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Background In Lao PDR, the epidemiology of varicella infection is uncertain, since it is not a notifiable disease and VZV outbreaks are rarely reported as fever/rash (F/R) diseases. Methods We estimated the seroprevalence of VZV (IgG ELISA) in different age cohorts (9 months to 46 years; N = 3139) and investigated VZV and 6 other viruses in patients during F/R outbreaks and in an ad hoc sentinel site in the context of the national reporting system (IgM ELISA, PCR). Results At least 80% of the sampled population had evidence of VZV infection before the age of 15. The largest increase in seroprevalence occurred between the age groups 1 to 5 and 6 to 7 year-olds. A VZV outbreak (clade 2) also occurred in this age group mostly during the first year of primary school (median age 6 years, interquartile range 4.0–7.5). During a dengue outbreak, 6% had varicella. At our F/R sentinel site, 14% of children with viral etiology were laboratory diagnosed as varicella and among others, a sizeable number of measles (N = 12) and rubella cases (N = 25) was detected compared to those reported for the whole country (N = 56 and 45), highlighting nationwide a large challenge of underreporting or misdiagnosis of these notifiable diseases because of lack of diagnostic laboratory capacity. Conclusion We recommend strengthening the clinical and laboratory diagnosis of VZV, measles and rubella, the surveillance and reporting of notifiable F/R diseases by retraining of healthcare workers and by setting up sentinel sites and enhancing laboratory capacity. Electronic supplementary material The online version of this article (10.1186/s12879-019-3990-7) contains supplementary material, which is available to authorized users.

The molecular diagnosis of acute Borreliosis is complicated and better strategies to improve the diagnostic processes are warranted. High Throughput Sequencing (HTS) of human B cell repertoires after e.g., Dengue virus infection or influenza vaccination revealed antigen-associated "CDR3 signatures" which may have the potential to support diagnosis in infectious diseases. The human B cell immune response to Borrelia burgdorferi sensu lato-the causative agent of Borreliosis-has mainly been studied at the antibody level, while less attention has been given to the cellular part of the humoral immune response. There are indications that Borrelia actively influence the B cell immune response and that it is therefore not directly comparable to responses induced by other infections. The main goal of this study was to identify B cell features that could be used to support diagnosis of Borreliosis. Therefore, we characterized the B cell immune response in these patients by combining multicolor flow cytometry, single Borrelia-reactive B cell receptor (BCR) sequencing, and B cell repertoire deep sequencing. Our phenotyping experiments showed, that there is no significant difference between B cell subpopulations of acute Borreliosis patients and controls. BCR sequences from individual epitope-reactive B cells had little in common between each other. HTS showed, however, a higher complementarity determining region 3 (CDR3) amino acid (aa) sequence overlap between samples from different timepoints in patients as compared to controls. This indicates, that HTS is sensitive enough to detect ongoing B cell immune responses in these patients. Although each individual's repertoire was dominated by rather unique clones, clustering of bulk BCR repertoire sequences revealed a higher overlap of IgG BCR repertoire sequences between acute patients than controls. Even if we have identified a few Borrelia-associated CDR3aa sequences, they seem to be rather unique for each patient and therefore not suitable as biomarkers.

The identification and tracking of antigen-specific immunoglobulin (Ig) sequences within total Ig repertoires is central to high-throughput sequencing (HTS) studies of infections or vaccinations. In this context, public Ig sequences shared by different individuals exposed to the same antigen could be valuable markers for tracing back infections, measuring vaccine immunogenicity, and perhaps ultimately allow the reconstruction of the immunological history of an individual. Here, we immunized groups of transgenic rats expressing human Ig against tetanus toxoid (TT), Modified Vaccinia virus Ankara (MVA), measles virus hemagglutinin and fusion proteins expressed on MVA and the environmental carcinogen Benzo[a]Pyrene, coupled to TT. We showed that these antigens impose a selective pressure causing the Ig Heavy chain (IgH) repertoires of the rats to converge towards the expression of antibodies with highly similar IgH CDR3 amino acid sequences. We present a computational approach, similar to differential gene expression analysis, that selects for clusters of CDR3s with 80% similarity, significantly overrepresented within the different groups of immunized rats. These IgH clusters represent antigen-induced IgH signatures exhibiting stereotypic amino acid patterns including previously described TT and measles specific IgH sequences. Our data suggest, that with the presented methodology, transgenic Ig rats can be utilized as a model to identify antigen-induced, human IgH signatures to a variety of different antigens.

With the advent of high-throughput sequencing (HTS), profiling immunoglobulin (IG) repertoires has become an essential part of immunological research. Advances in sequencing technology enable the IonTorrent Personal Genome Machine (PGM) to cover the full-length of IG mRNA transcripts. Nucleotide insertions and deletions (indels) are the dominant errors of the PGM sequencing platform and can critically influence IG repertoire assessments. Here, we present a PGM-tailored IG repertoire sequencing approach combining error correction through unique molecular identifier (UID) barcoding and indel detection through ImMunoGeneTics (IMGT), the most commonly used sequence alignment database for IG sequences. Using artificially falsified sequences for benchmarking, we found that IMGT efficiently detects 98% of the introduced indels through gene-segment frameshifts. Undetected indels are either located at the ends of the sequences or produce masked frameshifts with an insertion and deletion in close proximity. IMGT’s indel correction algorithm resolves up to 87% of the tested insertions, but no deletions. The complementary determining regions 3 (CDR3s) are returned 100% correct for up to 3 insertions or 3 deletions through conservative culling. We further show, that our PGM-tailored unique molecular identifiers results in highly accurate HTS datasets if combined with the presented data processing. In this regard, considering sequences with at least two copies from datasets with UID families of minimum 3 reads result in correct sequences with over 99% confidence. The protocol and sample processing strategies described in this study will help to establish benchtop-scale sequencing of IG heavy chain transcripts in the field of IG repertoire research.

// Jean-Philippe Burckert 1 , William J. Faison 1 , Danielle E. Mustin 1 , Axel R.S.X. Dubois 1 , Regina Sinner 1 , Oliver Hunewald 1 , Anke Wienecke-Baldacchino 1 , Anne Brieger 1,* and Claude P. Muller 1,* 1 Department of Infection and Immunity, Luxembourg Institute of Health, Esch-sur-Alzette, Luxembourg * These authors share senior authorship Correspondence to: Jean-Philippe Burckert, email: jean-philippe.buerckert@lih.lu Claude P. Muller, email: claude.muller@lih.lu Keywords : high-throughput sequencing; murine IG repertoire; unique molecular barcoding; database benchmarking; IMGT; Immunology Received: August 31, 2017 Accepted: May 07, 2018 Published: July 13, 2018 Abstract With the advent of high-throughput sequencing (HTS), profiling immunoglobulin (IG) repertoires has become an essential part of immunological research. Advances in sequencing technology enable the IonTorrent Personal Genome Machine (PGM) to cover the full-length of IG mRNA transcripts. Nucleotide insertions and deletions (indels) are the dominant errors of the PGM sequencing platform and can critically influence IG repertoire assessments. Here, we present a PGM-tailored IG repertoire sequencing approach combining error correction through unique molecular identifier (UID) barcoding and indel detection through ImMunoGeneTics (IMGT), the most commonly used sequence alignment database for IG sequences. Using artificially falsified sequences for benchmarking, we found that IMGT’s underlying algorithms efficiently detect 98% of the introduced indels. Undetected indels are either located at the end of the sequences or produce masked frameshifts with an insertion and deletion in close proximity. The complementary determining regions 3 (CDR3s) are returned correct for up to 3 insertions or 3 deletions through conservative culling. We further show, that our PGM-tailored unique molecular identifiers result in highly accurate HTS data if combined with the presented processing strategy. In this regard, considering sequences with at least two copies from datasets with UID families of minimum 3 reads result in correct sequences with over 99% confidence. Finally, we show that the protocol can readily be used to generate homogenous datasets for bulk sequencing of murine bone marrow samples. Taken together, this approach will help to establish benchtop-scale sequencing of IG heavy chain transcripts in the field of IG repertoire research.

OBJECTIVE The hybridoma technology is one of the most important advances in clinical immunology. Little is known about the differences between the antibodies produced during the in vivo immune response and those recovered in hybridoma libraries. Here, we investigate a potential fusion bias inherent to the hybridoma production process. METHODS Transgenic rats carrying human Ig heavy and light chain loci were immunized with measles virus (MV) to generate human mAbs. Usin g high-throughput sequencing of IgH mRNA, we compared the IgH repertoire of lymph nodes and the derived hybridoma library using the sequences of the MV-specific hybridoma clones as a reference set with known specificity. RESULTS We observed that large clonotypes from the lymph nodes were not represented in the hybridoma library, but low-frequency B cell populations became highly enriched and most hybridoma clones were derived from these. Our data also showed that identical CDR3s evolved from diverse VDJ recombinations, indicating convergence of different B cells subpopulations towards expression of antibodies with similar paratopes. CONCLUSION The efficient generation of mAbs results from a fusion process highly selective for rare antigen-specific B cells rather than in vivo expanded populations. Antibodies of particular interest may therefore be missed during classical hybridoma production.

Background In Europe, Ixodes ricinus ticks are the most important vectors of diseases threatening humans, livestock, wildlife and companion animals. Nevertheless, genomic sequence information is missing and functional annotation of transcripts and proteins is limited. This lack of information is restricting studies of the vector and its interactions with pathogens and hosts. Here we present and integrate the first analysis of the I. ricinus genome with the transcriptome and proteome of the unfed I. ricinus midgut. Methods Whole genome sequencing was performed on I. ricinus ticks and the sequences were de novo assembled. In parallel, I. ricinus ticks were dissected and the midgut transcriptome sequenced. Both datasets were integrated by transcript discovery analysis to identify putative genes and genome contigs were screened for homology. An alignment-based and a motif-search-based approach were combined for the annotation of the midgut transcriptome. Additionally, midgut proteins were identified and annotated by mass spectrometry with public databases and the in-house built transcriptome database as references and results were cross-validated. Results The de novo assembly of 1 billion DNA sequences to a reference genome of 393 Mb length provides an unprecedented insight into the I. ricinus genome. A homology search revealed sequences in the assembled genome contigs homologous to 89 % of the I. scapularis genome scaffolds indicating coverage of most genome regions. We identified moreover 6,415 putative genes. More than 10,000 transcripts from naïve midgut were annotated with respect of predicted function and/or cellular localization. By combining an alignment-based with a motif-search-based annotation approach, we doubled the number of annotations throughout all functional categories. In addition, 574 gel spots were significantly identified by mass spectrometry (p