Tuksin Jearanaiwitayakul

The pathogenesis of dengue is complex and requires further study. Infection with the dengue virus can lead to severe dengue, in which patients experience severe plasma leakage and excessive bleeding. The dengue virus non-structural protein 1 (DENV NS1) is among several factors that affect the outcome. The secreted form of this protein induces severe cases through direct and indirect immunopathogenesis mechanisms. Previously, we discovered that the stimulation of blood monocytes by NS1 upregulates transcripts involved in inflammation, and suppresses transcripts involved in antiviral processes. In this study, we examined the specific responses unique to high-dose NS1 exposure of cultured human monocytes. We found that genes involved in hemostasis were altered following NS1 exposure and were associated with platelet activation, fibrinolysis, and hemostatic regulation. Some of these expression changes were also observed in patients with dengue fever (DF) or dengue hemorrhagic fever (DHF). The altered expression of these genes suggests that fibrinolysis is more likely balanced during DF, whereas coagulation may be impaired in DHF.

Bacillus Calmette-Guérin cell wall skeleton (BCG-CWS) has been shown to enhance vaccine effectiveness and antitumor immunity. In our previous study, we reported that co-administration of BCG-CWS with the encapsidated dengue antigens, UV-inactivated DENV2 and DENV2 NS1, synergistically induced DENV-specific adaptive immune responses in mice. As dendritic cells (DCs) are key immune players that mediate innate and adaptive immunity, we, here, asked how well the response of DCs to this adjuvant aligns with the immune responses elicited in vivo . The responses of primary monocyte-derived DCs to BCG-CWS-adjuvanted encapsidated dengue immunogens compared with the unadjuvanted vaccine were investigated. DCs stimulated by BCG-CWS and the dengue nanoparticle vaccine exhibited a superior response. This response correlated well with the stronger immune response observed in mice. This was evidenced by the marked elevation in expression levels of DC activation markers, such as CD80, CD83, CD86, and HLA-DR, and various innate immune cytokines. Additionally, this adjuvant markedly elevated the expression levels of miRNAs related to DC function, such as miR-146a, miR-147, miR-223, and miR-155. These immune components could suppress DENV2 multiplication in bystander skin cells. BCG-CWS exerted an adjuvant effect on DC responses by enhancing antigen-processing activity and activating several innate immune cytokines and immune-related miRNAs.

SARS-CoV-2 is classified as a containment level 3 (CL3) pathogen, limiting research access and antiviral testing. To address this, we developed a non-infectious viral surrogate system using reverse genetics to generate sub-genomic replicons. These replicons contained the nsp1 mutations K164A and H165A and had the spike, membrane, ORF6, and ORF7a coding sequences replaced with various reporter and selectable marker genes. Replicons based on the ancestral Wuhan Hu-1 strain and the Delta variant of concern were replication-competent in multiple cell lines, as assessed by Renilla luciferase activity, fluorescence, immunofluorescence staining, and single-molecule fluorescent in situ hybridization. Antiviral assays using transient replicon expression showed that remdesivir effectively inhibited both replicon and viral replication. Ritonavir and cobicistat inhibited Delta variant replicons similarly to wild-type virus but did not inhibit Wuhan Hu-1 replicon replication. To further investigate the impact of nsp1 mutations, we generated a recombinant SARS-CoV-2 virus carrying the K164A and H165A mutations. The virus exhibited attenuated replication across a range of mammalian cell lines, was restricted by the type I interferon response, and showed reduced cytopathic effects. These findings highlight the utility of sub-genomic replicons as reliable CL2-compatible surrogates for studying SARS-CoV-2 replication and drug activity mechanisms.

Nonstructural protein 1 (NS1) of dengue virus (DENV) can influence dengue severity. In this study, we used RNA sequencing analysis to assess the blood monocyte response to different concentrations of NS1. We showed that NS1 at the level found in severe dengue may be involved in severe dengue development through 2 potential mechanisms: induction of excessive inflammation and suppression of antiviral responses. At high levels, NS1 significantly up-regulated S100A8 and S100A9, ACOD1, and TREM1, which might help amplify the inflammatory loops. In terms of antiviral suppression, we found that high NS1 concentration significantly suppressed interferon signaling and major histocompatibililty complex class II transcripts. This potentially delayed the clearance of both DENV and NS1 protein. Our study highlighted the possible role of NS1-activated monocytes in dengue severity.

The intranasal vaccine against coronavirus disease 2019 (COVID‐19) has gained more attention because of its ability to induce both mucosal and systemic immune responses. We have recently developed a c‐GAMP‐adjuvanted bivalent receptor‐binding domain (RBD) vaccine, derived from the ancestral strain and the Omicron variant. We demonstrated here that intranasal administration of this vaccine candidate triggers not only the respiratory but also the systemic immune response against SARS‐CoV‐2. The immunized mice elicited the broadly neutralizing antibodies against the ancestral strain (Wuhan‐1) and variants of concern (Delta, Omicron BA.1, and Omicron BA.5). This route of vaccination also induced potent systemic T cell responses with strong cytotoxic activity against both the Wuhan‐1 and Omicron BA.1 strains. Additionally, intranasally immunized mice significantly suppressed SARS‐CoV‐2 RNA levels in circulation and spleens, indicating effective containment of the virus beyond the respiratory tract. These findings suggest that the intranasal bivalent RBD vaccine holds promise for combating SARS‐CoV‐2 infections.

The uneven immunogenicity of the attenuated tetravalent dengue vaccine has made it difficult to achieve balanced protection against all four serotypes of the dengue virus (DENV). To overcome this problem, non-replicative vaccines have come into focus, as their immunogenicity is adjustable. This approach is excellent for multivalent vaccines but commonly faces the issue of low immunogenicity. In this present study, we developed a non-replicating dengue vaccine composed of UV-inactivated dengue virus-2 (UV-DENV-2) and DENV-2 NS_1-279 protein encapsidated within nanoparticles. This vaccine candidate was administered in the presence of BCG cell wall cytoskeleton (BCG-CWS) as an adjuvant. We revealed, here, that encapsidated immunogens with BCG-CWS exerted potent activities on both B and T cells and elicited Th-1/Th-2 responses in mice. This was evidenced by BCG-CWS significantly augmenting antibody-mediated complement-fixing activity, strongly stimulating the antigen-specific polyfunctional T cell responses, and activating mixed Th-1/Th-2 responses specific to DENV-2- and NS_1-279 antigens. In conclusion, BCG-CWS potently adjuvanted the inactivated DENV-2 and DENV subunit immunogens. The mechanism of adjuvanticity remains unclear. This study revealed the potential use of BCG-CWS in vaccine development.

The uneven immunogenicity of the attenuated tetravalent dengue vaccine has created the diffi-culty to achieve a balanced protection against all four serotypes of dengue virus (DENV). To overcome this problem, non-replicative-based vaccines have come into focus as their immuno-genicity is adjustable. This approach is excellent for multivalent vaccines but commonly faces the issue of low immunogenicity. In this present study, we developed a non-replicating dengue vaccine composing of UV-inactivated dengue-2 virus (UV-DENV2) and DENV-2 NS1-279 protein encapsidated within nanoparticles. This vaccine candidate was administered in the presence of BCG cell wall cytoskeleton (BCG-CWS) as an adjuvant. We revealed, here, that encapsidated immunogens with BCG-CWS exerted potent activities on both B and T cells and elicited Th-1/Th-2 responses in mice. This was evidenced by strong neutralizing antibody activity with an unde-tectable antibody enhancing activity in a group of mice receiving encapsidated immunogens with BCG-CWS. Importantly, BCG-CWS significantly augmented antibody-mediated comple-ment-fixing activity, strongly stimulated the antigen-specific polyfunctional T cell responses as well as activated mixed Th-1/Th-2 responses specific to DENV2- and NS1-279 antigens. In conclu-sion, BCG-CWS potently adjuvanted the inactivated DENV-2 and subunit DENV immunogens. The mechanism of adjuvanticity remains unclear. This study revealed the potential use of BCG-CWS in vaccine development.

Dengue is a disease that poses a significant global public health concern. Although a tetravalent live-attenuated dengue vaccine has been licensed, its efficacy is still debated due to evidence of vaccine breakthrough infection. To avoid this issue, dengue vaccines should stimulate a high degree of serotype-specific response. Thus, envelope domain III (EDIII), which contains serotype-specific neutralizing epitopes, is an attractive target for dengue vaccine development. In this study, we investigated how EDIII encapsidated in N, N, N-trimethyl chitosan chloride nanoparticles (TMC NPs) stimulates a serotype-specific response and whether this response exerts a potential in vitro breakthrough infection. The immune response to DENV-2 elicited by EDIII TMC NP-immunized mice was monitored. We demonstrated that immunization with EDIII TMC NPs resulted in a high level of anti-EDIII antibody production. These antibodies included IgG, IgG1, and IgG2a subtypes. Importantly, antibodies from the immunized mice exerted efficient neutralizing activity with undetectable antibody dependent enhancement (ADE) activity. We also found that EDIII TMC NPs activated functional EDIII-specific CD4⁺ and CD8⁺ T cell responses. In conclusion, EDIII TMC NPs stimulated humoral immunity with a strong neutralizing antibody response, as well as a cellular immune response against DENV-2.

The respiratory organ serves as a primary target site for SARS-CoV-2. Thus, the vaccine-stimulating immune response of the respiratory tract is significant in controlling SARS-CoV-2 transmission and disease development. In this study, mucoadhesive nanoparticles were used to deliver SARS-CoV-2 spike proteins (S-NPs) into the nasal tracts of mice. The responses in the respiratory organ and the systemic responses were monitored. The administration of S-NPs along with cGAMP conferred a robust stimulation of antibody responses in the respiratory tract, as demonstrated by an increase of IgA and IgG antibodies toward the spike proteins in bronchoalveolar lavages (BALs) and the lungs. Interestingly, the elicited antibodies were able to neutralize both the wild-type and Delta variant strains of SARS-CoV-2. Significantly, the intranasal immunization also stimulated systemic responses. This is evidenced by the increased production of circulating IgG and IgA, which were able to neutralize and bind specifically to the SARS-CoV-2 virion and spike protein. Additionally, this intranasal administration potently activated a splenic T cell response and the production of Th-1 cytokines, suggesting that this vaccine may well activate a cellular response in the respiratory tract. The results demonstrate that STING agonist strongly acts as an adjuvant to the immunogenicity of S-NPs. This platform may be an ideal vaccine against SARS-CoV-2.