Long-term control of diabetes by tofacitinib-based immunosuppressive regimen after allo islet transplantation in diabetic rhesus monkeys that rejected previously transplanted porcine islets.

Porcine islet xenotransplantation has been highlighted as an alternative to allo islet transplantation. Despite the remarkable progress that has been made in porcine-islet pre-clinical studies in nonhuman primates, immunological tolerance to porcine islets has not been achieved to date. Therefore, allo islet transplantation could be required after the failure of porcine islet xenotransplantation. Here, we report the long-term control of diabetes by allogeneic pancreatic islet transplantation in diabetic rhesus monkeys that rejected previously transplanted porcine islets. Four diabetic male rhesus monkeys received the porcine islets and then allo islets (5700-19 000 IEQ/kg) were re-transplanted for a short or long period after the first xeno islet rejection. The recipient monkeys were treated with an immunosuppressive regimen consisting of ATG, humira, and anakinra for induction, and sirolimus and tofacitinib for maintenance therapy. The graft survival days of allo islets in these monkeys were >440, 395, >273, and 127, respectively, similar to that in allo islet transplanted cynomolgus monkeys that received the same immunosuppressive regimen without xeno sensitization. Taken together, it is likely that prior islet xenotransplantation does not affect the survival of subsequent allo islets under clinically applicable immunosuppressants.

Distinct transcriptomes and autocrine cytokines underpin maturation and survival of antibody-secreting cells in systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is an autoimmune disease characterized by multiple autoantibody types, some of which are produced by long-lived plasma cells (LLPC). Active SLE generates increased circulating antibody-secreting cells (ASC). Here, we examine the phenotypic, molecular, structural, and functional features of ASC in SLE. Relative to post-vaccination ASC in healthy controls, circulating blood ASC from patients with active SLE are enriched with newly generated mature CD19-CD138+ ASC, similar to bone marrow LLPC. ASC from patients with SLE displayed morphological features of premature maturation and a transcriptome epigenetically initiated in SLE B cells. ASC from patients with SLE exhibited elevated protein levels of CXCR4, CXCR3 and CD138, along with molecular programs that promote survival. Furthermore, they demonstrate autocrine production of APRIL and IL-10, which contributed to their prolonged in vitro survival. Our work provides insight into the mechanisms of generation, expansion, maturation and survival of SLE ASC.

Intranasal immunization with the recombinant measles virus encoding the spike protein of SARS-CoV-2 confers protective immunity against COVID-19 in hamsters.

BACKGROUND: As the nasal mucosa is the initial site of infection for COVID-19, intranasal vaccines are more favorable than conventional vaccines. In recent clinical studies, intranasal immunization has been shown to generate higher neutralizing antibodies; however, there is a lack of evidence on sterilizing immunity in the upper airway. Previously, we developed a recombinant measles virus encoding the spike protein of SARS-CoV-2 (rMeV-S), eliciting humoral and cellular immune responses against SARS-CoV-2. OBJECTIVES: In this study, we aim to provide an experiment on nasal vaccines focusing on a measles virus platform as well as injection routes. STUDY DESIGN: Recombinant measles viruses expressing rMeV-S were prepared, and 5 × 105 PFUs of rMeV-S were administered to Syrian golden hamsters via intramuscular or intranasal injection. Subsequently, the hamsters were challenged with inoculations of 1 × 105 PFUs of SARS-CoV-2 and euthanized 4 days post-infection. Neutralizing antibodies and RBD-specific IgG in the serum and RBD-specific IgA in the bronchoalveolar lavage fluid (BALF) were measured, and SARS-CoV-2 clearance capacity was determined via quantitative reverse-transcription PCR (qRT-PCR) analysis and viral titer measurement in the upper respiratory tract and lungs. Immunohistochemistry and histopathological examinations of lung samples from experimental hamsters were conducted. RESULTS: The intranasal immunization of rMeV-S elicits protective immune responses and alleviates virus-induced pathophysiology, such as body weight reduction and lung weight increase in hamsters. Furthermore, lung immunohistochemistry demonstrated that intranasal rMeV-S immunization induces effective SARS-CoV-2 clearance that correlates with viral RNA content, as determined by qRT-PCR, in the lung and nasal wash samples, SARS-CoV-2 viral titers in lung, nasal wash, BALF samples, serum RBD-specific IgG concentration, and RBD-specific IgA concentration in the BALF. CONCLUSION: An intranasal vaccine based on the measles virus platform is a promising strategy owing to the typical route of infection of the virus, the ease of administration of the vaccine, and the strong immune response it elicits.

Apigenin's Therapeutic Potential Against Viral Infection.

Several antiviral drugs are clinically approved to treat influenza that is a highly prevalent acute respiratory disease. However, emerging drug-resistant virus strains undermine treatment efficacy, highlighting the exigency for novel antiviral drugs to counter these drug-resistant strains. Plants and their derivates have been historically utilized as medicinal remedies, and extensive studies have evidenced the antiviral potential of phytochemicals. Notably, apigenin is a predominant flavonoid with minimal toxicity and substantial therapeutic effects in various disease models. Despite its many anti-inflammatory, anti-oxidant, anti-cancer, anti-bacterial, and other beneficial bioactivities, existing reviews have yet to focus on apigenin's antiviral effects. Therefore, this review elucidates apigenin's therapeutic and antiviral properties in vitro and in vivo, discussing its mode of action and future prospects. Apigenin's remarkable inhibition by modulating multiple mechanisms against viruses has promising potential for novel plant-derived antiviral drugs and further clinical study developments.

Analysis of the immunostimulatory effects of cytokine-expressing internal ribosome entry site-based RNA adjuvants and their applications.

Developing new adjuvants that can effectively induce both humoral and cellular immune responses while broadening the immune response is of great value. In this study, we aimed to develop GM-CSF- or IL-18-expressing single-stranded RNA (ssRNA) adjuvants based on the encephalomyocarditis virus (EMCV) internal ribosome entry site (IRES) and tested their efficacy in combination with ovalbumin (OVA) or inactivated influenza vaccines. Notably, cytokine-expressing RNA adjuvants increased the expression of antigen-presenting cell activation markers. Specifically, GM-CSF-expressing RNA adjuvants increased CD4+T cell responses, while IL-18-expressing RNA adjuvants increased CD8+T cell responses in mice when combined with OVA. In addition, cytokine-expressing RNA adjuvants increased the frequency of polyclonal T cells in combination with the influenza vaccine and reduced the clinical illness scores and weight loss of mice after viral challenge. Collectively, our results suggest that cytokine-expressing RNA adjuvants can be applied to protein-based or inactivated vaccines to increase their efficacy.

Disruption of type I interferon pathway and reduced production of IFN-α by parabens in virus-infected dendritic cells.

BACKGROUND: Parabens are widely used preservatives commonly found in foods, cosmetics, and industrial products. Several studies have examined the effects of parabens on human health owing to widespread and continuous exposure to them in daily life. However, little is known about their immune-regulatory effects. OBJECTIVE: Here, we aimed to investigate whether methylparaben, ethylparaben, and propylparaben affect the function of dendritic cells (DCs) as the most potent antigen-presenting cells that play a critical role in the initiation of adaptive immune responses. METHODS: Bone-marrow derived DCs (BMDCs) were treated with three types of parabens (methylparaben, ethylparaben, and propylparaben) for 12 h. Subsequently, the transcriptomic profile was analyzed using RNA sequencing with further gene set enrichment analysis based on commonly regulated differentially expressed genes (DEGs). To test whether parabens suppress the production of type-I interferons (IFN-I) in BMDCs during viral infection, BMDCs or paraben-treated BMDCs were infected with Lymphocytic Choriomeningitis Virus (LCMV) at 10 multiplicity of infection (MOI) and measured the production of IFN-α1. RESULTS: Transcriptomic analyses revealed that all three types of parabens reduced the transcription levels of genes in virus infection-associated pathways, such as IFN-I responses in BMDCs. Furthermore, parabens considerably reduced IFN-α1 production in the virus-infected BMDCs. CONCLUSION: Our study is the first to show that parabens may modulate anti-viral immune responses by regulating DCs.

SLE Antibody-Secreting Cells Are Characterized by Enhanced Peripheral Maturation and Survival Programs.

Systemic Lupus Erythematosus (SLE) is an autoimmune disease characterized by multiple autoantibodies, some of which are present in high titers in a sustained, B cell-independent fashion consistent with their generation from long-lived plasma cells (LLPC). Active SLE displays high numbers of circulating antibody-secreting cells (ASC). Understanding the mechanisms of generation and survival of SLE ASC would contribute important insight into disease pathogenesis and novel targeted therapies. We studied the properties of SLE ASC through a systematic analysis of their phenotypic, molecular, structural, and functional features. Our results indicate that in active SLE, relative to healthy post-immunization responses, blood ASC contain a much larger fraction of newly generated mature CD19- CD138+ ASC similar to bone marrow (BM) LLPC. SLE ASC were characterized by morphological and structural features of premature maturation. Additionally, SLE ASC express high levels of CXCR4 and CD138, and molecular programs consistent with increased longevity based on pro-survival and attenuated pro-apoptotic pathways. Notably, SLE ASC demonstrate autocrine production of APRIL and IL-10 and experience prolonged in vitro survival. Combined, our findings indicate that SLE ASC are endowed with enhanced peripheral maturation, survival and BM homing potential suggesting that these features likely underlie BM expansion of autoreactive PC.

Analyzing immune responses to varied mRNA and protein vaccine sequences.

In response to the COVID-19 pandemic, different types of vaccines, such as inactive, live-attenuated, messenger RNA (mRNA), and protein subunit, have been developed against SARS-CoV-2. This has unintentionally created a unique scenario where heterologous prime-boost vaccination against a single virus has been administered to a large human population. Here, we aimed to analyze whether the immunization order of vaccine types influences the efficacy of heterologous prime-boost vaccination, especially mRNA and protein-based vaccines. We developed a new mRNA vaccine encoding the hemagglutinin (HA) glycoprotein of the influenza virus using the 3'-UTR and 5'-UTR of muscle cells (mRNA-HA) and tested its efficacy by heterologous immunization with an HA protein vaccine (protein-HA). The results demonstrated higher IgG2a levels and hemagglutination inhibition titers in the mRNA-HA priming/protein-HA boosting (R-P) regimen than those induced by reverse immunization (protein-HA priming/mRNA-HA boosting, P-R). After the viral challenge, the R-P group showed lower virus loads and less inflammation in the lungs than the P-R group did. Transcriptome analysis revealed that the heterologous prime-boost groups had differentially activated immune response pathways, according to the order of immunization. In summary, our results demonstrate that the sequence of vaccination is critical to direct desired immune responses. This study demonstrates the potential of a heterologous vaccination strategy using mRNA and protein vaccine platforms against viral infection.

Heterologous vaccination utilizing viral vector and protein platforms confers complete protection against SFTSV.

Severe fever with thrombocytopenia syndrome virus was first discovered in 2009 as the causative agent of severe fever with thrombocytopenia syndrome. Despite its potential threat to public health, no prophylactic vaccine is yet available. This study developed a heterologous prime-boost strategy comprising priming with recombinant replication-deficient human adenovirus type 5 (rAd5) expressing the surface glycoprotein, Gn, and boosting with Gn protein. This vaccination regimen induced balanced Th1/Th2 immune responses and resulted in potent humoral and T cell-mediated responses in mice. It elicited high neutralizing antibody titers in both mice and non-human primates. Transcriptome analysis revealed that rAd5 and Gn proteins induced adaptive and innate immune pathways, respectively. This study provides immunological and mechanistic insight into this heterologous regimen and paves the way for future strategies against emerging infectious diseases.

Influence of Microbiota on Vaccine Effectiveness: "Is the Microbiota the Key to Vaccine-induced Responses?"

Vaccines are one of the most powerful tools for preventing infectious diseases. To effectively fight pathogens, vaccines should induce potent and long-lasting immune responses that are specific to the pathogens. However, not all vaccines can induce effective immune responses, and the responses vary greatly among individuals and populations. Although several factors, such as age, host genetics, nutritional status, and region, affect the effectiveness of vaccines, increasing data have suggested that the gut microbiota is critically associated with vaccine-induced immune responses. In this review, I discuss how gut microbiota affects vaccine effectiveness based on the clinical and preclinical data, and summarize possible underlying mechanisms related to the adjuvant effects of microbiota. A better understanding of the link between vaccine-induced immune responses and the gut microbiota using high-throughput technology and sophisticated system vaccinology approaches could provide crucial insights for designing effective personalized preventive and therapeutic vaccination strategies.

Recombinant measles virus encoding the spike protein of SARS-CoV-2 efficiently induces Th1 responses and neutralizing antibodies that block SARS-CoV-2 variants.

Owing to the rapid spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and its variants, the development of effective and safe vaccines has become a priority. The measles virus (MeV) vaccine is an attractive vaccine platform as it has been administered to children for more than 40 years in over 100 countries. In this study, we developed a recombinant MeV expressing the full-length SARS-CoV-2 spike protein (rMeV-S) and tested its efficacy using mouse and hamster models. In hCD46Tg mice, two-dose rMeV-S vaccination induced higher Th1 secretion and humoral responses than one-dose vaccination. Interestingly, neutralizing antibodies induced by one-dose and two-dose rMeV-S immunization effectively blocked the entry of the α, ß, γ, and δ variants of SARS-CoV-2. Furthermore, two-dose rMeV-S immunization provided complete protection against SARS-CoV-2 in the hamster model. These results suggest the potential of rMeV-S as a vaccine candidate for targeting SARS-CoV-2 and its variants.

Galectin-4 increases the ability of M2 macrophages to enhance antiviral CD4+ T-cell responses.

Galectin-4 (Gal-4) is a ß-galactoside-binding protein belonging to the galectin family. Although Gal-4 is known to be involved in several physiologic processes of the gastrointestinal tract, its immunomodulatory roles remain unclear. In this study, we investigated whether Gal-4 influences the function of M1 and M2 macrophages. Gal-4 treatment drove more robust changes in the gene expression of M2 macrophages compared to M1 macrophages. Antiviral immune response-related genes were significantly upregulated in Gal-4-treated M2 macrophages. Gal-4 significantly enhanced the immunostimulatory activity of M2 macrophages upon Toll-like receptor 7 stimulation or infection with lymphocytic choriomeningitis virus (LCMV). Moreover, the antibody production against LCMV infection and the antiviral CD4+ T-cell responses, but not the antiviral CD8+ T-cell responses, were greatly increased by Gal-4-treated M2 macrophages in vivo. The present results indicate that Gal-4 enhances the ability of M2 macrophages to promote antiviral CD4+ T-cell responses. Thus, Gal-4 could be used to boost antiviral immune responses.

Inhibition of KIF20A suppresses the replication of influenza A virus by inhibiting viral entry.

The influenza A virus (IAV) has caused several pandemics, and therefore there are many ongoing efforts to identify novel antiviral therapeutic strategies including vaccines and antiviral drugs. However, influenza viruses continuously undergo antigenic drift and shift, resulting in the emergence of mutated viruses. In turn, this decreases the efficiency of existing vaccines and antiviral drugs to control IAV infection. Therefore, this study sought to identify alternative therapeutic strategies targeting host cell factors rather than viruses to avoid infection by mutated viruses. Particularly, we investigated the role of KIF20A that is one of kinesin superfamily proteins in the replication of IAV. The KIF20A increased viral protein levels in IAV-infected cells by regulating the initial entry stage during viral infection. Furthermore, the KIF20A inhibitor significantly suppressed viral replication, which protected mice from morbidity and mortality. Therefore, our findings demonstrated that KIF20A is highly involved in the viral replication process and viral propagation both in vitro and in vivo, and could thus be used as a target for the development of novel antiviral drugs.

Adjuvant effect of IRES-based single-stranded RNA on melanoma immunotherapy.

BACKGROUND: Adjuvant therapies such as radiation therapy, chemotherapy, and immunotherapy are usually given after cancer surgery to improve the survival of cancer patients. However, despite advances in several adjuvant therapies, they are still limited in the prevention of recurrences. METHODS: We evaluated the immunological effects of RNA-based adjuvants in a murine melanoma model. Single-stranded RNA (ssRNA) were constructed based on the cricket paralysis virus (CrPV) internal ribosome entry site (IRES). Populations of immune cells in bone marrow cells and lymph node cells following immunization with CrPVIRES-ssRNA were determined using flow cytometry. Activated cytokine levels were measured using ELISA and ELISpot. The tumor protection efficacy of CrPVIRES-ssRNA was analyzed based on any reduction in tumor size or weight, and overall survival. RESULTS: CrPVIRES-ssRNA treatment stimulated antigen-presenting cells in the drain lymph nodes associated with activated antigen-specific dendritic cells. Next, we evaluated the expression of CD40, CD86, and XCR1, showing that immunization with CrPVIRES-ssRNA enhanced antigen presentation by CD8a+ conventional dendritic cell 1 (cDC1), as well as activated antigen-specific CD8 T cells. In addition, CrPVIRES-ssRNA treatment markedly increased the frequency of antigen-specific CD8 T cells and interferon-gamma (IFN-γ) producing cells, which promoted immune responses and reduced tumor burden in melanoma-bearing mice. CONCLUSIONS: This study provides evidence that the CrPVIRES-ssRNA adjuvant has potential for use in therapeutic cancer vaccines. Moreover, CrPVIRES-ssRNA possesses protective effects on various cancer cell models.

Anti-influenza A virus activity by Agrimonia pilosa and Galla rhois extract mixture.

Influenza A virus (IAV) continues to threaten human health. To date, two classes of antiviral drugs have been approved to treat IAV infection, but the continuous emergence of the drug-resistant IAV mutant reinforces the need to develop new antiviral drugs. In this study, we aimed to investigate the anti-IAV activity of an aqueous mixture of Agrimonia pilosa and Galla rhois extracts (APRG64). We demonstrated that APRG64 significantly reduced the IAV-induced cytopathic effect, the transcription/expression of viral proteins, and the production of infectious viral particles. Among nine major components of APRG64, apigenin was identified as the main ingredient responsible for the anti-IAV activity. Interestingly, APRG64 and apigenin inhibited the cell attachment and entry of virus and polymerase activity. Importantly, intranasal administration of APRG64 or apigenin strongly reduced viral loads in the lungs of IAV-infected mice. Furthermore, oral administration of APRG64 significantly reduced the level of viral RNAs and the expression level of pro-inflammatory cytokines in the lungs, which protected mice from IAV-induced mortality. In conclusion, APRG64 could be an attractive antiviral drug to treat IAV infection.

Single-stranded RNA adjuvant enhances the efficacy of 10-valent human papilloma virus-like particle vaccine.

Following the development of various types of vaccines, the use of adjuvants to boost vaccine efficacy has become a focus of research. Aluminum hydroxide (alum), the most commonly used adjuvant, induces a certain immune response and ensures safety in human trials. However, alum mainly induces only a Th2 response; its Th1 response is weak. Thus, we previously developed a single-stranded ribose nucleic acid (ssRNA) adjuvant that induces a Th1 response through toll-like receptors. Here, we explored whether 10-valent human papilloma virus (HPV)-like particle (VLP) vaccine formulated with ssRNA adjuvant and alum helped to enhance immune response and maintained memory response. The mice were immunized intramuscularly twice at 2 week intervals and were inoculated 4 days after the second boost (after about 1 year). The antibody response and T cell activation were measured by Elispot, ELISA using harvested serum and splenocytes. The 10-valent HPV VLP vaccine formulated with ssRNA adjuvant and alum increased the antigen-specific immune response more than alum used alone. It increased each type-specific IgG1/IgG2a titer, and antigen-specific IFN-γ cells. Furthermore, the ssRNA adjuvant with alum induced memory response. In memory response, each type-specific IgG1/IgG2c, IFN-γ, and IL-6 cytokine, and neutralizing antibodies were increased by the ssRNA adjuvant with alum. Overall, the ssRNA adjuvant with alum induced memory responses and balanced Th1/Th2 responses. The ssRNA adjuvant and alum may help to enhance prophylactic vaccine efficacy.

Peptides Derived From S and N Proteins of Severe Acute Respiratory Syndrome Coronavirus 2 Induce T Cell Responses: A Proof of Concept for T Cell Vaccines.

The emergence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants that escape vaccine-induced neutralizing antibodies has indicated the importance of T cell responses against this virus. In this study, we highlight the SARS-CoV-2 epitopes that induce potent T cell responses and discuss whether T cell responses alone are adequate to confer protection against SARS-CoV-2 and describe the administration of 20 peptides with an RNA adjuvant in mice. The peptides have been synthesized based on SARS-CoV-2 spike and nucleocapsid protein sequences. Our study demonstrates that immunization with these peptides significantly increases the proportion of effector memory T cell population and interferon-γ (IFN-γ)-, interleukin-4 (IL-4)-, tumor necrosis factor-α (TNF-α)-, and granzyme B-producing T cells. Of these 20 peptides, four induce the generation of IFN-γ-producing T cells, elicit CD8+ T cell (CTL) responses in a dose-dependent manner, and induce cytotoxic T lymphocytes that eliminate peptide-pulsed target cells in vivo. Although it is not statistically significant, these peptide vaccines reduce viral titers in infected hamsters and alleviate pulmonary pathology in SARS-CoV-2-infected human ACE2 transgenic mice. These findings may aid the design of effective SARS-CoV-2 peptide vaccines, while providing insights into the role of T cells in SARS-CoV-2 infection.

Effective inactivated influenza vaccine for the elderly using a single-stranded RNA-based adjuvant.

There is an unmet need for new influenza vaccine strategies that compensate for impaired vaccine responses in elderly individuals. Here, we evaluated the effectiveness of a single-stranded RNA (ssRNA) as an adjuvant to enhance the efficacy of inactivated influenza vaccine (IIV) in mouse models. Immunization with the ssRNA along with IIV reduced viral titers as well as pathological and inflammatory scores in the lungs after influenza challenge in aged mice. ssRNA induced balanced Th1/Th2 responses with an increase in IgA titers. Moreover, the ssRNA adjuvant markedly increased the frequency of influenza HA-specific T cells and IFN-γ production along with the expression of genes related to innate and adaptive immune systems that could overcome immunosenescence in aged mice. Our findings indicate that ssRNA is an efficient vaccine adjuvant that boosts cellular and humoral immunity in aged mice, demonstrating its potential as a novel adjuvant for currently available influenza virus vaccines for elderly individuals.

Long-term control of diabetes in a nonhuman primate by two separate transplantations of porcine adult islets under immunosuppression.

Porcine islet transplantation is an alternative to allo-islet transplantation. Retransplantation of islets is a routine clinical practice in islet allotransplantation in immunosuppressed recipients and will most likely be required in islet xenotransplantation in immunosuppressed recipients. We examined whether a second infusion of porcine islets could restore normoglycemia and further evaluated the efficacy of a clinically available immunosuppression regimen including anti-thymocyte globulin for induction; belimumab, sirolimus, and tofacitinib for maintenance and adalimumab, anakinra, IVIg, and tocilizumab for inflammation control in a pig to nonhuman primate transplantation setting. Of note, all nonhuman primates were normoglycemic after the retransplantation of porcine islets without induction therapy. Graft survival was >100 days for all 3 recipients, and 1 of the 3 monkeys showed insulin independence for >237 days. Serious lymphodepletion was not observed, and rhesus cytomegalovirus reactivation was controlled without any serious adverse effects throughout the observation period in all recipients. These results support the clinical applicability of additional infusions of porcine islets. The maintenance immunosuppression regimen we used could protect the reinfused islets from acute rejection.