COVID-19 Subunit Vaccine with a Combination of TLR1/2 and TLR3 Agonists Induces Robust and Protective Immunity.

The development of COVID-19 vaccines is critical in controlling global health issues under the COVID-19 pandemic. The subunit vaccines are the safest and most widely used vaccine platform and highly effective against a multitude of infectious diseases. An adjuvant is essential for subunit vaccines to enhance the magnitude and durability of immune responses. In this study, we determined whether a combination of toll-like receptor (TLR)1/2 and TLR3 agonists (L-pampo) can be a potent adjuvant for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) subunit vaccine. We measured a neutralizing antibody (nAb) and an angiotensin-converting enzyme 2 (ACE2) receptor-blocking antibody against SARS-CoV-2 receptor-binding domain (RBD). We also detected interferon-gamma (IFN-γ) production by using ELISPOT and ELISA assays. By employing a ferret model, we detected nAbs and IFN-γ producing cells and measured viral load in nasal wash after the challenge of SARS-CoV-2. We found that SARS-CoV-2 antigens with L-pampo stimulated robust humoral and cellular immune responses. The efficacy of L-pampo was higher than the other adjuvants. Furthermore, in the ferret model, SARS-CoV-2 antigens with L-pampo elicited nAb response and antigen-specific cellular immune response against SARS-CoV-2, resulting in substantially decreased viral load in their nasal wash. Our study suggests that SARS-CoV-2 antigens formulated with TLR agonists, L-pampo, can be a potent subunit vaccine to promote sufficient protective immunity against SARS-CoV-2.

Analysis of immune responses against nucleocapsid protein of the Hantaan virus elicited by virus infection or DNA vaccination.

Even though neutralizing antibodies against the Hantaan virus (HTNV) has been proven to be critical against viral infections, the cellular immune responses to HTNV are also assumed to be important for viral clearance. In this report, we have examined the cellular and humoral immune responses against the HTNV nucleocapsid protein (NP) elicited by virus infection or DNA vaccination. To examine the cellular immune response against HTNV NP, we used H-2K(b) restricted T-cell epitopes of NP. The NP-specific CD8(+) T cell response was analyzed using a (51)Cr-release assay, intracellular cytokine staining assay, enzyme-linked immunospot assay and tetramer binding assay in C57BL/6 mice infected with HTNV. Using these methods, we found that HTNV infection elicited a strong NP-specific CD8(+) T cell response at eight days after infection. We also found that several different methods to check the NP-specific CD8(+) T cell response showed a very high correlation among analysis. In the case of DNA vaccination by plasmid encoding nucleocapsid gene, the NP-specific antibody response was elicited 2 approximately 4 weeks after immunization and maximized at 6 approximately 8 weeks. NP-specific CD8(+) T cell response reached its peak 3 weeks after immunization. In a challenge test with the recombinant vaccinia virus expressing NP (rVV-HTNV-N), the rVV-HTNV-N titers in DNA vaccinated mice were decreased about 100-fold compared to the negative control mice.

TH2 and TH1 lung inflammation induced by airway allergen sensitization with low and high doses of double-stranded RNA.

BACKGROUND: Although respiratory viral infections in early childhood can enhance the development of airway allergen sensitization, the exact mechanisms of the effects of viral infections on the adaptive immune response to inhaled allergens are controversial. OBJECTIVE: We sought to evaluate the effects of double-stranded RNA (dsRNA) on airway sensitization to inhaled allergens. METHODS: Novel mouse models were created through simultaneous airway sensitization to an allergen and low or high doses of dsRNA. The mouse models were applied to Toll-like receptor 3-, IL-13-, IL-4-, signal transducer and activator of transcription (STAT) 6-, IFN-gamma-, and T-box expressed in T cells (T-bet)-deficient mice to evaluate underlying pathophysiologic mechanisms in the development of allergic lung inflammation. RESULTS: We found that airway allergen sensitization with dsRNA induced lung inflammation that was not present in Toll-like receptor 3-deficient mice. Moreover, lung inflammation enhanced by low-dose dsRNA was impaired in IL-13-deficient mice, whereas lung inflammation by high-dose dsRNA was impaired in IFN-gamma-deficient mice. The models also demonstrated that low-dose dsRNA enhanced IL-4 expression during allergen sensitization and that inflammation enhanced by low-dose dsRNA was not present in IL-4- or STAT6-deficient mice. In contrast, the present study showed that high-dose dsRNA enhanced IFN-gamma expression during allergen sensitization and that the development of lung inflammation enhanced by high-dose dsRNA was impaired in T-bet-deficient mice. CONCLUSION: These findings suggest that airway allergen exposure during respiratory viral infections might induce asthma induced by both T(H)1 and T(H)2 immune responses to inhaled allergens. CLINICAL IMPLICATIONS: Targeting both T(H)1 and T(H)2 lung inflammation might be important in the treatment of virus-associated asthma.

Recombinant basic fibroblast growth factor inhibits the airway hyperresponsiveness, mucus production, and lung inflammation induced by an allergen challenge.

BACKGROUND: IL-13 is believed to be a central mediator of asthma, and TGF-beta1 is a key downstream mediator in the development of IL-13-mediated asthma phenotypes. OBJECTIVE: To evaluate the biological roles of basic fibroblast growth factor (FGF2) in phenotype expression in transgenic (TG) mice overexpressing lung-specific TGF-beta1, and the therapeutic effects of recombinant FGF2 in the development of asthma phenotypes. METHODS: To evaluate the roles of FGF2 in airway hyperresponsiveness (AHR) expression induced by high levels of TGF-beta1, TGF-beta1 TG (+) mice were bred with FGF2-deficient mice. To evaluate the therapeutic effects of recombinant FGF2 (rFGF2) in the development of asthma, mice were given 10 mug of rFGF2 subcutaneously once a day, 1 hour before the allergen challenge in an asthma mouse model. AHR was evaluated using noninvasive whole-body plethysmography, mucus production by diastase-resistant periodic acid Schiff (DPAS) staining, and lung inflammation using bronchoalveolar lavage (BAL) cellularity and lung histology. RESULTS: AHR decreased in TGF-beta1 TG (+) mice and was accompanied by the upregulation of FGF2 mRNA expression in lung tissues, when compared with littermate wild-type control mice. Interestingly, AHR was enhanced markedly in TGF-beta1 (+) mice with homozygous FGF2 gene disruption. In an asthma mouse model, AHR, mucus production, and lung inflammation were inhibited markedly by rFGF2 treatment. This inhibition was accompanied by downregulation of the allergen-induced proliferation of T cells from regional lymph nodes. CONCLUSION: FGF2 seems to be a key inhibitor in the development of AHR, and rFGF2 treatment constrains the development of asthma phenotypes.

Airway exposure levels of lipopolysaccharide determine type 1 versus type 2 experimental asthma.

Allergic asthma is characterized by airway inflammation initiated by adaptive immune responses to aeroallergens. Recent data suggest that severe asthma may be a different form of asthma rather than an increase in asthma symptoms and that innate immune responses to LPS can modulate adaptive immune responses to allergens. In this study, we evaluated the hypothesis that airway exposure to different doses of LPS induces different form of asthma. Our study showed that neutrophilic inflammation and IFN-gamma expression were higher in induced sputum from severe asthma patients than from mild to moderate asthmatics. Animal experiments indicated that allergen sensitization with low-dose LPS (0.1 microg) induced type 2 asthma phenotypes, i.e., airway hyperresponsiveness, eosinophilic inflammation, and allergen-specific IgE up-regulation. In contrast, allergen sensitization with high-dose LPS (10 microg) induced asthma phenotypes, i.e., airway hyperresponsiveness and noneosinophilic inflammation that were not developed in IFN-gamma-deficient mice, but unaffected in the absence of IL-4. During the allergen sensitization period, TNF-alpha expression was found to be enhanced by both low- and high-dose LPS, whereas IL-12 expression was only enhanced by high-dose LPS. Interestingly, the asthma phenotypes induced by low-dose LPS, but not by high-dose LPS, were completely inhibited in TNF-alpha receptor-deficient mice, whereas the asthma phenotypes induced by high-dose LPS were abolished in the homozygous null mutation of the STAT4 gene. These findings suggest that airway exposure levels of LPS induces different forms of asthma that are type 1 and type 2 asthma phenotypes by high and low LPS levels, respectively.

Focal trichloroacetic acid peel method for benign pigmented lesions in dark-skinned patients.

BACKGROUND: Benign pigmented lesions, including seborrheic keratosis, solar lentigines, melasma, and freckles, are common disorders, and various treatment modalities have been tried. We suggest a technique consisting of focal trichloroacetic acid (TCA) peel applied by pressing firmly onto the focal lesions. OBJECTIVE: To evaluate the clinical effects of focal TCA peel on pigmented lesions in dark-skinned patients. METHODS: An analysis was conducted of 106 patients with benign pigmented lesions who were treated using focal TCA peel. Seborrheic keratosis was treated with 65% focal TCA peel, solar lentigines, and freckles with 50% to 65% focal TCA peel, and melasmas with 10% to 50% focal TCA peel. Patients had Fitzpatrick skin types IV-V. RESULTS: Patient treatment data indicated that 19 of 23 (83%) patients with seborrheic keratosis, 42 of 49 (86%) patients with solar lentigines, 8 of 14 (58%) patients with freckles, and 11 of 20 (55%) patients with melasma experienced a good clinical response. Good satisfaction rates in the seborrheic keratosis, solar lentigines, freckles, and melasma groups were recorded. No significant complications were observed. CONCLUSION: The focal TCA peel method presented in this study is a safe and effective modality for the treatment of benign pigmented lesions with no significant complications.