Belimumab concentration measurements using a homologous anti-idiotype immunoassay.

Monitoring belimumab concentrations in patients can be a valuable tool for assessing treatment response and for personalizing drug doses. Various assay formats may be used to measure concentrations of therapeutic monoclonal antibodies. A particularly useful format involves the use of anti-idiotype monoclonal antibodies, selected to be highly specific to the antibody of interest. Here, we describe the development of a specific, high-affinity anti-idiotype antibody to belimumab, and the application of this antibody in a homologous sandwich ELISA to measure belimumab concentrations.

A Novel Combination of Host Protein Biomarkers to Distinguish Bacterial From Viral Infections in Febrile Children in Emergency Care.

BACKGROUND: Distinguishing bacterial and viral infections based on clinical symptoms in febrile children attending the emergency department (ED) is challenging. The aim of this study is to determine a novel combination of host protein biomarkers and to assess its performance in distinguishing between bacterial and viral infection in febrile children attending EDs. METHODS: A literature search was performed to identify blood protein biomarkers able to distinguish bacterial and viral infections (May 2015-May 2019). We selected 7 protein biomarkers: Procalcitonin, TNF-related apoptosis-inducing ligand (TRAIL), interleukin (IL)-4, IL-6, Interferon gamma-induced protein-10 (CXCL-10), interferon-gamma and lipocalin 2 (LCN2). These were measured in blood plasma using a bead-based immunoassay in children with a confirmed bacterial or viral infection attending EDs in the Netherlands. We used generalized linear modeling to classify bacterial and viral infections and applied a previously developed feature selection algorithm to select the optimal combination of proteins. We performed a subgroup analysis of this protein signature in patients with C-reactive protein <60 mg/L, representing a clinically challenging diagnostic group. RESULTS: In total 102 children were included (N = 67 bacterial; N = 35 viral). Individual performance of the 7 biomarkers in classifying bacterial versus viral infections ranged from 60.8%-74.5% area under the receiver operator curve (AUC). TRAIL, LCN2 and IL-6 were identified as the best 3-protein signature with an AUC of 86% (95% CI: 71.3%-100%). In 57 patients with C-reactive protein levels <60 mg/L, the 3-protein signature had an AUC of 85.1% (95% CI: 75.3%-94.9%). CONCLUSION: We demonstrate a promising novel combination of 3 host protein biomarkers; TRAIL, LCN2 and IL-6, which performs well in classifying bacterial and viral infections in febrile children in emergency care.

Neisseria meningitidis Serogroup Z Meningitis in a Child With Complement C8 Deficiency and Potential Cross Protection of the MenB-4C Vaccine.

Complement deficient patients are susceptible to rare meningococcal serogroups. A 6-year-old girl presented with serogroup Z meningitis. This led to identification of a C8 deficiency. The MenB-4C vaccine induced cross-reactive antibodies to serogroup Z and increased in vitro opsonophagocytic killing and may thus protect complement deficient patients.

Eculizumab impairs Neisseria meningitidis serogroup B killing in whole blood despite 4CMenB vaccination of PNH patients.

Complement C5 inhibitor eculizumab has a great impact on the treatment of patients with paroxysmal nocturnal hemoglobinuria (PNH). However, this treatment success has a major drawback: a substantially increased susceptibility for life-threatening Neisseria meningitidis infections. Therefore, N meningitidis vaccination is strongly advised before initiating complement C5-blocking therapy. In this study, we show that the multicomponent N meningitidis serogroup B (4CMenB) vaccination of PNH patients treated with eculizumab results in a significant increase in anti-N meningitidis serogroup B (MenB) plasma immunoglobulin G (IgG) levels. Anti-MenB IgG was able to bind to the bacterial surface and initiate complement activation; however, inhibition of the membrane attack complex formation completely blocked whole blood-mediated killing of MenB. This would suggest that, despite 4CMenB vaccination, PNH patients taking C5 inhibitors are not sufficiently protected against MenB infection, which is in line with the fact that vaccinated PNH patients still experience meningococcal infections.

Common Genetic Variants in the Complement System and their Potential Link with Disease Susceptibility and Outcome of Invasive Bacterial Infection.

Streptococcus pneumoniae and Neisseria meningitidis are pathogens that frequently colonize the nasopharynx in an asymptomatic manner but are also a cause of invasive bacterial infections mainly in young children. The complement system plays a crucial role in humoral immunity, complementing the ability of antibodies to clear microbes, thereby protecting the host against bacterial infections, including S. pneumoniae and N. meningitidis. While it is widely accepted that complement deficiencies due to rare genetic variants increase the risk for invasive bacterial infection, not much is known about the common genetic variants in the complement system in relation to disease susceptibility. In this review, we provide an overview of the effects of common genetic variants on complement activation and on complement-mediated inflammation.

Streptococcus pneumoniae PspC Subgroup Prevalence in Invasive Disease and Differences in Contribution to Complement Evasion.

The pneumococcal capsular serotype is an important determinant of complement resistance and invasive disease potential, but other virulence factors have also been found to contribute. Pneumococcal surface protein C (PspC), a highly variable virulence protein that binds complement factor H to evade C3 opsonization, is divided into two subgroups: choline-bound subgroup I and LPxTG-anchored subgroup II. The prevalence of different PspC subgroups in invasive pneumococcal disease (IPD) and functional differences in complement evasion are unknown. The prevalence of PspC subgroups in IPD isolates was determined in a collection of 349 sequenced strains of Streptococcus pneumoniae isolated from adult patients. pspC deletion mutants and isogenic pspC switch mutants were constructed to study differences in factor H binding and complement evasion in relation to capsule thickness. Subgroup I pspC was far more prevalent in IPD isolates than subgroup II pspC The presence of capsule was associated with a greater ability of bound factor H to reduce complement opsonization. Pneumococcal subgroup I PspC bound significantly more factor H and showed more effective complement evasion than subgroup II PspC in isogenic encapsulated pneumococci. We conclude that variation in the PspC subgroups, independent of capsule serotypes, affects pneumococcal factor H binding and its ability to evade complement deposition.

Acquisition of C1 inhibitor by Bordetella pertussis virulence associated gene 8 results in C2 and C4 consumption away from the bacterial surface.

Whooping cough, or pertussis, is a contagious disease of the respiratory tract that is re-emerging worldwide despite high vaccination coverage. The causative agent of this disease is the Gram-negative Bordetella pertussis. Knowledge on complement evasion strategies of this pathogen is limited. However, this is of great importance for future vaccine development as it has become apparent that a novel pertussis vaccine is needed. Here, we unravel the effect of Virulence associated gene 8 (Vag8) of B. pertussis on the human complement system at the molecular level. We show that both recombinant and endogenously secreted Vag8 inhibit complement deposition on the bacterial surface at the level of C4b. We reveal that Vag8 binding to human C1-inhibitor (C1-inh) interferes with the binding of C1-inh to C1s, C1r and MASP-2, resulting in the release of active proteases that subsequently cleave C2 and C4 away from the bacterial surface. We demonstrate that the depletion of these complement components in the bacterial surrounding and subsequent decreased deposition on B. pertussis leads to less complement-mediated bacterial killing. Vag8 is the first protein described that specifically prevents C1s, C1r and MASP-2 binding to C1-inh and thereby mediates complement consumption away from the bacterial surface. Unravelling the mechanism of this unique complement evasion strategy of B. pertussis is one of the first steps towards understanding the interactions between the first line of defense complement and B. pertussis.

Hijacking Complement Regulatory Proteins for Bacterial Immune Evasion.

The human complement system plays an important role in the defense against invading pathogens, inflammation and homeostasis. Invading microbes, such as bacteria, directly activate the complement system resulting in the formation of chemoattractants and in effective labeling of the bacteria for phagocytosis. In addition, formation of the membrane attack complex is responsible for direct killing of Gram-negative bacteria. In turn, bacteria have evolved several ways to evade complement activation on their surface in order to be able to colonize and invade the human host. One important mechanism of bacterial escape is attraction of complement regulatory proteins to the microbial surface. These molecules are present in the human body for tight regulation of the complement system to prevent damage to host self-surfaces. Therefore, recruitment of complement regulatory proteins to the bacterial surface results in decreased complement activation on the microbial surface which favors bacterial survival. This review will discuss recent advances in understanding the binding of complement regulatory proteins to the bacterial surface at the molecular level. This includes, new insights that have become available concerning specific conserved motives on complement regulatory proteins that are favorable for microbial binding. Finally, complement evasion molecules are of high importance for vaccine development due to their dominant role in bacterial survival, high immunogenicity and homology as well as their presence on the bacterial surface. Here, the use of complement evasion molecules for vaccine development will be discussed.