A novel assay to measure B cell responses to keyhole limpet haemocyanin vaccination in healthy volunteers and subjects with systemic lupus erythematosus

Abstract

The aim of the study was to characterize performance of a complementary set of assays to measure antigen‐specific immune responses in subjects immunized with a neoantigen. Healthy volunteers (HV) (n = 8) and patients with systemic lupus erythematosus (SLE) (n = 6) were immunized with keyhole limpet haemocyanin (KLH) on days 1 and 29. Serum antibodies were detected using a flow cytometric bead array (CBA) that multiplexed the KLH response alongside pre‐existing anti‐tetanus antibodies. Peripheral blood mononuclear cells were studied by B cell ELISPOT. These assays were built upon precedent assay development in cynomolgus monkeys, which pointed towards their utility in humans. Primary anti‐KLH IgG responses rose to a mean of 65–93‐fold above baseline for HV and SLE patients, respectively, and secondary responses rose to a mean of 260‐170‐fold above baseline. High levels of anti‐tetanus IgG were detected in pre‐immunization samples and their levels did not change over the course of study. Anti‐KLH IgG1‐4 subclasses were characterized by a predominant IgG1 response, with no significant differences in subclass magnitude or distribution between HV and SLE subjects. Anti‐KLH IgM levels were detectable, although the overall response was lower. IgM was not detected in two SLE subjects whodid generate an IgG response. All subjects responded to KLH by B cell ELISPOT, with no significant differences observed between HV and SLE subjects. The CBA and B cell ELISPOT assays reliably measured anti‐KLH B cell responses, supporting use of this approach and these assays to assess the pharmacodynamic and potential safety impact of marketed/investigational immune‐therapeutics.

Keyhole limpet haemocyanin – a model antigen for human immunotoxicological studies

“…A measureable, robust antigen‐specific immune response can be generated following administration of KLH via a number of routes – intradermal , subcutaneous , intramuscular and inhalational . Studies that administered KLH intra‐muscularly used the deltoid muscle.…”

“…Studies that administered KLH intra‐muscularly used the deltoid muscle. The site of subcutaneous administration was documented over the deltoid or ‘arm’ , whilst intradermal immunization was given in the upper arm or forearm . In those studies assessing delayed type hypersensitivity (DTH) response, intradermal KLH was administered to the volar aspect of the forearm , upper arm or gluteal aspect of the leg .…”

“…Most studies that have used KLH immunization to assess an antigen‐specific immune response have measured KLH antibodies (14 of the 16 unique studies listed in Table ), with the majority utilizing indirect ELISA assays. A multiplex flow cytometric bead array platform has been recently reported that allows a semi‐quantitative assessment of multiple antibody targets simultaneously, an advance on previous single‐plex ELISA platforms . Studies have differed by the timing of serum sampling, immunoglobulin sub‐type targets (e.g.…”

“…KLH immunization has been used in a number of clinical studies to assess the influence of various physiological and disease states and exposures on immune function. These have included psychological states , cardiovascular exercise , cancer and/or chemotherapy , immunodeficiency states , atopy and asthma and autoimmune disease . Our objectives in this systematic review were to i) survey the formulations, modes of administration and doses used, ii) assess the available safety data and iii) summarize the measures of antigen‐specific immune response used, both antibody‐ and cell‐mediated.…”

Inducible T-cell co-stimulator ligand (ICOSL) blockade leads to selective inhibition of anti-KLH IgG responses in subjects with systemic lupus erythematosus

“…Detection of anti-KLH IgM and IgG antibodies in serum KLH-specific IgM and IgG levels in the serum were measured using a validated, semiquantitative flow cytometric bead array as described by Ferbas et al, 41 modified to include an anti-KLH IgG standard curve. Briefly, serum was incubated with a mixture of fluorescently labelled beads each individually conjugated to KLH, tetanus toxoid (TT), sperm whale myoglobin (SWM), IgG and IgM (manufactured by Becton Dickinson, La Jolla, California, USA).…”