Serotype-specific Antigens Research Articles

Genomic Islands Shape the Genetic Background of Both JP2 and Non-JP2 Aggregatibacter actinomycetemcomitans

Aggregatibacter actinomycetemcomitans is a periodontal pathogen associated with periodontitis. This species exhibits substantial variations in gene content among different isolates and has different virulence potentials. This study examined the distribution of genomic islands and their insert sites among genetically diverse A. actinomycetemcomitans strains by comparative genomic analysis. The results showed that some islands, presumably more ancient, were found across all genetic clades of A. actinomycetemcomitans. In contrast, other islands were specific to individual clades or a subset of clades and may have been acquired more recently. The islands for the biogenesis of serotype-specific antigens comprise distinct genes located in different loci for serotype a and serotype b–f strains. Islands that encode the same cytolethal distending toxins appear to have been acquired via distinct mechanisms in different loci for clade b/c and for clade a/d/e/f strains. The functions of numerous other islands remain to be elucidated. JP2 strains represent a small branch within clade b, one of the five major genetic clades of A. actinomycetemcomitans. In conclusion, the complex process of genomic island acquisition, deletion, and modification is a significant force in the genetic divergence of A. actinomycetemcomitans. Assessing the genetic distinctions between JP2 and non-JP2 strains must consider the landscape of genetic variations shaped by evolution.

PCP consensus protein/peptide alphavirus antigens stimulate broad spectrum neutralizing antibodies

Vaccines based on proteins and peptides may be safer and if calculated based on many sequences, more broad-spectrum than those designed based on single strains. Physicochemical Property Consensus (PCPcon) alphavirus (AV) antigens from the B-domain of the E2 envelope protein were designed, synthesized recombinantly and shown to be immunogenic (i.e. sera after inoculation detected the antigen in dotspots and ELISA). Antibodies in sera after inoculation with B-region antigens based on individual AV species (eastern or Venezuelan equine encephalitis (EEEVcon, VEEVcon), or chikungunya (CHIKVcon) bound only their cognate protein, while those designed against multiple species (Mosaikcon and EVCcon) recognized all three serotype specific antigens. The VEEVcon and EEEVcon sera only showed antiviral activity against their related strains (in plaque reduction neutralization assays (PRNT50/80). Peptides designed to surface exposed areas of the E2-A-domain of CHIKVcon were added to CHIKVcon inocula to provide anti-CHIKV antibodies. EVCcon, based on three different alphavirus species, combined with E2-A-domain peptides from AllAVcon, a PCPcon of 24 diverse AV, generated broad spectrum, antiviral antibodies against VEEV, EEEV and CHIKV, AV with less than 35% amino acid identity to each other (>65% diversity). This is a promising start to a molecularly defined vaccine against all AV. Further study with these antigens can illuminate what areas are most important for a robust immune response, resistant to mutations in rapidly evolving viruses. The validated computational methods can also be used to design broad spectrum antigens against many other pathogen families.

Comparative genomics of 26 complete circular genomes of 18 different serotypes of Actinobacillus pleuropneumoniae.

Actinobacillus pleuropneumoniae is a Gram-negative, rod-shaped bacterium of the family Pasteurellaceae causing pig pleuropneumonia associated with great economic losses worldwide. Nineteen serotypes with distinctive lipopolysaccharide (LPS) and capsular (CPS) compositions have been described so far, yet complete circular genomes are publicly available only for the reference strains of serotypes 1, 4 and 5b, and for field strains of serotypes 1, 3, 7 and 8. We aimed to complete this picture by sequencing the reference strains of 17 different serotypes with the MinION sequencer (Oxford Nanopore Technologies, ONT) and on an Illumina HiSeq (Illumina) platform. We also included two field isolates of serotypes 2 and 3 that were PacBio- and MinION-sequenced, respectively. Genome assemblies were performed following two different strategies, i.e. PacBio- or ONT-only de novo assemblies polished with Illumina reads or a hybrid assembly by directly combining ONT and Illumina reads. Both methods proved successful in obtaining accurate circular genomes with comparable qualities. blast-based genome comparisons and core-genome phylogeny based on core genes, SNP typing and multi-locus sequence typing (cgMLST) of the 26 circular genomes indicated well-conserved genomes across the 18 different serotypes, differing mainly in phage insertions, and CPS, LPS and RTX-toxin clusters, which, consistently, encode serotype-specific antigens. We also identified small antibiotic resistance plasmids, and complete subtype I-F and subtype II-C CRISPR-Cas systems. Of note, highly similar clusters encoding all those serotype-specific traits were also found in other pathogenic and commensal Actinobacillus species. Taken together with the presence of transposable elements surrounding these loci, we speculate a dynamic intra- and interspecies exchange of such virulence-related factors by horizontal gene transfer. In conclusion, our comprehensive genomics analysis provides useful information for diagnostic test and vaccine development, but also for whole-genome-based epidemiological studies, as well as for the surveillance of the evolution of antibiotic resistance and virulence genes in A. pleuropneumoniae .

Current methods of capsular typing of Streptococcus pneumoniae: possibilities and availability for local laboratories

Pneumococcal conjugate vaccines contain a limited number of serotype-specific antigens of S. pneumoniae. It is important for vaccination programmes effectiveness assessment to control a variety of circulating S. pneumoniae serotypes. This review provides an analysis of pneumococcal serotyping approaches and further ways of improving pneumococcal serotype detection within the microbiological surveillance. Serological methods and multiplex PCR can identify a limited number of pneumococcal serotypes only. Whole-genome sequencing-based approaches can predict almost all serotypes and sequence types as well as detect antimicrobial resistance and virulence genes.

Draft genome sequences of the type strains of Shigella flexneri held at Public Health England: comparison of classical phenotypic and novel molecular assays with whole genome sequence

BackgroundPublic Health England (PHE) holds a collection of Shigella flexneri Type strains isolated between 1949 and 1972 representing 15 established serotypes and one provisional type, E1037. In this study, the genomes of all 16 PHE Type strains were sequenced using the Illumina HiSeq platform. The relationship between core genome phylogeny and serotype was examined.ResultsThe most common target gene for the detection of Shigella species in clinical PCR assays, ipaH, was detected in all genomes. The type-specific target genes were correctly identified in each genome sequence. In contrast to the S. flexneri in serotype 5 strain described by Sun et al. (2012), the two PHE serotype 5 Type strains possessed an additional oac gene and were differentiated by the presence (serotype 5b) or absence (serotype 5a) of gtrX. The somatic antigen structure and phylogenetic relationship were broadly congruent for strains expressing serotype specific antigens III, IV and V, but not for those expressing I and II. The whole genome phylogenies of the 15 isolates sequenced showed that the serotype 6 Type Strain was phylogenetically distinct from the other S. flexneri serotypes sequenced. The provisional serotype E1037 fell within the serotype 4 clade, being most closely related to the Serotype 4a Type Strain.ConclusionsThe S. flexneri genome sequences were used to evaluate phylogenetic relationships between Type strains and validate genotypic and phenotypic assays. The analysis confirmed that the PHE S. flexneri Type strains are phenotypically and genotypically distinct. Novel variants will continue to be added to this archive.

Mucosal Immunization with Vibrio cholerae Outer Membrane Vesicles Provides Maternal Protection Mediated by Antilipopolysaccharide Antibodies That Inhibit Bacterial Motility

Vibrio cholerae is the causative agent of cholera, a severe diarrheal disease that remains endemic in many parts of the world and can cause outbreaks wherever sanitation and clean water systems break down. Prevention of disease could be achieved through improved sanitation and clean water provision supported by vaccination. V. cholerae serogroup O1 is the major cause of cholera; O1 serotypes Inaba and Ogawa have similar disease burdens, while O139 is the only non-O1 serogroup to cause epidemics. We showed previously that immunization of adult female mice with purified V. cholerae outer membrane vesicles (OMVs) elicits an antibody response that protect neonates from oral V. cholerae challenge and that suckling from an immunized dam accounts for the majority of protection from V. cholerae colonization. Here we report that lipopolysaccharide (LPS) is the major OMV protective antigen. Mucosal immunization with OMVs from Inaba or Ogawa provides significant cross-serotype protection from V. cholerae colonization, although serotype-specific antigens are dominant. OMVs from O1 or O139 do not provide cross-serogroup protection, but by immunization with a mixture of O1 and O139 OMVs, cross-serogroup protection was achieved. Neonatal protection is not associated with significant bacterial death but may involve inhibition of motility, as antibodies from OMV-immunized mice inhibit V. cholerae motility in vitro, with trends that parallel in vivo protection. Motility assays also reveal that a higher antibody titer is required to immobilize O139 compared to O1, a phenotype that is O139 capsule dependent.

Prevalence and distribution of serotype-specific genotypes of Aggregatibacter actinomycetemcomitans in chronic periodontitis Brazilian subjects

Objective Previous studies have suggested that Aggregatibacter actinomycetemcomitans is involved in the aetiology of aggressive periodontitis as well as chronic periodontitis. In addition, some authors have also reported that serotype-specific antigens of A. actinomycetemcomitans determine the severity of disease. This study aimed to elucidate the prevalence of A. actinomycetemcomitans and the distribution of A. actinomycetemcomitans serotypes in Brazilian subjects with chronic periodontitis. Design A total of 486 individuals were enrolled in this survey. All patients received clinical examinations that included periodontal pocket depth, clinical attachment loss, plaque, and gingival indexes. Subgingival samples were taken for microbial analysis. The genomic DNA of A. actinomycetemcomitans was provided by PCR. Results Out of 486 subjects examined, A. actinomycetemcomitans was isolated in 85 (17.5%) individuals. Out of 85 positive samples, 68 were infected by at least 1 serotype, 7 by mixed infection, and 10 were non-serotyped. Serotypes d and f were not detected. Serotype c showed the highest prevalence (52.9%), followed by serotype a (31.8%). Conclusions Intragroup analysis revealed that, in slight/moderate periodontitis, serotypes c and a were significantly more prevalent than serotypes b and d–f; the prevalence of serotype c in severe periodontitis was significantly greater than that of serotypes a and b. Our data were similar in Asian and Eurasian populations.

Cross-Reactive T-Cell Responses to the Nonstructural Regions of Dengue Viruses among Dengue Fever and Dengue Hemorrhagic Fever Patients in Malaysia

Dengue virus infections are a major cause of morbidity and mortality in tropical and subtropical areas in the world. Attempts to develop effective vaccines have been hampered by the lack of understanding of the pathogenesis of the disease and the absence of suitable experimental models for dengue viral infection. The magnitude of T-cell responses has been reported to correlate with dengue disease severity. Sixty Malaysian adults with dengue viral infections were investigated for their dengue virus-specific T-cell responses to 32 peptides antigens from the structural and nonstructural regions from a dengue virus isolate. Seventeen different peptides from the C, E, NS2B, NS3, NS4A, NS4B, and NS5 regions were found to evoke significant responses in a gamma interferon enzyme-linked immunospot (ELISPOT) assay of samples from 13 selected patients with dengue fever (DF) and dengue hemorrhagic fever (DHF). NS3 and predominantly NS3(422-431) were found to be important T-cell targets. The highest peaks of T-cell responses observed were in responses to NS3(422-431) and NS5(563-571) in DHF patients. We also found almost a sevenfold increase in T-cell response in three DHF patients compared to three DF patient responses to peptide NS3(422-431). A large number of patients' T cells also responded to the NS2B(97-106) region. The ELISPOT analyses also revealed high frequencies of T cells that recognize both serotype-specific and cross-reactive dengue virus antigens in patients with DHF.

Developing an ELISA using serotype-specific antigens from selected pneumococcal strains of own selection

Streptococcus pneumoniae (pneumococcus) is a significant cause of morbidity and mortality in infants and elderly people. Pneumococcal strains possess a polysaccharide capsule, and 90 different serotypes have been identified. The pneumococcal ELISA suggested by World Health Organization (WHO) guidelines is based on capsular serotypes included in the 23-valent polysaccharide vaccine (Pneumovax ®). Pneumococcal antigens were developed and applied in an ELISA, which elicited a low baseline cross-reaction with the common cell wall polysaccharide (C-Ps). The aim of this study was to develop an ELISA using type-specific antigens from S. pneumoniae strains with serotypes of own selection in order to quantify type-specific pneumococcal antibodies in human serum.

Failure of a low virulence Streptococcus gallolyticus serotype 1 strain to immunize pigeons against streptococcosis

Ten pigeons were inoculated intravenously with the low virulence Streptococcus gallolyticus strain PDH 827, which belongs to serotype 1, supernatant phenotype A - T2. The birds did not develop clinical disease but shed S. gallolyticus in their faeces, and antibodies against the bacterium were detected in post-inoculation plasma samples of all birds. Seven weeks later, these pigeons, as well as 14 control pigeons, were challenged intravenously with the highly virulent S. gallolyticus strain STR 357, which also belongs to serotype 1 but to the supernatant phenotype A + T1. Post-inoculation morbidity in the immunized group amounted to 90%, demonstrating that no protective immunity had been built up after the first infection. These findings indicate that serotype-specific antigens are not, or at least not solely, involved in induction of protection against S. gallolyticus septicaemia in pigeons.

Biochemical and genetic characterization of serologically untypable Streptococcus mutans strains isolated from patients with bacteremia.

Four out of 522 streptococcal isolates from the peripheral blood of patients with bacteremia exhibited typical properties of Streptococcus mutans in terms of sucrose-dependent adhesion, expression of glucosyltransferases, fermentation profiles of sugars, the presence of surface protein antigen, and DNA-DNA hybridization. Two strains were determined as serotype f and e by immunodiffusion, whereas the other two isolates did not react with the specific antiserum to S. mutans serotype c. e. or f of the eight different serotypes of mutans streptococci. The latter two untypable isolates, however, expressed a new antigenic determinant that was different from serotype c/e/f specificity as revealed by immunodiffusion. Analysis of the cell wall polysaccharides revealed very low contents of glucose in the untypable isolates. Furthermore, Southern blot analysis demonstrated that the untypable strains lacked at least one gene corresponding to a glucose-adding enzyme. These results indicate that the serologically untypable nature is due to the loss of glucosidic residue from the serotype-specific polysaccharide antigens of S. mutans.

Identification of Actinobacillus actinomycetemcomitans serotypes by multiplex PCR.

Oligonucleotide primers specific for gene clusters involved in the biosynthesis of serotype-specific polysaccharide antigens were designed to identify Actinobacillus actinomycetemcomitans serotypes a to e using the multiplex PCR. This method may be useful for serotype-specific genotyping rapidly and directly from clinical samples containing various organisms.

A novel serotype-specific gene cassette (gltA-gltB) is required for expression of teichoic acid-associated surface antigens in Listeria monocytogenes of serotype 4b.

Listeria monocytogenes serotype 4b strains account for about 40% of sporadic cases and many epidemics of listeriosis. Mutations in a chromosomal locus resulted in loss of reactivity with all three monoclonal antibodies (MAbs) which were specific to serotype 4b and the closely related serotypes 4d and 4e. Here we show that this locus contains a serotype 4b-4d-4e-specific gene cassette (3,071 bp) which consists of two genes, gltA and gltB, and is flanked by palindromic sequences (51 and 44 nucleotides). Complete loss of reactivity with the three serotype-specific MAbs resulted from insertional inactivation of either gltA or gltB. The gltA and gltB mutants were characterized by loss and severe reduction, respectively, of glucose in the teichoic acid, whereas galactose, the other serotype-specific sugar substituent in the teichoic acid, was not affected. Within L. monocytogenes, only strains of serotypes 4b, 4d, and 4e harbored the gltA-gltB cassette, whereas coding sequences on either side of the cassette were conserved among all serotypes. Comparative genomic analysis of a serotype 1/2b strain showed that the 3,071-bp gltA-gltB cassette was replaced by a much shorter (528-bp) and unrelated region, flanked by inverted repeats similar to their counterparts in serotype 4b. These findings indicate that in the evolution of different serotypes of L. monocytogenes, this site in the genome has become occupied by serotype-specific sequences which, in the case of serotype 4b, are essential for expression of serotype-specific surface antigens and presence of glucose substituents in the teichoic acids in the cell wall.

Study of antigenic heterogeneity among Actinobacillus pleuropneumoniae serotype 7 strains.

Actinobacillus pleuropneumoniae serotype 7 strains were studied for their antigenic heterogeneity using rabbit polyclonal hyperimmune sera against all the known twelve reference strains of A. pleuropneumoniae and a battery of different serological tests such as coagglutination (COA), immunodiffusion (ID), indirect hemagglutination (IHA), counterimmunoelectrophoresis (CIE), rapid dot-ELISA (RDE), serum soft-agar (SSA) and growth agglutination (GA). Reference serotype 7 strain (WF83) showed cross-reactivity with reference serotype 1B strain but not with other serotypes. Field serotype 7 strains showed cross-reactivities with serotypes 1A, 1B, 4, 9, 10, and 11 in COA, ID, and CIE tests, but not in IHA test. Two field strains of serotype 7 (90-3182 and 86-1411) which appeared to be different from the typical serotype 7 strains were selected for further antigenic characterization by SDS-PAGE, Western blot, and Tricine SDS-PAGE assays, and identified as serotypes 1 and 7, respectively. For serotyping atypical strains, it is suggested to use Western blot assay as a confirmatory test to identify serotype-specific capsular and somatic antigens.

Development of Monoclonal Antibodies against Ureaplasma urealyticum Serotypes and Their Use for Serotyping Clinical Isolates

Monoclonal antibodies (MAbs) against Ureaplasma urealyticum serotype 2, 5, 7, 8, 10, 11, 12, and 13 reference strains were developed. The reactivities of these MAbs with the 14 serotype reference strains was verified by colony immunofluorescence assay and Western blot assay. MAbs against serotypes 2, 7, 10, 11, and 12 were serotype specific, whereas MAbs against serotypes 5, 8, and 13 showed cross-reactivity. All MAbs against serotype 5 were cross-reactive with serotype 2, and one showed, in addition, cross-reactivity to serotypes 9 and 10. Mutual cross-reactivities were observed between MAbs against serotypes 8 and 13. The usefulness of the MAbs for the serotyping of U. urealyticum strains was evaluated by serotyping 21 selected clinical isolates. A complete set of MAbs (the newly developed MAbs and the previously described MAbs against serotypes 1, 3, 4, 6, 9, and 14) as well as a complete set of polyclonal antibodies (PAbs), PAbs 1 to 14, were used. MAbs were able to identify 18 of 21 isolates including 2 isolates with mixed serotypes. Polyreactivity, which occurred with 19 of the 21 isolates with PAbs, was not observed by the use of MAbs. MAbs seem to be a more valuable tool than PAbs for serotyping and could help in investigating a possible link between the expression or variability of the serotype-specific antigens and pathogenicity.

Monkeys immunized with intertypic chimeric dengue viruses are protected against wild-type virus challenge.

Dengue epidemics caused by the four dengue virus serotypes continue to pose a major public health problem in most tropical and subtropical regions. A safe and effective vaccine against dengue is still not available. The current strategy for dengue immunization favors the use of a vaccine containing each of the four serotypes. We previously employed full-length dengue type 4 virus (DEN4) cDNA to construct a viable intertypic dengue virus of type 1 or type 2 antigenic specificity that contained the genes for the capsid-premembrane-envelope (C-pre-M-E) structural proteins of DEN1 or pre-M and E structural proteins of DEN2 substituting for the corresponding DEN4 genes. Chimeras DEN1/DEN4 and DEN2/DEN4, which express the nonstructural proteins of DEN4 and the C-pre-M-E structural proteins of DEN1 or the pre-M-E structural proteins of DEN2, and therefore the antigenicity of type 1 or type 2, were used to immunize rhesus monkeys. Other monkeys were inoculated with parental DEN1, DEN2, or cDNA-derived DEN4. Three of four monkeys immunized with DEN1/DEN4 developed neutralizing antibodies against DEN1 and were protected against subsequent DEN1 challenge. All four monkeys immunized with DEN2/DEN4 developed antibodies against DEN2 and were protected against subsequent DEN2 challenge. DEN1- and DEN2-immunized monkeys were protected against homologous virus challenge, but DEN4-immunized animals became viremic on cross-challenge with DEN1 or DEN2. In a second experiment, eight monkeys were immunized with equal mixtures of DEN1/DEN4 and DEN2/DEN4. Each of these monkeys developed neutralizing antibodies against both DEN1 and DEN2 and were protected against subsequent challenge with DEN1 or DEN2. Chimeric dengue viruses similar to those described here could be used to express serotype-specific antigens in a live attenuated tetravalent human vaccine.

Purification of serotype d-- and e--specific antigens from Actinobacillus actinomycetemcomitans and seroclassification of clinical isolates from periodontal patients

Oral Actinobacillus actinomycetemcomitans strains have been classified into five serotypes. The aim of this study was to determine the compositions of A. actinomycetemcomitans serotype d- and e- specific antigens. The serodistribution of clinical isolates from the patients with periodontitis were also investigated. Serotype-specific polysaccharide antigens of A. actinomycetemcomitans IDH 781 (serotype d) and OMZ 534 (serotype e) were extracted from whole cells by autoclaving. The extracts were purified by chromatography on DEAE-Sepharose CL-6B and Sephacryl S-300HR columns. The serotype d antigen was composed of rhamnose (17.1%), mannose (45.5%), galactose (2.0%) and glucose (35.5%). On the other hand, the serotype e antigen was composed of rhamnose (23.9%), mannose (29.1%), galactose (11.0%), glucose (13.5%) and unidentified sugar (22.5%). Immunodiffusion tests revealed that the purified polysaccharide antigen form a single precipitin line with the corresponding rabbit antiserum. A total of 157 A. actinomycetemcomitans clinical isolates from diseased sites of 39 patients with periodontitis were serotyped by using serotype-specific rabbit antisera against A. actinomycetemcomitans serotype a, b, c, d and e strains. In the immunodiffusion assay, the autoclaved extracts of 42, 12, 34, 8 and 41 A. actinomycetemcomitans clinical isolates reacted with serotype a, b, c, d and e antisera, respectively. These findings indicate that the extraction of serotype antigens by autoclaving is useful and definite for the serotyping of A. actinomycetemcomitans clinical isolates.

An enzyme-linked immunosorbent assay for measuring antibodies to serotype-specific polysaccharide antigens of Actinobacillus actinomycetemcomitans

An enzyme-linked immunosorbent assay utilizing avidin-biotin binding has been developed for measuring antibodies to serotype-specific polysaccharide antigens of Actinobacillus actinomycetemcomitans. The serotype a, b and c polysaccharide antigens were coupled with biotin via an adipic acid hydrazide functional group. The biotinylated antigens immobilized on the avidin-precoated wells reacted specifically with rabbit antisera to whole cells of the corresponding serotype of A. actinomycetemcomitans. Antibodies to the antigens in 1:6,400 to 1:12,800 dilutions of human sera from subjects with periodontitis could be detected by using the assay.

Effect of passive immunization with serotype-specific monoclonal antibodies on Actinobacillus pleuropneumoniae infection of mice.

Specific monoclonal antibodies (MoAbs) to Actinobacillus pleuropneumoniae (APP) serotypes 1 and 2 which recognized serotype-specific antigens were produced. It was revealed that the two serotype-specific MoAbs H1-18 and H22-7 recognized O polysaccharides of the lipopolysaccharide (LPS) from APP serotypes 1 and 2, respectively, in the results of antigen analysis by means of SDS-PAGE and Western blotting. Furthermore, ddY mice immunized passively with the above type-specific MoAbs were protected against challenge infection by the homologous serotype of APP at 24 or 48 hr later. However, H1-9 and H3-2 MoAbs recognizing serotype-common protein antigens of APP did not show signs of any protective effect. These results showed that LPS from APP bacterial cells was one of the structural substances in the serotype-specific antigens, and an important component as one of the antigens protecting against the homologous serotype strain.

Stereochemical aspects of the antigenic determinants of bacterial polysaccharides: the Rhodococcus equi capsular polysaccharides

The antigenic specificity of bacterial capsular polysaccharides has been extensively investigated. The origin of this specificity arises from the diversity of the oligosaccharide epitopes of the polysaccharide. Nuclear magnetic resonance spectroscopy provides a very powerful tool for probing the structure of the oligosaccharide epitopes. Using this technique in conjunction with chemical methods, the structures of the serotype-specific capsular polysaccharide antigens of Rhodococcus equi have been investigated. These studies have led to the identification of several structural features which can be related to serologic type specificity. This review will focus on some stereochemical aspects of the antigen determinants. Nuclear magnetic resonance based methods for determination of bacterial polysaccharide primary structure and for stereochemical analysis of chiral acidic substituents are presented.