The human diploid cell line Medical Research Council -5 (MRC-5) is commonly utilized for vaccine development. Although a rabies vaccine developed in cultured MRC-5 cells exists, the poor susceptibility of MRC-5 cells to the rabies virus (RABV) infection limits the potential yield of this vaccine. The underlying mechanism of MRC-5 cell resistance to RABV infection remains unknown. In this study, we demonstrate that viral infection increased exosomal release from MRC-5 cells; conversely, blocking exosome release promoted RABV infection in MRC-5 cells. Additionally, RABV infection up-regulated microRNA (miR)-423-5p expression in exosomes, resulting in feedback inhibition of RABV replication by abrogating the inhibitory effect of suppressor of cytokine signaling 3 (SOCS3) on type I interferon (IFN) signaling. Furthermore, intercellular delivery of miR-423-5p by exosomes inhibited RABV replication in MRC-5 cells. We also show that RABV infection increased IFN-β production in MRC-5 cells and that blocking the type I IFN receptor promoted RABV infection. In conclusion, MRC-5 cells were protected from RABV infection by the intercellular delivery of exosomal miR-423-5p and the up-regulation of IFN-β. These findings reveal novel antiviral mechanisms in MRC-5 cells against RABV infection. miR-423-5p, exosomes, and IFN signaling pathways may therefore be potential targets for improving MRC-5 cell-based rabies vaccine production.

Inactivated vaccines based on cell culture are very useful in the prevention and control of many diseases. The most popular strategy for the production of inactivated vaccines is based on monkey-derived Vero cells, which results in high productivity of the virus but has a certain carcinogenic risk due to non-human DNA contamination. Since human diploid cells, such as MRC-5 cells, can produce a safer vaccine, efforts to develop a strategy for inactivated vaccine production using these cells have been investigated using MRC-5 cells. However, most viruses do not replicate efficiently in MRC-5 cells. In this study, we found that rabies virus (RABV) infection activated a robust interferon (IFN)-β response in MRC-5 cells but almost none in Vero cells, suggesting that the IFN response could be a key limiting factor for virus production. Treatment of the MRC-5 cells with IFN inhibitors increased RABV titers by 10-fold. Additionally, the RABV titer yield was improved five-fold when using IFN receptor 1 (IFNAR1) antibodies. As such, we established a stable IFNAR1-deficient MRC-5 cell line (MRC-5^IFNAR1−), which increased RABV production by 6.5-fold compared to normal MRC-5 cells. Furthermore, in a pilot-scale production in 1500 square centimeter spinner flasks, utilization of the MRC-5^IFNAR1− cell line or the addition of IFN inhibitors to MRC cells increased RABV production by 10-fold or four-fold, respectively. Thus, we successfully established a human diploid cell-based pilot scale virus production platform via inhibition of IFN response for rabies vaccines, which could also be used for other inactivated virus vaccine production.

Genital herpes (GH) has become one of the most common sexually transmitted diseases worldwide, and it is spreading rapidly in developing countries. Approximately 90% of GH cases are caused by HSV-2. Therapeutic HSV-2 vaccines are intended for people already infected with HSV-2 with the goal of reducing clinical recurrences and recurrent virus shedding. In our previous work, we evaluated recombinant adenovirus-based vaccines, including rAd-gD2ΔUL25, rAd-ΔUL25, and rAd-gD2, for their potency as prophylactic vaccines. In this study, we evaluated these three vaccines as therapeutic vaccines against acute and recurrent diseases in intravaginal challenged guinea pigs. Compared with the control groups, the recombinant vaccine rAd-gD2ΔUL25 induced a higher titer of the binding antibody, and rAd-gD2 + rAd-ΔUL25 induced a higher titer of the neutralizing antibody. Both rAd-gD2ΔUL25 and rAd-gD2 + rAd-ΔUL25 vaccines significantly enhanced the survival rate by 50% compared to rAd-gD2 and reduced viral replication in the genital tract and recurrent genital skin disease. Our findings provide a new perspective for HSV-2 therapeutic vaccine research and provide a new technique to curtail the increasing spread of HSV-2.

Spinal cord injury (SCI) has emerged as a major challenge to global public health due to its low recovery rate and high disability rate, making the development of novel therapeutic approaches imperative. Mesenchymal stem cell lysate (MSC-lysate) not only retains the regenerative potential of mesenchymal stem cells (MSC) but also avoids many potential risks associated with MSC, serving as a safe and effective novel strategy for SCI treatment. However, its therapeutic efficacy and underlying mechanisms in SCI remain unclear. First, a MSC-lysate (MSC derived from dental pulp stem cells) sustained-release nanohydrogel composite system (DL@ZIF-8@PF-127) was fabricated using MSC-lysate, ZIF-8, and PF-127. The anti-inflammatory, antiapoptotic, and ferroptosis-inhibitory effects of the DL@ZIF-8@PF-127 composite system were evaluated using a SCI animal model and a H₂O₂-induced cellular model. Subsequently, Western blot, immunohistochemistry, Enzyme-Linked Immunosorbent Assay, real-time quantitative polymerase chain reaction (RT-qPCR), and JC-1 assays were employed to detect the expression of proteins related to the Nrf2/HO-1/P65 pathway, mitochondrial membrane potential, and apoptosis-related markers. Additionally, the Basso, Beattie, Bresnahan locomotor rating scale, gait analysis, H&E staining, Nissl staining, TUNEL staining, and Annexin V/PI double staining were used to assess neurological function recovery and tissue damage repair. After treatment with DL@ZIF-8@PF-127, the mitochondrial intrinsic apoptosis and ferroptosis pathways induced by SCI were significantly inhibited. The tissue structure damage, inflammatory cell infiltration, and neuronal loss at the injury site were markedly alleviated, accompanied by an increased number of Nissl bodies and reduced neuronal degeneration and gliosis. The DL@ZIF-8@PF-127 composite system can targetedly regulate the Nrf2/HO-1/P65 pathway through the synergistic effect of carrier materials and MSC-lysate. It inhibits ferroptosis and mitochondrial intrinsic apoptosis, alleviates inflammatory responses, improves mitochondrial function, and promotes neuronal repair, thereby mitigating spinal cord injury. This study provides a safe, convenient, and potential therapeutic strategy for SCI.