Despite the ease of production and improved safety profiles of recombinant vaccines, the inherently low immunogenicity of unadjuvanted proteins remains an impediment to their widespread adoption. The covalent tethering of...

A diversity of vaccines is necessary to reduce the mortality and morbidity of SARS-CoV-2. Vaccines must be efficacious, easy to manufacture, and stable within the existing cold chain to improve their availability around the world. Recombinant protein subunit vaccines adjuvanted with squalene-based emulsions such as AS03™ and MF59™ have a long and robust history of safe, efficacious use with straightforward production and distribution. Here, subunit vaccines were made with squalene-based emulsions containing novel, synthetic toll-like receptor (TLR) agonists, INI-2002 (TLR4 agonist) and INI-4001 (TLR7/8 agonist), using the recombinant receptor-binding domain (RBD) of SARS-CoV-2 S protein as an antigen. The addition of the TLR4 and TLR7/8 agonists, alone or in combination, maintained the formulation characteristics of squalene-based emulsions, including a sterile filterable droplet size (<220 nm), high homogeneity, and colloidal stability after months of storage at 4, 25, and 40 °C. Furthermore, the addition of the TLR agonists skewed the immune response from Th2 towards Th1 in immunized C57BL/6 mice, resulting in an increased production of IgG2c antibodies and a lower antigen-specific production of IL-5 with a higher production of IFNγ by lymphocytes. As such, incorporating TLR4 and TLR7/8 agonists into emulsions leveraged the desirable formulation and stability characteristics of emulsions and can induce Th1-type humoral and cell-mediated immune responses to combat the continued threat of SARS-CoV-2.

Stroke is the fifth leading cause of death annually in the United States. Ischemic stroke occurs when a blood vessel supplying the brain is occluded. The hippocampus is particularly susceptible to AMPA receptor-mediated delayed neuronal death as a result of ischemic/reperfusion injury. AMPA receptors composed of a GluA2 subunit are impermeable to calcium due to a post-transcriptional modification in the channel pore of the GluA2 subunit. GluA2 undergoes internalization and is subsequently degraded following ischemia/reperfusion. The subsequent increase in the expression of GluA2-lacking, Ca2+-permeable AMPARs results in excitotoxicity and eventually delayed neuronal death. Following ischemia/reperfusion, there is increased production of superoxide radicals. This study describes how the internalization and degradation of GluA1 and GluA2 AMPAR subunits following ischemia/reperfusion is mediated through an oxidative stress signaling cascade. U251-MG cells were transiently transfected with fluorescently tagged GluA1 and GluA2, and different Rab proteins to observe AMPAR endocytic trafficking following oxygen glucose-deprivation/reperfusion (OGD/R), an in vitro model for ischemia/reperfusion. Pretreatment with Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin (MnTMPyP), a superoxide dismutase mimetic, ameliorated the OGD/R-induced, but not agonist-induced, internalization and degradation of GluA1 and GluA2 AMPAR subunits. Specifically, MnTMPyP prevented the increased colocalization of GluA1 and GluA2 with Rab5, an early endosomal marker, and with Rab7, a late endosomal marker, but did not affect the colocalization of GluA1 with Rab11, a marker for recycling endosomes. These data indicate that oxidative stress may play a vital role in AMPAR-mediated cell death following ischemic/reperfusion injury.