STRAIN: an R package for multi-locus sequence typing from whole genome sequencing data.

BACKGROUND: Multi-locus sequence typing (MLST) is a standard typing technique used to associate a sequence type (ST) to a bacterial isolate. When the output of whole genome sequencing (WGS) of a sample is available the ST can be assigned directly processing the read-set. Current approaches employ reads mapping (SRST2) against the MLST loci, k-mer distribution (stringMLST), selective assembly (GRAbB) or whole genome assembly (BIGSdb) followed by BLASTn sequence query. Here we present STRAIN (ST Reduced Assembly IdentificatioN), an R package that implements a hybrid strategy between assembly and mapping of the reads to assign the ST to an isolate starting from its read-sets. RESULTS: Analysis of 540 publicly accessible Illumina read sets showed STRAIN to be more accurate at correct allele assignment and new alleles identification compared to SRTS2, stringMLST and GRAbB. STRAIN assigned correctly 3666 out of 3780 alleles (capability to identify correct alleles 97%) and, when presented with samples containing new alleles, identified them in 3730 out of 3780 STs (capability to identify new alleles 98.7%) of the cases. On the same dataset the other tested tools achieved lower capability to identify correct alleles (from 28.5 to 96.9%) and lower capability to identify new alleles (from 1.1 to 97.1%). CONCLUSIONS: STRAIN is a new accurate method to assign the alleles and ST to an isolate by processing the raw reads output of WGS. STRAIN is also able to retrieve new allele sequences if present. Capability to identify correct and new STs/alleles, evaluated on a benchmark dataset, are higher than other existing methods. STRAIN is designed for single allele typing as well as MLST. Its implementation in R makes allele and ST assignment simple, direct and prompt to be integrated in wider pipeline of downstream bioinformatics analyses.

Sequence conservation analysis and in silico human leukocyte antigen-peptide binding predictions for the Mtb72F and M72 tuberculosis candidate vaccine antigens.

BACKGROUND: Requisites for an efficacious tuberculosis (TB) vaccine are a minimal genomic diversity among infectious Mycobacterium tuberculosis strains for the selected antigen, and the capability to induce robust T-cell responses in the majority of human populations. A tool in the identification of putative T-cell epitopes is in silico prediction of major histocompatibility complex (MHC)-peptide binding. Candidate TB vaccine antigen Mtb72F and its successor M72 are recombinant fusion proteins derived from Mtb32A and Mtb39A (encoded by Rv0125 and Rv1196, respectively). Adjuvanted Mtb72F and M72 candidate vaccines were shown to induce CD4(+) T-cell responses in European, US, African and Asian populations. METHODS: Sequence conservation of Mtb32A, Mtb39A, Mtb72F and M72 among 46 strains (prevalent Mycobacterium strains causing human TB disease, and H37Ra) was assessed by multiple alignments using ClustalX. For Mtb32A, Mtb39A and Mtb72F, 15-mer human leukocyte antigen (HLA)-class II-binding peptides were predicted for 158 DRB1 alleles prevailing in populations with high TB burden, 6 DRB3/4/5, 8 DQ and 6 DP alleles, using NetMHCII-pan-3.0. Results for 3 DRB1 alleles were compared with previously published allele-matched in vitro binding data. Additional analyses were done for M72. Nonameric MHC class I-binding peptides in Mtb72F were predicted for three alleles representative of class I supertypes A02, A03 and B07, using seven prediction algorithms. RESULTS: Sequence identity among strains was ≥98 % for each protein. Residue changes in Mtb39A comprised primarily single residue or nucleotide insertions and/or deletions in repeat regions, and were observed in 67 % of strains. For Mtb72F, 156 DRB1, 6 DRB3/4/5, 7 DQ and 5 DP alleles were predicted to contain at least one MHC class II-binding peptide, and class I-binding peptides were predicted for each HLA-A/B allele. Comparison of predicted MHC-II-binding peptides with experimental data indicated that the algorithm's sensitivity and specificity were variable among alleles. CONCLUSIONS: The sequences from which Mtb72F and M72 are derived are highly conserved among representative Mycobacterium strains. Predicted putative T-cell epitopes in M72 and/or Mtb72F covered a wide array of HLA alleles. In silico binding predictions for class I- and II-binding putative epitopes can be complemented with biochemical verification of HLA binding capacity, processing and immunogenicity of the predicted peptides.

Non-clinical evaluation of local and systemic immunity induced by different vaccination strategies of the candidate tuberculosis vaccine M72/AS01.

A new efficacious tuberculosis vaccine targeting adolescents/adults represents an urgent medical need. The M72/AS01E vaccine candidate protected half of the latently-infected adults against progression to pulmonary tuberculosis in a Phase IIb trial (NCT01755598). We report that three immunizations of mice, two weeks apart, with AS01-adjuvanted M72 induced polyfunctional, Th1-cytokine-expressing M72-specific CD4+/CD8+ T cells in blood and lungs, with the highest frequencies in lungs. Antigen-dose reductions across the three vaccinations skewed pulmonary CD4+ T-cell profiles towards IL-17 expression. In blood, reducing antigen and adjuvant doses of only the third injection (to 1/5th or 1/25th of those of the first injections) did not significantly alter CD4+ T-cell/antibody responses; applying a 10-week delay for the fractional third dose enhanced antibody titers. Delaying a full-dose booster enhanced systemic CD4+ T-cell and antibody responses. Cross-reactivity with PPE and non-PPE proteins was assessed, as Mycobacterium tuberculosis (Mtb) virulence factors and evasion mechanisms are often associated with PE/PPE proteins, to which Mtb39a (contained in M72) belongs. In silico/in vivo analyses revealed that M72/AS01 induced cross-reactive systemic CD4+ T-cell responses to epitopes in a non-vaccine antigen (putative latency-associated Mtb protein PPE24/Rv1753c). These preclinical data describing novel mechanisms of M72/AS01-induced immunity could guide future clinical development of the vaccine.

Impact of bacterial strain acquisition in the lung of patients with COPD: the AERIS study.

BACKGROUND: Bacterial infections are associated with acute exacerbations of chronic obstructive pulmonary disease (AECOPD), but the mechanism is incompletely understood. METHOD: In a COPD observational study (NCT01360398), sputum samples were collected monthly at the stable state and exacerbation. Post-hoc analyses of 1307 non-typeable Haemophilus influenzae (NTHi) isolates from 20 patients and 756 Moraxella catarrhalis isolates from 38 patients in one year of follow-up were conducted by multilocus sequence typing (MLST). All isolates came from cultured sputum samples that were analyzed for bacterial species presence, apparition (infection not detected at the preceding visit), or acquisition (first-time infection), with the first study visit as a baseline. Strain apparition or new strain acquisition was analyzed by MLST. The odds ratio (OR) of experiencing an exacerbation vs. stable state was estimated by conditional logistic regression modelling, stratified by patient. RESULTS: The culture results confirmed a significant association with exacerbation only for NTHi species presence (OR 2.28; 95% confidence interval [CI]: 1.12-4.64) and strain apparition (OR 2.38; 95% CI: 1.08-5.27). For M. catarrhalis, although confidence intervals overlapped, the association with exacerbation for first-time species acquisition (OR 5.99; 2.75-13.02) appeared stronger than species presence (OR 3.67; 2.10-6.40), new strain acquisition (OR 2.94; 1.43-6.04), species apparition (OR 4.18; 2.29-7.63), and strain apparition (OR 2.78; 1.42-5.42). This may suggest that previous M. catarrhalis colonization may modify the risk of exacerbation associated with M. catarrhalis infection. CONCLUSIONS: The results confirm that NTHi and M. catarrhalis infections are associated with AECOPD but suggest different dynamic mechanisms in triggering exacerbations.

Transcriptional regulation, occurrence and putative role of the Pht family of Streptococcus pneumoniae.

Restricted to the genus Streptococcus, the Pht protein family comprises four members: PhtA, PhtB, PhtD and PhtE. This family has the potential to provide a protein candidate for incorporation in pneumococcal vaccines. Based on sequence analysis and on RT-PCR experiments, we show here that the pht genes are organized in tandem but that their expression, except that of phtD, is monocistronic. PhtD, PhtE, PhtB and PhtA are present in 100, 97, 81 and 62 % of the strains, respectively, and, by analysing its sequence conservation across 107 pneumococcal strains, we showed that PhtD displays very little variability. To analyse the physiological function of these proteins, several mutants were constructed. The quadruple Pht-deficient mutant was not able to grow in a poor culture medium, but the addition of Zn(2+) or Mn(2+) restored its growth capacity. Moreover, the phtD mRNA expression level increased when the culture medium was depleted in zinc. Therefore, we suggest that these proteins are zinc and manganese scavengers, and are able to store these metals and to release them when the bacterium faces an ion-restricted environment. The data also showed that this protein family, and more particularly PhtD, is a promising candidate to be incorporated into pneumococcal vaccines.

UspA2 is a cross-protective Moraxella catarrhalis vaccine antigen.

Moraxella catarrhalis (Mcat) is a key pathogen associated with exacerbations of chronic obstructive pulmonary disease (COPD) in adults and playing a significant role in otitis media in children. A vaccine would help to reduce the morbidity and mortality associated with these diseases. UspA2 is an Mcat surface antigen considered earlier as vaccine candidate before the interest in this molecule vanished due to sequence variability. However, the observation that some conserved domains are the target of bactericidal antibodies prompted us to reconsider UspA2 as a potential vaccine antigen. We first determined its prevalence among the COPD patients from the AERIS study, as the prevalence of UspA2 in a COPD-restricted population had yet to be documented. The gene was found in all Mcat isolates either as UspA2 or UspA2H variant. The percentage of UspA2H variant was higher than in any report so far, reaching 51%. A potential link between the role of UspA2H in biofilm formation and this high prevalence is discussed. To study further UspA2 as a vaccine antigen, recombinant UspA2 molecules were designed and used in animal models and bactericidal assays. We showed that UspA2 is immunogenic and that UspA2 immunization clears Mcat pulmonary challenge in a mouse model. In a serum bactericidal assay, anti-UspA2 antibodies generated in mice, guinea pigs or rabbits were able to kill Mcat strains of various origins, including a subset of isolates from the AERIS study, cross-reacting with UspA2H and even UspA1, a closely related Mcat surface protein. In conclusion, UspA2 is a cross-reactive Mcat antigen presenting the characteristics of a vaccine candidate.

Patients with Chronic Obstructive Pulmonary Disease harbour a variation of Haemophilus species.

H. haemolyticus is often misidentified as NTHi due to their close phylogenetic relationship. Differentiating between the two is important for correct identification and appropriate treatment of infective organism and to ensure any role of H. haemolyticus in disease is not being overlooked. Speciation however is not completely reliable by culture and PCR methods due to the loss of haemolysis by H. haemolyticus and the heterogeneity of NTHi. Haemophilus isolates from COPD as part of the AERIS study (ClinicalTrials - NCT01360398) were speciated by analysing sequence data for the presence of molecular markers. Further investigation into the genomic relationship was carried out using average nucleotide identity and phylogeny of allelic and genome alignments. Only 6.3% were identified as H. haemolyticus. Multiple in silico methods were able to distinguish H. haemolyticus from NTHi. However, no single gene target was found to be 100% accurate. A group of omp2 negative NTHi were observed to be phylogenetically divergent from H. haemolyticus and remaining NTHi. The presence of an atypical group from a geographically and disease limited set of isolates supports the theory that the heterogeneity of NTHi may provide a genetic continuum between NTHi and H. haemolyticus.