A broad-spectrum pneumococcal vaccine induces mucosal immunity and protects against lethal Streptococcus pneumoniae challenge.

Pneumococcal disease is a major threat to public health globally, impacting individuals across all age groups, particularly infants and elderly individuals. The use of current vaccines has led to unintended consequences, including serotype replacement, leading to a need for a new approach to combat pneumococcal disease. A promising solution is the development of a broad-spectrum pneumococcal vaccine. In this study, we present the development of a broad-spectrum protein-based pneumococcal vaccine that contains three pneumococcal virulence factors: rlipo-PsaA (lipidated form), rPspAΔC (truncated form), and rPspCΔC (truncated form). Intranasal immunization with rlipo-PsaA, rPspAΔC, and rPspCΔC (LAAC) resulted in significantly higher IgG titres than those induced by administration of nonlipidated rPsaA, rPspAΔC, and rPspCΔC (AAC). Furthermore, LAAC immunization induced the production of higher IgA titres in vaginal washes, feces, and sera in mice, indicating that LAAC can induce systemic mucosal immunity. In addition, administration of LAAC also induced Th1/Th17-biased immune responses and promoted opsonic phagocytosis of Streptococcus pneumoniae strains of various serotypes, implying that the immunogenicity of LAAC immunization provides a protective effect against pneumococcal infection. Importantly, challenge data showed that the LAAC-immunized mice had a reduced bacterial load not only for several serotypes of the 13-valent conjugate pneumococcal vaccine (PCV13) but also for selected non-PCV13 serotypes. Consistently, LAAC immunization increased the survival rate of mice after bacterial challenge with both PCV13 and non-PCV13 serotypes. In conclusion, our protein-based pneumococcal vaccine provides protective effects against a broad spectrum of Streptococcus pneumoniae serotypes.

Cytotoxicity of Venoms and Cytotoxins from Asiatic Cobras (Naja kaouthia, Naja sumatrana, Naja atra) and Neutralization by Antivenoms from Thailand, Vietnam, and Taiwan.

Envenoming by cobras (Naja spp.) often results in extensive local tissue necrosis when optimal treatment with antivenom is not available. This study investigated the cytotoxicity of venoms and purified cytotoxins from the Monocled Cobra (Naja kaouthia), Taiwan Cobra (Naja atra), and Equatorial Spitting Cobra (Naja sumatrana) in a mouse fibroblast cell line, followed by neutralization of the cytotoxicity by three regional antivenoms: the Thai Naja kaouthia monovalent antivenom (NkMAV), Vietnamese snake antivenom (SAV) and Taiwanese Neuro bivalent antivenom (NBAV). The cytotoxins of N. atra (NA-CTX) and N. sumatrana (NS-CTX) were identified as P-type cytotoxins, whereas that of N. kaouthia (NK-CTX) is S-type. All venoms and purified cytotoxins demonstrated varying concentration-dependent cytotoxicity in the following trend: highest for N. atra, followed by N. sumatrana and N. kaouthia. The antivenoms moderately neutralized the cytotoxicity of N. kaouthia venom but were weak against N. atra and N. sumatrana venom cytotoxicity. The neutralization potencies of the antivenoms against the cytotoxins were varied and generally low across NA-CTX, NS-CTX, and NK-CTX, possibly attributed to limited antigenicity of CTXs and/or different formulation of antivenom products. The study underscores the need for antivenom improvement and/or new therapies in treating local tissue toxicity caused by cobra envenomings.

Development of a Broad-Spectrum Antiserum against Cobra Venoms Using Recombinant Three-Finger Toxins.

Three-finger toxins (3FTXs) are the most clinically relevant components in cobra (genus Naja) venoms. Administration of the antivenom is the recommended treatment for the snakebite envenomings, while the efficacy to cross-neutralize the different cobra species is typically limited, which is presumably due to intra-specific variation of the 3FTXs composition in cobra venoms. Targeting the clinically relevant venom components has been considered as an important factor for novel antivenom design. Here, we used the recombinant type of long-chain α-neurotoxins (P01391), short-chain α-neurotoxins (P60770), and cardiotoxin A3 (P60301) to generate a new immunogen formulation and investigated the potency of the resulting antiserum against the venom lethality of three medially important cobras in Asia, including the Thai monocled cobra (Naja kaouthia), the Taiwan cobra (Naja atra), and the Thai spitting cobra (Naja Siamensis) snake species. With the fusion of protein disulfide isomerase and the low-temperature settings, the correct disulfide bonds were built on these recombinant 3FTXs (r3FTXs), which were confirmed by the circular dichroism spectra and tandem mass spectrometry. Immunization with r3FTX was able to induce the specific antibody response to the native 3FTXs in cobra venoms. Furthermore, the horse and rabbit antiserum raised by the r3FTX mixture is able to neutralize the venom lethality of the selected three medically important cobras. Thus, the study demonstrated that the r3FTXs are potential immunogens in the development of novel antivenom with broad neutralization activity for the therapeutic treatment of victims involving cobra snakes in countries.

Discovery of M Protease Inhibitors Encoded by SARS-CoV-2.

The coronavirus (CoV) disease 2019 (COVID-19) pandemic caused by severe acute respiratory syndrome CoV-2 (SARS-CoV-2) is a health threat worldwide. Viral main protease (Mpro, also called 3C-like protease [3CLpro]) is a therapeutic target for drug discovery. Herein, we report that GC376, a broad-spectrum inhibitor targeting Mpro in the picornavirus-like supercluster, is a potent inhibitor for the Mpro encoded by SARS-CoV-2, with a half-maximum inhibitory concentration (IC50) of 26.4 ± 1.1 nM. In this study, we also show that GC376 inhibits SARS-CoV-2 replication with a half-maximum effective concentration (EC50) of 0.91 ± 0.03 µM. Only a small portion of SARS-CoV-2 Mpro was covalently modified in the excess of GC376 as evaluated by mass spectrometry analysis, indicating that improved inhibitors are needed. Subsequently, molecular docking analysis revealed that the recognition and binding groups of GC376 within the active site of SARS-CoV-2 Mpro provide important new information for the optimization of GC376. Given that sufficient safety and efficacy data are available for GC376 as an investigational veterinary drug, expedited development of GC376, or its optimized analogues, for treatment of SARS-CoV-2 infection in human is recommended.

Immunological evaluation of a novel HLA-A2 restricted phosphopeptide of tumor associated Antigen, TRAP1, on cancer therapy.

The tumor necrosis factor receptor associated protein 1 (TRAP1) is a mitochondria chaperon protein that has been previously implicated as a target for cancer therapy due to its expression level is linked to tumor progression. In this study, an immunodominant phosphopeptide of TRAP1 was identified from an HLA-A2 gene transfected mouse cancer cell line using mass spectrometry, and a synthetic phosphopeptide was generated to evaluate the potency on cancer immunotherapy. In the transporter associated with antigen processing (TAP) deficient cell, the conjugated phosphate group plays a critical role to enhance the binding affinity of phosphopeptide with HLA-A2 molecule. On the basis of immunological assay, immunization of synthetic phosphopeptide could induce a high frequency of IFN-γ-secreting CD8+ T cells in HLA-A2 transgenic mice, and the stimulated cytotoxic T lymphocytes showed a high target specificity to lysis the epitope-pulsed splenocytes in vivo and the human lung cancer cell in vitro. In a tumor challenge assay, vaccination of the HLA-A2 restricted phosphopeptide appeared to suppress the tumor growth and prolong the survival period of tumor-bearing mice. These results suggest that novel phosphopeptide is naturally presented as a HLA-A2-restricted CTL epitope and capable of being a potential candidate for the development of therapeutic vaccine against high TRAP1-expressing cancers.

Cobra venom proteome and glycome determined from individual snakes of Naja atra reveal medically important dynamic range and systematic geographic variation.

Recent progress in snake venomics has shed much light on the intra-species variation among the toxins from different geographical regions and has provided important information for better snakebite management. Most previous reports on snake venomics were based on venoms pooled from different snakes. In this study, we present the proteomic and glycomic profiles of venoms from individual Naja atra snakes. The results reveal wide dynamic range of three-finger toxins. Systematic classification based on cardiotoxin (CTX-) profiles of A2/A4 and A6, respectively, allowed the identification of two putative subspecies of Taiwan cobra from the eastern and western regions. We also identified four major N-glycan moieties on cobra snake venom metalloproteinase on the bi-antennary glycan core. ELISA showed that these glycoproteins (<3%) could elicit much higher antibody response in antiserum when compared to other high-abundance cobra venom toxins such as small molecular weight CTXs (~60%). By removing these high-molecular weight glycoproteins from the immunogen, we demonstrated better protection than that achieved with conventional crude venom immunization in mice challenged by crude venom. We conclude that both intra-species and inter-individual variations of proteomic and glycomic profiles of snake venomics should be considered to provide better antivenomic approach for snakebite management. BIOLOGICAL SIGNIFICANCE: Based on the proteomic and glycomic profiles of venoms obtained from individual snakes, we demonstrated a surprisingly wide dynamic range and geographical variation of three-finger toxins in cobra venomics. This provides a reasonable explanation for the variable neutralization effects of antivenom treatment on victims suffering from cobra snakebite and suggests a simple and economic method to produce potent antivenom with better efficacy. Since two major venomic profiles with distinct dynamic ranges were observed for Taiwan cobra venoms isolated from the eastern and western regions, the current venomic profile should be used as a quality control for future production of antivenom in clinical applications.

Mesenchymal stem cells promote neutrophil activation by inducing IL-17 production in CD4+ CD45RO+ T cells.

Mesenchymal stem cells (MSCs) are multi-potent with numerous mesenchymal-lineage differentiation potential and immunomodulatory capabilities. However, the immunoregulatory properties of MSCs are not clearly defined. The objective of the present study was to elucidate the role(s) of MSCs in IL-17 production and the subsequent effect(s) on neutrophil activation. We have demonstrated that human bone marrow-derived MSCs (BM-MSCs) instruct anti-CD3/anti-CD28 antibody-activated CD4(+) CD45RO(+) memory T cells, but not other CD4(+) subsets or CD8(+) T cells, to produce IL-17 after cell-cell contact. After the addition of IL-17, neutrophil phagocytic activity was increased. This is the first report on the ability of BM-MSCs to induce IL-17 production in memory CD4(+) T cells that, in turn, promotes enhanced phagocytic activity of neutrophils. These results suggest that MSCs regulate the functional activation of neutrophils via their role in modulating IL-17 from CD4(+) CD45RO(+) memory T cells.