1. Effect of the Eculizumab (Soliris  ), on the meningococcal serogroup B serum bactericidal antibody activity and opsonophagocytic activity Jamie Findlow 1, Xilian Bai 1, Dominic Carr 1, Holly Humphries 3, Ann Holland 1, Anu S. Varghese 2, Helen Findlow 1, Stephen M. Hughes 2, Stephen Taylor 3, Andrew Gorringe 3, Ray Borrow 1 1. Vaccine Evaluation Unit, Public Health England, Manchester Royal Infirmary, Manchester, UK. 2. Department of Paediatric Allergy and Immunology, Royal Manchester Children’s Hospital, Manchester, UK 3. Public Health England, Porton Down, Salisbury, UK INTRODUCTIONMETHODS Eculizumab (Soliris  ), a monoclonal antibody, targets complement protein C5 and inhibits terminal complement-mediated haemolysis associated with Paroxysmal Nocturnal Haemoglobinuria (PNH) 1 and atypical Haemolytic Uremic Syndrome (aHUS) 2. The concentrations of Eculizumab in PNH peak and trough are 194±76 µg/mL and 97±60 µg/mL, respectively. Following the licensure of Bexsero  in the UK, patients with PNH and aHUS are offered Bexsero 3. Therefore it is important to know whether these patients have protective serogroup B (MenB) serum bactericidal antibody (SBA) titres during Soliris therapy. The MenB SBA assay is a functional measure of the ability of antibodies in conjunction with human complement to kill the meningococcus. No formation of the lytic terminal C5b-9 complement complex will take place in the absence of C5. Opsonophagocytic activity (OPA) of meningococci is initiated by the binding of C3, OPA should not be impaired in C5-deficient blood, since C5 is downstream of C3 in the complement cascade. All test samples spiked with Soliris were heat inactivated before doubling diluted in the microtitre plate wells in the SBA assay. Colony Forming Unit (CFU)s were calculated in each of the following experiment conditions and then normalised to the CFU counts in the correspondence control well. 1. Effect of Soliris on the SBA killing activity in the SBA assay Two samples with unknown meningococcal vaccine history against the MenB target strain NZ 98/254 were assayed in the MenB SBA assay in the presence of Soliris, at varying concentrations from 0.0003 µg/mL to 1250 µg/mL. 2. Inhibition of Soliris effect in the SBA assay with different concentrations of C5 from 100 µg/mL to 3.125 µg/mL. One sample was tested in the presence of Soliris (40 µg/mL) with different concentrations of C5 from 3.125 µg/mL to 100 µg/mL. 3. High concentration of human complement C5 blocking the Soliris inhibition activity in the SBA assay Each of four samples were tested at the following conditions: spiked with 40 µg/mLSoliris, 40 µg/mL Soliris and 125 µg/mL C5, 125 µg/mL C5 and Bactericidal buffer in the SBA assay. 4. Effect of Soliris on the OPA activity in the OPA assay One sample from a subject following Bexsero  was assyed in the OPA assay with concentrations of Soliris varying from 10 µg/mL to 200 µg/mL. OPA activity measured the presence of killed fluorescently (BCECF) labelled menB target strain NZ 98/254 within human granulocytes (differentiated HL60 cells or fresh polymorphonuclear granulocytes (PMNs)) by flow cytometry, using IgG-depleted pooled human plasma as the exogenous source of complement. CONCLUSIONS Patients on Soliris therapy are at increased risk of meningococcal disease. Soliris inhibits SBA activity. Human complement protein C5 can be used in the SBA assay to block Soliris inhibition. Soliris does not appear to affect OPA when using human PMNLs and complement. These data suggest meningococcal vaccination of patients on Soliris therapy is still beneficial. Correspondence REFERENCES RESULTS 1.Hematology Am Soc Hematol Educ Program. 2008:116-23 2.Pediatr Transplantation 2012: 16: E246– E250 3.Expert Opin Biol Ther. 2011 Jul;11(7):969-85 Figure 1. Minimum concentration of Soliris required for inhibiting activity in the SBA assay Figure 2. Inhibition of Soliris function with different concentrations of C5 Figure 3. Human complement C5 blocking the Soliris inhibition activity in the SBA assay xilian.bai@phe.gov.uk AIM The aim of the study is to exam the effect of Eculizumab on the MenB SBA and OPA activity. Figure 4. Effect of Soliris at varying concentrations on meningococcal OPA (using human PMNLs and complement) Figure 5. Effect of Soliris at varying concentrations on meningococcal OPA – Soliris added prior to pre-opsonisation step The number of CFUs increased sharply in the presence of Soliris at concentrations greater than 5 µg/mL (Figure 1). Soliris at concentration 40 µg/mL in a serum can be blocked by C5 at concentration of 100 µg/mL (Figure 2). Figure 3 is an example of a Soliris spiking serum sample (Soliris 40 µg/mL in the serum) treated with and without complement C5 (125 µg/mL). Low killing activity was observed when Soliris in the serum prevented the bacteria being lysed at the concentration of 40 µg/mL of Soliris. Soliris could be inhibited by C5 at the concentration of 125 µg/mL as over 50% of killing activity was observed in columns 1 to 5 which were similar to C5 alone and control (without Soliris and C5). Soliris has no effect on the OPA even at the highest concentration up to 40 µg/mL when Soliris was added at the same point as the complement (Figure 4). Also no effect was seen when the concentration of Soliris was increased to 200 µg/mL (data not shown). Figure 5 shows that there is also no effect on OPA when Soliris was added with serum at first stage of the assay.