Vaccination With Detoxified Leukocidin AB Reduces Bacterial Load in a Staphylococcus aureus Minipig Deep Surgical Wound Infection Model.

Vaccines against Staphylococcus aureus have eluded researchers for >3 decades while the burden of staphylococcal diseases has increased. Early vaccine attempts mainly used rodents to characterize preclinical efficacy, and all subsequently failed in human clinical efficacy trials. More recently, leukocidin AB (LukAB) has gained interest as a vaccine antigen. We developed a minipig deep surgical wound infection model offering 3 independent efficacy readouts: bacterial load at the superficial and at the deep-seated surgical site, and dissemination of bacteria. Due to similarities with humans, minipigs are an attractive option to study novel vaccine candidates. With this model, we characterized the efficacy of a LukAB toxoid as vaccine candidate. Compared to control animals, a 3-log reduction of bacteria at the deep-seated surgical site was observed in LukAB-treated minipigs and dissemination of bacteria was dramatically reduced. Therefore, LukAB toxoids may be a useful addition to S. aureus vaccines and warrant further study.

Epidemiology of Escherichia coli Bacteremia: A Systematic Literature Review.

BACKGROUND: Escherichia coli is the most common cause of bacteremia in high-income countries. To enable the development and implementation of effective prevention strategies, a better understanding of the current epidemiology of invasive E. coli infections is needed. METHODS: A systematic review of literature published between 1 January 2007 and 31 March 2018 on the burden and epidemiology of E. coli bacteremia in populations that include adults in high-income countries was conducted. Meta-analysis was performed for descriptive purposes. RESULTS: During the studied time interval, the estimated incidence rate of E. coli bacteremia was 48 per 100 000 person-years, but this increased considerably with age: rates per 100 000 person-years were >100 in 55-to-75-year-olds and >300 in 75-to-85-year-olds. Overall, E. coli accounted for 27% of documented bacteremia episodes: 18% if hospital acquired, 32% if community-onset healthcare associated, and 33% if community acquired. The estimated case fatality rate was 12%. Approximately 44% of episodes were community acquired, 27% community-onset healthcare associated, and 27% hospital acquired. Urinary tract infection (UTI) was the primary source for 53% of episodes. CONCLUSIONS: This systematic review confirms the substantial burden of E. coli bacteremia in older adults and justifies the implementation of community-level programs to prevent E. coli bacteremia and ideally UTI in this age group.

Extraintestinal Pathogenic Escherichia coli, a Common Human Pathogen: Challenges for Vaccine Development and Progress in the Field.

Extraintestinal pathogenic Escherichia coli (ExPEC) is the most common gram-negative bacterial pathogen in humans. ExPEC causes the vast majority of urinary tract infections (UTIs), is a leading cause of adult bacteremia, and is the second most common cause of neonatal meningitis. Increasing multidrug resistance among ExPEC strains constitutes a major obstacle to treatment and is implicated in increasing numbers of hospitalizations and deaths and increasing healthcare costs associated with ExPEC infections. An effective vaccine against ExPEC infection is urgently needed. The O antigen, a component of the surface lipopolysaccharide, has been identified as a promising vaccine target. With the availability of a novel bioconjugation technology it is expected that multivalent O antigen conjugate vaccines can be produced at industrial scale. Clinical proof of concept of a 4-valent O antigen conjugate vaccine is ongoing. An ExPEC vaccine effective against strains that are associated with major diseases and resistant to multiple drugs could be routinely delivered to individuals at risk of developing severe E. coli infection, such as elderly people, individuals undergoing abdominal surgery and prostatic biopsy procedures, and persons at risk of recurrent and/or complicated UTI.

Zinc piracy as a mechanism of Neisseria meningitidis for evasion of nutritional immunity.

The outer membrane of Gram-negative bacteria functions as a permeability barrier that protects these bacteria against harmful compounds in the environment. Most nutrients pass the outer membrane by passive diffusion via pore-forming proteins known as porins. However, diffusion can only satisfy the growth requirements if the extracellular concentration of the nutrients is high. In the vertebrate host, the sequestration of essential nutrient metals is an important defense mechanism that limits the growth of invading pathogens, a process known as "nutritional immunity." The acquisition of scarce nutrients from the environment is mediated by receptors in the outer membrane in an energy-requiring process. Most characterized receptors are involved in the acquisition of iron. In this study, we characterized a hitherto unknown receptor from Neisseria meningitidis, a causative agent of sepsis and meningitis. Expression of this receptor, designated CbpA, is induced when the bacteria are grown under zinc limitation. We demonstrate that CbpA functions as a receptor for calprotectin, a protein that is massively produced by neutrophils and other cells and that has been shown to limit bacterial growth by chelating Zn²âº and Mn²âº ions. Expression of CbpA enables N. meningitidis to survive and propagate in the presence of calprotectin and to use calprotectin as a zinc source. Besides CbpA, also the TonB protein, which couples energy of the proton gradient across the inner membrane to receptor-mediated transport across the outer membrane, is required for the process. CbpA was found to be expressed in all N. meningitidis strains examined, consistent with a vital role for the protein when the bacteria reside in the host. Together, our results demonstrate that N. meningitidis is able to subvert an important defense mechanism of the human host and to utilize calprotectin to promote its growth.

ZnuD, a potential candidate for a simple and universal Neisseria meningitidis vaccine.

Neisseria meningitidis serogroup B (MenB) is a major cause of bacterial sepsis and meningitis, with the highest disease burden in young children. Available vaccines are based on outer membrane vesicles (OMVs) obtained from wild-type strains. However, particularly in toddlers and infants, they confer protection mostly against strains expressing the homologous protein PorA, a major and variable outer membrane protein. In the quest for alternative vaccine antigens able to provide broad MenB strain coverage in younger populations, but potentially also across all age groups, ZnuD, a protein expressed under zinc-limiting conditions, may be considered a promising candidate. Here, we have investigated the potential value of ZnuD and show that it is a conserved antigen expressed by all MenB strains tested except for some strains of clonal complex ST-8. In mice and guinea pigs immunized with ZnuD-expressing OMVs, antibodies were elicited that were able to trigger complement-mediated killing of all the MenB strains and serogroup A, C, and Y strains tested when grown under conditions of zinc limitation. ZnuD is also expressed during infection, since anti-ZnuD antibodies were detected in sera from patients. In conclusion, we confirm the potential of ZnuD-bearing OMVs as a component of an effective MenB vaccine.

Description and nomenclature of Neisseria meningitidis capsule locus.

Pathogenic Neisseria meningitidis isolates contain a polysaccharide capsule that is the main virulence determinant for this bacterium. Thirteen capsular polysaccharides have been described, and nuclear magnetic resonance spectroscopy has enabled determination of the structure of capsular polysaccharides responsible for serogroup specificity. Molecular mechanisms involved in N. meningitidis capsule biosynthesis have also been identified, and genes involved in this process and in cell surface translocation are clustered at a single chromosomal locus termed cps. The use of multiple names for some of the genes involved in capsule synthesis, combined with the need for rapid diagnosis of serogroups commonly associated with invasive meningococcal disease, prompted a requirement for a consistent approach to the nomenclature of capsule genes. In this report, a comprehensive description of all N. meningitidis serogroups is provided, along with a proposed nomenclature, which was presented at the 2012 XVIIIth International Pathogenic Neisseria Conference.

Meningococcal surface fibril (Msf) binds to activated vitronectin and inhibits the terminal complement pathway to increase serum resistance.

Complement evasion is an important survival strategy of Neisseria meningitidis (Nm) during colonization and infection. Previously, we have shown that Nm Opc binds to serum vitronectin to inhibit complement-mediated killing. In this study, we demonstrate meningococcal interactions with vitronectin via a novel adhesin, Msf (meningococcal surface fibril, previously NhhA or Hsf). As with Opc, Msf binds preferentially to activated vitronectin (aVn), engaging at its N-terminal region but the C-terminal heparin binding domain may also participate. However, unlike Opc, the latter binding is not heparin-mediated. By binding to aVn, Msf or Opc can impart serum resistance, which is further increased in coexpressers, a phenomenon dependent on serum aVn concentrations. The survival fitness of aVn-binding derivatives was evident from mixed population studies, in which msf/opc mutants were preferentially depleted. In addition, using vitronectin peptides to block Msf-aVn interactions, aVn-induced inhibition of lytic C5b-9 formation and of serum killing could be reversed. As Msf-encoding gene is ubiquitous in the meningococcal strains examined and is expressed in vivo, serum resistance via Msf may be of significance to meningococcal pathogenesis. The data imply that vitronectin binding may be an important strategy for the in vivo survival of Nm for which the bacterium has evolved redundant mechanisms.

An outer membrane receptor of Neisseria meningitidis involved in zinc acquisition with vaccine potential.

Since the concentration of free iron in the human host is low, efficient iron-acquisition mechanisms constitute important virulence factors for pathogenic bacteria. In Gram-negative bacteria, TonB-dependent outer membrane receptors are implicated in iron acquisition. It is far less clear how other metals that are also scarce in the human host are transported across the bacterial outer membrane. With the aim of identifying novel vaccine candidates, we characterized in this study a hitherto unknown receptor in Neisseria meningitidis. We demonstrate that this receptor, designated ZnuD, is produced under zinc limitation and that it is involved in the uptake of zinc. Upon immunization of mice, it was capable of inducing bactericidal antibodies and we could detect ZnuD-specific antibodies in human convalescent patient sera. ZnuD is highly conserved among N. meningitidis isolates and homologues of the protein are found in many other Gram-negative pathogens, particularly in those residing in the respiratory tract. We conclude that ZnuD constitutes a promising candidate for the development of a vaccine against meningococcal disease for which no effective universal vaccine is available. Furthermore, the results suggest that receptor-mediated zinc uptake represents a novel virulence mechanism that is particularly important for bacterial survival in the respiratory tract.

Preclinical evaluation of the Pht proteins as potential cross-protective pneumococcal vaccine antigens.

Current pneumococcal vaccines are composed of capsular polysaccharides (PS) of various serotypes, either as free PS or as protein-PS conjugates. The use of pneumococcus protein antigens that are able to afford protection across the majority of serotypes is envisaged as a relevant alternative and/or complement to the polysaccharides. In this context, based on several studies, the Pht protein family emerged as relevant vaccine candidates. The purpose of the present study was to evaluate the Pht protein family in several preclinical mouse models. Immunization with these antigens was compared with immunization with other pneumococcal antigens, such as CbpA, PspA, and PsaA. In a nasopharyngeal colonization model and in a lung colonization model, the Phts were found to be superior to the other candidates in terms of efficacy of protection and serotype coverage. Likewise, vaccination with PhtD allowed higher animal survival rates after lethal intranasal challenge. Finally, a passive transfer model in which natural anti-PhtD human antibodies were transferred into mice demonstrated significant protection against lethal intranasal challenge. This indicates that natural anti-PhtD human antibodies are able to protect against pneumococcal infection. Our findings, together with the serotype-independent occurrence of the Phts, designate this protein family as valid candidate antigens to be incorporated in protein-based pneumococcal vaccines.

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.

Expanding the role of bacterial vaccines into life-course vaccination strategies and prevention of antimicrobial-resistant infections.

A crisis in bacterial infections looms as ageing populations, increasing rates of bacteraemia and healthcare-associated infections converge with increasing antimicrobial resistance and a paucity of new antimicrobial classes. New initiatives are needed to develop bacterial vaccines for older adults in whom immune senescence plays a critical role. Novel vaccines require an expanded repertoire to prevent mucosal diseases such as pneumonia, skin and soft tissue infections and urinary tract infections that are major causes of morbidity and mortality in the elderly, and key drivers of antimicrobial resistance. This review considers the challenges inherent to the prevention of bacterial diseases, particularly mucosal infections caused by major priority bacterial pathogens against which current vaccines are sub-optimal. It has become clear that prevention of many lung, urinary tract and skin infections requires more than circulating antibodies. Induction of Th1/Th17 cellular responses with tissue-resident memory (Trm) cells homing to mucosal tissues may be a pre-requisite for success.

[Bacterial vaccines]. / Bacteriële vaccins.

Vaccines were originally developed to prevent potentially deadly childhood diseases, but during the 21st century attention broadened to include prevention of infection in all stages of life. Prevention and treatment of bacterial infections are two of the biggest public health challenges of the 21st century. A crisis threatens to arise as the ageing of the population and the associated increase in cases of life-threatening bacteraemia and healthcare-associated infection coincides with an increase in antimicrobial resistance.

Genetically modified L3,7 and L2 lipooligosaccharides from Neisseria meningitidis serogroup B confer a broad cross-bactericidal response.

Currently available Neisseria meningitidis serogroup B (MenB) vaccines are based on outer membrane vesicles (OMVs) that are obtained from wild-type strains. They are purified with the aim of decreasing the lipooligosaccharide (LOS) content and hence reduce the reactogenicity of the vaccine even though LOS is a potential protective antigen. In <2-year-old children, these MenB vaccines confer protection only against strains expressing homologous PorA, a major and variable outer membrane protein. Our objective was to develop a safe LOS-based vaccine against MenB. To this end, we used modified porA knockout strains expressing genetically detoxified (msbB gene-deleted) L2 and L3,7 LOSs, allowing the production of LOS-enriched OMVs. The vaccine-induced antibodies were found to be bactericidal against nearly all invasive strains, irrespective of capsular serogroup. In addition, we have also demonstrated that LOS lacking the terminal galactose (with a lgtB mutation; truncated L3 LOS), but not LOS produced without the galE gene, induced a bactericidal antibody response in mice similar to that seen for LOS containing the full lacto-N-neotetraose (L3,7 LOS). In conclusion, a bivalent detoxified LOS OMV-based vaccine demonstrated the potential to afford a broad cross-protection against meningococcal disease.

Escherichia coli and Staphylococcus aureus: leading bacterial pathogens of healthcare associated infections and bacteremia in older-age populations.

INTRODUCTION: Bacteremia and healthcare-associated infections (HAIs) continue to rise in industrialized countries, in line with population aging and increased healthcare needs. Escherichia coli and Staphylococcus aureus are the most common pathogens causing HAIs and bacteremia. AREAS COVERED: This review discusses risk factors in the healthcare setting contributing to the rise in bacteremia and other invasive bacterial HAIs in older populations in general, and attributed to E. coli and S. aureus. Treatment of infections is becoming increasingly difficult due to emerging antimicrobial resistance. Prophylactic vaccines may be the solution to lowering the burden of serious infections in the elderly. EXPERT COMMENTARY: E. coli and S. aureus vaccines routinely administered to older adults and to people undergoing prescheduled procedures with high infection risks could prevent a significant proportion of severe disease, and could potentially also limit the further emergence of antimicrobial resistance.

Development and Qualification of an Opsonophagocytic Killing Assay To Assess Immunogenicity of a Bioconjugated Escherichia coli Vaccine.

The global burden of disease caused by extraintestinal pathogenic Escherichia coli (ExPEC) is increasing as the prevalence of multidrug-resistant strains rises. A multivalent ExPEC O-antigen bioconjugate vaccine could have a substantial impact in preventing bacteremia and urinary tract infections. Development of an ExPEC vaccine requires a readout to assess the functionality of antibodies. We developed an opsonophagocytic killing assay (OPA) for four ExPEC serotypes (serotypes O1A, O2, O6A, and O25B) based on methods established for pneumococcal conjugate vaccines. The performance of the assay was assessed with human serum by computing the precision, linearity, trueness, total error, working range, and specificity. Serotypes O1A and O6A met the acceptance criteria for precision (coefficient of variation for repeatability and intermediate precision, ≤50%), linearity (90% confidence interval of the slope of each strain, 0.80, 1.25), trueness (relative bias range, -30% to 30%), and total error (total error range, -65% to 183%) at five serum concentrations and serotypes O2 and O25B met the acceptance criteria at four concentrations (the lowest concentration for serotypes O2 and O25B did not meet the system suitability test of maximum killing of ≥85% of E. coli cells). All serotypes met the acceptance criteria for specificity (opsonization index value reductions of ≤20% for heterologous serum preadsorption and ≥70% for homologous serum preadsorption). The assay working range was defined on the basis of the lowest and highest concentrations at which the assay jointly fulfilled the target acceptance criteria for linearity, precision, and accuracy. An OPA suitable for multiple E. coli serotypes has been developed, qualified, and used to assess the immunogenicity of a 4-valent E. coli bioconjugate vaccine (ExPEC4V) administered to humans.

Predicting future trends in the burden of pertussis in the 21st century: implications for infant pertussis and the success of maternal immunization.

Support is growing for maternal immunization using acellular pertussis (aP) vaccines to prevent severe pertussis disease and deaths among very young, unvaccinated infants. Vaccine effectiveness of maternal immunization is 91% in preventing laboratory-confirmed pertussis in infants aged <3 months. To date, most mothers were primed in childhood with whole-cell pertussis vaccines. Soon, the generation of aP-primed individuals will become the new mothers-to-be. The shorter duration of protection afforded by aP vaccines, which is more pronounced with repeated aP boosters, may lead to increased pertussis circulation among aP-primed parents. Maternal Tdap immunization in aP-primed mothers-to-be may become less effective. Additional measures to protect young infants may eventually be needed, along with new vaccines that induce higher quality and more durable responses.

Immunogenicity and safety of a tetravalent E. coli O-antigen bioconjugate vaccine in animal models.

BACKGROUND: Extra-intestinal pathogenic Escherichia coli (ExPEC) are major human pathogens; however, no protective vaccine is currently available. We assessed in animal models the immunogenicity and safety of a 4-valent E. coli conjugate vaccine (ExPEC-4V, serotypes O1, O2, O6 and O25 conjugated to Exotoxin A from Pseudomonas aeruginosa (EPA)) produced using a novel in vivo bioconjugation method. METHODS: Three doses of ExPEC-4V (with or without aluminum hydroxide) were administered to rabbits (2µg or 20µg per O-antigen, subcutaneously), mice (0.2µg or 2µg per O-antigen, subcutaneously) and rats (0.4µg or 4µg per O-antigen, intramuscularly). Antibody persistence and boostability were evaluated in rats using O6-EPA monovalent conjugate (0.4µg O-antigen/dose, intramuscularly). Toxicity was assessed in rats (16µg total polysaccharide, intramuscularly). Serum IgG and IgM antibodies were measured by ELISA. RESULTS: Robust antigen-specific IgG responses were observed in all animal models, with increased responses in rabbits when administered with adjuvant. O antigen-specific antibody responses persisted up to 168days post-priming. Booster immunization induced a rapid recall response. Toxicity of ExPEC-4V when administered to rats was considered to be at the no observed adverse effect level. CONCLUSIONS: ExPEC-4V conjugate vaccine showed good immunogenicity and tolerability in animal models supporting progression to clinical evaluation.

Cloning and molecular analysis of the galE gene of Neisseria meningitidls and its role in lipopolysaccharide biosynthesis.

The galE gene from Haemophilus influenzae was used as a hybridization probe for the galE gene of Neisseria meningitidis Group B, identifying two different homologous loci. Each of the loci was cloned and nucleotide sequence analysis revealed that both loci contained sequences similar to galE. One contained a functional galE gene and mapped to the capsule biosynthetic locus. The second contained only a partial galE-coding sequence, which did not express a functional gene product. A galE mutant meningococcal strain was constructed by transformation with an inactivated galE gene. Analysis of the LPS from the galE mutant strain revealed an apparent reduction in molecular weight and a loss of reactivity with monoclonal antibodies specific for structures known to contain galactose. These results are consistent with an essential role for galE in the incorporation of galactose into meningococcal lipopolysaccharide.

Multivalent meningococcal serogroup B vaccines: challenges in predicting protection and measuring effectiveness.

Vaccines targeting Neisseria meningitidis serogroup B (MenB) have been attempted for 40 years. Monovalent outer membrane vesicle vaccines targeted at epidemic outbreaks have been successfully developed. Newer vaccines aim to induce antibodies to cross-reactive antigens, such as factor H binding protein (rLP2086) or a mix of outer membrane vesicle, factor H binding protein and other minor antigens (4CMenB). The true protective coverage among circulating MenB isolates afforded by these vaccines is unknown. Carefully conducted Phase IV post-implementation evaluations designed to measure specific effectiveness against major circulating MenB clonal lineages are needed to address the critical question of which antigens are linked to protection. Progress with whole-genome sequencing and bio-informatics may allow the composition of antigen mozaics based on two major outer membrane proteins: PorA and FetA.

Adjuvant system AS02V enhances humoral and cellular immune responses to pneumococcal protein PhtD vaccine in healthy young and older adults: randomised, controlled trials.

BACKGROUND: The protection elicited by polysaccharide pneumococcal vaccines against community-acquired pneumonia in older adults remains debatable. Alternative vaccine targets include well-conserved pneumococcal protein antigens, such as pneumococcal histidine triad protein D (PhtD). OBJECTIVE: To evaluate humoral and cellular immune responses and safety/reactogenicity following immunisation with PhtD vaccine with or without adjuvant (alum or AS02V) in older (≥65 years) and young (18-45 years) healthy adults. METHODS: Two phase I/II, single-blind, parallel-group studies were conducted in 150 older and 147 young adults. Participants were randomised to receive 2 doses (months 0 and 2) of PhtD 30 µg, PhtD 10 µg plus alum, PhtD 30 µg plus alum, PhtD 10 µg plus AS02V or PhtD 30 µg plus AS02V, or the 23-valent polysaccharide pneumococcal vaccine (23PPV) at month 0 with placebo (saline solution) at month 2. Safety/reactogenicity was assessed. PhtD-specific antibody, T cell and memory B cell responses were evaluated. RESULTS: Solicited adverse events were more common in young participants and with adjuvanted vaccines. No vaccine-related serious adverse events were reported. Although anti-PhtD geometric mean antibody concentrations (GMCs) were consistently lower in the older adult cohort than in young adults, GMCs in the older cohort following PhtD 30 µg plus AS02V were comparable to those induced by plain PhtD or PhtD 30 µg plus alum in the young cohort. Compared with alum adjuvant, AS02V adjuvant system was associated with an increased frequency of PhtD-specific CD4 cells in both cohorts and a significantly higher specific memory B cell response in the older cohort, similar to responses obtained in the young cohort. CONCLUSION: The improved immune response to PhtD vaccine containing the AS02V adjuvant system in comparison to alum suggests that the reduced immune response to vaccines in older adults can be partially restored to the response level observed in young adults. ClinicalTrials.gov identifiers: NCT00307528/NCT01767402.