AIRRSHIP: simulating human B cell receptor repertoire sequences.

SUMMARY: Adaptive Immune Receptor Repertoire Sequencing is a rapidly developing field that has advanced understanding of the role of the adaptive immune system in health and disease. Numerous tools have been developed to analyse the complex data produced by this technique but work to compare their accuracy and reliability has been limited. Thorough, systematic assessment of their performance is dependent on the ability to produce high quality simulated datasets with known ground truth. We have developed AIRRSHIP, a flexible and fast Python package that produces synthetic human B cell receptor sequences. AIRRSHIP uses a comprehensive set of reference data to replicate key mechanisms in the immunoglobulin recombination process, with a particular focus on junctional complexity. Repertoires generated by AIRRSHIP are highly similar to published data and all steps in the sequence generation process are recorded. These data can be used to not only determine the accuracy of repertoire analysis tools but can also, by tuning of the large number of user-controllable parameters, give insight into factors that contribute to inaccuracies in results. AVAILABILITY AND IMPLEMENTATION: AIRRSHIP is implemented in Python. It is available via https://github.com/Cowanlab/airrship and on PyPI at https://pypi.org/project/airrship/. Documentation can be found at https://airrship.readthedocs.io/.

A Conserved TCRß Signature Dominates a Highly Polyclonal T-Cell Expansion During the Acute Phase of a Murine Malaria Infection.

CD4+ αß T-cells are key mediators of the immune response to a first Plasmodium infection, undergoing extensive activation and splenic expansion during the acute phase of an infection. However, the clonality and clonal composition of this expansion has not previously been described. Using a comparative infection model, we sequenced the splenic CD4+ T-cell receptor repertoires generated over the time-course of a Plasmodium chabaudi infection. We show through repeat replicate experiments, single-cell RNA-seq, and analyses of independent RNA-seq data, that following a first infection - within a highly polyclonal expansion - T-effector repertoires are consistently dominated by TRBV3 gene usage. Clustering by sequence similarity, we find the same dominant clonal signature is expanded across replicates in the acute phase of an infection, revealing a conserved pathogen-specific T-cell response that is consistently a hallmark of a first infection, but not expanded upon re-challenge. Determining the host or parasite factors driving this conserved response may uncover novel immune targets for malaria therapeutic purposes.

In Human Autoimmunity, a Substantial Component of the B Cell Repertoire Consists of Polyclonal, Barely Mutated IgG+ve B Cells.

B cells are critical for promoting autoimmunity and the success of B cell depletion therapy in rheumatoid arthritis (RA) confirms their importance in driving chronic inflammation. Whilst disease specific autoantibodies are useful diagnostically, our understanding of the pathogenic B cell repertoire remains unclear. Defining it would lead to novel insights and curative treatments. To address this, we have undertaken the largest study to date of over 150 RA patients, utilizing next generation sequencing (NGS) to analyze up to 200,000 BCR sequences per patient. The full-length antigen-binding variable region of the heavy chain (IgGHV) of the IgG B cell receptor (BCR) were sequenced. Surprisingly, RA patients do not express particular clonal expansions of B cells at diagnosis. Rather they express a polyclonal IgG repertoire with a significant increase in BCRs that have barely mutated away from the germline sequence. This pattern remains even after commencing disease modifying therapy. These hypomutated BCRs are expressed by TNF-alpha secreting IgG+veCD27-ve B cells, that are expanded in RA peripheral blood and enriched in the rheumatoid synovium. A similar B cell repertoire is expressed by patients with Sjögren's syndrome. A rate limiting step in the initiation of autoimmunity is the activation of B cells and this data reveals that a sizeable component of the human autoimmune B cell repertoire consists of polyclonal, hypomutated IgG+ve B cells, that may play a critical role in driving chronic inflammation.

Abnormal proliferation and spontaneous differentiation of myoblasts from a symptomatic female carrier of X-linked Emery-Dreifuss muscular dystrophy.

Emery-Dreifuss muscular dystrophy (EDMD) is a neuromuscular disease characterized by early contractures, slowly progressive muscular weakness and life-threatening cardiac arrhythmia that can develop into cardiomyopathy. In X-linked EDMD (EDMD1), female carriers are usually unaffected. Here we present a clinical description and in vitro characterization of a mildly affected EDMD1 female carrying the heterozygous EMD mutation c.174_175delTT; p.Y59* that yields loss of protein. Muscle tissue sections and cultured patient myoblasts exhibited a mixed population of emerin-positive and -negative cells; thus uneven X-inactivation was excluded as causative. Patient blood cells were predominantly emerin-positive, but considerable nuclear lobulation was observed in non-granulocyte cells - a novel phenotype in EDMD. Both emerin-positive and emerin-negative myoblasts exhibited spontaneous differentiation in tissue culture, though emerin-negative myoblasts were more proliferative than emerin-positive cells. The preferential proliferation of emerin-negative myoblasts together with the high rate of spontaneous differentiation in both populations suggests that loss of functional satellite cells might be one underlying mechanism for disease pathology. This could also account for the slowly developing muscle phenotype.

A novel malaria vaccine candidate antigen expressed in Tetrahymena thermophila.

Development of effective malaria vaccines is hampered by the problem of producing correctly folded Plasmodium proteins for use as vaccine components. We have investigated the use of a novel ciliate expression system, Tetrahymena thermophila, as a P. falciparum vaccine antigen platform. A synthetic vaccine antigen composed of N-terminal and C-terminal regions of merozoite surface protein-1 (MSP-1) was expressed in Tetrahymena thermophila. The recombinant antigen was secreted into the culture medium and purified by monoclonal antibody (mAb) affinity chromatography. The vaccine was immunogenic in MF1 mice, eliciting high antibody titers against both N- and C-terminal components. Sera from immunized animals reacted strongly with P. falciparum parasites from three antigenically different strains by immunofluorescence assays, confirming that the antibodies produced are able to recognize parasite antigens in their native form. Epitope mapping of serum reactivity with a peptide library derived from all three MSP-1 Block 2 serotypes confirmed that the MSP-1 Block 2 hybrid component of the vaccine had effectively targeted all three serotypes of this polymorphic region of MSP-1. This study has successfully demonstrated the use of Tetrahymena thermophila as a recombinant protein expression platform for the production of malaria vaccine antigens.

Antibody responses to a novel Plasmodium falciparum merozoite surface protein vaccine correlate with protection against experimental malaria infection in Aotus monkeys.

The Block 2 region of the merozoite surface protein-1 (MSP-1) of Plasmodium falciparum has been identified as a target of protective immunity by a combination of seroepidemiology and parasite population genetics. Immunogenicity studies in small animals and Aotus monkeys were used to determine the efficacy of recombinant antigens derived from this region of MSP-1 as a potential vaccine antigen. Aotus lemurinus griseimembra monkeys were immunized three times with a recombinant antigen derived from the Block 2 region of MSP-1 of the monkey-adapted challenge strain, FVO of Plasmodium falciparum, using an adjuvant suitable for use in humans. Immunofluorescent antibody assays (IFA) against erythrocytes infected with P. falciparum using sera from the immunized monkeys showed that the MSP-1 Block 2 antigen induced significant antibody responses to whole malaria parasites. MSP-1 Block 2 antigen-specific enzyme-linked immunosorbent assays (ELISA) showed no significant differences in antibody titers between immunized animals. Immunized animals were challenged with the virulent P. falciparum FVO isolate and monitored for 21 days. Two out of four immunized animals were able to control their parasitaemia during the follow-up period, whereas two out of two controls developed fulminating parasitemia. Parasite-specific serum antibody titers measured by IFA were four-fold higher in protected animals than in unprotected animals. In addition, peptide-based epitope mapping of serum antibodies from immunized Aotus showed distinct differences in epitope specificities between protected and unprotected animals.

A malaria vaccine based on the polymorphic block 2 region of MSP-1 that elicits a broad serotype-spanning immune response.

Polymorphic parasite antigens are known targets of protective immunity to malaria, but this antigenic variation poses challenges to vaccine development. A synthetic MSP-1 Block 2 construct, based on all polymorphic variants found in natural Plasmodium falciparum isolates has been designed, combined with the relatively conserved Block 1 sequence of MSP-1 and expressed in E.coli. The MSP-1 Hybrid antigen has been produced with high yield by fed-batch fermentation and purified without the aid of affinity tags resulting in a pure and extremely thermostable antigen preparation. MSP-1 hybrid is immunogenic in experimental animals using adjuvants suitable for human use, eliciting antibodies against epitopes from all three Block 2 serotypes. Human serum antibodies from Africans naturally exposed to malaria reacted to the MSP-1 hybrid as strongly as, or better than the same serum reactivities to individual MSP-1 Block 2 antigens, and these antibody responses showed clear associations with reduced incidence of malaria episodes. The MSP-1 hybrid is designed to induce a protective antibody response to the highly polymorphic Block 2 region of MSP-1, enhancing the repertoire of MSP-1 Block 2 antibody responses found among immune and semi-immune individuals in malaria endemic areas. The target population for such a vaccine is young children and vulnerable adults, to accelerate the acquisition of a full range of malaria protective antibodies against this polymorphic parasite antigen.

Identification of the high affinity binding site in the Streptococcus intermedius toxin intermedilysin for its membrane receptor, the human complement regulator CD59.

The unique species specificity of the bacterial cytolysin intermedilysin is explained by its requirement for the human complement regulator CD59 as the primary receptor. Binding studies using individual domains of intermedilysin mapped the CD59-binding site to domain 4 and swap mutants between human and rabbit (non-intermedilysin-binding) CD59 implicated a short sequence (residues 42-59) in human CD59 in binding intermedilysin. We set out to map more closely the CD59 binding site in intermedilysin. We first looked for regions of homology between domain 4 in intermedilysin and the terminal complement components that bind CD59, C8 and C9. A nine amino acid sequence immediately adjacent the undecapeptide segment in intermedilysin domain 4 matched (5 of 9 identical, 3 of 9 conserved) a sequence in C9. A peptide containing this sequence caused dose-dependent inhibition of intermedilysin-mediated lysis of human erythrocytes and rendered erythrocytes more susceptible to complement lysis. Surface plasmon resonance analysis of intermedilysin binding to immobilized CD59 revealed saturable fast-on, fast-off binding and a calculated affinity of 4.9 nM. Substitution of three residues from the putative binding site caused a 5-fold reduction in lytic potency of intermedilysin and reduced affinity for immobilized CD59 by 2.5-fold. The demonstration that a peptide modeled on the CD59-binding site inhibits intermedilysin-mediated haemolysis leads us to suggest that such peptides might be useful in treating infections caused by intermedilysin-producing bacteria.

Immunisation with anthrolysin O or a genetic toxoid protects against challenge with the toxin but not against Bacillus anthracis.

Anthrolysin O (ALO) is a toxin produced by Bacillus anthracis, the causative agent of anthrax. It is a member of the cholesterol-dependent cytolysin (CDC) group of toxins, many of which are potential vaccine candidates that protect against their producing organisms. Pore formation by ALO was studied by transmission electron microscopy and pores were found to be consistent with those formed by other members of this toxin family. We constructed and characterised a novel genetic toxoid of anthrolysin O, Delta6mALO, which was able to bind to cells but was incapable of pore-formation or haemolysis. The capacity of the haemolytic and non-haemolytic forms of ALO to protect against challenge with the toxin or B. anthracis was determined. Immunisation with both active and non-haemolytic forms of ALO elicited protection against lethal i.v. challenge with ALO but neither was protective against B. anthracis in a murine i.p. challenge model. Immunisation with another CDC, pneumolysin, did not confer cross-protection against challenge with ALO. Histopathological investigation following lethal i.v. challenge with ALO revealed acute pathology in the lungs with occlusion of alveolar vessels by fibrin deposits.

Presence of nonhemolytic pneumolysin in serotypes of Streptococcus pneumoniae associated with disease outbreaks.

Pneumolysin is an important virulence factor of the human pathogen Streptococcus pneumoniae. Sequence analysis of the ply gene from 121 clinical isolates of S. pneumoniae uncovered a number of alleles. Twenty-two strains were chosen for further analysis, and 14 protein alleles were discovered. Five of these had been reported previously, and the remaining 9 were novel. Cell lysates were used to determine the specific hemolytic activities of the pneumolysin proteins. Six strains showed no hemolytic activity, and the remaining 16 were hemolytic, to varying degrees. We report that the nonhemolytic allele reported previously in serotype 1, sequence type (ST) 306 isolates is also present in a number of pneumococcal isolates of serotype 8 that belong to the ST53 lineage. Serotype 1 and 8 pneumococci are known to be associated with outbreaks of invasive disease. The nonhemolytic pneumolysin allele is therefore associated with the dominant clones of outbreak-associated serotypes of S. pneumoniae.