Herman Groen

Human Monoclonal Antibodies against Anthrax Lethal Factor and Protective Antigen Act Independently To Protect against Bacillus anthracis Infection and Enhance Endogenous Immunity to Anthrax

ABSTRACT The unpredictable nature of bioterrorism and the absence of real-time detection systems have highlighted the need for an efficient postexposure therapy for Bacillus anthracis infection. One approach is passive immunization through the administration of antibodies that mitigate the biological action of anthrax toxin. We isolated and characterized two protective fully human monoclonal antibodies with specificity for protective antigen (PA) and lethal factor (LF). These antibodies, designated IQNPA (anti-PA) and IQNLF (anti-LF), were developed as hybridomas from individuals immunized with licensed anthrax vaccine. The effective concentration of IQNPA that neutralized 50% of the toxin in anthrax toxin neutralization assays was 0.3 nM, while 0.1 nM IQNLF neutralized the same amount of toxin. When combined, the antibodies had additive neutralization efficacy. IQNPA binds to domain IV of PA containing the host cell receptor binding site, while IQNLF recognizes domain I containing the PA binding region in LF. A single 180-μg dose of either antibody given to A/J mice 2.5 h before challenge conferred 100% protection against a lethal intraperitoneal spore challenge with 24 50% lethal doses [LD50s] of B. anthracis Sterne and against rechallenge on day 20 with a more aggressive challenge dose of 41 LD50s. Mice treated with either antibody and infected with B. anthracis Sterne developed detectable murine anti-PA and anti-LF immunoglobulin G antibody responses by day 17 that were dependent on which antibody the mice had received. Based on these results, IQNPA and IQNLF act independently during prophylactic anthrax treatment and do not interfere with the establishment of endogenous immunity.

The capsular overgrowth on microencapsulated pancreatic islet grafts in streptozotocin and autoimmune diabetic rats

Abstracts This study investigates whether capsular overgrowth on alginate‐polylysine microencapsulated islets is influenced by (1) the presence of islet tissue, (2) MHC incompatibility between donor and recipient, or (3) the presence of autoimmune diabetes. Encapsulated Albino Oxford (AO, n= 6, isografts) and Lewis (n= 6, allografts) rat islets, and encapsulated human islets (n= 5, xenografts) were implanted intraperitoneally into streptozotocin‐dia‐betic AO rats. Also, encapsulated AO islets were implanted into autoimmune diabetic Bio Breeding/Organon (BB/O) rats (n= 5, allografts). Five isografts, five allografts, and three xenografts in AO recipients and five allografts in BB/O recipients resulted in normoglycemia. Two weeks after implantation, islets containing capsules were retrieved by peritoneal lavage, after which all animals that had become normoglycemic after transplantation returned to a state of hyperglycemia. Recovery rates of the capsules of these successful grafts, expressed as percentages of the initially implanted graft volume, varied from 72%± 7% to 80%± 9%. The associated pericapsular infiltrates (PCI) were similar in all groups and varied from 3.2%± 1.4% to 8.3%+ 2.6% Similar recovery rates and PCI were also found with empty capsules. However, the recovery rates of recipients with graft failures were lower and showed more PCI. Immunohistological staining of PCI showed no differences in the types of cells in the PCI on capsules with or without islets. We conclude that this early PCI is a capsule‐induced foreign body reaction that is not influenced by MHC incompatibility or by the presence of autoimmune diabetes, and it should be avoided by improving the biocompatibility of the capsules.

High Anti-Staphylococcal Antibody Titers in Patients with Epidermolysis Bullosa Relate to Long-Term Colonization with Alternating Types of Staphylococcus aureus

Abbreviations: EB, epidermolysis bullosa; ET, exfoliative toxin; HlgB, gamma-hemolysin B; IsaA, immunodominant antigen A; Isd, iron-responsive surface determinant; Luk, leukocidin; LytM, peptidoglycan hydrolase; MFI, median fluorescence intensity; MLVA, multiple-locus variable number of tandem repeats analysis; Nuc, endonuclease; SAgs, superantigens; SasG, S. aureus surface protein G; SCIN, staphylococcal complement inhibitor; SE, staphylococcal enterotoxin

Surface shaving as a versatile tool to profile global interactions between human serum proteins and the Staphylococcus aureus cell surface

The human commensal bacterium Staphylococcus aureus is renowned as a causative agent of severe invasive diseases. Upon entering the bloodstream, S. aureus can infect almost every tissue and organ system in the human body. To withstand insults from the immune system upon invasion, several immune‐evasive mechanisms have evolved in S. aureus, such as complement inhibition by secreted proteins and IgG‐binding by surface‐exposed protein A. While it is generally accepted that S. aureus cells bind a range of host factors for various purposes, no global analyses to profile staphylococcal host factor binding have so far been performed. Therefore, we explored the possibility to profile the binding of human serum proteins to S. aureus cells by “surface shaving” with trypsin and subsequent MS analysis of liberated peptides. This resulted in the identification of several components of the complement system, the platelet factor 4 and the isoform 1 of the inter‐α‐trypsin inhibitor heavy chain H4 on the staphylococcal cell surface. We conclude that surface shaving is a versatile tool to profile global interactions between human serum proteins and the S. aureus cell surface.

A human monoclonal antibody targeting the conserved staphylococcal antigen IsaA protects mice against Staphylococcus aureus bacteremia

Due to substantial therapy failure and the emergence of antibiotic-resistant Staphylococcus aureus strains, alternatives for antibiotic treatment of S. aureus infections are urgently needed. Passive immunization using S. aureus-specific monoclonal antibodies (mAb) could be such an alternative to prevent and treat severe S. aureus infections. The invariantly expressed immunodominant staphylococcal antigen A (IsaA) is a promising target for passive immunization. Here we report the development of the human anti-IsaA IgG1 mAb 1D9, which was shown to bind to all 26 S. aureus isolates tested. These included both methicillin-susceptible and methicillin-resistant S. aureus (MSSA and MRSA, respectively). Immune complexes consisting of IsaA and 1D9 stimulated human as well as murine neutrophils to generate an oxidative burst. In a murine bacteremia model, the prophylactic treatment with a single dose of 5 mg/kg 1D9 improved the survival of mice challenged with S. aureus isolate P (MSSA) significantly, while therapeutic treatment with the same dose did not influence animal survival. Neither prophylactic nor therapeutic treatment with 5 mg/kg 1D9 resulted in improved survival of mice with S. aureus USA300 (MRSA) bacteremia. Importantly, our studies show that healthy S. aureus carriers elicit an immune response which is sufficient to generate protective mAbs against invariant staphylococcal surface antigens. Human mAb 1D9, possibly conjugated to for example another antibody, antibiotics, cytokines or chemokines, may be valuable to fight S. aureus infections in patients.

Timing of pentoxifylline treatment determines its protective effect on diabetes development in the Bio Breeding rat

Diabetes-prone Bio Breeding (DP-BB) rats spontaneously develop diabetes between 60 and 120 days of age. Diabetes-resistant (DR)-BB rats can be induced to develop diabetes by poly(I:C) and anti-RT6. Here, we studied the effect of pentoxifylline, a potent anti-inflammatory agent, on diabetes development in both BB rat models of insulin-dependent diabetes mellitus and investigated whether these effects were related to differential modulation of tumour necrosis factor (TNF)-alpha and interleukin-10. When DP-BB rats received pentoxifylline from day 60 onwards, diabetes development was delayed and reduced. The other treatment protocols had no effect. In DR-BB rats, pentoxifylline treatment resulted only in a delay of diabetes development. In both BB rat models, in vivo pentoxifylline treatment potently suppressed TNF-alpha, but only moderately affected interleukin-10 production in vitro. These results show that timing of pentoxifylline treatment determines its protective effect on diabetes development in DP-BB rats. The observed pentoxifylline-induced increase of the interleukin-10/TNF-alpha ratio might be a mechanism for protection or delay of the diabetes development.

Temporary, but essential requirement of CD8+ T cells early in the pathogenesis of diabetes in BB rats as revealed by thymectomy and CD8 depletion.

Autoimmunity-prone BB rats demonstrate a T lymphocytopenia and abnormal T cell subset distribution. To test whether the life span of all T cells or only of certain subsets is reduced in BB rats, we thymectomised 8-week-old BB and PVG rats and subsequently assessed size and composition of the T cell population over a 6-week-period. In both strains, thymectomy (Tx) was followed by a decrease in peripheral T cell numbers, which was proportionally larger in BB rats. The decline of the Thy-1+ recent thymic migrant (RTM) T cell phenotype was similar in both strains. BB rats showed a rapid preferential loss of CD8+ and CD45RC+ T cells, whereas the relative loss of RT6+ T cells was proportional to that of all T cells and not significantly different from that in PVG rats. Tx at 8-week did not prevent diabetes. Tx of 4-week-old BB rats revealed essentially the same changes in peripheral T cell subset distribution as in 8-week-old animals. However, Tx at week 4 did prevent diabetes. Since this raised the possibility of a temporary requirement of CD8+ T cells for the development of diabetes, we performed CD8 depletions during different pre-diabetic intervals. We found that CD8 depletion from 4 to 8 and 4 to 14 weeks, but not from 8 to 14 weeks of age prevented diabetes. We conclude that the protective effect of early adult Tx is, at least in part, due to the rapid loss of CD8+ T cells, and that these cells are only required between 4 and 8 weeks of age for diabetes to develop in BB rats.

Simultaneous transplantation and intrathymic tolerance induction : a method with clinical potential

It has been shown that donor-specific tolerance to cardiac allografts can be induced by pretreating the prospective recipient with injections of donor splenocytes (intrathymically) and antilymphocyte serum (intraperitoneally) weeks or days before the actual transplantation. This procedure, however, lacks clinical relevance in the case of cadaver donors due to the obligatory interval between the start of the tolerance induction protocol and transplantation. We have tried to devise a protocol in which this interval is eliminated, thus allowing allotransplantation simultaneously with tolerance induction. Our results show that simultaneous cardiac allotransplantation and intrathymic tolerance induction by intrathymic injection of donor splenocytes and treatment with antilymphocyte serum is indeed possible in the PVG to AO high-responder rat strain combination, provided that low doses of cyclosporine are given intramuscularly on day 1, 2, and 3 after transplantation. As we now are able to combine the start of tolerance induction with the actual allotransplantation, this procedure may indeed have clinical potential.

The diabetes prone BB rat model of IDDM shows duration of breastfeeding to influence Type 1 diabetes development later in life

To the Editor: Besides genetic background, environmental factors such as food ingredients and viral infections have been indicated as important and possibly decisive elements for the induction of insulin-dependent diabetes mellitus (IDDM). Our data show that diabetes development in diabetes-prone (DP)BB rats can be delayed and prevented by prolonging the nursing period of these rats. These results provide experimental evidence strengthening human epidemiological data that the duration of exclusive breastfeeding influences diabetes development later in life. Most human epidemiological data suggest that a short, as opposed to a long, duration of exclusive breastfeeding is related with an increased risk for IDDM development [1, 2, 3, 4, 5]. The introduction of cow’s milk and wheat proteins at a young age has been suggested as potential diabetogenic risk factors for infants at risk of developing diabetes [1, 2, 3, 4, 5]. The importance of food ingredients in IDDM is further illustrated by the fact that the development of diabetes is reduced in DP-BB rats receiving a special diet in which hydrolysed casein is the sole source of protein [6]. Since, in epidemiological studies the period of nursing is established using questionnaires, the exact duration of breastfeeding remains unclear and duration of breastfeeding varies considerably between the siblings [1, 2, 3, 4, 5]. DP-BB rats normally spontaneously develop diabetes between 60 and 160 days of age and are used as an animal model for the study of Type 1 diabetes. In the DP-BB rat model for the study of diabetes, the period of suckling can be established precisely. To investigate whether a relation exists between the duration of exclusive breastfeeding and the onset of diabetes DOI 10.1007/s00125-003-1239-9 Received: 14 April 2003 / Revised: 8 July 2003 Published online: 1 November 2003

A human monoclonal antibody that specifically binds and inhibits the staphylococcal complement inhibitor protein SCIN

ABSTRACT Staphylococcus aureus is a serious public health burden causing a wide variety of infections. Earlier detection of such infections could result in faster and more directed therapies that also prevent resistance development. Human monoclonal antibodies (humAbs) are promising tools for diagnosis and therapy owing to their relatively straightforward synthesis, long history of safe clinical use and high target specificity. Here we show that the humAb 6D4, which was obtained from a random screen of B-cells producing antibodies that bind to whole cells of S. aureus, targets the staphylococcal complement inhibitor (SCIN). The epitope recognized by 6D4 was localized to residues 26 to 36 in the N-terminus of SCIN, which overlap with the active site. Accordingly, 6D4 can inhibit SCIN activity as demonstrated through the analysis of C3b deposition on S. aureus cells and complement-induced lysis of rabbit erythrocytes. Importantly, while SCIN is generally regarded as a secreted virulence factor, 6D4 allowed detection of strongly increased SCIN binding to S. aureus cells upon exposure to human serum, relating to the known binding of SCIN to C3 convertases deposited on the staphylococcal cell surface. Lastly, we show that labeling of humAb 6D4 with a near-infrared fluorophore allows one-step detection of SCIN-producing S. aureus cells. Together, our findings show that the newly described humAb 6D4 specifically recognizes S. aureus SCIN, which can potentially be used for detection of human serum-incubated S. aureus strains expressing SCIN.