Erratum: Rotavirus Genotypes and Vaccine Effectiveness from a Sentinel, Hospital-Based, Surveillance Study for Three Consecutive Rotavirus Seasons in Lebanon.

[This corrects the article DOI: 10.1371/journal.pone.0161345.].

The impact of vaccination on the burden of invasive pneumococcal disease from a nationwide surveillance program in Lebanon: an unexpected increase in mortality driven by non-vaccine serotypes.

BACKGROUND: The impact of pneumococcal conjugate vaccines (PCVs) on the burden of invasive pneumococcal disease (IPD) and serotype distribution was examined across age groups from data collected by the Lebanese Inter-Hospital Pneumococcal Surveillance Program. METHODS: Between 2005 and 2020, 593 invasive Streptococcus pneumoniae isolates were collected from 79 hospitals throughout Lebanon. Serotypes and antimicrobial resistance (AMR) profiles were identified, and trends compared over 3 eras: PCV7, post-PCV7/ pre-PCV13, and PCV13 eras. RESULTS: The prevalence of PCV7 serotypes decreased significantly from 43.6% in the PCV7 era to 17.8% during the PCV13 era (p<0.001). PCV13-only serotypes remained stable in the PCV13 compared to the post-PCV7 eras, especially serotypes 1 and 3, whereas non-vaccine types (NVT) increased throughout the study period, especially 24 and 16F. The mortality rate increased substantially from 12.5% (PCV7 era) to 24.8% (PCV13 era). A significant decrease in AMR was observed across the three study eras. CONCLUSION: PCVs substantially impacted IPD and AMR in vaccinated and unvaccinated populations despite an increase in mortality driven by NVT. Broadening the recommendation of vaccination to include older age-groups, using higher valency vaccines, and implementing stringent antimicrobial stewardship are likely to further impact the burden of IPD.

Wide Variability in the Sensitivity and Specificity of Rotavirus Immunoassay Diagnostic Kits in Practice.

INTRODUCTION: Most hospitals rely on rapid antigen-detection kits for the diagnosis of rotavirus infection. Several small studies reviewed the sensitivity and specificity of some of these kits. These studies showed discrepancy in results obtained for sensitivity and specificity that varied according to the type of kit used, area of study, and type of test used as standard for diagnosis of rotavirus infection. The objective of the study is to determine the sensitivity and specificity of five commonly used rotavirus immunoassay kits in comparison to RT-PCR as standard. METHODOLOGY: Stool samples (N = 1,414) collected from children under 5 years of age hospitalized with gastroenteritis were tested for rotavirus by immunoassay kits and RT-PCR in a prospective hospital-based surveillance study conducted at 7 centers in Lebanon. Concordance and discrepancy between the two methods was used to calculate sensitivity and specificity, using RT-PCR as the "gold standard". RESULTS: The sensitivity and specificity were respectively 95.08% and 86.62% for the SD Bioline® (Standard Diagnostics, Inc, South Korea) kit calculated on 645 samples, 65.86% and 45.90% for the VIROTECT® (Trinity Biotech, Ireland) kit calculated on 327 samples, 83.9% and 64.2% for the Rota-Strip (C-1001) (Coris Bioconcept, Belgium) calculated on 95 samples, 52.3% and 10.9% for the Acon® (Acon Laboratories, Inc, California, USA) kit calculated on 122 samples, 68.1% and 20% for the VIKIA® Rota-Adéno (Biomerieux, France) kit calculated on 32 samples. CONCLUSION: A wide discrepancy was detected between the calculated and advertised sensitivity and specificity for most of the kits.

The Emergence of Invasive Streptococcus pneumoniae Serotype 24F in Lebanon: Complete Genome Sequencing Reveals High Virulence and Antimicrobial Resistance Characteristics.

BACKGROUND: Invasive pneumococcal disease (IPD) remains a global health problem. IPD incidence has significantly decreased by the use of pneumococcal conjugate vaccines (PCV). Nevertheless, non-PCV serotypes remain a matter of concern. Eight Streptococcus pneumoniae serotype 24F isolates, belonging to a non-PCV serotype, were detected through the Lebanese Inter-Hospital Pneumococcal Surveillance Program. The aim of the study is to characterize phenotypic and genomic features of the 24F isolates in Lebanon. METHODS: WGS using long reads sequencing (PacBio) was performed to produce complete circular genomes and to determine clonality, antimicrobial resistance and virulence determinants. RESULTS: The sequencing results yielded eight closed circular genomes. Three multilocus sequence typing (MLST) types were identified (ST11618, ST14184, ST15253). Both MLST and WGS analyses revealed that these isolates from Lebanon were genetically homogenous belonging to clonal complex CC230 and clustered closely with isolates originating from Canada, United States of America, United Kingdom and Iceland. Their penicillin binding protein profiles correlated with both ß-lactam susceptibility patterns and MLST types. Moreover, the isolates harbored the macrolide and tetracycline resistance genes and showed a similar virulence gene profile. To our knowledge, this study represents the first report of complete phenotypic and genomic characterization of the emerging Streptococcus pneumoniae, serotype 24F, in the Middle East and North Africa region.

Molecular Characterization of Candida auris Isolates at a Major Tertiary Care Center in Lebanon.

BACKGROUND: The globally emerging Candida auris pathogens poses heavy burden to the healthcare system. Their molecular analyses assist in understanding their epidemiology, dissemination, treatment, and control. This study was warranted to describe the genomic features and drug resistance profiles using whole genome sequencing (WGS) among C. auris isolates from Lebanon. METHODS: A total of 28 C. auris clinical isolates, from different hospital units, were phenotypically identified by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) and tested for antifungal resistance using Vitek-2 system and E test. The complete genomes were determined by WGS using long reads sequencing (PacBio) to reveal the clade distribution and antifungal resistance genes. RESULTS: Candida auris revealed uniform resistance to fluconazole and amphotericin B, with full susceptibility to echinocandins. Among key resistance genes studied, only two mutations were detected: Y132F in ERG11 gene and a novel mutation, D709E, found in CDR1 gene encoding for an ABC efflux pump. Phylogenetically, C. auris genomes belonged to South Asian clade I and showed limited genetic diversity, suggesting person to person transmission. CONCLUSION: This characterization of C. auris isolates from Lebanon revealed the exclusivity of clade I lineage together with uniform resistance to fluconazole and amphotericin B. The control of such highly resistant pathogen necessitates an appropriate and rapid recovery and identification to contain spread and outbreaks.

The origins of G12P[6] rotavirus strains detected in Lebanon.

The G12 rotaviruses are an increasingly important cause of severe diarrhoea in infants and young children worldwide. Seven human G12P[6] rotavirus strains were detected in stool samples from children hospitalized with gastroenteritis in Lebanon during a 2011-2013 surveillance study. Complete genomes of these strains were sequenced using VirCapSeq-VERT, a capture-based high-throughput viral-sequencing method, and further characterized based on phylogenetic analyses with global RVA and vaccine strains. Based on the complete genomic analysis, all Lebanese G12 strains were found to have Wa-like genetic backbone G12-P[6]-I1-R1-C1-M1-A1-N1-T1-E1-H1. Phylogenetically, these strains fell into two clusters where one of them might have emerged from Southeast Asian strains and the second one seems to have a mixed backbone between North American and Southeast Asian strains. Further analysis of these strains revealed high antigenic variability compared to available vaccine strains. To our knowledge, this is the first report on the complete genome-based characterization of G12P[6] emerging in Lebanon. Additional studies will provide important insights into the evolutionary dynamics of G12 rotaviruses spreading in Asia.

Spoligotyping of Mycobacterium tuberculosis isolates using Luminex®-based method in Lebanon.

INTRODUCTION: Data about the genotypes of circulating Mycobacterium tuberculosis isolates (MTB) in Lebanon are scarce. This study was undertaken to reveal the spoligotypes of MTB isolates recovered from patients in Lebanon. METHODOLOGY: MTB isolates from 49 patients living in Lebanon were recovered and identified. The samples were heat killed and subjected to DNA extraction. Spoligotyping was performed using microbeads from TB-SPOL Kit and the fluorescence intensity was measured using Luminex 200®. Generated patterns were assigned to families using the SITVIT2 international database of the Pasteur Institute of Guadeloupe and compared. RESULTS: The spoligotyping of the 49 MTB isolates revealed that 31 isolates belonged to Lineage 4 (Euro-American, 63.3%), 12 to Lineage 3 (East- African Indian, 24.5%), 3 to Lineage 2 (East Asian, 6%) and 2 were unknown. Over half of the genotypes (16 of 30) harbored SIT127 supposed to belong to the L4.5 sublineage. One isolate belonging to the rare Manu-Ancestor SIT523 was recovered for the first time in Lebanon, being associated with highly virulent extensively drug-resistant (XDR) MTB phenotype. CONCLUSION: The application of the Spoligotyping Multiplex Luminex® method is an efficient, discriminatory and rapid method to use for first-lane genotyping of MTB isolates. Though humble numbers were tested, this study is one of the first to describe the genomic diversity and epidemiology of MTB isolates of Lebanon, and suggests an increasing prevalence of SIT127 in the country.

Genetic Diversity of Human Rotavirus A Among Hospitalized Children Under-5 Years in Lebanon.

Human rotavirus remains a major cause of gastroenteritis worldwide despite the availability of effective vaccines. In this study, we investigated the genetic diversity of rotaviruses circulating in Lebanon. We genetically characterized the VP4 and VP7 genes encoding the outer capsid proteins of 132 rotavirus-associated gastroenteritis specimens, previously identified in hospitalized children (<5 years) from 2011 to 2013 in Lebanon. These included 43 vaccine-breakthrough specimens and the remainder were from non-vaccinated subjects. Phylogenetic analysis of VP4 and VP7 genes revealed distinct clustering compared to the vaccine strains, and several substitutions were identified in the antigenic epitopes of Lebanese specimens. No unique changes were identified in the breakthrough specimens compared to non-breakthroughs that could explain the occurrence of infection in vaccinated children. Further, we report the emergence of a rare P[8] OP354-like strain with a G9 VP7 in Lebanon, possessing high genetic variability in their VP4 compared to vaccine strains. Therefore, human rotavirus strains circulating in Lebanon and globally have accumulated numerous substitutions in their antigenic sites compared to those currently used in the licensed vaccines. The successful spread and continued genetic drift of these strains over time might undermine the effectiveness of the vaccines. The effect of such changes in the antigenic sites on vaccine efficacy remains to be assessed.

Epidemiology of invasive and non-invasive pneumococcal infections in hospitalised adult patients in a Lebanese medical centre, 2006-2015.

This is a retrospective medical file review of adult inpatients with Streptococcus pneumoniae infections admitted to a Lebanese hospital between 2006 and 2015. We revisited the clinical scenarios of these infections in view of increasing antibiotic resistance in Lebanon. One hundred and three patients were included; 92% were eligible for pneumococcal vaccination, yet none were vaccinated. Non-invasive pneumococcal disease (non-IPD) represented 64% of these infections. Superinfections caused by antibiotic-resistant bacteria were documented in 17.5% of the patients, with the predominance of ventilator-associated pneumonia (12.6%). Kidney disease and septic shock were positive predictors for mortality [adjusted odds ratio (OR) = 14.96, 95% confidence interval (CI) 2.34-95.45, P = 0.004; OR = 5.09, 95% CI 1.33-19.51, P = 0.02, respectively]. Herein, the differences in clinical success, S. pneumoniae infection-related death, and total mortality were not statistically significant between invasive pneumococcal disease (IPD) and non-IPD subgroups (59.5% vs. 77.3%, P = 0.056; 21.6% vs. 9.1%, P = 0.08; and 35.1% vs. 22.7%, P = 0.174; respectively). Upon comparing antibiotic susceptibility of S. pneumoniae during the first two years of the study (2006-2007) (n = 32 isolates) and the last two (2014-2015) (n = 14 isolates), there was an increasing non-susceptibility to penicillin (34.4%-50.0%, P = 0.25), and a decreasing susceptibility to erythromycin and clindamycin (81.3%-78.6%, P = 0.67 and 90.6%-85.7%, P = 0.65; respectively).

Full genome characterization of human G3P[6] and G3P[9] rotavirus strains in Lebanon.

Rotaviruses are the most common infectious agents causing severe diarrheal diseases in young children globally. Three rare human rotavirus strains, two G3P[9] and one G3P[6], were detected in stool samples of children under 5 years of age hospitalized for gastroenteritis in Lebanon during the course of a surveillance study. Complete genomes of these strains were sequenced using VirCapSeq-VERT, a capture based high-throughput sequencing method. Genomic sequences were further characterized by using phylogenetic analyses with global RVA G3P[6]/P[9] strains, other vaccine and reference strains. Genetic analysis revealed that the G3P[6] strain emerged as a DS-1/Wa-like mono-reassortant strain with a potential Ethiopian origin. The two G3P[9] strains possessed a mixed DS-1/Wa/AU-1-like origin indicating that these may have evolved via multiple reassortment events involving feline, human and bovine rotaviruses. Furthermore, analysis of these strains revealed high antigenic variability compared to the vaccine strains. Additional studies are essential to fully understand the evolutionary dynamics of G3P[6]/P[9] strains spreading worldwide and their implications on vaccine effectiveness.

Genotyping of Mycobacterium tuberculosis in Lebanon using a novel rapid spoligotyping multiplex luminex method.

INTRODUCTION: Incidence of Tuberculosis (TB) in Lebanon, according to the WHO, is estimated to be 35 cases per 100,000 people. However, data about the genotypes of circulating Mycobacterium tuberculosis isolates (MTB) in this country is lacking. This study aims to reveal the genotypes of TB isolates recovered from patients in Lebanon. METHODOLOGY: Fifty M. tuberculosis isolates from patients in Lebanon were recovered and identified at the reference TB center of the Ministry of Public Health. All isolates were heat killed and subjected to DNA extraction. Spoligotyping method (TB-Spol, Beamedex, France) was used to identify the presence of 43 spacers via a multi-analyte profiling system (Luminex, Bio-Rad). Generated patterns were assigned to families using the SITVIT2 international database of the Pasteur Institute of Guadeloupe. RESULTS: The spoligotyping of the 50 MTB isolates revealed 13 lineages, one being novel. The most frequent shared-types (SIT) identified lineage was the Ural (34%), followed by the Central Asian lineage (10%) and a single isolate (2%) belonging to the rare Manu-Ancestor SIT523 lineage, associated with a highly virulent XDR MTB phenotype. The rest of the SIT isolates (18%) were equally distributed along 9 different lineages. The 13th non-SIT lineage is a novel one constituting 36% of the total isolates. CONCLUSION:  The application of Spoligotyping Multiplex Luminex method is a novel, discriminatory and rapid method to use for genotyping of MTB isolates employing the multi-spacer analysis system. Our study showed genomic diversification of MTB isolates from Lebanon.

Epidemiology of pneumococcal infections in hospitalised adult patients in Lebanon with a highlight on non-invasive disease.

INTRODUCTION: Streptococcus pneumoniae causes a wide range of infections classified as invasive and non-invasive pneumococcal disease (non-IPD). METHODOLOGY: We retrospectively reviewed over a decade the clinical course and outcome of 103 adult subjects infected with S. pneumoniae. RESULTS: The majority of the subjects (92%) were eligible for pneumococcal vaccination, however none were vaccinated. Most of the infective strains caused non-IPD (64%), with CAP being the leading primary infection (49%). Clinical success was achieved in 71% of the cases and microbiological success in 94% of the cases with available documented follow-up cultures. Yet, 19% of the subjects developed superinfections caused by extensive-drug resistant bacteria with the predominance of ventilator-associated pneumonia (13%). Total in-hospital mortality reached 27% and S. pneumoniae infection attributed mortality was 20%. Using multivariate logistic regression, kidney disease and septic shock were independent risk factors for mortality [Odd's Ratio (OR) = 14.96 (2.34-95.45), p = 0.004; OR = 5.09 (1.33-19.51), p = 0.02, respectively]. On comparing outcome between subjects with IPD and those with non-IPD, death attributed to S. pneumoniae infection was found to be significantly higher in subjects with IPD (23%, p = 0.023). Nevertheless, clinical success and total in-hospital mortality rates were not statistically different between the two groups (p = 0.056, p = 0.174, respectively). CONCLUSION: S. pneumoniae remains a pathogen causing considerable mortality. In adults, non-IPD should be considered of comparable importance as IPD. Increasing pneumococcal vaccine awareness at the healthcare professional and patient levels is essential for increasing vaccine uptake, thus decreasing the incidence, severity and sequelea of pneumococcal disease.

Characterization of astrovirus-associated gastroenteritis in hospitalized children under five years of age.

PURPOSE: The aim of this study was to determine the incidence and genetic diversity of astrovirus (AstV) detected in children hospitalized for gastroenteritis (GE). METHODS: A multi-center, hospital-based surveillance study was conducted across Lebanon to investigate the incidence of AstV among diarrheal hospitalizations. Viral RNA was extracted from stool samples collected between 2011 and 2013 from children, below the age of 5years, hospitalized for GE at six medical centers across Lebanon. Demographic and clinical data were collected and analyzed. RNA of eligible samples (n=739) was screened by two AstV-specific PCR assays followed by genotype-specific PCR. Sanger sequencing and phylogenetic analysis were performed for genotypic characterization. RESULTS: Overall, 5.5% (41/739) of rotavirus-negative stool samples collected from hospitalized children <5years old tested positive for AstV infection. AstV infections were detected all year long. Diarrhea, dehydration, vomiting and fever were the most common symptoms associated with AstV infections. Children aged 48-59months had the highest incidence of AstV. Using the Vesikari Scoring System to assess clinical severity, 85.4% of children with AstV had a score>11, indicating severe GE. Genotype-specific PCR identified 22 classical and 4 MLB-like AstV specimens. Further sequencing and phylogenetic analysis of orf1b and orf2 genes revealed that AstV classical 1-3, 5, 6, and 8, MLB-1, VA-1 and -2 genotypes circulated in Lebanon. Recombination between classical AstV strains was detected in several cases as evident by the lack of congruency in the tree topologies of the orf1b and orf2. Two cases of mixed infections between classical and non-classical genotypic strains were recorded. CONCLUSION: High genetic diversity was detected among AstVs in Lebanon. AstVs are associated with 5.5% of non-rotavirus GE-associated hospitalizations in children under five years in Lebanon.

Rotavirus Genotypes and Vaccine Effectiveness from a Sentinel, Hospital-Based, Surveillance Study for Three Consecutive Rotavirus Seasons in Lebanon.

INTRODUCTION: Globally, rotavirus (RV) is the leading cause of gastroenteritis (GE) in children. Longitudinal data about changes in RV genotype distribution and vaccine effectiveness (VE) are scarce. This study was conducted in Lebanon over 3 consecutive RV seasons to estimate the rate of RVGE hospitalization, identify RV genotypes, determine the seasonal and geographical variations, and calculate RV VE. MATERIALS AND METHODS: This prospective, multicenter, hospital-based surveillance study was conducted between 2011 and 2013 and enrolled children (<5 years) admitted for GE. Socio-demographic and clinical data about the current episode of GE at admission were collected. Genotypes were determined from stool samples testing positive for RV by PCR. RESULTS: Of 1,414 cases included in the final analysis, 83% were <2 years old and 55.6% were boys. Median duration of hospitalization was 4 days and 91.6% of GE cases were severe (Vesikari score ≥11). PCR testing showed that 30.3% of subjects were RV-positive of which 62.1% had fever versus 71.1% of RV-negative subjects (P = 0.001). RV was predominantly detected in the cold season from November till March (69.9%). G and P genotype pairs for all RV-positive stool specimens showed a predominance of G1P[8] in 36% (n = 154) of specimens, G9P[8] in 26.4% (n = 113), and G2P[4] in 17.8% (n = 76). RV-negative subjects were more likely to be RV-vaccinated (21%) compared to the RV-positive subjects (11.3%) (P<0.001), with a vaccine breakthrough rate of 18.8%. The ratio of RV1-vaccinated for each RV5-vaccinated subject was 7.8 and VE against RV disease was 68.4% (95%CI, 49.6%-80.2%). CONCLUSION: RV is a major cause of GE requiring hospitalization of children under 5 years of age in Lebanon. A few genotypes predominated over the three RV seasons studied. Mass RV vaccination will likely decrease the burden of hospitalization due to RV. VE is similar to what has been observed for other middle-income countries.

The Novel PKCθ from Benchtop to Clinic.

The protein kinases C (PKCs) are a family of serine/threonine kinases involved in regulating multiple essential cellular processes such as survival, proliferation, and differentiation. Of particular interest is the novel, calcium-independent PKCθ which plays a central role in immune responses. PKCθ shares structural similarities with other PKC family members, mainly consisting of an N-terminal regulatory domain and a C-terminal catalytic domain tethered by a hinge region. This isozyme, however, is unique in that it translocates to the immunological synapse between a T cell and an antigen-presenting cell (APC) upon T cell receptor-peptide MHC recognition. Thereafter, PKCθ interacts physically and functionally with downstream effectors to mediate T cell activation and differentiation, subsequently leading to inflammation. PKCθ-specific perturbations have been identified in several diseases, most notably autoimmune disorders, and hence the modulation of its activity presents an attractive therapeutic intervention. To that end, many inhibitors of PKCs and PKCθ have been developed and tested in preclinical and clinical studies. And although selectivity remains a challenge, results are promising for the future development of effective PKCθ inhibitors that would greatly advance the treatment of several T-cell mediated diseases.

Preclinical activity of the type II CD20 antibody GA101 (obinutuzumab) compared with rituximab and ofatumumab in vitro and in xenograft models.

We report the first preclinical in vitro and in vivo comparison of GA101 (obinutuzumab), a novel glycoengineered type II CD20 monoclonal antibody, with rituximab and ofatumumab, the two currently approved type I CD20 antibodies. The three antibodies were compared in assays measuring direct cell death (AnnexinV/PI staining and time-lapse microscopy), complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cell-mediated phagocytosis (ADCP), and internalization. The models used for the comparison of their activity in vivo were SU-DHL4 and RL xenografts. GA101 was found to be superior to rituximab and ofatumumab in the induction of direct cell death (independent of mechanical manipulation required for cell aggregate disruption formed by antibody treatment), whereas it was 10 to 1,000 times less potent in mediating CDC. GA101 showed superior activity to rituximab and ofatumumab in ADCC and whole-blood B-cell depletion assays, and was comparable with these two in ADCP. GA101 also showed slower internalization rate upon binding to CD20 than rituximab and ofatumumab. In vivo, GA101 induced a strong antitumor effect, including complete tumor remission in the SU-DHL4 model and overall superior efficacy compared with both rituximab and ofatumumab. When rituximab-pretreated animals were used, second-line treatment with GA101 was still able to control tumor progression, whereas tumors escaped rituximab treatment. Taken together, the preclinical data show that the glyoengineered type II CD20 antibody GA101 is differentiated from the two approved type I CD20 antibodies rituximab and ofatumumab by its overall preclinical activity, further supporting its clinical investigation.

Preclinical studies on the mechanism of action and the anti-lymphoma activity of the novel anti-CD20 antibody GA101.

GA101 is a novel glycoengineered Type II CD20 monoclonal antibody. When compared with rituximab, it mediates less complement-dependent cytotoxicity (CDC). As expected for a Type II antibody, GA101 appears not to act through CDC and is more potent than the Type I antibody rituximab in inducing cell death via nonclassical induction of apoptosis cytotoxicity, with more direct cytotoxicity and more antibody-dependent cell-mediated cytotoxicity. We evaluated the antitumor activity of GA101 against the human-transformed follicular lymphoma RL model in vivo in severe combined immunodeficient mice (SCID) mice. GA101 induced stronger inhibition of tumor growth than rituximab. Combination of GA101 with cyclophosphamide in vivo confirmed the superiority of GA101 over rituximab. Neutralizing the complement system with cobra venom factor partially impaired the antitumor activity of rituximab, but had no impact on the efficacy of GA101. In vitro GA101 more potently induced cell death of RL cells than rituximab. The expression of a limited number of genes was found to be induced by both antibodies after exposure in vitro. Among these, early growth response 1 and activation transcription factor 3 were confirmed to be increased at the protein level, suggesting a possible role of these proteins in the apoptotic signalling of anti-CD20 antibodies. These data imply that GA101 is superior to rituximab not only as a single agent, but also in combination with chemotherapy. These data suggest the presence of novel signalization pathways activated after exposure to anti-CD20 antibodies.

Identification of Mycoplasma mycoides subsp. mycoides small colony genes coding for T-cell antigens.

Genes of the Mycoplasma mycoides subsp. mycoides small colony biotype (MmmSC) coding for proteins capable of eliciting protective T-cell memory responses have potential for incorporation into a recombinant subunit vaccine against contagious bovine pleuropneumonia (CBPP). Here we used lymphocytes from cattle that had completely recovered from infection to screen products of MmmSC genes for recognition by CD4(+) effector memory (Tem) and central memory (Tcm) T lymphocytes. Six MmmSC genes (abc, gapN, glpO, lppA, lppB, and ptsG) were expressed as histidine-tagged recombinant polypeptides, or synthetic overlapping peptides, before inclusion in proliferation and gamma interferon (IFN-gamma) assays. Only two MmmSC antigens, LppA and PtsG, consistently induced recall proliferation from immune CD4(+) T cells and IFN-gamma production in all animals tested. Moreover, LppA and PtsG were shown to possess epitopes recognized by both short-lived CD4(+) Tem and long-lived CD4(+) Tcm cells.

Transfection of cells in suspension by ultrasound cavitation.

Sonoporation holds many promises in developing an efficient, reproducible and permanent gene delivery vector. In this study, we evaluated sonoporation as a method to transfect nucleic acids in suspension cells, including the human follicular lymphoma cell line RL and fresh human Chronic Lymphocytic Leukemia (CLL) cells. RL and CLL cells were exposed to continuous ultrasound waves (445 kHz) in the presence of either plasmid DNA coding for green fluorescent protein (GFP) or fluorescent siRNA directed against BCL2L1. Transfection efficiency and cell viability were assessed using fluorescent microscopy and flow cytometry analysis, respectively. Knock-down of target protein by siRNA was assessed by immunoblotting. Moreover, sonoporation was used to stably transfect RL cells with a plasmid coding for luciferase (pGL3). These cells were then used for the non-invasive monitoring of tumorigenesis in immunodeficient SCID mice. Sonoporation allows a highly efficient transfection of nucleic acid in suspension cells with a low rate of mortality, both in a tumor cell line and in fresh human leukemic cells. It also allowed efficient transfection of BCL2L1 siRNA with efficient reduction of the target protein level. In conclusion, ultrasound cavitation represents an efficient method for the transfection of cells in suspension, including fresh human leukemic cells.