Lectin

Clostridial neurotoxins potently and specifically inhibit neurotransmitter release in defined cell types. Here we report that a catalytically active derivative (termed LHN/A) of the type A neurotoxin from Clostridium botulinum has been coupled to a lectin obtained from Erythrina cristagalli to form a novel conjugate. This conjugate exhibits anin vitro selectivity for nociceptive afferents compared with the anatomically adjacent spinal neurons, as assessed usingin vitro primary neuronal culture systems to measure inhibition of release of neurotransmitters. Chemical conjugates prepared between E. cristagalli lectin and either natively sourced LHN/A or recombinant LHN/A purified from Escherichia coli are assessed, and equivalence of the recombinant material are demonstrated. Furthermore, the dependence of inhibition of neurotransmitter release on the cleavage of SNAP-25 is demonstrated through the use of an endopeptidase-deficient LHN/A conjugate variant. The duration of action of inhibition of neurotransmitter released by the conjugate in vitro is assessed and is comparable with that observed withClostridium botulinum neurotoxin. Finally, in vivo electrophysiology shows that these in vitroactions have biological relevance in that sensory transmission from nociceptive afferents through the spinal cord is significantly attenuated. These data demonstrate that the potent endopeptidase activity of clostridial neurotoxins can be selectively retargeted to cells of interest and that inhibition of release of neurotransmitters from a neuronal population of therapeutic relevance to the treatment of pain can be achieved.

Retargeted clostridial endopeptidases: inhibition of nociceptive neurotransmitter release in vitro, and antinociceptive activity in in vivo models of pain.

Clostridial neurotoxins potently and specifically inhibit neurotransmitter release in defined cell types. Previously reported data have demonstrated that the catalytically active LHN endopeptidase fragment of botulinum neurotoxin type A (termed LHN/A) can be retargeted to a range of cell types in vitro to lead to inhibition of secretion of a range of transmitters. Here, we report the synthesis of endopeptidase conjugates with in vitro selectivity for nociceptive afferents compared to spinal neurons. Chemical conjugates prepared between Erythrina cristagalli lectin and LHN/A are assessed in vitro and in in vivo models of pain. Chemical conjugates prepared between E. cristagalli lectin and either natively sourced LHN/A, or recombinant LHN/A purified from Escherichia coli are assessed, and equivalence of the recombinant material is demonstrated. The duration of action of inhibition of neurotransmitter release by the conjugate in vitro is also assessed and is comparable to that observed with Clostridium botulinum neurotoxin. Selectivity of targeting and therapeutic potential have been confirmed by in vivo electrophysiology studies. Furthermore, the analgesic properties of the conjugate have been assessed in in vivo models of pain and extended duration effects observed. These data provide proof of principle for the concept of retargeted clostridial endopeptidases as novel analgesics.

Evaluation of new vaccines for tuberculosis in the guinea pig model

The guinea pig is a very useful animal model for evaluating new tuberculosis candidate vaccines. In addition to established methods for bacterial load determinations, new technologies are emerging that allow us to specifically evaluate effects of vaccines on the pathology of the disease process and the expression by the host of cell mediated immunity. Limitations to the model include housing and related costs, which often contribute to issue with study design and adequate statistical power, and the use of laboratory strains of Mycobacterium tuberculosis which lack the high virulence and immune evasion properties of newly emerging clinical isolates.

The ring vaccination trial: a novel cluster randomised controlled trial design to evaluate vaccine efficacy and effectiveness during outbreaks, with special reference to Ebola

A World Health Organization expert meeting on Ebola vaccines proposed urgent safety and efficacy studies in response to the outbreak in West Africa. One approach to communicable disease control is ring vaccination of individuals at high risk of infection due to their social or geographical connection to a known case. This paper describes the protocol for a novel cluster randomised controlled trial design which uses ring vaccination. In the Ebola ça suffit ring vaccination trial, rings are randomised 1:1 to (a) immediate vaccination of eligible adults with single dose vaccination or (b) vaccination delayed by 21 days. Vaccine efficacy against disease is assessed in participants over equivalent periods from the day of randomisation. Secondary objectives include vaccine effectiveness at the level of the ring, and incidence of serious adverse events. Ring vaccination trials are adaptive, can be run until disease elimination, allow interim analysis, and can go dormant during inter-epidemic periods.

Vaccines designed to protect against Mycobacterium tuberculosis infection may aid the identification of novel vaccine constructs and diagnostic antigens for bovine tuberculosis

Vaccination has been identified as a promising control strategy for tuberculosis in both humans and cattle. Recent heterologous prime-boost approaches combining BCG vaccination with subunit boosts have shown considerable promise in both fields. However, the identification of further protective antigens is still a research priority. In this paper we have established the response hierarchy in Mycobacterium bovis infected or BCG vaccinated cattle of 6 Mycobacterium tuberculosis-derived proteins that were protective against M. tuberculosis in the guinea pig aerosol challenge model. Two of these proteins, Rv1806 and Rv3812, were recognised most frequently in cattle and therefore constitute potential subunit vaccine candidates that merit further evaluation in cattle. Their epitopes were mapped in infected cattle and were shown to be located primarily in the non-conserved regions of these PE/PE-PGRS protein family members. The aim of this study was to ascertain the presence of any correlation between the immunogenicity of defined antigens in different animal species. A weak association between guinea pig immunogenicity (as measured by protection) and antigenicity in M. bovis infected or BCG vaccinated cattle was found.

Enhancing the Biological Relevance of Machine Learning Classifiers for Reverse Vaccinology

Reverse vaccinology (RV) is a bioinformatics approach that can predict antigens with protective potential from the protein coding genomes of bacterial pathogens for subunit vaccine design. RV has become firmly established following the development of the BEXSERO® vaccine against Neisseria meningitidis serogroup B. RV studies have begun to incorporate machine learning (ML) techniques to distinguish bacterial protective antigens (BPAs) from non-BPAs. This research contributes significantly to the RV field by using permutation analysis to demonstrate that a signal for protective antigens can be curated from published data. Furthermore, the effects of the following on an ML approach to RV were also assessed: nested cross-validation, balancing selection of non-BPAs for subcellular localization, increasing the training data, and incorporating greater numbers of protein annotation tools for feature generation. These enhancements yielded a support vector machine (SVM) classifier that could discriminate BPAs (n = 200) from non-BPAs (n = 200) with an area under the curve (AUC) of 0.787. In addition, hierarchical clustering of BPAs revealed that intracellular BPAs clustered separately from extracellular BPAs. However, no immediate benefit was derived when training SVM classifiers on data sets exclusively containing intra- or extracellular BPAs. In conclusion, this work demonstrates that ML classifiers have great utility in RV approaches and will lead to new subunit vaccines in the future.