Human Antibody Responses against Virulence Factors of Staphylococcus aureus during Infection

_Staphylococcus aureus_ is an infamous bacterial pathogen which can produce a wide array of virulence factors, enabling it to cause many different infections with significant morbidity and mortality in humans. The ability of _S. aureus_ to form biofilm and its rising resistance against antibiotics further complicate treatment of many infections. Therefore, alternative treatment strategies, such as vaccination, are receiving great scientific and clinical interest. However, despite the many different virulence factors that have been targeted by vaccines and their promising effects in animal models, so far all clinical trials failed to demonstrate any favourable effect in humans. Thus, there remains a need for more insights into the presence of _S. aureus_ virulence factors and their ability to induce an antibody response during infection in humans. The general aim of this thesis was twofold; to provide further insights into the presence of a wide range of well-characterized virulence factors of _S. aureus_ during growth in _in vitro_ and _ex vivo_ infection models, mimicking the _in vivo_ situation during different infection in humans, and to further characterize the human antibody response during these different infections. The high-throughput, bead-based Luminex assay was used together with confirmation by additional techniques such as RT-PCR and mass-spectrometry throughout this thesis to achieve these aims. Data concerning _in vitro_ presence of -and _in vivo_ antibody responses against _S. aureus_ virulence factors are compared and, together, can help in identifying potential targets for novel vaccination strategies

Factors Associated with Worse Lung Function in Cystic Fibrosis Patients with Persistent Staphylococcus aureus

Background Staphylococcus aureus is an important pathogen in cystic fibrosis (CF). However, it is not clear which factors are associated with worse lung function in patients with persistent S. aureus airway cultures. Our main hypothesis was that patients with high S. aureus density in their respiratory specimens would more likely experience worsening of their lung disease than patients with low bacterial loads. Methods Therefore, we conducted an observational prospective longitudinal multi-center study and assessed the association between lung function and S. aureus bacterial density in respiratory samples, co-infection with other CF-pathogens, nasal S. aureus carriage, clinical status, antibiotic therapy, IL-6- and IgG-levels against S. aureus virulence factors. Results 195 patients from 17 centers were followed; each patient had an average of 7 visits. Data were analyzed using descriptive statistics and generalized linear mixed models. Our main hypothesis was only supported for patients providing throat specimens indicating that patients with higher density experienced a steeper lung function decline (p<0.001). Patients with exacerbations (n = 60), S. aureus small-colony variants (SCVs, n = 84) and co-infection with Stenotrophomonas maltophilia (n = 44) had worse lung function (p = 0.0068; p = 0.0011; p = 0.0103). Patients with SCVs were older (p = 0.0066) and more often treated with trimethoprim/sulfamethoxazole (p = 0.0078). IL-6 levels positively correlated with decreased lung function (p<0.001), S. aureus density in sputa (p = 0.0016), SCVs (p = 0.0209), exacerbations (p = 0.0041) and co-infections with S. maltophilia (p = 0.0195) or A. fumigatus (p = 0.0496). Conclusions In CF-patients with chronic S. aureus cultures, independent risk factors for worse lung function are high bacterial density in throat cultures, exacerbations, elevated IL-6 levels, presence of S. aureus SCVs and co-infection with S. maltophilia. Trial Registration ClinicalTrials.gov NCT0066976

Combining in vitro protein detection and in vivo antibody detection identifies potential vaccine targets against Staphylococcus aureus during osteomyelitis

Currently, little is known about the in vivo human immune response against Staphylococcus aureus during a biofilm-associated infection, such as osteomyelitis, and how this relates to protein production in biofilms in vitro. Therefore, we characterized IgG responses in 10 patients with chronic osteomyelitis against 50 proteins of S. aureus, analyzed the presence of these proteins in biofilms of the infecting isolates on polystyrene (PS) and human bone in vitro, and explored the relation between in vivo and in vitro data. IgG levels against 15 different proteins were significantly increased in patients compared to healthy controls. Using a novel competitive Luminex-based assay, eight of these proteins [alpha toxin, Staphylococcus aureus formyl peptide receptor-like 1 inhibitor (FlipR), glucosaminidase, iron-responsive surface determinants A and H, the putative ABC transporter SACOL0688, staphylococcal complement inhibitor (SCIN), and serine–aspartate repeat-containing protein E (SdrE)] were also detected in a majority of the infecting isolates during biofilm formation in vitro. However, 4 other proteins were detected in only a minority of isolates in vitro while, vice versa, 7 proteins were detected in multiple isolates in vitro but not associated with significantly increased IgG levels in patients. Detection of proteins was largely confirmed using a transcriptomic approach. Our data provide further insights into potential therapeutic targets, such as for vaccination, to reduce S. aureus virulence and biofilm formation. At the same time, our data suggest that either in vitro or immunological in vivo data alone should be interpreted cautiously and that combined studies are necessary to identify potential targets

Human immunoglobulin G cannot inhibit fibrinogen binding by the genetically diverse A domain of Staphylococcus aureus fibronectinbinding protein A

The fibronectin-binding protein A (FnBPA) is a cell surface-associated protein of Staphylococcus aureus which mediates adherence to the host extracellular matrix and is important for bacterial virulence. Previously, substantial sequence diversity was found among strains in the fibrinogen-binding A domain of this protein, and 7 different isotypes were described. The effect of this sequence diversity on the human antibody response, in terms of both antibody production and antibody function, remains unclear. In this study, we identify five different FnBPA A domain isotypes based on the sequence results of 22 clinical S. aureus isolates, obtained from the same number of patients suffering from bacteremia. Using a bead-based Luminex technique, we measure the patients' total immunoglobulin G (IgG) against the 7 FnBPA isotypes at the onset and during the time course of bacteremia (median of 10 serum samples per patient over a median of 35 days). A significant increase in IgG against the FnBPA A domain, including the isotype carried by the infecting strain, is observed in only three out of 22 patients (14%) after the onset of bacteremia. Using a Luminex-based FnBPA-fibrinogen-binding assay, we find that preincubation of recombinant FnBPA isotypes with IgG from diverse patients does not interfere with binding to fibrinogen. This observation is confirmed using an alternative Luminex-based assay and enzyme-linked immunosorbent assay (ELISA)

Staphylococcus aureus pathogenicity in cystic fibrosis patients-results from an observational prospective multicenter study concerning virulence genes, phylogeny, and gene plasticity

Staphylococcus aureus and cystic fibrosis (CF) are closely interlinked. To date, however, the impact of S. aureus culture in CF airways on lung function and disease progression has only been elucidated to a limited degree. This analysis aims to identify bacterial factors associated to clinical deterioration. Data were collected during an observational prospective multi-center study following 195 patients from 17 centers. The average follow-up time was 80 weeks. S. aureus isolates (n = 3180) were scanned for the presence of 25 virulence genes and agr-types using single and multiplex PCR. The presence of specific virulence genes was not associated to clinical deterioration. For the agr-types 1 and 4, however, a link to the subjects' clinical status became evident. Furthermore, a significant longitudinal decrease in the virulence gene quantity was observed. Analyses of the plasticity of the virulence genes revealed significantly increased plasticity rates in the presence of environmental stress. The results suggest that the phylogenetic background defines S. aureus pathogenicity rather than specific virulence genes. The longitudinal loss of virulence genes most likely reflects the adaptation process directed towards a persistent and colonizing rather than infecting lifestyle

Characterization of the Humoral Immune Response during Staphylococcus aureus Bacteremia and Global Gene Expression by Staphylococcus aureus in Human Blood

Attempts to develop an efficient anti-staphylococcal vaccine in humans have so far been unsuccessful. Therefore, more knowledge of the antigens that are expressed by Staphylococcus aureus in human blood and induce an immune response in patients is required. In this study we further characterize the serial levels of IgG and IgA antibodies against 56 staphylococcal antigens in multiple serum samples of 21 patients with a S. aureus bacteremia, compare peak IgG levels between patients and 30 non-infected controls, and analyze the expression of 3626 genes by two genetically distinct isolates in human blood. The serum antibody levels were measured using a bead-based flow cytometry technique (xMAP®, Luminex corporation). Gene expression levels were analyzed using a microarray (BμG@s microarray). The initial levels and time taken to reach peak IgG and IgA antibody levels were heterogeneous in bacteremia patients. The antigen SA0688 was associated with the highest median initial-to-peak antibody fold-increase for IgG (5.05-fold) and the second highest increase for IgA (2.07-fold). Peak IgG levels against 27 antigens, including the antigen SA0688, were significantly elevated in bacteremia patients versus controls (P≤0.05). Expression of diverse genes, including SA0688, was ubiquitously high in both isolates at all time points during incubation in blood. However, only a limited number of genes were specifically up- or downregulated in both isolates when cultured in blood, compared to the start of incubation in blood or during incubation in BHI broth. In conclusion, most staphylococcal antigens tested in this study, including many known virulence factors, do not induce uniform increases in the antibody levels in bacteremia patients. In addition, the expression of these antigens by S. aureus is not significantly altered by incubation in human blood over time. One immunogenic and ubiquitously expressed antigen is the putative iron-regulated ABC transporter SA0688