[COVID-19 vaccines: the facts]. / COVID-19-vaccins: hoe zit het?

Not all physicians advocate for large-scale vaccination programmes against COVID-19. In this article, we respond on some of their reflections. Moreover, we explain that there are strong arguments for these large-scale vaccination programmes, aimed to prevent COVID-19 associated morbidity, mortality and overwhelmed health care systems, and to hinder the emergence of new strains of SARS-CoV-2 by reducing the virus transmission.

Hepatitis B vaccination strategies tailored to different endemicity levels: some considerations.

Hepatitis B is a serious public health problem. Worldwide three different levels of hepatitis B endemicity (high, intermediate and low) can be distinguished. Areas with different levels of endemicity require tailored vaccination strategies to fit the needs for individuals at risk and/or countries, depending on the infection risk per age group, vaccination rate, duration of protection after vaccination, cost effectiveness of vaccination strategies and ease of implementation in the national immunization schedules.This opinion paper evaluates these factors and proposes a combination of infant risk group and universal adolescent vaccination for low endemic countries thus targeting the different groups at risk. A universal infant vaccination schedule starting with a newborn vaccination within 24h after birth is more appropriate in intermediate- and high-endemic regions.

On the design of national vaccination programmes.

The decision to include a vaccine in a national vaccination programme (or not) is usually evidence-based. Thereby, it is essential that the target disease causes a high burden of disease and that vaccination reduces this burden considerably. Furthermore, vaccination should be considered to be cost-effective by a government. Vaccines are usually administered according to standard vaccination schedules, which have been established on historical grounds. We argue and demonstrate with examples (meningococci C, Haemophilus influenzae, pneumococci and Bordetella pertussis) that adaptation of these standard vaccination schedules can be cost-saving and lead to better protection. To facilitate the improvement of vaccination programmes, a better understanding of protective immune responses (correlates of protection) and immunologic memory are required.

DNA rearrangements in the flagellin locus of an flaA mutant of Campylobacter jejuni during colonization of chicken ceca.

Campylobacter jejuni is an enteropathogen for humans but commensal for chickens. In both hosts, the flagella and motility are important colonization factors. The flagellin gene is duplicated in Campylobacter, but only one flagellin gene, flaA, is sufficient for motility. We found that, during colonization of the chicken intestine, a nonmotile flaA mutant of C. jejuni underwent rearrangements within its flagellin locus, thereby regaining its motility and colonization capacity. In contrast, in vitro motile revertants isolated from liquid culture showed different flagellin DNA rearrangements than after reversion in the chicken.

Inactivation of the flagellin gene of Salmonella enterica serotype enteritidis strongly reduces invasion into differentiated Caco-2 cells.

A nonflagellated mutant of Salmonella enterica serotype Enteritidis was constructed by disrupting the flagellin gene (fliC). Northern blot analysis indicated that the mutation did not affect expression of the downstream fliU gene. Infection experiments with differentiated Caco-2 cells revealed that the mutant was about 50-fold less invasive than the wild-type strain, while bacterial adherence was unaffected. Complementation of the mutant with an intact fliC copy restored flagella formation and efficient bacterial invasion. Our data demonstrate that the fliC gene of S. enterica serotype Enteritidis is essential for the invasion of Caco-2 cells.

Genetic variation and evolutionary origin of the direct repeat locus of Mycobacterium tuberculosis complex bacteria.

The direct repeat region in Mycobacterium tuberculosis complex strains is composed of multiple direct variant repeats (DVRs), each of which is composed of a 36-bp direct repeat (DR) plus a nonrepetitive spacer sequence of similar size. It has been shown previously that clinical isolates show extensive polymorphism in the DR region by the variable presence of DVRs, and this polymorphism has been used in the epidemiology of tuberculosis. In an attempt to better understand the evolutionary scenario leading to polymorphic DR loci and to improve strain differentiation by spoligotyping, we characterized and compared the DNA sequences of the complete DR region and its flanking DNA of M. tuberculosis complex strains. We identified 94 different spacer sequences among 26 M. tuberculosis complex strains. No sequence homology was found between any of these spacers and M. tuberculosis DNA outside of the DR region or with any other known bacterial sequence. Although strains differed extensively in the presence or absence of DVRs, the order of the spacers in the DR locus was found to be well conserved. The data strongly suggest that the polymorphism in clinical isolates is the result of successive deletions of single discrete DVRs or of multiple contiguous DVRs from a primordial DR region containing many more DVRs than seen in present day isolates and that virtually no scrambling of DVRs took place during evolution. Because the majority of the novel spacer sequences identified in this study were confined to isolates of the rare Mycobacterium canettii taxon, the use of the novel spacers in spoligotyping led only to a slight improvement of strain differentiation by spoligotyping.

Detection of spirochetes by polymerase chain reaction and its relation to the course of digital dermatitis after local antibiotic treatment in dairy cattle.

The aim of the study was to monitor the course of digital dermatitis after local antibiotic treatment in an experimental group (treated on diagnosis) and a control group (treated 5 days later). The present study was carried out on 2 farms involving 18 animals. Monitoring was performed by means of clinical findings and detection of spirochetes on the surface of the lesions, using a polymerase chain reaction. Superficial wound smears were taken before and after treatment. Twelve animals on both farms followed the classical healing process, but six animals responded poorly to treatment. We observed that without treatment, there was no self-cure in the control group within 5 days. There was a significant improvement in the clinical condition of all animals after treatment on both farms, during the follow-up period. The time until reappearance of new digital dermatitis lesions was not significantly different between the experimental and control group, but it was different between the two farms which could be due to the influence of farm factors. Using primers specific for Treponema denticola and Treponema vincentii, all the disease stages had at least one positive polymerase chain reaction result indicating the presence of spirochetes in samples of all the disease stages during the healing process. This implies that the spirochetes are not completely eradicated from the surface of the lesions after treatment. It was also observed that the classical ulcerative disease stage (M2) had relatively more positive polymerase chain reaction results compared to any other disease stage, showing a possible link between the presence of spirochetes and clinical disease.

A serological study of cohorts of young dogs, naturally exposed to Ixodes ricinus ticks, indicates seasonal reinfection by Borrelia burgdorferi sensu lato.

Thirty-three family dogs were monitored for antibodies to Borrelia burgdorferi sensu lato over a 3-year period. Serum samples were collected before and during the season of high tick activity. Antibody levels were measured with an ELISA based on whole-cell antigens and an ELISA with a purified recombinant flagellin (r410). Antibody levels measured with the whole-cell ELISA increased after the first exposure to ticks. Following the first seasonal period of tick quiescence, antibody levels decreased, and subsequently increased again in the second tick season. Thereafter whole-cell ELISA titres persisted at moderate levels and did not decrease between tick seasons. The recombinant flagellin ELISA did not show a strong response in the first tick season, but did in the second tick season and levels of antibodies continued to fluctuate thereafter. We conclude that most dogs in this study developed an antibody response against Borrelia burgdorferi sensu lato after their first tick infestation and were thereafter repeatedly immunologically stimulated, probably reinfected, during the consecutive tick seasons.

Diversity of capsular polysaccharide synthesis gene clusters in Streptococcus pneumoniae.

Streptococcus pneumoniae comprises 90 serotypes, each one having its own specific polysaccharide capsule. In order to explore the diversity of capsular polysaccharide synthesis (cps) gene clusters in S. pneumoniae, we performed cross-hybridizations between the 12 cps genes of S. pneumoniae serotype 14 and chromosomal DNA of 26 strains comprising 26 different capsule types. Large variations in the hybridization patterns were observed. The genes cps14A to cps14D are conserved in most serotypes. Sequences homologous to cps14I to cps14L were only observed in the four types of serogroup 15, which all have a capsule structure similar to that of type 14. By using a cps14E knock-out construct, cpsE mutants of the pneumococcal types 9N, 13, and 15B were obtained. These mutants were unencapsulated and showed reduced glycosyltransferase activity, indicating that the pneumococcal types 9N, 13, and 15B express a glucosyl-1-phosphate transferase which is homologous to Cps14E. Glycosyltransferase assays showed that among 21 pneumococcal types which contain glucose in the core of their capsule polysaccharide, 19 types express glucosyl-1-phosphate transferase activity. However, not all of these types hybridized strongly with Cps14E, the type 14 glucosyl-1-phosphate transferase gene. Thus, pneumococci possess glucosyltransferase genes distinct from cps14E, but encoding enzymes with identical activity. All serotypes which synthesized lipid-linked lactose intermediates in glycosyltransferase activity assays (type 11B, 13, 15F, 15A, 15B, 15C) hybridized with cps14G. This gene encodes a galactosyltransferase which catalyzes the addition of 1,4-linked beta-galactose to lipid-linked glucose. The cps14G homologues in type 11B, 13, 15F, 15A, 15B, and 15C may encode a similar beta-galactosyltransferase activity as cps14G in type 14.

Cloning and characterization of the gene encoding the primary sigma-factor of Campylobacter jejuni.

The rpoD gene encoding the primary sigma-factor of Campylobacter jejuni was amplified from genomic DNA with degenerate oligonucleotide primers. The complete gene encodes a polypeptide of 622 amino acids and has a deduced M(r) of 72.6 kDa. This polypeptide is 40% identical to the RpoD (sigma 70) protein of Escherichia coli and has 66% identity with the Helicobacter pylori RpoD protein. A C. jejuni sigma 70 promoter, not recognized by the E. coli sigma 70 factor, could be activated in this bacterium in the presence of the cloned C. jejuni RpoD protein.

Identification of Campylobacter jejuni promoter sequences.

A promoterless lacZ shuttle vector, which allowed screening of promoters by beta-galactosidase activity in Campylobacter jejuni and Escherichia coli, was developed. Chromosomal DNA fragments from C. jejuni were cloned into this vector; 125 of 1,824 clones displayed promoter activity in C. jejuni. Eleven clones with strong promoter activity in C. jejuni were further characterized. Their nucleotide sequences were determined, and the transcriptional start sites of the putative promoters in C. jejuni were determined by primer extension. Only 6 of these 11 promoters were functional in E. coli. The 11 newly characterized and 10 previously characterized C. jejuni promoters were used to establish a consensus sequence for C. jejuni promoters. The 21 promoters were found to be very similar. They contain three conserved regions, located approximately 10, 16, and 35 bp upstream of the transcriptional start point. The -10 region resembles that of a typical sigma70 E. coli promoter, but the -35 region is completely different. In addition a -16 region typical for gram-positive bacteria was identified.

Functional analysis of glycosyltransferases encoded by the capsular polysaccharide biosynthesis locus of Streptococcus pneumoniae serotype 14.

Bacteria belonging to the species Streptococcus pneumoniae vary in their capsule. Presently, 90 capsular serotypes are known, all possessing their own specific polysaccharide structure. Little is known about the biosynthesis of these capsular polysaccharides. The cps locus of S. pneumoniae serotype 14 was cloned. So far, 7 open reading frames have been sequenced, cps14B to cps14H. The gene products are similar to proteins involved in bacterial polysaccharide biosynthesis, both of Gram-negative and -positive micro-organisms. Gene-specific mutants were created for cps14D to cps14H by insertional mutagenesis. All mutants no longer agglutinated with a monoclonal antibody against type 14 capsule polysaccharides. The biosynthetic function of cps14E and cps14G was determined by analysis of the intermediates in the synthesis of the oligosaccharide subunit, formed in membrane preparations of the wild-type and mutant strains and in membrane preparations of Escherichia coli expressing the pneumococcal glycosyltransferases. The enzyme encoded by cps14E is a glucosyl-1-phosphate transferase that links glucose to a lipid carrier, the first step in the biosynthesis of the type 14 repeating unit. The gene product of cps14G encodes a beta-1,4-galactosyltransferase, the enzyme responsible for the second step in the subunit synthesis, the transfer of galactose to lipid-linked glucose.

Molecular discrimination between Campylobacter jejuni, Campylobacter coli, Campylobacter lari and Campylobacter upsaliensis by polymerase chain reaction based on a novel putative GTPase gene.

Polymerase chain reaction (PCR) mediated DNA fingerprinting has resulted in the identification of a novel Campylobacter jejuni gene, encoding a GTPase protein. The gene, consisting of 383 amino acids contained semi-conserved GTP-binding sites (designated G-1 to G-4), that are characteristic for members of the GTPase protein superfamily. Remarkably, this gene from C. Jejuni appears to encode a member of a novel family of GTP-binding proteins, containing two separate putative GTP-binding domains, each comprising a series of semi-conserved GTP-binding motifs. Spacing between these motifs is highly conserved. Based on this novel gene, a general PCR strategy for the identification of C. jejuni, C. coli, C. lari and C. upsaliensis was developed. PCR primers were deduced from GTP-binding motifs G-1 and G-3 of the first GTP-binding domain. These GTP-binding sites flank a variable region of precisely 117 bp in the four Campylobacter spp. that allowed the development of species-specific probes. This PCR-hybridization assay offers a novel tool for rapid molecular detection and specific identification of the thermophilic Campylobacter spp.

Capsular polysaccharide synthesis in Streptococcus pneumoniae serotype 14: molecular analysis of the complete cps locus and identification of genes encoding glycosyltransferases required for the biosynthesis of the tetrasaccharide subunit.

We have reported previously on seven genes (cps14B-H) of Streptococcus pneumoniae serotype 14, which are part of the type 14 capsular polysaccharide synthesis (cps14) locus. This study describes the cloning and sequencing of the remaining part of the cps14 locus. The entire cps14 gene cluster consists of 12 open reading genes (cps14A to cps14L), which appear to be arranged as a single transcriptional unit. The flanking regions of the cps14 locus contain vestiges of insertion elements. Moreover, a 115-bp-long repeated DNA element, which is also present in several other intergenic regions on the pneumococcal chromosome, has been identified upstream of cps14A. All 12 open reading frames (ORFs) were inactivated by the insertion of a tetracycline resistance cassette. The cps14A to cps14J and cps14L mutants were unencapsulated, whereas only a limited amount of capsular polysaccharide was expressed by a cps14K insertion mutant. Comparison with DNA and protein sequences available in databases allowed us to predict functions for four out of the five new cps14 gene products. The biosynthetic function of Cps14I was determined experimentally by analysis of intermediates in the synthesis of the type 14 tetrasaccharide subunit, catalysed by membrane preparations of Escherichia coli expressing pneumococcal glycosyltransferases. The cps14I gene encodes the beta-1,3-N-acetylglucosaminyltransferase activity necessary for the addition of the third sugar in the synthesis of the type 14 repeating unit. The activity encoded by cps14J was established using a synthetic glycosyltransferase acceptor: cps14J encodes a beta-1,4-galactosyltransferase, which requires beta-linked GlcNAc as an acceptor. Thus, Cps14J is responsible for the addition of the last (fourth) sugar in the synthesis of the type 14 subunit.

Cloning and characterization of the lytB gene of Campylobacter jejuni.

LytB of Escherichia coli is an essential gene involved in penicillin tolerance and the stringent response. The lytB gene of Campylobacter jejuni was cloned and characterized. It could complement a temperature-sensitive E. coli lytB mutant. The C. jejuni lytB gene encodes a protein of 277 amino acids that has 34, 36 and 40% amino acid identity with the LytB proteins of E. coli, Haemophilus influenzae, and Synechocystis sp. PCC6803, respectively. The lytB gene is situated between the aroA gene and a gene that encodes ribosomal protein S1.

Characterization of the fim2 and fim3 fimbrial subunit genes of Bordetella bronchiseptica: roles of Fim2 and Fim3 fimbriae and flagella in adhesion.

With DNA probes derived from the fimbrial subunit genes fim2 and fim3 of Bordetella pertussis, two homologous subunit genes of Bordetella bronchiseptica were identified and cloned. The nucleotide sequences of these genes were determined. Comparison of these nucleotide sequences with the B. pertussis fimbrial fim2 and fim3 subunit genes showed a pronounced homology. Therefore, the B. bronchiseptica genes were also designated fim2 and fim3. Expression of the two B. bronchiseptica genes was demonstrated by Western blotting (immunoblotting) with polyclonal antiserum directed against the Fim2 and Fim3 fimbrial subunit proteins of B. pertussis and by enzyme-linked immunosorbent assay with monoclonal antibodies. After growth of B. bronchiseptica in the presence of MgSO4, no expression of both fimbrial subunit genes was observed. While no fimbriae were expressed, expression of flagella was observed under these circumstances. A longer C-stretch (up to 19 cytosine residues) than the one in front of the fim2 and fim3 genes of B. pertussis is present in front of the B. bronchiseptica fimbrial genes. In adherence experiments, fimbriated (Bvg+) as well as flagellated (Bvg-) B. bronchiseptica bacteria were able to adhere to HeLa cells, whereas nonflagellated B. pertussis did not. This suggests that fimbriae as well as other factors (possibly flagella) contribute to adherence of B. bronchiseptica to eukaryotic cells.

The aroA gene of Campylobacter jejuni.

The gene for 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase (aroA) cloned from Campylobacter jejuni (Cj) strain 81116 was identified by complementation of an Escherichia coli (Ec) auxotrophic aroA mutant. The Cj aroA gene has been sequenced. It encodes an enzyme of 428 amino acids (aa), that is homologous to other bacterial EPSP synthases, especially that of Bacillus subtilis with which it has a 39% aa identity. The transcriptional start point was mapped. It is present in an upstream open reading frame (ORF) that has a strong homology to the gene encoding phenylalanine tRNA synthetase (pheS). Downstream from aroA another ORF is present which is homologous to the lytB gene of Ec. The stop codon of the aroA gene overlaps the start codon of lytB.

The capsule polysaccharide synthesis locus of streptococcus pneumoniae serotype 14: Identification of the glycosyl transferase gene cps14E.

To identify a chromosomal region of Streptococcus pneumoniae serotype 14 involved in capsule polysaccharide synthesis, two strategies were used: (i) Tn916 mutagenesis, followed by the characterization of four unencapsulated mutants, and (ii) cross-hybridization with a capsule polysaccharide synthesis gene (cps) probe from S. agalactiae, which has a structurally similar capsule. The two approaches detected the same chromosomal region consisting of two adjacent EcoRI fragments. One of these EcoRI fragments was cloned and hybridized with a cosmid library. This resulted in clone cMKO2. A similar cosmid clone was obtained from an unencapsulated Tn916 mutant, Spnl4.H. Sequence analysis of the two cosmid clones revealed that in the Tn916 mutant, a gene, cps14E, which is homologous to other bacterial genes encoding glycosyl transferases, had been inactivated. An open reading frame immediately downstream of cps14E, designated cps14F, shows no significant homology with any known genes or proteins. A functional assay showed that cps14E encodes a glycosyl transferase and that a gene-specific knockout mutant lacks this enzyme activity, whereas inactivation of cps14F does not have this effect.

Recombinant expression and use in serology of a specific fragment from the Cowdria ruminantium MAP1 protein.

The major antigenic protein (MAP1) of Cowdria ruminantium was screened for immunogenic regions by expression of overlapping recombinant DNA clones of the gene encoding the MAP1 protein. Two regions, designated MAP1-A and MAP1-B, were recognized by all antisera to 9 different isolates of C. ruminantium. MAP1-A contained one or more epitopes responsible for false-positive reactions with Ehrlichia antisera in several serological tests for cowdriosis. Cross-reactivity with MAP1-B was limited to antisera to Ehrlichia chaffeensis and Ehrlichia canis. Antisera to Ehrlichia species that infect ruminants (E. bovis, E. ovina, and E. phagocytophila) did not recognize MAP1-B. The sensitivity of an indirect ELISA based on MAP1-B was found to be excellent, since all sera from animals experimentally infected with C. ruminantium (64 out of 64) reacted with MAP1-B. Validation of this ELISA was carried out with field sera obtained from sheep raised in heartwater-free areas in Zimbabwe and from several Caribbean islands. Only 9 out of 111 samples from Zimbabwe, and 1 out of 58 samples from the Caribbean islands, which were considered to be false positives by immunoblot or indirect ELISA, reacted with MAP1-B. Thus, the ELISA based on MAP1-B is at present the most specific and sensitive serological test for cowdriosis.

Differences between Taylorella equigenitalis strains in their invasion of and replication in cultured cells.

The ability of Taylorella equigenitalis, the causative agent of contagious equine metritis, to invade and replicate in equine derm cells was studied. The kinetics of invasion and replication were determined for four T. equigenitalis strains. On the basis of these experiments, a simpler assay in which the invasive as well as the replicative properties of a particular strain could be determined was developed. This assay was used to characterize 32 strains, which had previously been typed by field inversion gel electrophoresis of genomic restriction fragments. The invasiveness of T. equigenitalis strains ranged from 3 to 0.015 bacteria per cell and seemed to be associated with the contagiousness of the infection. The replication index (number of intracellular bacteria per cell at 24 h after inoculation divided by the number of intracellular bacteria per cell at 4 h after inoculation) varied from 1 to 857 and seemed to be associated with the severity of the symptoms of contagious equine metritis. There was no association between the invasiveness and the replication index of the strains, nor was there an association of invasion and replication with field inversion gel electrophoresis grouping.