CpG Adjuvant Research Articles

Systemic administration of polymersomal oncolytic peptide LTX-315 combining with CpG adjuvant and anti-PD-1 antibody boosts immunotherapy of melanoma

Oncolytic peptide LTX-315 while showing clinical promise in treating solid tumors is limited to intratumoral administration, which is not applicable for inaccessible or metastatic tumors. The cationic and amphipathic nature of oncolytic peptides engenders formidable challenges to developing systems for their systemic delivery. Here, we describe cRGD-functionalized chimaeric polymersomes (cRGD-CPs) as a robust systemic delivery vehicle for LTX-315, which in combination with CpG adjuvant and anti-PD-1 boost immunotherapy of malignant B16F10 melanoma in mice. cRGD-CPs containing 14.9 wt% LTX-315 (cRGD-CPs-L) exhibited a size of 53 nm, excellent serum stability, and strong and selective killing of B16F10 cells (versus L929 fibroblasts) in vitro, which provoked similar immunogenic effects to free LTX-315 as revealed by release of danger-associated molecular pattern molecules. The systemic administration of cRGD-CPs-L gave a notable tumor accumulation of 4.8% ID/g and significant retardation of tumor growth. More interestingly, the treatment of B16F10 tumor-bearing mice was further boosted by co-administration of polymersomal CpG and anti-PD-1 antibody, in which two out of seven mice were cured as a result of strong immune response and long-term immune memory protection. The immunotherapeutic effect was evidenced by secretion of IL-6, IFN-γ and TNF-α, tumor infiltration of CD8+ CTLs and Th, and induction of TEM and TCM in spleen. This study opens a new avenue to oncolytic peptides, which enables durable immunotherapy of tumors via systemic administration.

Development of a Virus-Like Particle-Based Anti-HER2 Breast Cancer Vaccine.

Simple SummaryVirus-like particles (VLPs) have attracted significant interest as immunotherapy platforms and cancer vaccines for inducing antigen-specific immune responses against tumors. We prepared a human epidermal growth factor receptor-2 (HER2) cancer vaccine, by conjugating the HER2-derived CH401 epitope to the external surface of Physalis mottle virus (PhMV)-like particles via copper-free click chemistry. Another candidate was prepared by loading Toll-like receptor 9 (TLR9) agonists as adjuvant into the interior cavity of PhMV-CH401—although the addition of the adjuvant conferred no additional immune priming. The VLP-based anti-HER2 vaccine candidate was administered subcutaneously, using a prime-boost immunization schedule and BALB/c mice. The vaccine candidate elicited a strong immune response, including high titers of HER2-specific immunoglobulins and increased the toxicity of antisera to DDHER2 tumor cells. DDHER2 tumor challenge studies demonstrated efficacy, as evident from the delayed onset of tumor growth and the prolonged survival of the vaccinated vs. naïve BALB/C mice.To develop a human epidermal growth factor receptor-2 (HER2)-specific cancer vaccine, using a plant virus-like particle (VLP) platform. Copper-free click chemistry and infusion encapsulation protocols were developed to prepare VLPs displaying the HER2-derived CH401 peptide epitope, with and without Toll-like receptor 9 (TLR9) agonists loaded into the interior cavity of the VLPs; Physalis mottle virus (PhMV)-based VLPs were used. After prime-boost immunization of BALB/c mice through subcutaneous administration of the vaccine candidates, sera were collected and analyzed by enzyme-linked immunosorbent assay (ELISA) for the CH401-specific antibodies; Th1 vs. Th2 bias was determined by antibody subtyping and splenocyte assay. Efficacy was assessed by tumor challenge using DDHER2 tumor cells. We successful developed two VLP-based anti-HER2 vaccine candidates—PhMV-CH401 vs. CpG-PhMV-CH401; however, the addition of the CpG adjuvant did not confer additional immune priming. Both VLP-based vaccine candidates elicited a strong immune response, including high titers of HER2-specific immunoglobulins and increased toxicity of antisera to DDHER2 tumor cells. DDHER2 tumor growth was delayed, leading to prolonged survival of the vaccinated vs. naïve BALB/C mice. The PhMV-based anti-HER2 vaccine PhMV-CH401, demonstrated efficacy as an anti-HER2 cancer vaccine. Our studies highlight that VLPs derived from PhMV are a promising platform to develop cancer vaccines.

GRA8 DNA vaccine formulations protect against chronic toxoplasmosis

Toxoplasma gondii has a very wide host range and infects all warm-blooded animals including humans. The disease causes great economic losses both in animals and humans. Vaccination is the most effective approach to fight against toxoplasmosis however an effective vaccine has not been developed yet. In the present study, GRA8 protein of T. gondii that showed high immunogenicity in our previous microarray screening study was used to develop a DNA vaccine using pcDNA 3.3 vector for the first time. In order to increase the potency of the DNA vaccine, 10 times lower amount of GRA8 DNA vaccine was combined with molecular adjuvant CpG and formulated into a commercial liposome (pcDNA3.3-GRA8+CpG+Escort). Mice were vaccinated intramuscularly two times at three-week intervals and challenged orally with the T. gondii PRU strain tissue cysts. The humoral immune response was determined by Western Blot and ELISA. The cellular immune response was analyzed by flow cytometry, cytokine ELISA and MTT assay. Among the vaccine groups, pcDNA3.3-GRA8 and pcDNA3.3-GRA8+CpG+Escort induced strong IgG response compared to controls (P < 0.001). The IgG1 and IgG2a responses showed a balanced Th1–Th2 polarization. The ratio of CD4+ and CD8+ T lymphocytes secreting IFN-γ increased, and significantly higher extracellular IFN-γ secretion was achieved compared to the controls (P < 0.01). The amount of tissue cysts in the group of mice vaccinated with pcDNA3.3-GRA8 decreased significantly compared to control groups (P < 0.0001). In the group vaccinated with pcDNA3.3-GRA8+CpG+Escort, the amount of tissue cysts also decreased significantly compared to PBS (P = 0.0086) and Empty plasmid+CpG+Escort (P = 0.0007) groups. This study showed for the first time that pcDNA 3.3. vector encoding GRA8 with or without CpG and Liposome can induce strong cellular and humoral immune responses and confer strong protection against mouse model of chronic toxoplasmosis.

Targeting the tumor microenvironment with amphiphilic near-infrared cyanine nanoparticles for potentiated photothermal immunotherapy

Despite the potential of photothermal therapy (PTT) for cancer treatments, PTT alone has limitations in treating metastatic tumors and preventing tumor recurrence, highlighting the need to combine PTT with immunotherapy. This study reports tumor microenvironment (TME)-targeting, near-infrared (NIR) dye derivative—based nanomedicine for effective combined PTT—immunotherapy. Amphiphilic NIR dye cyanine derivatives are used not only for constructing the nanoparticle mass, but also for creating a stable complex with CpG adjuvant; a peptide specific to fibronectin extra domain B (APTEDB) is also introduced as a TME-targeting ligand, yielding the TME-targeting nanomedicine, APTEDB-cyNP@CpG. APTEDB-cyNP@CpG shows cancer-targeting ability in EDB-overexpressing CT26 colon tumor-bearing mice. When combined with laser irradiation, it induces immunogenic cell death (ICD) and subsequently leads to significant increase in CD8+ T cell population in the tumor, resulting in greater antitumor therapeutic efficacy than does cyNP@CpG lacking the TME-targeting ligand. Moreover, the combination of APTEDB-cyNP@CpG–based PTT and an immune checkpoint blockade (ICB) antibody leads to remarkable antitumor efficacy against the laser-irradiated primary tumor as well as distant tumor through potentiation of systemic cancer cell-specific T cell immunity. Furthermore, the PTT-immunotherapy combination regimen is highly effective in inhibiting tumor recurrence and metastasis.

Biomimetic sonodynamic therapy-nanovaccine integration platform potentiates Anti-PD-1 therapy in hypoxic tumors

Immune checkpoint blockade (ICB) therapy has emerged as novel therapeutic modality for hard-to-treat solid tumors. However, inadequate T cell infiltration in hypoxic solid tumors limits its anticancer efficacy. Here we develop a sonodynamic therapy (SDT)-nanovaccine integration platform constructed by binding a manganese porphyrin-based metal-organic frameworks (Mn-MOF) with an immune adjuvant CpG and then coating with cell membranes derived from ovalbumin (OVA)-overexpressing melanoma B16 cells (cMn-MOF@CM) for potentiating anti-programmed death receptor 1 (PD-1) antibody in malignant melanoma therapy. cMn-MOF@CM with prolonged blood circulation and enhanced tumor targeting efficiently relieves tumor hypoxia and generates strong SDT effects and immunogenic cell death. The tumor-associated antigens both in situ derived from SDT and OVA exhibit vaccine-like functions together with the immune adjuvant CpG, eliciting a strong tumor-specific immune response by promoting DC maturation and T cell activation. Importantly, the combination of cMn-MOF@CM-triggered SDT and anti-PD-1 antibody induces stronger systemic immune response and long-term immunological memory function to prevent tumor growth and recurrence. These findings reveal that cMn-MOF@CM with US irradiation may be a potential candidate to potentiate ICB therapy for hypoxic cancer therapy.

Intranasal immunization with inactivated chlamydial elementary bodies formulated in VCG-chitosan nanoparticles induces robust immunity against intranasal Chlamydia psittaci challenge

Vaccines based on live attenuated Chlamydia elementary bodies (EBs) can cause disease in vaccinated animals and the comparably safer inactivated whole EBs are only marginally protective. Recent studies show that a vaccine formulation comprising UV-inactivated EBs (EB) and appropriate mucosal delivery systems and/or adjuvants induced significant protective immunity. We tested the hypothesis that intranasal delivery of UV-inactivated C. psittaci EB formulated in Vibrio cholerae ghosts (VCG)-chitosan nanoparticles will induce protective immunity against intranasal challenge in SPF chickens. We first compared the impact of VCG and CpG adjuvants on protective immunity following IN mucosal and IM systemic delivery of EB formulated in chitosan hydrogel/microspheres. Immunologic analysis revealed that IN immunization in the presence of VCG induced higher levels of IFN-γ response than IM delivery or the CpG adjuvanted groups. Also, vaccine efficacy evaluation showed enhanced pharyngeal bacterial clearance and protection against lung lesions with the VCG adjuvanted vaccine formulation, thereby establishing the superior adjuvanticity of VCG over CpG. We next evaluated the impact of different concentrations of VCG on protective immunity following IN mucosal immunization. Interestingly, the adjuvanticity of VCG was concentration-dependent, since protective immunity induced following IN mucosal immunization showed dose-dependent immune responses and protection. These studies reveal that formulation of inactivated chlamydial antigens with adjuvants, such as VCG and chitosan increases their ability to induce protective immune responses against challenge.

Combined PET and whole-tissue imaging of lymphatic-targeting vaccines in non-human primates

Antigen accumulation in lymph nodes (LNs) is critical for vaccine efficacy, but understanding of vaccine biodistribution in humans or large animals remains limited. Using the rhesus macaque model, we employed a combination of positron emission tomography (PET) and fluorescence imaging to characterize the whole-animal to tissue-level biodistribution of a subunit vaccine comprised of an HIV envelope trimer protein nanoparticle (trimer-NP) and lipid-conjugated CpG adjuvant (amph-CpG). Following immunization in the thigh, PET imaging revealed vaccine uptake primarily in inguinal and iliac LNs, reaching distances up to 17 cm away from the injection site. Within LNs, trimer-NPs exhibited striking accumulation on the periphery of follicular dendritic cell (FDC) networks in B cell follicles. Comparative imaging of soluble Env trimers (not presented on nanoparticles) in naïve or previously-immunized animals revealed diffuse deposition of trimer antigens in LNs following primary immunization, but concentration on FDCs in pre-immunized animals with high levels of trimer-specific IgG. These data demonstrate the capacity of nanoparticle or “albumin hitchhiking” technologies to concentrate vaccines in genitourinary tract-draining LNs, which may be valuable for promoting mucosal immunity.

A Versatile and Robust Platform for the Scalable Manufacture of Biomimetic Nanovaccines.

Biomimetic strategies are useful for designing potent vaccines. Decorating a nanoparticulate adjuvant with cell membrane fragments as the antigen‐presenting source exemplifies, such as a promising strategy. For translation, a standardizable, consistent, and scalable approach for coating nanoadjuvant with the cell membrane is important. Here a turbulent mixing and self‐assembly method called flash nanocomplexation (FNC) for producing cell membrane‐coated nanovaccines in a scalable manner is demonstrated. The broad applicability of this FNC technique compared with bulk‐sonication by using ten different core materials and multiple cell membrane types is shown. FNC‐produced biomimetic nanoparticles have promising colloidal stability and narrow particle polydispersity, indicating an equal or more homogeneous coating compared to the bulk‐sonication method. The potency of a nanovaccine comprised of B16‐F10 cancer cell membrane decorating mesoporous silica nanoparticles loaded with the adjuvant CpG is then demonstrated. The FNC‐fabricated nanovaccines when combined with anti‐CTLA‐4 show potency in lymph node targeting, DC antigen presentation, and T cell immune activation, leading to prophylactic and therapeutic efficacy in a melanoma mouse model. This study advances the design of a biomimetic nanovaccine enabled by a robust and versatile nanomanufacturing technique.

Personalized combination nano-immunotherapy for robust induction and tumor infiltration of CD8+ T cells

Identification of tumor-specific mutations, called neoantigens, offers new exciting opportunities for personalized cancer immunotherapy. However, it remains challenging to achieve robust induction of neoantigen-specific T cells and drive their infiltration into the tumor microenvironment (TME). Here, we have developed a novel polyethyleneimine (PEI)-based personalized vaccine platform carrying neoantigen peptides and CpG adjuvants in a compact nanoparticle (NP) for their spatio-temporally concerted delivery. The NP vaccine significantly enhanced activation and antigen cross-presentation of dendritic cells, resulting in strong priming of neoantigen-specific CD8+ T cells with the frequency in the systemic circulation reaching as high as 23 ± 7% after a single subcutaneous administration. However, activated CD8+ T cells in circulation exhibited limited tumor infiltration, leading to poor anti-tumor efficacy. Notably, local administration of stimulator of interferon genes (STING) agonist promoted tumor infiltration of vaccine-primed CD8+ T cells, thereby overcoming one of the major challenges in achieving strong anti-tumor efficacy with cancer vaccination. The NP vaccination combined with STING agonist therapy eliminated tumors in murine models of MC-38 colon carcinoma and B16F10 melanoma and established long-term immunological memory. Our approach provides a novel therapeutic strategy based on combination nano-immunotherapy for personalized cancer immunotherapy.

CpG-Adjuvanted Hepatitis B Vaccine (HEPLISAV-B®) Update

ABSTRACT Introduction: HEPLISAV-B is a hepatitis B vaccine composed of rHBsAg mixed with a synthetic oligonucleotide containing CpG motifs that stimulate innate immunity through TLR9. This vaccine was recently approved by FDA in view of its superior efficacy. Areas covered: Published literature on HEPLISAV-B was critically reviewed. Four randomized controlled trials among 7,056 subjects receiving 2 doses of HEPLISAV-B and 3,214 subjects receiving 3 doses of Engerix-B showed superior seroprotection rate (SPR) (anti-HBs ≥10 mIU/mL) of 90–100%, compared with 71–90% in those receiving Engerix-B. Furthermore, the seroprotection rate was also significantly higher in HEPLISAV-B compared with Engerix-B recipients in persons with traditionally poor vaccine responses such as older adults, diabetics, and those with chronic kidney disease. The safety profiles among 9,871 subjects were similar between HEPLISAV-B and Engerix-B . Expert opinion: HEPLISAV-B, a CpG adjuvant mixed with HBsAg, is more efficacious and produced earlier seroprotection compared to existing vaccines, with a favorable safety profile. The shorter, two-dose regimen, earlier seroprotection, higher adherence, and a higher seroprotection rate, especially in populations with traditionally poor vaccine response, makes this an important therapeutic option in hepatitis B vaccination.

In vitro naphthylquinoxaline thymidine conjugate and UVA treated cancer cells are effective therapeutic vaccines for tumors in vivo with CpG as the adjuvant

IntroductionCancer cells induced into immunogenic cell death (ICD) in vitro can be directly used as a whole cell vaccine for tumor immunotherapy with many advantages, especially enacting immediate and intense ‘eat me’ signals to engage immune system. Unfortunately, there have been few successes with in vitro ICD cancer cells as a treatment vaccine. ObjectiveTo demonstrate that cancer cells treated in vitro with a new class of potent ICD inducer, naphthylquinoxaline thymidine conjugate (NAP) followed by UVA irradiation would be able to act as an effective tumor immunotherapy directly. MethodsThe therapeutic potentials of treated cancer cell plus different vaccine adjuvants were assessed by in vivo liver tumor model and in vitro mixed lymphocyte reaction studies. The elicited activated T cells were determined with immunohistochemistry and T cell induced cytotoxicity studies. ResultsTreatment of established H22 tumor with in vitro NAP and UVA treated cancer cell vaccine led to significantly improved survival. Further mixed lymphocyte reaction study implied that adjuvants alum and CpG would improve the therapeutic potential whereas poly IC would not be as effective. Subsequent in vivo validation of alum and CpG adjuvants indicated that only CpG in NAP and UVA treated cell vaccine resulted in markedly enhanced survival (median at 71 days and 50% tumor-free) as compared with PBS group (14.5 days, 0%) and CpG alone (36 days, 0%). It was revealed that the enhanced efficacy by CpG was specific to NAP and UVA treated cells. Moreover, the effective tumor immunotherapy was achieved through the infiltration of active CD4 and CD8 T cells in tumors and acquisition of cancer cell-specific cytotoxic CD8 T cells. ConclusionIn vitro NAP and UVA treated cancer cells plus CpG adjuvant are effective tumor therapeutic vaccines per se.

Exploiting albumin as a mucosal vaccine chaperone for robust generation of lung-resident memory T cells.

Tissue-resident memory T cells (TRMs) can profoundly enhance mucosal immunity, but parameters governing TRM induction by vaccination remain poorly understood. Here, we describe an approach exploiting natural albumin transport across the airway epithelium to enhance mucosal TRM generation by vaccination. Pulmonary immunization with albumin-binding amphiphile conjugates of peptide antigens and CpG adjuvant (amph-vaccines) increased vaccine accumulation in the lung and mediastinal lymph nodes (MLNs). Amph-vaccines prolonged antigen presentation in MLNs over 2 weeks, leading to 25-fold increased lung-resident T cell responses over traditional immunization and enhanced protection from viral or tumor challenge. Mimicking such prolonged exposure through repeated administration of soluble vaccine revealed that persistence of both antigen and adjuvant was critical for optimal TRM induction, mediated through T cell priming in MLNs after prime, and directly in the lung tissue after boost. Thus, vaccine persistence strongly promotes TRM induction, and amph-conjugates may provide a practical approach to achieve such kinetics in mucosal vaccines.

Nanosonosensitizer-Augmented Sonodynamic Therapy Combined with Checkpoint Blockade for Cancer Immunotherapy.

IntroductionSonodynamic therapy (SDT) has good targeting and non-invasive advantages in the treatment of solid cancers, and checkpoint blockade immunotherapy is also a promising treatment to cure cancer. However, their antitumor effects are not sufficient due to some inherent factors. Some studies that combined SDT with immunotherapy or nanoparticles have managed to enhance its efficiency to treat cancers.MethodsIn this work, an effective therapeutic strategy that can potentiate the antitumor efficacy of anti-PD-L1 antibody (aPD-L1) is developed by the use of cascade immuno-sonodynamic therapy (immuno-SDT). Titanium dioxide (TiO2), a nanostructured agent for SDT, sonosensitizer Chlorin e6 (Ce6), and immunological adjuvant CpG oligonucleotide (CpG ODN), are used to construct a multifunctional nanosonosensitizer (TiO2-Ce6-CpG). Then, we conducted in vitro and in vivo experiments to explore the antitumor effect of TiO2-Ce6-CpG under ultrasound (US) treatment.ResultsThe characterization tests showed that the nanosonosensitizers are polycrystalline structure with homogeneous sizes, resulting in a good drug loading efficiency. The innovative nanosonosensitizers (TiO2-Ce6-CpG) can not only effectively inhibit tumor growth but also stimulate the immune system to activate the adaptive immune responses, using the TiO2-Ce6 to augment SDT and the immune adjuvant CpG to enhance the immune response. After combined with the aPD-L1, the synergistic effect could not only efficiently inhibit the primary tumor growth but also lead to an inhibition of the non-irradiated pre-existing distant tumors by inducing a strong tumor-specific immune response.ConclusionIn this study, we present an effective strategy for tumor treatment by combining nanosonosensitizer-augmented SDT and aPD-L1 checkpoint blockade. This work provides a promising strategy and offers a new vision for treating malignant tumors.

CpG Adjuvant in Allergen-Specific Immunotherapy: Finding the Sweet Spot for the Induction of Immune Tolerance.

Prevalence and incidence of IgE-mediated allergic diseases have increased over the past years in developed and developing countries. Allergen-specific immunotherapy (AIT) is currently the only curative treatment available for allergic diseases that has long-term efficacy. Although AIT has been proven successful as an immunomodulatory therapy since its beginnings, it still faces several unmet needs and challenges today. For instance, some patients can experience severe side effects, others are non-responders, and prolonged treatment schedules can lead to lack of patient adherence and therapy discontinuation. A common strategy to improve AIT relies on the use of adjuvants and immune modulators to boost its effects and improve its safety. Among the adjuvants tested for their clinical efficacy, CpG oligodeoxynucleotide (CpG-ODN) was investigated with limited success and without reaching phase III trials for clinical allergy treatment. However, recently discovered immune tolerance-promoting properties of CpG-ODN place this adjuvant again in a prominent position as an immune modulator for the treatment of allergic diseases. Indeed, it has been shown that the CpG-ODN dose and concentration are crucial in promoting immune regulation through the recruitment of pDCs. While low doses induce an inflammatory response, high doses of CpG-ODN trigger a tolerogenic response that can reverse a pre-established allergic milieu. Consistently, CpG-ODN has also been found to stimulate IL-10 producing B cells, so-called B regulatory cells (Bregs). Accordingly, CpG-ODN has shown its capacity to prevent and revert allergic reactions in several animal models showing its potential as both preventive and active treatment for IgE-mediated allergy. In this review, we describe how CpG-ODN-based therapies for allergic diseases, despite having shown limited success in the past, can still be exploited further as an adjuvant or immune modulator in the context of AIT and deserves additional attention. Here, we discuss the past and current knowledge, which highlights CpG-ODN as a potential adjuvant to be reevaluated for the enhancement of AIT when used in appropriate conditions and formulations.

The immunity of the recombinant prokaryotic and eukaryotic subunit vaccines against cutaneous leishmaniasis

Leishmaniasis counts as one of the most neglected tropical diseases. Despite the amount of research perceived in this field, no fully effective and approved vaccine against this disease is yet available in humans. In this study, LACK and KMP11 antigens were constructed simultaneously by recombinant methods in prokaryotic and eukaryotic expression systems and were compared and assessed along with the CpG adjuvant in BALB/c mice.In the prokaryotic method, LACK and KMP11 protein gene sequences were synthesized in pET28a-TEV vector. In order to extract these two proteins after expression, His-tag and S-tag sequences were added to the constructs, respectively for LACK and KMP11. The pET28a-TEV-LACK/KMP11 construct was transformed into Escherichia coli, and the inserts were verified by Colony PCR. Pure proteins were verified by western blot, and groups of BALB/c mice were injected with the created prokaryotic recombinant proteins along with an ODN CpG adjuvant. In the eukaryotic method, antigen sequences were constructed in the pLEXSY-neo 2.1 vector, E.coli Top10 strain was cloned in the bacteria, and after being linearized were transfected into Leishmania tarentolae genome. After recombinant strains were selected, they were verified by molecular methods. After the extraction and purification of the protein using the method above, groups of mice were injected with the recombinant antigens and ODN CpG adjuvant.Eukaryotic subunit vaccines showed more effective immunization compared with prokaryotic vaccines and caused an immune system shift towards Th1 and protection. Protein expression in L. tarentolae by the constructs created in this host contains Post-Translational Modifications.The constructed protein will be significantly similar to eukaryotic proteins, considering that they are identical epitopes. More comprehensive studies aiming to improve the effectiveness of this vaccine are being conducted to improve immune profiles and immunological memory stimulation in future designs.

Bacterial-Derived Outer Membrane Vesicles are Potent Adjuvants that Drive Humoral and Cellular Immune Responses.

Discovery and development of novel adjuvants that can improve existing or next generation vaccine platforms have received considerable interest in recent years. In particular, adjuvants that can elicit both humoral and cellular immune responses would be particularly advantageous because the majority of licensed vaccines are formulated with aluminum hydroxide (alum) which predominantly promotes antibodies. We previously demonstrated that bacterial-derived outer membrane vesicles (OMV) possess inherent adjuvanticity and drive antigen-specific antibody and cellular immune responses to OMV components. Here, we investigated the ability of OMVs to stimulate innate and adaptive immunity and to function as a stand-alone adjuvant. We show that OMVs are more potent than heat-inactivated and live-attenuated bacteria in driving dendritic cell activation in vitro and in vivo. Mice immunized with OMVs admixed with heterologous peptides generated peptide-specific CD4 and CD8 T cells responses. Notably, OMV adjuvant induced much greater antibody and B cell responses to co-delivered ovalbumin compared to the responses elicited by the adjuvants alum and CpG DNA. Additionally, pre-existing antibodies raised against the OMVs did not impair OMV adjuvanticity upon repeat immunization. These results indicate that vaccines adjuvanted with OMVs elicit robust cellular and humoral immune responses, supporting further development of OMV adjuvant for use in next-generation vaccines.

Modularly Programmable Nanoparticle Vaccine Based on Polyethyleneimine for Personalized Cancer Immunotherapy.

Nanoparticles (NPs) can serve as a promising vaccine delivery platform for improving pharmacological property and codelivery of antigens and adjuvants. However, NP‐based vaccines are generally associated with complex synthesis and postmodification procedures, which pose technical and manufacturing challenges for tailor‐made vaccine production. Here, modularly programmed, polyethyleneimine (PEI)‐based NP vaccines are reported for simple production of personalized cancer vaccines. Briefly, PEI is conjugated with neoantigens by facile coupling chemistry, followed by electrostatic assembly with CpG adjuvants, leading to the self‐assembly of nontoxic, sub‐50 nm PEI NPs. Importantly, PEI NPs promote activation and antigen cross‐presentation of antigen‐presenting cells and cross‐priming of neoantigen‐specific CD8+ T cells. Surprisingly, after only a single intratumoral injection, PEI NPs with optimal PEGylation elicit as high as ≈30% neoantigen‐specific CD8+ T cell response in the systemic circulation and sustain elevated CD8+ T cell response over 3 weeks. PEI‐based nanovaccines exert potent antitumor efficacy against pre‐established local tumors as well as highly aggressive metastatic tumors. PEI engineering for modular incorporation of neoantigens and adjuvants offers a promising strategy for rapid and facile production of personalized cancer vaccines.

Comprehensive mapping of immune tolerance yields a regulatory TNF receptor 2 signature in a murine model of successful Fel d 1-specific immunotherapy using high-dose CpG adjuvant.

BackgroundThe prevalence of allergy to cat is expanding worldwide. Allergen‐specific immunotherapy (AIT) has advantages over symptomatic pharmacotherapy and promises long‐lasting disease control in allergic patients. However, there is still a need to improve cat AIT regarding efficacy, safety, and adherence to the treatment. Here, we aim to boost immune tolerance to the major cat allergen Fel d 1 by increasing the anti‐inflammatory activity of AIT with the established immunomodulatory adjuvant CpG, but at a higher dose than previously used in AIT.MethodsTogether with CpG, we used endotoxin‐free Fel d 1 as therapeutic allergen throughout the study in a BALB/c model of allergy to Fel d 1, thus mimicking the conditions of human AIT trials. Multidimensional immune phenotyping including mass cytometry (CyTOF) was applied to analyze AIT‐specific immune signatures.ResultsWe show that AIT with high‐dose CpG in combination with endotoxin‐free Fel d 1 reverts all major hallmarks of allergy. High‐dimensional CyTOF analysis of the immune cell signatures initiating and sustaining the AIT effect indicates the simultaneous engagement of both, the pDC‐Treg and B‐cell axis, with the emergence of a systemic GATA3+ FoxP3hi biTreg population. The regulatory immune signature also suggests the involvement of the anti‐inflammatory TNF/TNFR2 signaling cascade in NK and B cells at an early stage and in Tregs later during AIT.ConclusionOur results highlight the potential of CpG adjuvant in a novel formulation to be further exploited for inducing allergen‐specific tolerance in patients with cat allergy or other allergic diseases.

Effects of rotavirus NSP4 protein on the immune response and protection of the SR69A-VP8* nanoparticle rotavirus vaccine

Rotavirus causes severe diarrhea and dehydration in young children. Even with the implementation of the current live vaccines, rotavirus infections still cause significant mortality and morbidity, indicating a need for new rotavirus vaccines with improved efficacy. To this end, we have developed an SR69A-VP8*/S60-VP8* nanoparticle rotavirus vaccine candidate that will be delivered parenterally with Alum adjuvant. In this study, as parts of our further development of this nanoparticle vaccine, we evaluated 1) roles of rotavirus nonstructural protein 4 (NSP4) that is the rotavirus enterotoxin, a possible vaccine target, and an immune stimulator, and 2) effects of CpG adjuvant that is a toll-like receptor 9 (TLR9) ligand and agonist on the immune response and protection of our SR69A-VP8*/S60-VP8* nanoparticle vaccine. The resulted vaccine candidates were examined for their IgG responses in mice. In addition, the resulted mouse sera were assessed for i) blocking titers against interactions of rotavirus VP8* proteins with their glycan ligands, ii) neutralization titers against rotavirus replication in cell culture, and iii) passive protection against rotavirus challenge with diarrhea in suckling mice. Our data showed that the Alum adjuvant appeared to work better with the SR69A-VP8*/S60-VP8* nanoparticles than the CpG adjuvant, while an addition of the NSP4 antigen to the SR69A-VP8*/S60-VP8* vaccine may not help to further increase the immune response and protection of the resulted vaccine.

A nanoparticle vaccine that targets neoantigen peptides to lymphoid tissues elicits robust antitumor T cell responses

Cancer vaccines using synthetic long peptides (SLP) targeting tumor antigens have been tested in the clinic but the outcomes have been unimpressive, perhaps because these peptides elicit predominantly CD4+ T cell responses. We hypothesized that enhanced delivery of peptide antigens to, and uptake in, secondary lymphoid tissues should elicit more robust CD8+ and CD4+ T cell responses and improved anti-tumor responses. Here, we have designed SLP-containing cationic lipoplexes (SLP–Lpx) that improve delivery of peptides to myeloid cells in the spleen and lymphatics. Using the G12D KRAS mutations as neoantigens, we found that vaccination of mice with naked synthetic peptides harboring the G12D mutation with CpG adjuvant stimulated mainly CD4+ T cell responses with limited tumor growth inhibition. On the other hand, immunization with SLP–Lpx stimulated both CD4+ and CD8+ T cells and suppressed tumor growth in a CD8+ T cell-dependent manner. Combination of the SLP–Lpx vaccines with a checkpoint inhibitor led to profound growth suppression of established tumors. These studies suggest that preferential targeting of peptides derived from neoantigens to the spleen via lipoplexes elicits potent CD4+ and CD8+ T cell responses that inhibit tumor growth.