An eight-plex immunoassay for Group A streptococcus serology and vaccine development.

Group A Streptococcus (GAS) is a major human pathogen responsible for superficial infections through to life-threatening invasive disease and the autoimmune sequelae acute rheumatic fever (ARF). Despite a significant global economic and health burden, there is no licensed vaccine available to prevent GAS disease. Several pre-clinical vaccines that target conserved GAS antigens are in development. Assays that measure antigen-specific antibodies are essential for vaccine research. The aim of this study was to develop a multiplex beadbased immunoassay that can detect and quantify antibody responses to multiple GAS antigen targets in small volume blood samples. This builds on our existing triplex assay comprised of antigens used in clinical serology for the diagnosis of ARF (SLO, DNase B and SpnA). Five additional conserved putative GAS vaccine antigens (Spy0843, SCPA, SpyCEP, SpyAD and the Group A carbohydrate), were coupled to spectrally unique beads to form an 8-plex antigen panel. After optimisation of the assay protocol, standard curves were generated, and assessments of assay specificity, precision and reproducibility were conducted. A broad range of antibody (IgG) titres were able to be quickly and accurately quantified from a single serum dilution. Assay utility was assessed using a panel of 62 clinical samples including serum from adults with GAS bacteraemia and children with ARF. Circulating IgG to all eight antigens was elevated in patients with GAS disease (n = 23) compared to age-matched controls (n = 39) (P < 0.05). The feasibility of using dried blood samples to quantify antigen-specific IgG was also demonstrated. In summary, a robust and reproducible 8-plex assay has been developed that simultaneously quantifies IgG antibodies to GAS vaccine and diagnostic antigens.

Immunomic Investigation of Holocyclotoxins to Produce the First Protective Anti-Venom Vaccine Against the Australian Paralysis Tick, Ixodes holocyclus.

Venom producing animals are ubiquitously disseminated among vertebrates and invertebrates such as fish, snakes, scorpions, spiders, and ticks. Of the ~890 tick species worldwide, 27 have been confirmed to cause paralysis in mammalian hosts. The Australian paralysis tick (Ixodes holocyclus) is the most potent paralyzing tick species known. It is an indigenous three host tick species that secretes potent neurotoxins known as holocyclotoxins (HTs). Holocyclotoxins cause a severe and harmful toxicosis leading to a rapid flaccid paralysis which can result in death of susceptible hosts such as dogs. Antivenins are generally polyclonal antibody treatments developed in sheep, horses or camels to administer following bites from venomous creatures. Currently, the methods to prevent or treat tick paralysis relies upon chemical acaricide preventative treatments or prompt removal of all ticks attached to the host followed by the administration of a commercial tick-antiserum (TAS) respectively. However, these methods have several drawbacks such as poor efficacies, non-standardized dosages, adverse effects and are expensive to administer. Recently the I. holocyclus tick transcriptome from salivary glands and viscera reported a large family of 19 holocyclotoxins at 38-99% peptide sequence identities. A pilot trial demonstrated that correct folding of holocyclotoxins is needed to induce protection from paralysis. The immunogenicity of the holocyclotoxins were measured using commercial tick antiserum selecting HT2, HT4, HT8 and HT11 for inclusion into the novel cocktail vaccine. A further 4 HTs (HT1, HT12, HT14 and HT17) were added to the cocktail vaccine to ensure that the sequence variation among the HT protein family was encompassed in the formulation. A second trial comparing the cocktail of 8 HTs to a placebo group demonstrated complete protection from tick challenge. Here we report the first successful anti-venom vaccine protecting dogs from tick paralysis.

Optimising a 6-plex tetanus-diphtheria-pertussis fluorescent bead-based immunoassay.

Small volume assays are required for large-scale research studies and in particular paediatric trials, where multiple measures are required from a single sample. Fluorescent bead-based technology (Bioplex/Luminex) allows high through-put and simultaneous quantification of multiple analytes in a single test. This technology uses sets of microspheres, each with a unique spectral address that can be coated with a different antigen of interest. Following the addition of a detector antibody, specific for the isotype of interest and labelled with R-Phycoerythrin, the bioplex reader determines the amounts of antigen-specific antibodies in each test sample relative to a reference standard. Here we outline the optimisations undertaken to establish a 6-plex fluorescent bead-based immunoassay that can accurately measure human IgG to individual tetanus-diphtheria-acellular pertussis (Tdap) antigens from 2 to 4 ul of human serum/ plasma. This protocol was adapted from previously published methods and aligns with current recommendations for developing pertussis-serological assays. To our knowledge, this is the first Tdap-specific multiplex immunoassay (MIA) established in Australia. All components were optimised and validated in-house including: microsphere preparation conditions, reference serum and QC development, and assay running.•Determining optimal antigen coating dose and conjugation method.•Optimising an in-house reference serum with clinically relevant titres.•Determining assay specificity and reproducibility.

An observational study of antibody responses to a primary or subsequent pertussis booster vaccination in Australian healthcare workers.

Adult pertussis vaccination is increasingly recommended to control pertussis in the community. However, there is little data on the duration and kinetics of immunity to pertussis boosters in adults. We compared IgG responses to vaccination with a tetanus, low-dose diphtheria, low-dose acellular pertussis (Tdap) booster at 1 week, 1 month and 1 year post-vaccination in whole-cell (wP)-primed Australian paediatric healthcare workers who had received an adult Tdap booster 5-12 years previously, to those who received their first Tdap booster. Tdap vaccination was well tolerated in both groups. Previously boosted adults had significantly higher pre-vaccination IgG concentrations for all vaccine-antigens, and more were seropositive for pertussis toxin (PT)-specific IgG (≥ 5 IU/mL) (69.5%; 95% confidence interval (CI) 59.5-79.5) than adults in the naïve group (45.2%; 95% CI 32.8-57.5). Tdap vaccination significantly increased IgG responses 1 month post-vaccination in both groups. This increase was more rapid in previously boosted than in naïve adults, with geometric mean fold-increases in PT-IgG at 1 week post vaccination of 3.6 (95% CI 2.9-4.3) and 2.6 (95% CI 2.2-3.2), respectively. Antibody waning between 1 month and 1 year post-vaccination was similar between groups for IgG specific to PT and filamentous haemagglutinin (FHA), but was faster for IgG against pertactin (PRN) in the naïve group (GMC ratio 0.36; 95% CI 0.31-0.42) than the previously boosted group (GMC ratio 0.45; 95% CI 0.39-0.50). At baseline, all but one adult had protective IgG titres against tetanus toxin (TT) (≥ 0.1 IU/mL), and 75.6% in the previously boosted and 61.3% in the naïve group had protective IgG titres against diphtheria toxoid (DT) of ≥ 0.1 IU/mL. This study shows that pertussis immune memory is maintained up to 12 years after Tdap vaccination in wP-primed Australian adults. There was no evidence that pertussis immune responses waned faster after a booster dose. These findings support current recommendations of repeating Tdap booster vaccination in paediatric healthcare workers at least every 10 years. Clinical trials registry: ACTRN12615001262594.

Corrigendum: Pediatric Burn Survivors Have Long-Term Immune Dysfunction With Diminished Vaccine Response.

[This corrects the article DOI: 10.3389/fimmu.2020.01481.].

Pediatric Burn Survivors Have Long-Term Immune Dysfunction With Diminished Vaccine Response.

Epidemiological studies have demonstrated that survivors of acute burn trauma are at long-term increased risk of developing a range of morbidities. The mechanisms underlying this increased risk remain unknown. This study aimed to determine whether burn injury leads to sustained immune dysfunction that may underpin long-term morbidity. Plasma and peripheral blood mononuclear cells were collected from 36 pediatric burn survivors >3 years after a non-severe burn injury (<10% total body surface area) and from age/sex-matched non-injured controls. Circulating cytokine and vaccine antibody levels were assessed using multiplex immunoassays and cell profiles compared using a panel of 40 metal-conjugated antibodies and mass cytometry. TNF-α (1.31-fold change from controls), IL-2 (1.18-fold), IL-7 (1.63-fold), and IFN-γ (1.18-fold) were all significantly elevated in the burn cohort. Additionally, burn survivors demonstrated diminished antibody responses to the diphtheria, tetanus, and pertussis vaccine antigens. Comparisons between groups using unsupervised clustering identified differences in proportions of clusters within T-cells, B-cells and myeloid cells. Manual gating confirmed increased memory T-regulatory and central memory CD4+ T-cells, with altered expression of T-cell, B-cell, and dendritic cell markers. Conclusions: This study demonstrates a lasting change to the immune profile of pediatric burn survivors, and highlights the need for further research into post-burn immune suppression and regulation.