In 2017, Monash University previously tested a non-immune colostrum, an anti-toxin B colostrum and the clinical product colostrum IMM529 by ELISA to determine the titre of C. difficile-specific antibodies in colostrum from cows immunised with C. difficile antigens. Furthermore, the colostrum IgG antibodies were isolated and used to determine the efficacy of the various colostrum batches at neutralising Toxin B activity in vitro.To determine the antibody titres, ELISA plates were coated with 100 µl per well of C.difficile exosporium, SLP, or recombinant Toxin B at a final concentration of 10‐3 mg/ml. Antigens were incubated with four-fold serial dilutions (starting at 1:250) of each colostrum product in comparison to a positive control (colostrum processed by DIAL) and colostrum from non‐immune cows.Summary of results:For the exosporium antigen, the following colostrum samples were tested and the resulting antibody titres obtained:•Exosporium- HBC = 64,000•Exosporium- HBC R&D = 64,000•IMM529 = 16,000•Exosporium-HBC (positive control) = 64,000For the SLP antigen, the following colostrum samples were tested and the resulting antibody titres obtained: •SLP‐HBC = 64,000•SLP-HBC R&D = 64,000•IMM529 = 16,000•SLP-HBC (positive control) = 64,000For the Toxin B antigen, the following colostrum samples were tested and the resulting antibody titres obtained: •Toxin B-HBC = 64,000•Toxin B‐HBC R&D = 64,000•IMM529 = 16,000•Toxin B-HBC (positive control) = 64,000To determine whether the colostrum contained Toxin B neutralising antibodies, IgG antibody purified from non-immune colostrum, anti-toxin B colostrum, and IMM529 was assessed for the ability to neutralise the cytopathic effects (CPE) of C. difficile toxin B on Vero cells. For this, toxin B was incubated with IgG purified from each colostrum product before being applied to Vero cells. Cytopathic effects were scored following 24 hours incubation in comparison to Vero cells treated with toxin B alone. Each sample was tested in duplicate per assay, with a total of three biological replicates performed. Statistical significance of the data was analysed using the Mann Whitney U test. Results showed CPE nearing 100% for Vero cells treated with toxin B alone, and for toxin B incubated with IgG from non-immune colostrum. This CPE is neutralised by approximately 50% when toxin B is incubated with IgG from anti-toxin B colostrum, and from IMM529. Results presented represented a concentration of approximately 256 pg/ml of toxin B (verified for cytotoxicity on Vero cells down to at least 50 pg/ml). n = 3; ** = p££0.01; error bars = mean ± SEM.
Immuron
now would like to generate SLP and exosporium cow vaccines to generate a new
batch of clinical product (Aim 1). The clinical product colostrum powder
(previously tested by Monash in 2017) has been stored since then under various
conditions (different storage temperatures and length of storage, see Table 1)
to allow for stability testing to be performed (Aim 2). This stability testing
will involve testing the stored samples for antibody titre and ability to
neutralise Toxin B activity in vitro.
Immuron
also has more of the clinical product stored as frozen liquid colostrum, which
will be processed to powder by CSIRO. During this processing, samples will be
collected at various stages of the process to assay at what stage (if any)
antibody titres or activity are being lost (Aim 3).