CpG Adjuvant Research Articles

Type I photodynamic therapy with a self-degradable conjugated polyelectrolyte in combination with CpG adjuvant for cancer immunotherapy

In Photodynamic therapy (PDT), reactive oxygen species (ROS) generated by photosensitizers (PSs) can cause endoplasmic reticulum oxidative stress response to induce immunogenic cell death (ICD). However, most PDT-induced ICD cannot generate enough antitumor immune responses to inhibit metastatic cancer. Here, we developed a self-degradable conjugated polyelectrolyte (CP+) to load the immune adjuvant (CpG) for the combination of PDT and immunotherapy. Interestingly, the final nanoparticles (CP+-CpG NPs) can effectively generate superoxide radical (O2−•) through type-I process under light irradiation, which is ideal for hypoxic tumor treatment. Meanwhile, CP+-CpG NPs can be degraded by the self-generated O2−• in return to release the CpG. The release of CpG can further promote PDT-induced ICD, activating immune responses and inducing systemic antitumor immunity. Taken together, a novel strategy has been developed to enhance PDT therapeutic effects and induce immunotherapy, which is greatly promising for cancer treatment in the future.

Potent anti-tumor immune response and tumor growth inhibition induced by HER2 subdomain fusion protein in a mouse tumor model.

Several approaches have so far been employed to establish anti-tumor immunity by targeting HER2 protein. Active immunization with recombinant HER2 subdomains has previously been demonstrated to induce potent immune response and tumor growth inhibition. In the present study, we investigated the immunogenicity and tumor inhibitory effect of a fusion protein consisting of human HER2 extracellular subdomain (ECD-DI + II) together with T-helper cell epitopes of Tetanus toxin (p2 and p30). BALB/c mice were immunized with two recombinant proteins (DI + II and p2p30-DI + II) emulsified in 4 different adjuvants. Anti-DI + II antibody response, cytokine profile, frequency of splenic CD25+FOXP3+ regulatory T cells (Tregs) and CD8+CD107a+ cytotoxic T lymphocytes (CTLs) were assessed in the immunized mice. To assess the anti-tumor effect, the immunized mice were subcutaneously challenged with HER2-overexpressing tumor cells and the tumor growth was determined. Both recombinant proteins were able to induce comparable levels of ECD-DI + II-specific antibodies. Immunization with p2p30-DI + II resulted in a significant increase in the level of Interferon-gamma (IFN-γ) secretion compared to DI + II protein and significantly higher frequency of CTLs and lower frequency of Tregs. The number of mice that remained tumor-free until day 120 was significantly higher in p2p30-DI + II vaccinated groups. Our data suggest that the p2p30-DI + II fusion protein together with CpG adjuvant induces more potent anti-tumor immune responses in a mouse tumor model. Accordingly, this formulation might be considered as a potential immunotherapeutic approach in HER2+ cancers.

Abstract LB531: The addition of IFNα and 5-Aza-2'deoxycytidine treatments to a dendritic cell-targeting DNA vaccine enhance the expression of Th1-related genes and the infiltration of CD11c+ CD8+ T-cells

Abstract Background: DNA vaccines fusing the chemokine MIP-3α (CCL20), which binds to CCR6 on immature dendritic cells (DCs), to melanoma-associated antigens have enhanced anti-tumor immunity and efficacy compared to vaccines lacking the chemokine. To further enhance the therapeutic effects of the vaccine, our laboratory has added type-I interferon (IFNα) and 5-Aza-2’-deoxycitidine (5Aza) to the therapy. Here, we report that the efficacy seen in this combination results in deeper intratumoral penetration of CD8+ cells, enhanced expression of Th1 t-cell related genes, and increased influx of CD11c+ CD8+ T-cells. Methods: Beginning on day five post-transplantation of B16F10 melanoma, the vaccine was administered intramuscularly (i.m.) by electroporation. CpG adjuvant was given two days later. 5Aza was given intraperitoneally at 1mg/kg and IFNα therapy intratumorally as noted. Tumor sizes over time were assessed. For immunohistochemistry (IHC), formalin-fixed tumors were processed, embedded, cut into slides, and stained for CD8 by on-site core facility. Stained slides were analyzed by a pathologist in a blinded fashion. Tumor lysate gene expression levels and tumor-infiltrating lymphocytes (TILs) were assessed by qRT-PCR and flow cytometry respectively. Results: Previous work has shown that the combination of IFNα, 5Aza, and vaccine led to significantly reduced tumor burden and overall increases in mouse survival dependent upon all three components. We have since discovered by IHC that CD8+ cells are more abundant and penetrate further into the tumor with the combination treatment compared to vaccine or therapeutics alone. The tumor immune genetic profile shows broad increases of expression for genes responsible for T-cell activation, efficacy, and trafficking. Of note, the combination therapy doubled the proportion of CD8+ T cells in the tumor microenvironment expressing CD11c (p = 0.027), and the proportions of these cells correlated more strongly to tumor size than do the proportions of CD11c- CD8+ T-cells (r2=0.51; p<0.0001 compared to r2=0.19; p = 0.02). Conclusions: Efficient targeting of antigen to immature dendritic cells with a chemokine-fusion vaccine offers a potential alternative approach to classic and dendritic cell-based vaccines. Combining this approach with IFNα and 5Aza treatments significantly improved vaccine efficacy. This treatment creates a complex intratumoral network that facilitates T-cell trafficking, Th1-associated gene expression, and an increase of CD11c+CD8+ T-cells, which have recently been shown in the literature to have greater anti-tumor activity than CD11c- T-cells. Further analysis of the pathways engaged by our treatment protocol should provide insights into how to more efficiently activate this recently described, highly effective T cell population. Citation Format: James T. Gordy, Kaitlyn Comstock, Avinaash K. Sandhu, Yinan Hui, Styliani Karanika, Joel Sunshine, Petros C. Karakousis, Richard B. Markham. The addition of IFNα and 5-Aza-2'deoxycytidine treatments to a dendritic cell-targeting DNA vaccine enhance the expression of Th1-related genes and the infiltration of CD11c+ CD8+ T-cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr LB531.

Protective Effect against Neosporosis Induced by Intranasal Immunization with Neospora caninum Membrane Antigens Plus Carbomer-Based Adjuvant.

Neospora caninum is an obligate intracellular protozoan responsible for abortion and stillbirths in cattle. We previously developed a mucosal vaccination approach using N. caninum membrane proteins and CpG adjuvant that conferred long-term protection against neosporosis in mice. Here, we have extended this approach by alternatively using the carbomer-based adjuvant Carbigen™ in the immunizing preparation. Immunized mice presented higher proportions and numbers of memory CD4+ and CD8+ T cells. Stimulation of spleen, lungs and liver leukocytes with parasite antigens induced a marked production of IFN-γ and IL-17A and, less markedly, IL-4. This balanced response was also evident in that both parasite-specific IgG1 and IgG2c were raised by immunization, together with specific intestinal IgA. Upon intraperitoneal infection with N. caninum, immunized mice presented lower parasitic burdens than sham-immunized controls. In the infected immunized mice, memory CD4+ T cells predominantly expressed T-bet and RORγt, and CD8+ T cells expressing T-bet were found increased. While spleen, lungs and liver leukocytes of both immunized and sham-immunized infected animals produced high amounts of IFN-γ, only the cells from immunized mice responded with high IL-17A production. Since in cattle both IFN-γ and IL-17A have been associated with protective mechanisms against N. caninum infection, the elicited cytokine profile obtained using CarbigenTM as adjuvant indicates that it could be worth exploring for bovine neosporosis vaccination.

Comparison of the efficacy and safety of different immunization routes induced by human respiratory syncytial virus F protein with CpG adjuvant in mice

Human respiratory syncytial virus (HRSV) is a leading cause worldwide of severe respiratory illness in infants and the elderly. The ideal HRSV vaccine should induce a systemic immune response, especially mucosal immunity. In this study, mice were immunized twice with F protein combined with CpG adjuvant to compare the safety and efficacy of 4 immunization routes, including intranasal primed/intramuscular boosted immunization (CpG + F/in+im), intramuscular primed/intranasal boosted immunization (CpG + F/im+in), intramuscular primed/intramuscular boosted immunization (CpG + F/im + im) and intranasal primed/intranasal boosted immunization (CpG + F/in+in). Compared with the control group (CpG/in+im, CpG/im+in, CpG/im + im and CpG/in+in), all 4 immunization routes induced a high titer of neutralizing antibodies and a strong cellular immune response. Mice in the CpG + F/in+in group induced the highest antibody neutralization titer, and IgA antibody in bronchoalveolar lavage fluid (BALF) was the highest. The copy of HRSVs in the lung decreased by approximately 3 log10. As seen from the IgG1/IgG2a and IFN-γ/IL-4-secreting lymphocyte ratios, compared with the mice in the CpG + F/im + im group, mice in the CpG + F/in+in group induced Th1-baised humoral and cellular immune responses and significantly reduced lung pathological injury. In conclusion, among the 4 immunization routes, the safety and efficacy induced by the mice in the CpG + F/in+in group were the best. We can conclude that intranasal immunization is superior to intramuscular immunization using F protein with CpG adjuvant as vaccine candidates.

Antigen-adjuvant interactions, stability, and immunogenicity profiles of a SARS-CoV-2 receptor-binding domain (RBD) antigen formulated with aluminum salt and CpG adjuvants

ABSTRACT Low-cost, refrigerator-stable COVID-19 vaccines will facilitate global access and improve vaccine coverage in low- and middle-income countries. To this end, subunit-based approaches targeting the receptor-binding domain (RBD) of SARS-CoV-2 Spike protein remain attractive. Antibodies against RBD neutralize SARS-CoV-2 by blocking viral attachment to the host cell receptor, ACE2. Here, a yeast-produced recombinant RBD antigen (RBD-L452K-F490W or RBD-J) was formulated with various combinations of aluminum-salt (Alhydrogel®, AH; AdjuPhos®, AP) and CpG 1018 adjuvants. We assessed the effect of antigen-adjuvant interactions on the stability and mouse immunogenicity of various RBD-J preparations. While RBD-J was 50% adsorbed to AH and <15% to AP, addition of CpG resulted in complete AH binding, yet no improvement in AP adsorption. ACE2 competition ELISA analyses of formulated RBD-J stored at varying temperatures (4, 25, 37°C) revealed that RBD-J was destabilized by AH, an effect exacerbated by CpG. DSC studies demonstrated that aluminum-salt and CpG adjuvants decrease the conformational stability of RBD-J and suggest a direct CpG-RBD-J interaction. Although AH+CpG-adjuvanted RBD-J was the least stable in vitro, the formulation was most potent at eliciting SARS-CoV-2 pseudovirus neutralizing antibodies in mice. In contrast, RBD-J formulated with AP+CpG showed minimal antigen-adjuvant interactions, a better stability profile, but suboptimal immune responses. Interestingly, the loss of in vivo potency associated with heat-stressed RBD-J formulated with AH+CpG after one dose was abrogated by a booster. Our findings highlight the importance of elucidating the key interrelationships between antigen-adjuvant interactions, storage stability, and in vivo performance to enable successful formulation development of stable and efficacious subunit vaccines.

Immunization with a mucosal, post-fusion F/G protein-based polyanhydride nanovaccine protects against BRSV infection in neonatal calves

Abstract Human respiratory syncytial virus (HRSV) is a leading cause of severe acute lower respiratory tract (LRT) disease in young children worldwide. Currently, there is no approved vaccine for HRSV. Bovine RSV (BRSV) is closely related to HRSV and is the cause of severe acute LRT disease in young cattle. A neonatal calf model presents an opportunity to test HRSV vaccines and study a naturally susceptible host-pathogen interaction similar to HRSV infection. We tested the efficacy of a polyanhydride nanoparticle-vaccine (nanovaccine) encapsulating the post-fusion F and G proteins from BRSV and CpG adjuvant in neonatal calves with maternally-derived antibodies (MDA). A prime-boost immunization of neonatal calves with the BRSV post-fusion F/G CpG nanovaccine induced clinical protection in the presence of MDA. Vaccinated calves developed moderate or no clinical symptoms and were mostly protected from virus-associated lung pathology compared to unvaccinated controls. Calves that received a mucosal/systemic nanovaccine immunization had complete virological protection against BRSV infection in the lungs, which correlated with BRSV-specific immune responses in the bronchoalveolar lavage. Vaccination with the nanovaccine induced anamnestic BRSV-specific IgA titers in the lungs and systemic T cell responses with BRSV-specific CD4+ T cell proliferation and antigen-specific IL-17 and IFNγ secretion. Overall, the BRSV post-fusion F/G CpG nanovaccine is a promising candidate for further studies to protect neonatal calves and infants from RSV infection.

Cutaneous CpG adjuvant conditioning to enhance vaccine responses

Adjuvant activity of the Toll receptor 9 agonist CpG 1826 was compared when given subcutaneously (s.c.) together with ovalbumin (s.c.[CpG + Ova]), or when given by either s.c. or intradermally (i.d.) routes two days prior to s.c. ovalbumin. Frequencies of CD8 + effector (TEFF) and central memory (TCM) T cells along with total IgG, IgG2c, and IgG1 titres were measured to ascertain how timing and location of CpG conditioning influenced vaccination outcome. Prior treatment with CpG enhanced TEFF, TCM, as well as total IgG responses. TEFF and TCM responses were greatest when CpG was given intradermally and prior to s.c. ovalbumin, conditions that eliminated the fraction of TCM ‘non-responders’ observed after s.c.[CpG + Ova] vaccination. IgG responses were polarized toward IgG2c after early s.c. CpG but toward IgG1 after early i.d. CpG. Separating CpG adjuvant and antigen application in time and space can improve vaccination outcome.

An aluminum hydroxide:CpG adjuvant enhances protection elicited by a SARS-CoV-2 receptor binding domain vaccine in aged mice.

Global deployment of vaccines that can provide protection across several age groups is still urgently needed to end the COVID-19 pandemic, especially in low- and middle-income countries. Although vaccines against SARS-CoV-2 based on mRNA and adenoviral vector technologies have been rapidly developed, additional practical and scalable SARS-CoV-2 vaccines are required to meet global demand. Protein subunit vaccines formulated with appropriate adjuvants represent an approach to address this urgent need. The receptor binding domain (RBD) is a key target of SARS-CoV-2 neutralizing antibodies but is poorly immunogenic. We therefore compared pattern recognition receptor (PRR) agonists alone or formulated with aluminum hydroxide (AH) and benchmarked them against AS01B and AS03-like emulsion-based adjuvants for their potential to enhance RBD immunogenicity in young and aged mice. We found that an AH and CpG adjuvant formulation (AH:CpG) produced an 80-fold increase in anti-RBD neutralizing antibody titers in both age groups relative to AH alone and protected aged mice from the SARS-CoV-2 challenge. The AH:CpG-adjuvanted RBD vaccine elicited neutralizing antibodies against both wild-type SARS-CoV-2 and the B.1.351 (beta) variant at serum concentrations comparable to those induced by the licensed Pfizer-BioNTech BNT162b2 mRNA vaccine. AH:CpG induced similar cytokine and chemokine gene enrichment patterns in the draining lymph nodes of both young adult and aged mice and enhanced cytokine and chemokine production in human mononuclear cells of younger and older adults. These data support further development of AH:CpG-adjuvanted RBD as an affordable vaccine that may be effective across multiple age groups.

CpG-Containing Oligodeoxynucleotides and Freund Adjuvant in Combination with Alum Augment the Production of Monoclonal Antibodies Against Recombinant HBsAg.

Background:Adjuvants are essential to potentiate the immune response to inoculated antigens and play a central role in vaccine development. Alum is generally used as a classic adjuvant, although it does not stimulate proper immunity, and some of the immunized subjects have low or no antibody response. Efforts have been continued to find more efficient adjuvants for better antibody responses. In the present study, the efficacy of three formulations of adjuvants, i.e. Cysteine p Guanine Oligodeoxynucleotide (CpG ODN), alum, and Freund, in the production of monoclonal anti Hepatitis B Surface Antigen (HBsAg) antibodies was investigated.Methods:To immunize mice, regular hepatitis B vaccine containing recombinant HBsAg and alum was used with CpG ODN or Freund adjuvants, and splenocytes of hyperimmunized mice were fused with murine myeloma Sp2/0 cells. Positive hybridomas were selected by Enzyme-Linked Immunosorbent Assay (ELISA) using HBsAg as coating antigen followed by a limited dilution process.Results:The results showed that by using all three formulations of adjuvants, monoclonal antibody (mAb) specific to HBsAg was successfully generated. It was also found that the mice immunized with (HBsAg + Alum) + CpG had the highest concentration of antibody production in serum and hybridoma supernatants as well as positive clones. Based on these findings, the addition of CpG ODN also induced a higher antibody response compared with Complete Freund’s Adjuvant (CFA).Conclusion:Results of this study showed that CpG and Freund adjuvants could be efficient partners for alum in the immunization period of the process of monoclonal antibody production.

RNA Hydrogel Combined with MnO2 Nanoparticles as a Nano-Vaccine to Treat Triple Negative Breast Cancer.

Hypoxia is not only the reason of tumor metastasis but also enhances the spread of cancer cells from the original tumor site, which results in cancer recurrence. Herein, we developed a self-assembled RNA hydrogel that efficiently delivered synergistic DNA CpG and short hairpin RNA (shRNA) adjuvants, as well as MnO2 loaded-photodynamic agent chlorine e6 (MnO2@Ce6), and a chemotherapy drug doxorubicin (DOX) into MDA-MB-231cells. The RNA hydrogel consists of one tumour suppressor miRNA (miRNA-205) and one anti-metastatic miRNA (miRNA-182), both of which showed an outstanding effect in synergistically abrogating tumours. The hydrogel would be dissociated by endogenous Dicer enzyme to release loaded therapeutic molecules, and in the meantime induce decomposition of tumor endogenous H2O2 to relieve tumor hypoxia. As a result, a remarkable synergistic therapeutic effect is achieved through the combined chemo-photodynamic therapy, which simultaneously triggers a series of anti-tumor immune responses. Besides, the hydrogel as the carrier which modified aptamer to targeted MDA-MB-231 has the advantages of good biocompatibility and low cytotoxicity. This strategy could be implemented to design any other microRNA (miRNA) as the carrier, combined with other treatment methods to treat human cancer, thereby overcoming the limitations of current cancer therapies.

Polyactin A and CpG enhance inactivated Pseudomonas plecoglossicida vaccine potency in large yellow croaker (Larimichthys crocea)

Pseudomonas plecoglossicida is the causative agent of visceral granulomas disease (VGD) in large yellow croaker (LYC, Larimichthys crocea) farming. However, multi-antibiotic resistant of P. plecoglossicida creates an urgent need of an efficient vaccine to combat this pathogen. In this study, an inactivated vaccine added polyactin (PA), CpG-riched plasmid (pCpG) and aluminum adjuvant (Al) was developed. As a result, its relative percentage survival (RPS) against P. plecoglossicida were up to 64%. Comparatively, RPS of groups that vaccinated with vaccines adjuvanted with PA and Al or CpG and Al were 49% and 39%. However, an interesting result that the vaccine combined with PA, CpG and Al did not show the strongest activation of total serum protein and antibody levels in serum among three vaccinated groups. According to expressions of some cellular immune related genes, we found that the inactivated vaccine combined with PA, CpG and Al was more likely to induce a cellular immune response rather than humoral immune response. Totally, our study demonstrated that the mixture of PA, CpG and aluminum adjuvant is a potential adjuvant system for LYC vaccine development.

Polymersomes as Stable Nanocarriers for a Highly Immunogenic and Durable SARS-CoV-2 Spike Protein Subunit Vaccine.

Multiple successful vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are urgently needed to address the ongoing coronavirus disease 2019 (Covid-19) pandemic. In the present work, we describe a subunit vaccine based on the SARS-CoV-2 spike protein coadministered with CpG adjuvant. To enhance the immunogenicity of our formulation, both antigen and adjuvant were encapsulated with our proprietary artificial cell membrane (ACM) polymersome technology. Structurally, ACM polymersomes are self-assembling nanoscale vesicles made up of an amphiphilic block copolymer comprising poly(butadiene)-b-poly(ethylene glycol) and a cationic lipid, 1,2-dioleoyl-3-trimethylammonium-propane. Functionally, ACM polymersomes serve as delivery vehicles that are efficiently taken up by dendritic cells (DC1 and DC2), which are key initiators of the adaptive immune response. Two doses of our formulation elicit robust neutralizing antibody titers in C57BL/6 mice that persist at least 40 days. Furthermore, we confirm the presence of functional memory CD4+ and CD8+ T cells that produce T helper type 1 cytokines. This study is an important step toward the development of an efficacious vaccine in humans.

Engineered porous/hollow Burkholderia pseudomallei loading tumor lysate as a vaccine

Due to its size, shape, and inherent expression of pathogen-associated molecular patterns and invasion-assistant adhesion proteins, Burkholderia pseudomallei can easily attach to, and then be internalized by, dendritic cells (DCs), leading to more efficient antigen cross-presentation if modified as carrier. Herein, we engineered Burkholderia pseudomallei as a porous/hollow carrier (SB) for loading tumor lysates (L) and adjuvant CpG (C) to be used as a tumor vaccine (SB-LC). We found that the adhesion proteins of Burkholderia pseudomallei promote internalization of the SB-LC vaccine by DCs, and result in enhanced DC maturation and antigen cross-presentation. SB-LC induces robust cellular and humoral antitumor responses that synergistically inhibit tumor growth with minimal adverse side effects in several tumor models. Moreover, SB-LC vaccination reverses the immunosuppressive tumor microenvironment, apparently as a result of CD8+-induced tumor ferroptosis. Thus, SB-LC is a potential model tumor vaccine for translating into a clinically viable treatment option.

FcRn-Targeted Mucosal Vaccination against Influenza Virus Infection.

The respiratory tract is constantly exposed to various airborne pathogens. Most vaccines against respiratory infections are designed for the parenteral routes of administration; consequently, they provide relatively minimal protection in the respiratory tract. A vaccination strategy that aims to induce the protective mucosal immune responses in the airway is urgently needed. The FcRn mediates IgG Ab transport across the epithelial cells lining the respiratory tract. By mimicking this natural IgG transfer, we tested whether FcRn delivers vaccine Ags to induce a protective immunity to respiratory infections. In this study, we designed a monomeric IgG Fc fused to influenza virus hemagglutinin (HA) Ag with a trimerization domain. The soluble trimeric HA-Fc were characterized by their binding with conformation-dependent HA Abs or FcRn. In wild-type, but not FcRn knockout, mice, intranasal immunization with HA-Fc plus CpG adjuvant conferred significant protection against lethal intranasal challenge with influenza A/PR/8/34 virus. Further, mice immunized with a mutant HA-Fc lacking FcRn binding sites or HA alone succumbed to lethal infection. Protection was attributed to high levels of neutralizing Abs, robust and long-lasting B and T cell responses, the presence of lung-resident memory T cells and bone marrow plasma cells, and a remarkable reduction of virus-induced lung inflammation. Our results demonstrate for the first time, to our knowledge, that FcRn can effectively deliver a trimeric viral vaccine Ag in the respiratory tract and elicit potent protection against respiratory infection. This study further supports a view that FcRn-mediated mucosal immunization is a platform for vaccine delivery against common respiratory pathogens.

Intratumoral Injection of a Human Papillomavirus Therapeutic Vaccine-Induced Strong Anti-TC-1-Grafted Tumor Activity in Mice.

PurposeThe route of administration of a therapeutic tumor vaccine is a critical factor in inducing antitumor activity. In this study, we explored the effects of three vaccination routes (subcutaneous, peritumoral, and intratumoral injection) on antitumor activity induced by a human papillomavirus (HPV) therapeutic vaccine containing HPV16 E7 peptide combined with the adjuvant CpG ODN in established TC-1 grafted tumors.MethodsWe used flow cytometry to evaluate splenic and tumor-infiltrating immune cells. We also assessed transcriptional changes in a sequence of immune-related genes in tumors of different treatment groups using quantitative real-time polymerase chain reaction. Immunohistochemistry was used to determine the expression of molecules related to tumor infiltrating immune cells, angiogenesis, and cancer-associated fibroblasts in tumor tissues.ResultsOur results suggested that intratumoral and peritumoral vaccination generated enhanced antitumor activity compared to subcutaneous delivery. In particular, intratumoral vaccination elicited a stronger antitumor effect, with two of the six treated mice being nearly tumor-free at day 28. Three vaccination routes induced increases in splenic CD4+ and/or CD8+ T lymphocytes, and marked decreases in immunosuppressive cells. Peritumoral vaccination increased the tumor-infiltrating CD8+T cells in tumors, while intratumoral vaccination enhanced the tumor-infiltrating CD4+ and CD8+ T lymphocytes, as well as decreased the tumor-infiltrating of immunosuppressive cells, which may result in stronger inhibition of tumor growth and prolonged survival in mice bearing tumors. Furthermore, compared to the subcutaneous route, intratumoral vaccination led to a significant increase in antitumor cytokines and chemokines. In addition, our data showed marked downregulation of MMP-2, MMP-9, VEGF, CD31, and α-SMA in the intratumoral vaccination group, which might contribute to the suppression of tumor invasion, angiogenesis, and metastasis.ConclusionOverall, intratumoral vaccination is superior to subcutaneous delivery and has the potential to inhibit tumor growth by improving the tumor microenvironment.

Immunopotentiation by Lymph-Node Targeting of a Malaria Transmission-Blocking Nanovaccine.

A successful malaria transmission blocking vaccine (TBV) requires the induction of a high antibody titer that leads to abrogation of parasite traversal of the mosquito midgut following ingestion of an infectious bloodmeal, thereby blocking the cascade of secondary human infections. Previously, we developed an optimized construct UF6b that elicits an antigen-specific antibody response to a neutralizing epitope of Anopheline alanyl aminopeptidase N (AnAPN1), an evolutionarily conserved pan-malaria mosquito midgut-based TBV target, as well as established a size-controlled lymph node targeting biodegradable nanoparticle delivery system that leads to efficient and durable antigen-specific antibody responses using the model antigen ovalbumin. Herein, we demonstrate that co-delivery of UF6b with the adjuvant CpG oligodeoxynucleotide immunostimulatory sequence (ODN ISS) 1018 using this biodegradable nanoparticle vaccine delivery system generates an AnAPN1-specific immune response that blocks parasite transmission in a standard membrane feeding assay. Importantly, this platform allows for antigen dose-sparing, wherein lower antigen payloads elicit higher-quality antibodies, therefore less antigen-specific IgG is needed for potent transmission-reducing activity. By targeting lymph nodes directly, the resulting immunopotentiation of AnAPN1 suggests that the de facto assumption that high antibody titers are needed for a TBV to be successful needs to be re-examined. This nanovaccine formulation is stable at -20°C storage for at least 3 months, an important consideration for vaccine transport and distribution in regions with poor healthcare infrastructure. Together, these data support further development of this nanovaccine platform for malaria TBVs.

Francisella tularensis induces Th1 like MAIT cells conferring protection against systemic and local infection

Mucosal-associated Invariant T (MAIT) cells are recognized for their antibacterial functions. The protective capacity of MAIT cells has been demonstrated in murine models of local infection, including in the lungs. Here we show that during systemic infection of mice with Francisella tularensis live vaccine strain results in evident MAIT cell expansion in the liver, lungs, kidney and spleen and peripheral blood. The responding MAIT cells manifest a polarised Th1-like MAIT-1 phenotype, including transcription factor and cytokine profile, and confer a critical role in controlling bacterial load. Post resolution of the primary infection, the expanded MAIT cells form stable memory-like MAIT-1 cell populations, suggesting a basis for vaccination. Indeed, a systemic vaccination with synthetic antigen 5-(2-oxopropylideneamino)-6-d-ribitylaminouracil in combination with CpG adjuvant similarly boosts MAIT cells, and results in enhanced protection against both systemic and local infections with different bacteria. Our study highlights the potential utility of targeting MAIT cells to combat a range of bacterial pathogens.

Abstract 1916: Lymph node targeting double stranded CpG act effective adjuvant in cancer peptide vaccine

Abstract Background: Adjuvants can enhance immune responses of vaccinated antigen by inducing host innate immune responses. While cancer peptide vaccines were largely studied in recent years, effective cancer peptide vaccines have not been found yet so far because of their poor immunogenicity. Developing an effective vaccine adjuvant suitable for peptide antigen is a promising approach. CpG oligodeoxynucleotides(CpG) are a toll-like receptor 9 agonists and effectively elicit innate immune responses. In order to improve adjuvanticity of CpG, we designed a novel lymph-node targeting CpG, which is composed from a single stranded CpG and a modified complementary strand with amphiphilic chain. Methods: S-540956 is a double stranded oligodeoxynucleotide composed from a single stranded phosphorothioate oligonucleotide and the complementary strand with amphiphilic chain at the 5' end. Accumulating capability to lymph nodes of fluorescent labeled S-540956 was analyzed by flow cytometer, IVIS imaging system, and 2-photon microscopy. Incorporation of S-540956 by antigen-presenting cells in lymph nodes was assessed by Image Stream® imaging cytometer. To test cellular immune responses, three types of MHC class-I epitope peptides were employed as antigenic peptides. Therapeutic effect of S-540956 adjuvanted HPV E7 peptide vaccine was evaluated in TC-1 tumor model. Results:S-540956 exhibited higher accumulating capacity to lymph nodes in mice compared to a typical CpG adjuvant, ODN2006. Systemic proinflammatory cytokine responses were decreased by combining the complementary strand with amphiphilic chain. S-540956 was efficiently incorporated into plasmacytoid DCs and induced the expression of costimulatory molecules. Cellular immune responses induced by S-540956 adjuvanted peptide vaccine were significantly higher than ODN2006 adjuvanted peptide vaccine. And this enhanced adjuvant effect was observed also in protein vaccine. In TC-1 tumor model, therapeutically administered S-540956 adjuvanted HPV-E7 peptide vaccine lead to significant tumor shrinkage compared to ODN2006 adjuvanted peptide vaccine. Conclusion: S-540956 acted as a potent adjuvant for peptide vaccine by inducing cellular immune responses effectively. Accumulating capacity to lymph nodes of S-540956 is a promising adjuvant for not only for peptide but for protein or other biologic antigens. Citation Format: Takayuki Nakagawa, Motoyasu Onishi, Soichi Tofukuji, Shinya Omoto, Kazufumi Katayama, Akira Kugimiya, Morio Nagira, Ken J. Ishii. Lymph node targeting double stranded CpG act effective adjuvant in cancer peptide vaccine [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1916.

Astragalus Polysaccharides Enhance the Immune Response to OVA Antigen in BALB/c Mice.

Vaccination has been identified as one of the most effective ways to prevent the transmission of infectious diseases in humans and animals. One of the most critical steps in vaccine development is the selection of a suitable adjuvant. Although various adjuvant candidates have been evaluated in the past few decades, only a limited amount of them are nontoxic and safe for human use. Astragalus polysaccharide (APS), due to its lack of toxicity, has been used as an immunomodulator to enhance immune responses. On the other hand, the immune effects of APS on ovalbumin are yet to be examined. Thus, in this study, we analyzed APS's effects on the immune response to ovalbumin in BALB/c mice. We have also used the classic adjuvant CpG oligodeoxynucleotide as the positive control.