No abstract

Yersinia pestis (Yp) causes the re-emerging disease plague, and is classified by the CDC and NIAID as a highest priority (Category A) pathogen. Currently, there is no approved human vaccine available and advances in early diagnostics and effective therapeutics are urgently needed. A deep understanding of the mechanisms of host response to Yp infection can significantly advance these three areas. We employed the Reverse Phase Protein Microarray (RPMA) technology to reveal the dynamic states of either protein level changes or phosphorylation changes associated with kinase-driven signaling pathways during host cell response to Yp infection. RPMA allowed quantitative profiling of changes in the intracellular communication network of human lung epithelial cells at different times post infection and in response to different treatment conditions, which included infection with the virulent Yp strain CO92, infection with a derivative avirulent strain CO92 (Pgm-, Pst-), treatment with heat inactivated CO92, and treatment with LPS. Responses to a total of 111 validated antibodies were profiled, leading to discovery of 12 novel protein hits. The RPMA analysis also identified several protein hits previously reported in the context of Yp infection. Furthermore, the results validated several proteins previously reported in the context of infection with other Yersinia species or implicated for potential relevance through recombinant protein and cell transfection studies. The RPMA results point to strong modulation of survival/apoptosis and cell growth pathways during early host response and also suggest a model of negative regulation of the autophagy pathway. We find significant cytoplasmic localization of p53 and reduced LC3-I to LC3-II conversion in response to Yp infection, consistent with negative regulation of autophagy. These studies allow for a deeper understanding of the pathogenesis mechanisms and the discovery of innovative approaches for prevention, early diagnosis, and treatment of plague.

Despite recent success in vaccinating populations against SARS-CoV-2, concerns about immunity duration, continued efficacy against emerging variants, protection from infection and transmission, and worldwide vaccine availability, remain. Although mRNA, pDNA, and viral-vector based vaccines are being administered, no protein subunit-based SARS-CoV-2 vaccine is approved. Molecular adjuvants targeting pathogen-recognition receptors (PRRs) on antigen-presenting cells (APCs) could improve and broaden the efficacy and durability of vaccine responses. Native SARS-CoV-2 infection stimulate various PRRs, including toll-like receptors (TLRs) and retinoic-acid-inducible gene I-like receptors (RIG-I). We hypothesized that targeting the same PRRs using adjuvants on nanoparticles along with a stabilized spike (S) protein antigen could provide broad and efficient immune responses. Formulations targeting TLR4 (MPLA), TLR7/8 (R848), TLR9 (CpG), and RIG-I (PUUC) delivered on degradable polymer-nanoparticles (NPs) were combined with the S1 subunit of S protein and assessed in vitro with isogeneic mixed lymphocyte reactions (iso-MLRs). For in vivo studies, the adjuvanted nanoparticles were combined with stabilized S protein and assessed using intranasal and intramuscular prime-boost vaccination models in mice. Combination NP-adjuvants targeting both TLR and RIG-I (MPLA+PUUC, CpG+PUUC, or R848+PUUC) differentially increased proinflammatory cytokine secretion (IL-1β, IL-12p70, IL-27, IFN-β) by APCs cultured in vitro, and induced differential T cell proliferation. When delivered intranasally, MPLA+PUUC NPs enhanced local CD4+CD44+ activated memory T cell responses while MPLA NPs increased anti-S-protein-specific IgG and IgA in the lung. Following intramuscular delivery, PUUC-carrying NPs induced strong humoral immune responses, characterized by increases in anti-S-protein IgG and neutralizing antibody titers and germinal center B cell populations (GL7+ and BCL6+ B cells). MPLA+PUUC NPs further boosted S-protein-neutralizing antibody titers and T follicular helper cell populations in draining lymph nodes. These results suggest that SARS-CoV-2-mimicking adjuvants and subunit vaccines could lead to robust and unique route-specific adaptive immune responses and may provide additional tools against the pandemic.GRAPHICAL ABSTRACT