HSP60 as a Target of Anti-Ergotypic Regulatory T Cells

The 60 kDa heat shock protein (HSP60) has been reported to influence T-cell responses in two ways: as a ligand of toll-like receptor 2 signalling and as an antigen. Here we describe a new mechanism of T-cell immuno-regulation focused on HSP60: HSP60 is up-regulated and presented by activated T cells (HSP60 is an ergotope) to regulatory (anti-ergotypic) T cells. Presentation of HSP60 by activated T cells was found to be MHC-restricted and dependent on accessory molecules - CD28, CD80 and CD86. Anti-ergotypic T cells responded to T-cell HSP60 by proliferation and secreted IFNγ and TGFβ1. In vitro, the anti-ergotypic T cells inhibited IFNγ production by their activated T-cell targets. In vivo, adoptive transfer of an anti-ergotypic HSP60-specific T-cell line led to decreased secretion of IFNγ by arthritogenic T cells and ameliorated adjuvant arthritis (AA). Thus, the presentation of HSP60 by activated T cells turns them into targets for anti-ergotypic regulatory T cells specific for HSP60. However, the direct interaction between the anti-ergotypic T regulators (anti-HSP60) and the activated T cells also down-regulated the regulators. Thus, by functioning as an ergotope, HSP60 can control both the effector T cells and the regulatory HSP60-specific T cells that control them

Enhanced production yields of rVSV-SARS-CoV-2 vaccine using Fibra-Cel® macrocarriers

The COVID-19 pandemic has led to high global demand for vaccines to safeguard public health. To that end, our institute has developed a recombinant viral vector vaccine utilizing a modified vesicular stomatitis virus (VSV) construct, wherein the G protein of VSV is replaced with the spike protein of SARS-CoV-2 (rVSV-ΔG-spike). Previous studies have demonstrated the production of a VSV-based vaccine in Vero cells adsorbed on Cytodex 1 microcarriers or in suspension. However, the titers were limited by both the carrier surface area and shear forces. Here, we describe the development of a bioprocess for rVSV-ΔG-spike production in serum-free Vero cells using porous Fibra-Cel® macrocarriers in fixed-bed BioBLU®320 5p bioreactors, leading to high-end titers. We identified core factors that significantly improved virus production, such as the kinetics of virus production, the use of macrospargers for oxygen supply, and medium replenishment. Implementing these parameters, among others, in a series of GMP production processes improved the titer yields by at least two orders of magnitude (2e9 PFU/mL) over previously reported values. The developed process was highly effective, repeatable, and robust, creating potent and genetically stable vaccine viruses and introducing new opportunities for application in other viral vaccine platforms

MHC class II-restricted recognition of activated T cells by HSP60-specific T-cells.

<p>A. Anti-ergotypic proliferative response of Anti-HSP60 or Anti-MBP T cell lines. Proliferative responses are presented as the ΔCPM±SEM of quadruplicate cultures. B. Anti-ergotypic proliferative response of Anti-p277 or Anti-MBP T cell lines. Proliferative responses are presented as the ΔCPM±SEM of quadruplicate cultures. C. Monoclonal antibodies to MHC-II/RT1.B, MHC-II/RT1.D or MHC-I were assayed for their ability to block the anti-ergotypic proliferative response of the Anti-HSP60 and the Anti-p277 T cell lines. Results are presented as the percent of inhibition of proliferation±SEM of quadruplicate cultures. D. IFNγ (IFNg), TGFβ1 (TGFb1), IL-10 and IL-4 were quantified in the culture supernatants after 72 hr of stimulation of the Anti-MBP, Anti-p277 or Anti-HSP60 T cell lines with 10⁵ activated or resting, irradiated, A2b cells per well. The results are presented as pg/ml±SEM of triplicate cultures. Three to five independent experiments produced similar results.</p

HSP60-specific anti-ergotypic T-cells control arthritogenic T-cells <i>in vivo</i>.

<p>A and B. Anti-MBP or Anti-p277 T cells were injected ip into naïve Lewis rats and three days later AA was induced. Twenty-six days after AA induction, at the peak of AA, the AA clinical score (A) and the hind paw diameter (B) were determined. The bars represent the mean values ± SEM for each group of 8 rats. C. LNC were collected on day 26 after AA induction and the secretion of IFNγ upon stimulation with Mt176-90 was studied. The results are presented as pg/ml±SEM of triplicate cultures. Three independent experiments produced similar results. * p<0.05 and ** p<0.005 compared to the Anti-MBP group.</p