Hyaluronic acid-bilirubin nanomedicine-based combination chemoimmunotherapy.

Despite significant advances in immune checkpoint blockade (ICB), immunosuppression mediated by tumor-associated myeloid cells (TAMCs) poses a major barrier to cancer immunotherapy. In addition, while immunogenic cell death (ICD) provides a viable approach to inducing anti-tumor immune response, it remains unknown how to effectively trigger ICD while addressing immunosuppressive TAMCs. Here, we show that SC144, a gp130 inhibitor that blocks the IL-6/gp130/STAT3 pathway, induces ICD of tumor cells and polarizes macrophages to M1-phenotype in vitro. However, as SC144 also induces killing of CD8+ T-cells, we sought to deliver SC144 selectively to tumor cells and TAMCs. Toward this goal, we have developed hyaluronic acid-bilirubin nanoparticles (HABN) that accumulate in CD44hi tumor cells and TAMCs. Systemic administration of SC144 loaded in HABN (SC144@HABN) induces apoptosis and ICD of tumor cells, increases the ratio of M1-like to M2-like macrophages, and decreases the frequency of myeloid-derived suppressor cells and CD4+ regulatory T-cells, while promoting anti-tumor CD8+ T-cells. Moreover, SC144@HABN combined with anti-PD-L1 ICB efficiently eliminates MC38 tumors and ICB-resistant 4T1 tumors. Overall, our work demonstrates a therapeutic strategy based on coordinated ICD induction and TAMC modulation and highlights the potential of combination chemoimmunotherapy.

STING agonist-loaded mesoporous manganese-silica nanoparticles for vaccine applications.

Cyclic dinucleotides (CDNs), as one type of Stimulator of Interferon Genes (STING) pathway agonist, have shown promising results for eliciting immune responses against cancer and viral infection. However, the suboptimal drug-like properties of conventional CDNs, including their short in vivo half-life and poor cellular permeability, compromise their therapeutic efficacy. In this study, we have developed a manganese-silica nanoplatform (MnOx@HMSN) that enhances the adjuvant effects of CDN by achieving synergy with Mn2+ for vaccination against cancer and SARS-CoV-2. MnOx@HMSN with large mesopores were efficiently co-loaded with CDN and peptide/protein antigens. MnOx@HMSN(CDA) amplified the activation of the STING pathway and enhanced the production of type-I interferons and other proinflammatory cytokines from dendritic cells. MnOx@HMSN(CDA) carrying cancer neoantigens elicited robust antitumor T-cell immunity with therapeutic efficacy in two different murine tumor models. Furthermore, MnOx@HMSN(CDA) loaded with SARS-CoV-2 antigen achieved strong and durable (up to one year) humoral immune responses with neutralizing capability. These results demonstrate that MnOx@HMSN(CDA) is a versatile nanoplatform for vaccine applications.

Cancer stem cell antigen nanodisc cocktail elicits anti-tumor immune responses in melanoma.

One of the major reasons for poor cancer outcomes is the existence of cancer stem cells (CSCs). CSCs are a small subpopulation of tumor cells that can self-renew, differentiate into the majority of tumor cells, and maintain tumorigenicity. As CSCs are resistant to traditional chemotherapy and radiation, they contribute to metastasis and relapse. Thus, new approaches are needed to target and eliminate CSCs. Here, we sought to target and reduce the frequency of CSCs in melanoma by therapeutic vaccination against CSC-associated transcription factors, such as Sox2 and Nanog, and aldehyde dehydrogenase (ALDH). Toward this goal, we have identified novel immunogenic peptide epitopes derived from CSC-associated Sox2 and Nanog and synthesized synthetic high-density lipoprotein (sHDL) nanodisc vaccine formulated with Sox2, Nanog, and ALDH antigen peptides together with CpG, a Toll-like receptor 9 agonist. Vaccination with nanodiscs containing six CSC antigen peptides elicited robust T cell responses against CSC-associated antigens and promoted intratumoral infiltration of CD8+ T cells, while reducing the frequency of CSCs and CD4+ regulatory T cells within melanoma tumors. Nanodisc vaccination effectively reduced tumor growth and significantly extended animal survival without toxicity toward normal stem cells. Overall, our therapeutic strategy against CSCs represents a cost-effective, safe, and versatile approach that may be applied to melanoma and other cancer types, as well as serve as a critical component in combined therapies to target and eliminate CSCs.

Distinctive populations of CD4+T cells associated with vaccine efficacy.

Memory T cells underpin vaccine-induced immunity but are not yet fully understood. To distinguish features of memory cells that confer protective immunity, we used single cell transcriptome analysis to compare antigen-specific CD4+T cells recalled to lungs of mice that received a protective or nonprotective subunit vaccine followed by challenge with a fungal pathogen. We unexpectedly found populations specific to protection that expressed a strong type I interferon response signature, whose distinctive transcriptional signature appeared unconventionally dependent on IFN-γ receptor. We also detected a unique population enriched in protection that highly expressed the gene for the natural killer cell marker NKG7. Lastly, we detected differences in TCR gene use and in Th1- and Th17-skewed responses after protective and nonprotective vaccine, respectively, reflecting heterogeneous Ifng- and Il17a-expressing populations. Our findings highlight key features of transcriptionally diverse and distinctive antigen-specific T cells associated with protective vaccine-induced immunity.

SLAMF1 Is Dispensable for Vaccine-Induced T Cell Development but Required for Resistance to Fungal Infection.

Homotypic signaling lymphocyte activation molecule (SLAM) receptor-ligand cell surface interactions between myeloid and lymphoid cells regulate innate and adaptive immune responses. In this article, we report that SLAMF1 is indispensable for host resistance to primary and vaccine-induced protection against fungal infection. Because vaccine immunity is dependent on cell-mediated immunity, we investigated the development of Ag-specific T cells. We studied the T cell-intrinsic and -extrinsic role of SLAMF1. We generated SLAMF1-/- TCR transgenic mice and analyzed the responses of adoptively transferred T cells. We also tracked endogenous Ag-specific T cells by using a tetramer. Intrinsic and extrinsic SLAMF1 signaling was dispensable for the development of antifungal Th1 and Th17 cells, which are requisite for the acquisition of vaccine-induced immunity. Despite intact T cell development, vaccinated SLAMF1-/- mice failed to control fungal infection. Failed accumulation of Ag-specific T cells in the lung on infection of vaccinated mice was due to uncontrolled early infection and inflammation, revealing a role for SLAMF1 in innate host immunity.

Targeting cancer stem cells via integrin ß4.

Integrins mediate cell-cell interactions and communication with the extracellular matrix (ECM). These transmembrane protein receptors allow binding between a cell and its surroundings, initiating a breadth of intracellular signaling resulting in proliferation, differentiation, survival, or migration. Such responses have made integrins an attractive target for cancer therapy. Self-renewing and highly tumorigenic cancer stem cells (CSCs) are most resistant to traditional radiation treatment and chemotherapy, and therefore may contribute directly to the metastasis and relapse of the disease. In both the 4T1 mouse metastatic mammary tumor model and SCC7 head and neck squamous cell carcinoma model, integrin ß4 (ITGB4) was expressed on ALDHhigh 4T1 and SCC7 CSCs. Using two immunological approaches, we targeted ITGB4 through 1) ITGB4 protein-pulsed dendritic cell (ITGB4-DC) vaccination or 2) via anti-CD3/anit-ITGB4 bispecific antibody (ITGB4 BiAb)-armed T cell adoptive transfer. These two therapies reduced ITGB4-expressing CSCs and inhibited local tumor growth and lung metastasis through ITGB4 specific cellular and humoral immune responses. Additionally, the combination of anti-PD-L1 immunotherapy with our two ITGB4-targeted approaches significantly improved treatment efficacy. We also found increased concentrations of serum IFN-γ and IL-6 in the 4T1 and SCC7 models which may help define future directions of this ITGB4-targeted study. Together, these results emphasize ITGB4 as a practical CSC immunological target with possible therapeutic benefits across tumor types with high ITGB4 expression.

Combination Adjuvants Enhance Recombinant Protein Vaccine Protection against Fungal Infection.

The development of effective vaccines against fungal infections requires the induction of protective, pathogen-specific cell-mediated immune responses. Here, we asked whether combination adjuvants based on delta inulin (Advax) formulated with Toll-like receptor (TLR) agonists could improve vaccine protection mediated by a fungal recombinant protein, Bl-Eng2 (i.e., Blastomyces endoglucanase 2), which itself harbors an immunodominant antigen and dectin-2 agonist/adjuvant. We found that Bl-Eng2 formulated with Advax3 containing TLR9 agonist or Advax8 containing TLR4 agonist provided the best protection against pulmonary infection with Blastomyces dermatitidis, being more effective than complete Freund's adjuvant or Adjuplex. Advax3 was most efficient in inducing gamma interferon (IFN-γ)- and interleukin-17 (IL-17)-producing antigen-specific T cells that migrated to the lung upon Blastomyces dermatitidis infection. Mechanistic studies revealed Bl-Eng2/Advax3 protection was tempered by neutralization of IL-17 and particularly IFN-γ. Likewise, greater numbers of lung-resident T cells producing IFN-γ, IL-17, or both IFN-γ and IL-17 correlated with fewer fungi recovered from lung. Protection was maintained after depletion of CD4+ T cells, partially reduced by depletion of CD8+ T cells, and completely eliminated after depletion of both CD4+ and CD8+ T cells. We conclude that Bl-Eng2 formulated with Advax3 is promising for eliciting vaccine-induced antifungal immunity, through a previously uncharacterized mechanism involving CD8+ and also CD4+ T cells producing IFN-γ and/or IL-17. Although no licensed vaccine exists as yet against any fungal disease, these findings indicate the importance of adjuvant selection for the development of effective fungal vaccines. IMPORTANCE Fungal disease remains a challenging clinical and public health problem. Despite medical advances, invasive fungal infections have skyrocketed over the last decade and pose a mounting health threat in immunocompetent and -deficient hosts, with worldwide mortality rates ranking 7th, even ahead of tuberculosis. The development of safe, effective vaccines remains a major hurdle for fungi. Critical barriers to progress include the lack of defined fungal antigens and suitable adjuvants. Our research is significant in identifying adjuvant combinations that elicit optimal vaccine-induced protection when formulated with a recombinant protective antigen and uncovering the mechanistic bases of the underlaying vaccine protection, which will foster the strategic development of antifungal vaccines.

HER2-targeted antibody-drug conjugate induces host immunity against cancer stem cells.

We previously tested HER2-targeted antibody-drug conjugates (ADCs) in immunocompromised (SCID) mice, precluding evaluation of host immunity, impact on cancer stem cells (CSCs), and potential benefit when combined with PD-L1 blockade. In this study, we tested HER2-targeted ADC in two immunocompetent mouse tumor models. HER2-targeted ADC specifically inhibited the growth of HER2-expressing tumors, prolonged animal survival, and reduced HER2+ and PD-L1+ cells. ADC + anti-PD-L1 antibody augmented therapeutic efficacy, modulated immune gene signatures, increased the number and function of CD3+ and CD19+ tumor-infiltrating lymphocytes (TILs), induced tumor antigen-specific immunological memory, stimulated B cell activation, differentiation, and IgG1 production both systemically and in the tumor microenvironment. In addition, ADC therapy modulated T cell subsets and their activation in TILs. Furthermore, HER2-targeted ADC reduced the number and tumorigenicity of ALDHhi CSCs. This study demonstrates that HER2-targeted ADC effectively targets ALDHhi CSCs and this effect is augmented by co-administration of anti-PD-L1 antibody.

Structural basis of Blastomyces Endoglucanase-2 adjuvancy in anti-fungal and -viral immunity.

The development of safe subunit vaccines requires adjuvants that augment immunogenicity of non-replicating protein-based antigens. Current vaccines against infectious diseases preferentially induce protective antibodies driven by adjuvants such as alum. However, the contribution of antibody to host defense is limited for certain classes of infectious diseases such as fungi, whereas animal studies and clinical observations implicate cellular immunity as an essential component of the resolution of fungal pathogens. Here, we decipher the structural bases of a newly identified glycoprotein ligand of Dectin-2 with potent adjuvancy, Blastomyces endoglucanase-2 (Bl-Eng2). We also pinpoint the developmental steps of antigen-specific CD4+ and CD8+ T responses augmented by Bl-Eng2 including expansion, differentiation and tissue residency. Dectin-2 ligation led to successful systemic and mucosal vaccination against invasive fungal infection and Influenza A infection, respectively. O-linked glycans on Bl-Eng2 applied at the skin and respiratory mucosa greatly augment vaccine subunit- induced protective immunity against lethal influenza and fungal pulmonary challenge.

Antigen discovery unveils resident memory and migratory cell roles in antifungal resistance.

Priming at the site of natural infection typically elicits a protective T cell response against subsequent pathogen encounter. Here, we report the identification of a novel fungal antigen that we harnessed for mucosal vaccination and tetramer generation to test whether we can elicit protective, antigen-specific tissue-resident memory (Trm) CD4+ T cells in the lung parenchyma. In contrast to expectations, CD69+, CXCR3+, CD103- Trm cells failed to protect against a lethal pulmonary fungal infection. Surprisingly, systemic vaccination induced a population of tetramer+ CD4+ T cells enriched within the pulmonary vasculature, and expressing CXCR3 and CX3CR1, that migrated to the lung tissue upon challenge and efficiently protected mice against infection. Mucosal vaccine priming of Trm may not reliably protect against mucosal pathogens.

Virally-vectored vaccine candidates against white-nose syndrome induce anti-fungal immune response in little brown bats (Myotis lucifugus).

White-nose syndrome (WNS) caused by the fungus, Pseudogymnoascus destructans (Pd) has killed millions of North American hibernating bats. Currently, methods to prevent the disease are limited. We conducted two trials to assess potential WNS vaccine candidates in wild-caught Myotis lucifugus. In a pilot study, we immunized bats with one of four vaccine treatments or phosphate-buffered saline (PBS) as a control and challenged them with Pd upon transfer into hibernation chambers. Bats in one vaccine-treated group, that received raccoon poxviruses (RCN) expressing Pd calnexin (CAL) and serine protease (SP), developed WNS at a lower rate (1/10) than other treatments combined (14/23), although samples sizes were small. The results of a second similar trial provided additional support for this observation. Bats vaccinated orally or by injection with RCN-CAL and RCN-SP survived Pd challenge at a significantly higher rate (P = 0.01) than controls. Using RT-PCR and flow cytometry, combined with fluorescent in situ hybridization, we determined that expression of IFN-γ transcripts and the number of CD4 + T-helper cells transcribing this gene were elevated (P < 0.10) in stimulated lymphocytes from surviving vaccinees (n = 15) compared to controls (n = 3). We conclude that vaccination with virally-vectored Pd antigens induced antifungal immunity that could potentially protect bats against WNS.