An mRNA-based T-cell-inducing antigen strengthens COVID-19 vaccine against SARS-CoV-2 variants.

Herd immunity achieved through mass vaccination is an effective approach to prevent contagious diseases. Nonetheless, emerging SARS-CoV-2 variants with frequent mutations largely evaded humoral immunity induced by Spike-based COVID-19 vaccines. Herein, we develop a lipid nanoparticle (LNP)-formulated mRNA-based T-cell-inducing antigen, which targeted three SARS-CoV-2 proteome regions that enriched human HLA-I epitopes (HLA-EPs). Immunization of HLA-EPs induces potent cellular responses to prevent SARS-CoV-2 infection in humanized HLA-A*02:01/DR1 and HLA-A*11:01/DR1 transgenic mice. Of note, the sequences of HLA-EPs are highly conserved among SARS-CoV-2 variants of concern. In humanized HLA-transgenic mice and female rhesus macaques, dual immunization with the LNP-formulated mRNAs encoding HLA-EPs and the receptor-binding domain of the SARS-CoV-2 B.1.351 variant (RBDbeta) is more efficacious in preventing infection of SARS-CoV-2 Beta and Omicron BA.1 variants than single immunization of LNP-RBDbeta. This study demonstrates the necessity to strengthen the vaccine effectiveness by comprehensively stimulating both humoral and cellular responses, thereby offering insight for optimizing the design of COVID-19 vaccines.

DosR Regulates the Transcription of the Arginine Biosynthesis Gene Cluster by Binding to the Regulatory Sequences in Mycobacterium bovis Bacille Calmette-Guerin.

l-Arginine serves as a carbon and nitrogen source and is critical for Mycobacterium tuberculosis (Mtb) survival in the host. Generally, ArgR acts as a repressor regulating arginine biosynthesis by binding to the promoter of the argCJBDFGH gene cluster. In this study, we report that the dormancy regulator DosR is a novel arginine regulator binding to the promoter region of argC (rv1652), which regulates arginine synthesis. Phosphorylation modification promoted DosR binding to a region upstream of the promoter. Cofactors, including arginine and metal ions, had an inhibitory effect on this association. Furthermore, DosR regulatory function relies on the interaction of the 167, 181, 182, and 197 amino acid residues with an inverse complementary sequence. Arginine also binds to DosR and directly affects its DNA-binding ability. Together, the results demonstrate that DosR acts as a novel transcriptional regulator of arginine synthesis in Mycobacterium bovis bacille Calmette-Guerin.

Extracellular DNase MAP3916c attacks the neutrophil extracellular traps and is needed for Mycobacterium avium subsp. paratuberculosis virulence.

Extracellular DNases/nucleases are important virulence factors in many bacteria. However, no DNase/nucleases have been reported in Mycobacterium avium subsp. paratuberculosis (MAP), which is a pathogen of paratuberculosis. Genome analyses of MAP K-10 revealed that the map3916c gene putatively encodes a nuclease. In this study, we show that MAP3916c is an extracellular nonspecific DNase requiring a divalent cation, especially Mg2+. The optimum DNase activity of MAP3916c was exhibited at 41 °C and pH 9.0. Site-directed mutagenesis studies indicated that 125-Histidine is necessary for MAP3916c DNase activity. In addition, MAP3916c DNase could destroy the neutrophil extracellular traps (NETs) induced by Phorbol 12-myristate 13-acetate in vitro and degrade the NETs induced by MAP K-10 upon infection. Furthermore, MAP3916c DNase promoted the colonization of MAP K-10, induced the formation of granulomas in the liver and small intestine and promoted the release of IL-1ß, IL-6 and TNF-α inflammatory cytokines during the infection of mice. These results indicated that MAP3916c is relevant to NETs escape and the pathogenicity of MAP. It also provides a basis for further study of the function of nuclease activity on the MAP immune evasion.

Re-burying Artificially Exposed Surface of Viral Subunit Vaccines Through Oligomerization Enhances Vaccine Efficacy.

Viral subunit vaccines often suffer low efficacy. We recently showed that when taken out of the context of whole virus particles, recombinant subunit vaccines contain artificially exposed surface regions that are non-neutralizing and reduce their efficacy, and thus these regions need to be re-buried in vaccine design. Here we used the envelope protein domain III (EDIII) of Japanese encephalitis virus (JEV), a subunit vaccine candidate, to further validate this important concept for subunit vaccine designs. We constructed monomeric EDIII, dimeric EDIII via a linear space, dimeric EDIII via an Fc tag, and trimeric EDIII via a foldon tag. Compared to monomeric EDIII or linearly linked dimeric EDIII, tightly packed EDIII oligomers via the Fc or foldon tag induce higher neutralizing antibody titers in mice and also protect mice more effectively from lethal JEV challenge. Structural analyses demonstrate that part of the artificially exposed surface areas on recombinant EDIII becomes re-buried in Fc or foldon-mediated oligomers. This study further establishes the artificially exposed surfaces as an intrinsic limitation of subunit vaccines, and suggests that re-burying these surfaces through tightly packed oligomerization is a convenient and effective approach to overcome this limitation.

Anti-Influenza Effect and Mechanisms of Lentinan in an ICR Mouse Model.

Influenza virus is a serious threat to global human health and public health security. There is an urgent need to develop new anti-influenza drugs. Lentinan (LNT) has attracted increasing attention in recent years. As potential protective agent, LNT has been shown to have anti-tumor, anti-inflammatory, and antiviral properties. However, there has been no further research into the anti-influenza action of lentinan in vivo, and the mechanism is still not fully understood. In this study, the anti-influenza effect and mechanism of Lentinan were studied in the Institute of Cancer Research (ICR) mouse model. The results showed that Lentinan had a high degree of protection in mice against infection with influenza A virus, delayed the emergence of clinical manifestations, improved the survival rate of mice, significantly prolonged the middle survival days, attenuated the weight loss, and reduced the lung coefficient of mice. It alleviated the pathological damage of mice infected with the influenza virus and improved blood indices. Lentinan treatment considerably inhibited inflammatory cytokine (TNF-α, IL-1ß, IL-4, IL-5, IL-6) levels in the serum and lung and improved IFN-γ cytokine levels, which reduced cytokine storms caused by influenza virus infection. The underlying mechanisms of action involved Lentinan inhibiting the inflammatory response by regulating the TLR4/MyD88 signaling pathway. This study provides a foundation for the clinical application of Lentinan, and provides new insight into the development of novel immunomodulators.

Characterization and pathogenicity of extracellular serine protease MAP3292c from Mycobacterium avium subsp. paratuberculosis.

Serine protease is the virulence factor of many pathogens. However, there are no prevailing data available for serine protease as a virulence factor derived from Mycobacterium avium subsp. paratuberculosis (MAP). The MAP3292c gene from MAP, the predicted serine protease, was expressed in Escherichia coli and characterized by biochemical methods. MAP3292c protein efficiently hydrolyzed casein at optimal temperature and pH of 41 °C and 9.0, respectively. Furthermore, divalent metal ions of Ca2+ significantly promoted the protease activity of MAP3292c, and MAP3292c had autocleavage activity between serine 86 and asparagine 87. Site-directed mutagenesis studies showed that the serine 238 residue had catalytic roles in MAP3292c. Furthermore, a BALB/c mouse model confirmed that MAP3292c significantly promoted the survival of Mycobacterium smegmatis in vivo; caused damage to the liver, spleen, and lung; and promoted the release of inflammatory cytokines IL-1ß, IL-6, and TNF-α in mice. Finally, we confirmed that MAP3292c was relevant to mycobacterial pathogenicity.