Formation and clearance of TNF-TNF inhibitor complexes during TNF inhibitor treatment.

BACKGROUND AND PURPOSE: Millions of patients with inflammatory diseases are treated with tumour necrosis factor (TNF) inhibitors (TNFi). Individual treatment response varies, in part related to variable drug clearance. The role of TNF-TNFi complexes in clearance of the different TNFi is controversial. Moreover, mechanistic insight into the structural aspects and biological significance of TNF-TNFi complexes is lacking. We hypothesized a role for Fc-mediated clearance of TNF-TNFi immune complexes. Therefore, we investigated circulating TNF-TNFi complexes upon treatment with certolizumab-lacking Fc tails-in comparison with adalimumab, golimumab, infliximab and etanercept. EXPERIMENTAL APPROACH: Drug-tolerant ELISAs were developed and used to quantify TNF during adalimumab, golimumab, etanercept, certolizumab and infliximab treatment in patients with inflammatory arthritis or ulcerative colitis for a maximum follow-up of 1 year. Effects on in vitro TNF production and Fc-mediated uptake of TNF-TNFi complexes were investigated for all five TNFi. KEY RESULTS: Circulating TNF concentrations were >20-fold higher during certolizumab treatment compared with adalimumab, reaching up to 23.1 ng·ml-1 . Internalization of TNF-TNFi complexes by macrophages depended on Fc valency, with efficient uptake for the full antibody TNFi (three Fc tails), but little or no uptake for etanercept and certolizumab (one and zero Fc tail, respectively). TNF production was not affected by TNFi. Total TNF load did not affect clearance rate of total TNFi. CONCLUSIONS AND IMPLICATIONS: Differences in TNFi structure profoundly affect clearance of TNF, while it is unlikely that TNF itself significantly contributes to target-mediated drug disposition of TNFi.

At Critically Low Antigen Densities, IgM Hexamers Outcompete Both IgM Pentamers and IgG1 for Human Complement Deposition and Complement-Dependent Cytotoxicity.

IgM is secreted as a pentameric polymer containing a peptide called the joining chain (J chain). However, integration of the J chain is not required for IgM assembly and in its absence IgM predominantly forms hexamers. The conformations of pentameric and hexameric IgM are remarkably similar with a hexagonal arrangement in solution. Despite these similarities, hexameric IgM has been reported to be a more potent complement activator than pentameric IgM, but reported relative potencies vary across different studies. Because of these discrepancies, we systematically investigated human IgM-mediated complement activation. We recombinantly generated pentameric and hexameric human IgM (IgM+J and IgM-J, respectively) mAbs and measured their ability to induce complement deposition and complement-dependent cytotoxicity when bound to several Ags at varying densities. At high Ag densities, hexameric and pentameric IgM activate complement to a similar extent as IgG1. However, at low densities, hexameric IgM outcompeted pentameric IgM and even more so IgG1. These differences became progressively more pronounced as antigenic density became critically low. Our findings highlight that the differential potency of hexameric and pentameric IgM for complement activation is profoundly dependent on the nature of its interactions with Ag. Furthermore, it underscores the importance of IgM in immunity because it is a more potent complement activator than IgG1 at low Ag densities.

Dynamics of antibodies to SARS-CoV-2 in convalescent plasma donors.

OBJECTIVES: Characterisation of the human antibody response to SARS-CoV-2 infection is vital for serosurveillance purposes and for treatment options such as transfusion with convalescent plasma or immunoglobulin products derived from convalescent plasma. In this study, we longitudinally and quantitatively analysed antibody responses in RT-PCR-positive SARS-CoV-2 convalescent adults during the first 250 days after onset of symptoms. METHODS: We measured antibody responses to the receptor-binding domain (RBD) of the SARS-CoV-2 spike protein and the nucleocapsid protein in 844 longitudinal samples from 151 RT-PCR-positive SARS-CoV-2 convalescent adults. With a median of 5 (range 2-18) samples per individual, this allowed quantitative analysis of individual longitudinal antibody profiles. Kinetic profiles were analysed by mixed-effects modelling. RESULTS: All donors were seropositive at the first sampling moment, and only one donor seroreverted during follow-up analysis. Anti-RBD IgG and anti-nucleocapsid IgG levels declined with median half-lives of 62 and 59 days, respectively, 2-5 months after symptom onset, and several-fold variation in half-lives of individuals was observed. The rate of decline of antibody levels diminished during extended follow-up, which points towards long-term immunological memory. The magnitude of the anti-RBD IgG response correlated well with neutralisation capacity measured in a classic plaque reduction assay and in an in-house developed competitive assay. CONCLUSION: The result of this study gives valuable insight into the long-term longitudinal response of antibodies to SARS-CoV-2.

Development of a SARS-CoV-2 Total Antibody Assay and the Dynamics of Antibody Response over Time in Hospitalized and Nonhospitalized Patients with COVID-19.

Severe acute respiratory syndrome coronavirus (SARS-CoV)-2 infections often cause only mild disease that may evoke relatively low Ab titers compared with patients admitted to hospitals. Generally, total Ab bridging assays combine good sensitivity with high specificity. Therefore, we developed sensitive total Ab bridging assays for detection of SARS-CoV-2 Abs to the receptor-binding domain (RBD) and nucleocapsid protein in addition to conventional isotype-specific assays. Ab kinetics was assessed in PCR-confirmed, hospitalized coronavirus disease 2019 (COVID-19) patients (n = 41) and three populations of patients with COVID-19 symptoms not requiring hospital admission: PCR-confirmed convalescent plasmapheresis donors (n = 182), PCR-confirmed hospital care workers (n = 47), and a group of longitudinally sampled symptomatic individuals highly suspect of COVID-19 (n = 14). In nonhospitalized patients, the Ab response to RBD is weaker but follows similar kinetics, as has been observed in hospitalized patients. Across populations, the RBD bridging assay identified most patients correctly as seropositive. In 11/14 of the COVID-19-suspect cases, seroconversion in the RBD bridging assay could be demonstrated before day 12; nucleocapsid protein Abs emerged less consistently. Furthermore, we demonstrated the feasibility of finger-prick sampling for Ab detection against SARS-CoV-2 using these assays. In conclusion, the developed bridging assays reliably detect SARS-CoV-2 Abs in hospitalized and nonhospitalized patients and are therefore well suited to conduct seroprevalence studies.

Divergent chemokine receptor expression and the consequence for human IgG4 B cell responses.

IgG4 antibodies are unique to humans. IgG4 is associated with tolerance during immunotherapy in allergy, but also with pathology, as in pemphigus vulgaris and IgG4-related disease. Its induction is largely restricted to nonmicrobial antigens, and requires repeated or prolonged antigenic stimulation, for reasons poorly understood. An important aspect in generating high-affinity IgG antibodies is chemokine receptor-mediated migration of B cells into appropriate niches, such as germinal centers. Here, we show that compared to IgG1 B cells, circulating IgG4 B cells express lower levels of CXCR3, CXCR4, CXCR5, CCR6, and CCR7, chemokine receptors involved in GC reactions and generation of long-lived plasma cells. This phenotype was recapitulated by in vitro priming of naive B cells with an IgG4-inducing combination of TFH /TH2 cytokines. Consistent with these observations, we found a low abundance of IgG4 B cells in secondary lymphoid tissues in vivo, and the IgG4 antibody response is substantially more short-lived compared to other IgG subclasses in patient groups undergoing CD20+ B cell depletion therapy with rituximab. These results prompt the hypothesis that factors needed to form IgG4 B cells restrain at the same time the induction of a robust migratory phenotype that could support a long-lived IgG4 antibody response.

Reference Intervals of Factor H and Factor H-Related Proteins in Healthy Children.

Complement is activated as part of the innate immune defense against invading pathogens. Also, it helps to remove apoptotic debris and immune complexes from the circulation. Impaired complement function due to aberrant plasma levels of complement proteins may be indicative for complement-mediated diseases or can be involved in susceptibility for infections. To determine whether plasma levels are abnormal, reference intervals (RIs) are used from adult healthy donors. Since many complement-mediated diseases have an onset during childhood, it is important to know whether these RIs can be extrapolated to children. RIs of Factor H (FH), the crucial fluid-phase regulator, and the FH-related proteins (FHRs), its homologous counterparts, are unknown in healthy children. While FH is measured to diagnose and monitor therapy of patients with atypical hemolytic uremic syndrome, recent studies also implicated increased plasma levels of FHRs in disease. Here, we investigated the levels of FH and FHRs in healthy children using recently developed specific ELISAs. We found that levels of FH, FHR-2, and FHR-3 were equal to those found in healthy adults. Levels of FHR-4A and FHR-5 were lower in children than in adults. However, only the FHR-5 levels associated with age. The RIs of these FH family proteins now serve to support the interpretation of plasma levels in prospective and retrospective studies that can be used for routine diagnostic and monitoring purposes including pediatric patient samples.

Restricted immune activation and internalisation of anti-idiotype complexes between drug and antidrug antibodies.

OBJECTIVES: Therapeutic antibodies can provoke an antidrug antibody (ADA) response, which can form soluble immune complexes with the drug in potentially high amounts. Nevertheless, ADA-associated adverse events are usually rare, although with notable exceptions including infliximab. The immune activating effects and the eventual fate of these 'anti-idiotype' complexes are poorly studied, hampering assessment of ADA-associated risk of adverse events. We investigated the in vitro formation and biological activities of ADA-drug anti-idiotype immune complexes using patient-derived monoclonal anti-infliximab antibodies. METHODS: Size distribution and conformation of ADA-drug complexes were characterised by size-exclusion chromatography and electron microscopy. Internalisation of and immune activation by complexes of defined size was visualised with flow imaging, whole blood cell assay and C4b/c ELISA. RESULTS: Size and conformation of immune complexes depended on the concentrations and ratio of drug and ADA; large complexes (>6 IgGs) formed only with high ADA titres. Macrophages efficiently internalised tetrameric and bigger complexes in vitro, but not dimers. Corroborating these results, ex vivo analysis of patient sera demonstrated only dimeric complexes in circulation.No activation of immune cells by anti-idiotype complexes was observed, and only very large complexes activated complement. Unlike Fc-linked hexamers, anti-idiotype hexamers did not activate complement, demonstrating that besides size, conformation governs immune complex potential for triggering effector functions. CONCLUSIONS: Anti-idiotype ADA-drug complexes generally have restricted immune activation capacity. Large, irregularly shaped complexes only form at high concentrations of both drug and ADA, as may be achieved during intravenous infusion of infliximab, explaining the rarity of serious ADA-associated adverse events.

Dimeric IgG complexes from IVIg are incapable of inducing in vitro neutrophil degranulation or complement activation.

PURPOSE: Intravenous immunoglobulin (IVIg) products contain various amounts of dimeric IgG complexes. Current insights into the possible biological activities of these dimers remain controversial, and both immunemodulating and immune-activating effects have been reported. Here, we analyzed the putative immune-activating effects of dimers isolated from IVIg. METHODS: Dimers isolated from IVIg were purified by high-performance size-exclusion chromatography (HP-SEC) and tested for the ability to induce neutrophil degranulation in vitro. RESULTS: Dimers isolated from IVIg were found to be incapable of inducing in vitro neutrophil degranulation or complement activation, even at concentrations exceeding those expected to be reached upon administration in patients. These results depend on the removal of artefactual activation by using 0.1 micron filtration and the use of poloxamer to prevent adsorption of IgG onto the solid phase. CONCLUSIONS: The data suggest dimeric IgG found in IVIg may bind to Fc-receptors without causing activation.

Adaptive antibody diversification through N-linked glycosylation of the immunoglobulin variable region.

A hallmark of B-cell immunity is the generation of a diverse repertoire of antibodies from a limited set of germline V(D)J genes. This repertoire is usually defined in terms of amino acid composition. However, variable domains may also acquire N-linked glycans, a process conditional on the introduction of consensus amino acid motifs (N-glycosylation sites) during somatic hypermutation. High levels of variable domain glycans have been associated with autoantibodies in rheumatoid arthritis, as well as certain follicular lymphomas. However, the role of these glycans in the humoral immune response remains poorly understood. Interestingly, studies have reported both positive and negative effects on antibody affinity. Our aim was to elucidate the role of variable domain glycans during antigen-specific antibody responses. By analyzing B-cell repertoires by next-generation sequencing, we demonstrate that N-glycosylation sites are introduced at positions in which glycans can affect antigen binding as a result of a specific clustering of progenitor glycosylation sites in the germline sequences of variable domain genes. By analyzing multiple human monoclonal and polyclonal (auto)antibody responses, we subsequently show that this process is subject to selection during antigen-specific antibody responses, skewed toward IgG4, and positively contributes to antigen binding. Together, these results highlight a physiological role for variable domain glycosylation as an additional layer of antibody diversification that modulates antigen binding.

Infusion reactions during infliximab treatment are not associated with IgE anti-infliximab antibodies.

OBJECTIVES: Controversy exists on the role of IgE antidrug antibodies (IgE-ADA) in infusion reactions (IR) on infliximab treatment, partly due to the lack of a positive control used for assay validation. We sought to (1) develop a robust assay to measure IgE-ADA, including a positive control, (2) determine the association between IgE-ADA and IR and (3) determine the incidence of IgE-ADA in infliximab treated patients. METHODS: A recombinant human IgE anti-infliximab monoclonal antibody was developed as standard and positive control. With this antibody, we set up a novel robust assay to measure IgE-ADA. IgE-ADA was determined in three retrospective cohorts (n=159) containing IR+ (n=37) and IR- (n=39), and longitudinal sera of 83 spondyloarthritis. RESULTS: IgE-ADA was found in 0/39 IR-, whereas 4/37 (11%) IR+ showed low levels (0.1-0.3 IU/mL, below the 0.35 IU/mL threshold associated with elevated risk of allergic symptoms). All patients who were IgE-ADA positive also had (very) high IgG-ADA levels. The incidence of IgE-ADA in patients with infliximab-treated spondyloarthritis was estimated at less than approximately 1%. CONCLUSIONS: IgE-ADA is rarely detected in infliximab-treated patients. Moreover, the absence of IgE-ADA in the majority of IR+ patients suggests that IgE-ADA is not associated with infusion reactions.

Therapeutic TNF Inhibitors can Differentially Stabilize Trimeric TNF by Inhibiting Monomer Exchange.

Tumor necrosis factor (TNF) is a homotrimeric cytokine that is a key mediator of inflammation. It is unstable at physiological concentrations and slowly converts into an inactive form. Here, we investigated the mechanism of this process by using a Förster resonance energy transfer (FRET) assay that allowed monitoring of monomeric subunit exchange in time. We observed continuous exchange of monomeric subunits even at concentrations of TNF high enough to maintain its bioactivity. The kinetics of this process closely corresponds with the appearance of monomeric subunits and disappearance of trimeric TNF in time at ng/ml concentrations as monitored by high-performance size-exclusion chromatography (HP-SEC). Furthermore, of the five therapeutic TNF inhibitors that are currently used in the clinic, three (adalimumab, infliximab, etanercept) were found to completely inhibit the monomer exchange reaction and stabilize TNF trimers, whereas golimumab and certolizumab could not prevent monomer exchange, but did slow down the exchange process. These differences were not correlated with the affinities of the TNF inhibitors, measured with both surface plasmon resonance (SPR) and in fluid phase using fluorescence-assisted HP-SEC. The stabilizing effect of these TNF inhibitors might result in prolonged residual TNF bioactivity under conditions of incomplete blocking, as observed in vitro for adalimumab.

Using monoclonal antibodies as an international standard for the measurement of anti-adalimumab antibodies.

Comparing studies investigating anti-drug antibody (ADA) formation is hampered by the lack of comparability between study protocols, assay formats, and standardized reference materials. In this respect, the use of an international standard would mean a major step forward. Here we compared 11 fully human monoclonal antibodies against adalimumab in two assays commonly used for ADA measurement; the bridging ELISA and the antigen binding test (ABT). Our results show non-parallel titration of the monoclonal antibodies in both assays, which we also find for polyclonal ADA sources. Moreover, we observed that the output of the bridging ELISA depends to a large degree on the affinity of the monoclonal antibody. For the ABT, results reflect a combination of affinity and avidity. This suggests that rather than reporting ADA values in nanogram per milliliter, arbitrary units may be more appropriate. Together our data highlight the difficulty of ADA standardization by identifying several pitfalls that should be taken into account when selecting a standard for ADA testing.

Functional analysis of the anti-adalimumab response using patient-derived monoclonal antibodies.

The production of antibodies to adalimumab in autoimmune patients treated with adalimumab is shown to diminish treatment efficacy. We previously showed that these antibodies are almost exclusively neutralizing, indicating a restricted response. Here, we investigated the characteristics of a panel of patient-derived monoclonal antibodies for binding to adalimumab. Single B-cells were isolated from two patients, cultured, and screened for adalimumab specificity. Analysis of variable region sequences of 16 clones suggests that the immune response against adalimumab is broad, involving multiple B-cell clones each using different combinations of V(D)J segments. A strong bias for replacement mutations in the complementarity determining regions was found, indicating an antigen-driven response. We recombinantly expressed 11 different monoclonal antibodies and investigated their affinity and specificity. All clones except one are of high affinity (Kd between 0.6 and 233 pm) and compete with TNF as well as each other for binding to adalimumab. However, binding to a panel of single-point mutants of adalimumab indicates markedly different fine specificities that also result in a differential tendency of each clone to form dimeric and multimeric immune complexes. We conclude that although all anti-adalimumab antibodies compete for binding to TNF, the response is clonally diverse and involves multiple epitopes on adalimumab. These results are important for understanding the relationship between self and non-self or idiotypic determinants on therapeutic antibodies and their potential immunogenicity.

Nanomolar to sub-picomolar affinity measurements of antibody-antigen interactions and protein multimerizations: fluorescence-assisted high-performance liquid chromatography.

Although several techniques exist for the measurement of high-affinity interactions, it is still challenging to determine dissociation constants around or even below 1pM. During the analysis of several human-derived monoclonal antibodies to adalimumab, we found a clone with a very high affinity that could not be measured using conventional surface plasmon resonance assays. We developed a straightforward and robust method to measure affinities in the nanomolar to sub-picomolar range. The assay is based on separation of bound and free fluorescently labeled antigen using size exclusion chromatography and quantification by in-line fluorescence detection. We describe optimal conditions and procedures that result in a very sensitive assay that can be used to reliably determine ultra-high affinities. Using the method described in this article, a dissociation constant of 0.78pM could be determined for the anti-adalimumab antibody.

Adalimumab elicits a restricted anti-idiotypic antibody response in autoimmune patients resulting in functional neutralisation.

OBJECTIVES: Millions of patients worldwide are treated with therapeutic monoclonal antibodies. These biological therapeutics can be immunogenic, resulting in anti-drug antibody formation which leads to loss of response. Fully human biological agents, such as the anti-tumour necrosis factor α (anti-TNFα) antibody adalimumab, are considered to be weakly immunogenic, but anti-adalimumab antibodies (AAA) were recently detected in more than half of treated patients with rheumatoid arthritis (RA) within 28 weeks of treatment. A study was undertaken to determine the mechanism by which AAA lead to loss of response. METHODS: The specificity of the repertoire of AAA was investigated in a cohort of 50 AAA-positive RA patients. Inhibition experiments using TNFα and patient-derived anti-adalimumab monoclonal antibodies were performed. RESULTS: The antibody response against adalimumab is highly restricted: Fab fragments of a single monoclonal antibody specific for the idiotype of adalimumab inhibited 98.65% (25th-75th percentiles: 98.25-99.90) of the total anti-adalimumab reactivity in serum from 50 AAA-positive patients. The anti-adalimumab response was confined to the TNFα binding region of adalimumab, thereby neutralising its therapeutic efficacy. In line with this restricted specificity, small immune complexes were found in the circulation of AAA-forming patients. CONCLUSIONS: The humoral immune response against adalimumab is highly restricted and limited to the idiotype of the therapeutic antibody. All antibodies result in functional neutralisation of the drug, thereby providing a mechanism by which AAA formation leads to clinical non-response.

Monoclonal anti-citrullinated protein antibodies selected on citrullinated fibrinogen have distinct targets with different cross-reactivity patterns.

OBJECTIVE: ACPAs are thought to play a pathogenic role in RA. Because of their polyclonal nature it is difficult to study characteristics of ACPAs such as cross-reactivity or affinity. This study aimed to analyse the ACPA response at clonal level. METHODS: Citrullinated fibrinogen-specific B cells were isolated from blood derived from an RA patient by fluorescent automated cell sorting (FACS). Antigen specificity was verified by ELISA of culture supernatant. RNA of antigen-specific B cells was isolated and VH and VL chains were cloned and subsequently expressed as IgG1 antibodies. RESULTS: Two human recombinant antibodies were obtained that bind to citrullinated fibrinogen peptide (cFib). Both monoclonal antibodies originate from different naive B cells, undergo extensive somatic hypermutation and bind to cFib (but not to Fib) with moderate avidity. Furthermore, they show distinct cross-reactivity patterns towards other citrullinated peptides, suggesting that both antibodies have different primary targets. CONCLUSION: Together these data suggest that ACPAs are formed by antigen-driven maturation, and that multiple citrullinated antigens are involved in activating the B-cell response.

Effective treatment of refractory CMV reactivation after allogeneic stem cell transplantation with in vitro-generated CMV pp65-specific CD8+ T-cell lines.

To treat patients with refractory cytomegalovirus (CMV) reactivation after allogeneic stem cell transplantation, a phase I/II clinical study on adoptive transfer of in vitro-generated donor-derived or patient-derived CMV pp65-specific CD8* T-cell lines was performed. Peripheral blood mononuclear cells from CMV seropositive donors or patients were stimulated with HLA-A*0201-restricted and/or HLA-B*0702-restricted CMV pp65 peptides (NLV/TPR) and 1 day after stimulation interferon-γ)-producing cells were enriched using the CliniMACS Cytokine Capture System (interferon-γ), and cultured with autologous feeders and low-dose interluekin-2. After 7-14 days of culture, quality controls were performed and the CMV-specific T-cell lines were administered or cryopreserved. The T-cell lines generated contained 0.6-17 × 10(6) cells, comprising 54%-96% CMV pp65-specific CD8 T cells, and showed CMV-specific lysis of target cells. Fifteen CMV-specific T-cell lines were generated of which 8 were administered to patients with refractory CMV reactivation. After administration, no acute adverse events and no graft versus host disease were observed and CMV load disappeared. In several patients, a direct relation between administration of the T-cell line and the in vivo appearance of CMV pp65-specific T cells could be documented. In conclusion, administration of CMV pp65-specific CD8* T-cell lines was found to be feasible and safe, and enduring efficacy of administered CMV pp65-specific CD8* T-cell lines could be demonstrated.

Simultaneous isolation of CD8(+) and CD4(+) T cells specific for multiple viruses for broad antiviral immune reconstitution after allogeneic stem cell transplantation.

Opportunistic viral infections can cause serious morbidity and mortality in immunocompromised patients after allogeneic stem cell transplantation. Clinical studies have shown that adoptive transfer of donor-derived T cells specific for cytomegalovirus (CMV), Epstein-Barr virus (EBV), or human adenovirus (HAdV) can be a safe and effective treatment of infections with these major viral pathogens. The aim of this study was to develop a method for the simultaneous isolation of coordinated CD8(+) and CD4(+) memory T-cell responses against a broad repertoire of viral epitopes. To ensure that the method was applicable to a wide variety of virus-specific T cells that may differ in phenotypic and functional properties, we focused on T cells specific for the persistent viruses, CMV and EBV, and T cells specific for HAdV and influenza (FLU), which are not repetitively activated in vivo after initial viral clearance. Following in vitro activation, nearly all T cells specific for these viruses produced interferon γ (IFN-γ) and tumor necrosis factor α, and expressed CD137, whereas the populations varied in the production of interleukin-2, degranulation, and expression of phenotypic markers. Different kinetics of IFN-γ production were observed in CMV/EBV-specific T cells and HAdV/FLU-specific T cells. However, after the stimulation of peripheral blood from seropositive donors with viral protein-spanning peptide pools, the activated virus-specific CD8(+) and CD4(+) T cells could be simultaneously isolated by either IFN-γ-based or CD137-based enrichment. This study provides an efficient and widely applicable strategy for the isolation of virus-specific T cells, which may be used for the reconstitution of virus-specific immunity in allogeneic stem cell transplantation recipients.

Combinations of maggot excretions/secretions and antibiotics are effective against Staphylococcus aureus biofilms and the bacteria derived therefrom.

OBJECTIVES: Maggots of the blowfly Lucilia sericata are used for the treatment of chronic wounds. Previously we reported that maggot excretions/secretions (ES) break down Staphylococcus aureus biofilms but do not kill the bacteria. As many antibiotics are not effective against biofilms we assessed the effect of combinations of ES and antibiotics on S. aureus biofilms and on the survival of the bacteria released from the biofilms. METHODS: Effects of ES, antibiotics (vancomycin, daptomycin or clindamycin) and combinations thereof on S. aureus ATCC 29 213 biofilms and bacterial viability were determined using microtitre plates and in vitro killing assays. RESULTS: Vancomycin and daptomycin dose-dependently enhanced biofilm formation, whereas clindamycin reduced S. aureus biofilm size. Adding ES to antibiotic incubations caused a complete biofilm breakdown. After a lag time the bacteria derived from biofilms became susceptible to vancomycin and clindamycin, provided that the medium was refreshed. Daptomycin dose-dependently eliminated the biofilm-derived bacteria immediately. Furthermore, it was significantly more effective against bacteria derived from ES-exposed biofilms than those from control biofilms. ES did not affect the activity of the antibiotics against log-phase S. aureus. CONCLUSIONS: Combinations of maggot ES and antibiotics eliminate S. aureus biofilms and the bacteria derived therefrom.