mRNA-HPV vaccine encoding E6 and E7 improves therapeutic potential for HPV-mediated cancers via subcutaneous immunization.

The E6 and E7 proteins of specific subtypes of human papillomavirus (HPV), including HPV 16 and 18, are highly associated with cervical cancer as they modulate cell cycle regulation. The aim of this study was to investigate the potential antitumor effects of a messenger RNA-HPV therapeutic vaccine (mHTV) containing nononcogenic E6 and E7 proteins. To achieve this, C57BL/6j mice were injected with the vaccine via both intramuscular and subcutaneous routes, and the resulting effects were evaluated. mHTV immunization markedly induced robust T cell-mediated immune responses and significantly suppressed tumor growth in both subcutaneous and orthotopic tumor-implanted mouse model, with a significant infiltration of immune cells into tumor tissues. Tumor retransplantation at day 62 postprimary vaccination completely halted progression in all mHTV-treated mice. Furthermore, tumor expansion was significantly reduced upon TC-1 transplantation 160 days after the last immunization. Immunization of rhesus monkeys with mHTV elicited promising immune responses. The immunogenicity of mHTV in nonhuman primates provides strong evidence for clinical application against HPV-related cancers in humans. All data suggest that mHTV can be used as both a therapeutic and prophylactic vaccine.

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.

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.

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.

Cricket paralysis virus internal ribosome entry site-derived RNA promotes conventional vaccine efficacy by enhancing a balanced Th1/Th2 response.

An ideal adjuvant should increase vaccine efficacy through balanced Th1/Th2 responses and be safe to use. Recombinant protein-based vaccines are usually formulated with aluminum (alum)-based adjuvants to ensure an adequate immune response. However, use of alum triggers a Th2-biased immune induction, and hence is not optimal. Although the adjuvanticity of RNA has been reported, a systematic and overall investigation on its efficacy is lacking. We found that single strand RNA (termed RNA adjuvant) derived from cricket paralysis virus intergenic region internal ribosome entry site induced the expression of various adjuvant-function-related genes, such as type 1 and 2 interferon (IFN) and toll-like receptor (TLR), T cell activation, and leukocyte chemotaxis in human peripheral blood mononuclear cells; furthermore, its innate and IFN transcriptome profile patterns were similar to those of a live-attenuated yellow fever vaccine. This suggests that protein-based vaccines formulated using RNA adjuvant function as live-attenuated vaccines. Application of the RNA adjuvant in mouse enhanced the efficacy of Middle East respiratory syndrome spike protein, a protein-subunit vaccine and human papillomavirus L1 protein, a virus-like particle vaccine, by activating innate immune response through TLR7 and enhancing pAPC chemotaxis, leading to a balanced Th1/Th2 responses. Moreover, the combination of alum and the RNA adjuvant synergistically induced humoral and cellular immune responses and endowed long-term immunity. Therefore, RNA adjuvants have broad applicability and can be used with all conventional vaccines to improve vaccine efficacy qualitatively and quantitively.

Effect of apoptosis-associated speck-like protein containing a caspase recruitment domain on vaccine efficacy: Overcoming the effects of its deficiency with aluminum hydroxide adjuvant.

Host factors such as nutritional status and immune cell state are important for vaccine efficacy. Inflammasome activation may be important for triggering vaccine-induced humoral and cell-mediated immune responses. Formulations with alum as a typical adjuvant to overcome the effects of host factors have recently been shown to induce inflammasome activation, which augments vaccine efficacy. Apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC) is one of the main components of inflammasomes, but it is not clear whether ASC affects the vaccine-induced immune response. Herein, we used two types of vaccines: inactivated influenza vaccine not formulated with alum, and HPV vaccine formulated with alum. We gave the vaccines to ASC knockout (ASC-/- ) mice to investigate the role of ASC in vaccine efficacy. Influenza vaccine-immunized ASC-/- mice did not show antibody titers in week 2 after the first vaccination. After boosting, the antibody titer in ASC-/- mice was about half that in wild type (WT) mice. Furthermore, a cytotoxic T-lymphocyte response against influenza vaccine was not induced in ASC-/- mice. Therefore, vaccinated ASC-/- mice did not show effective protection against viral challenge. ASC-/- mice immunized with alum-formulated HPV vaccine showed similar antibody titers and T-cell proliferation compared with immunized WT mice. However, the HPV vaccine without alum induced up to threefold lower titers of HPV-specific antibody titers in ASC-/- mice compared with those in WT mice. These findings suggest that alum in vaccine can overcome the ASC-deficient condition.

Macrophage-derived insulin-like growth factor-1 affects influenza vaccine efficacy through the regulation of immune cell homeostasis.

The level of antibody production induced by a vaccine involves a variety of host factors. One of these, insulin-like growth factor-1 (IGF-1), plays an important role in lymphocyte maturation and antibody expression. Here, we investigated the role of macrophage-derived IGF-1 in the induction of influenza vaccine-specific antibodies using macrophage-derived IGF-1 gene knockout (MIKO) mice. The titers of vaccine-specific total immunoglobulin G (IgG) and IgG1 after immunization were about two- to fourfold lower in MIKO mice than in WT mice. Moreover, MIKO mice showed a relatively weak booster effect of repeated immunization. In contrast, antigen-nonspecific total IgG was about threefold higher in MIKO mice than in WT mice. After viral challenge, the viral titer and the pathological damage in lungs of MIKO mice were higher than those in WT mice despite vaccination. Interestingly, the proportions of proinflammatory immune cells including M1 macrophages, Th1 and Th17 cells was higher in unvaccinated MIKO mice than in unvaccinated WT mice. This suggests that nonspecific activation of immune cells may paradoxically impair the response to the vaccine. In addition, although the proportions of T follicular helper (Tfh) cells and GL-7+ germinal center (GC) B cells were higher in MIKO mice than in WT mice, the population of CD138+B220+ antibody-secreting plasmablasts was lower in MIKO mice, which may be a cause of the low influenza-specific antibody titer in MIKO mice. Taken together, these results suggest that macrophage-derived IGF-1 might play an important role in the vaccine-triggered immune response by regulating immune cell homeostasis.