Fc-silent anti-TIGIT antibodies potentiate anti-tumor immunity without depleting regulatory T cells.

TIGIT is an inhibitory receptor on immune cells that outcompetes an activating receptor, CD226, for shared ligands. Tumor-infiltrating lymphocytes express TIGIT and CD226 on regulatory T cells (Treg) and on CD8+ T cells with tumor-reactive or exhausted phenotypes, supporting the potential of therapeutically targeting TIGIT to enhance anti-tumor immunity. To optimize the efficacy of therapeutic antibodies against TIGIT, it is necessary to understand whether there is therapeutic benefit from Fcγ receptor (FcγR) binding. Here, we showed that combining Fc-enabled (Fce) or Fc-silent (Fcs) anti-TIGIT with anti-PD-1 in mice resulted in enhanced control of tumors by differential mechanisms: Fce anti-TIGIT promoted depletion of intratumoral Treg, whereas Fcs anti-TIGIT did not. Despite leaving Treg numbers intact, Fcs anti-TIGIT potentiated activation of tumor-specific exhausted CD8+ populations in a lymph node-dependent manner. Fce anti-TIGIT induced antibody-dependent cell-mediated cytotoxicity against human Treg in vitro, and significant decreases in Treg were measured in the peripheral blood of Phase I solid tumor cancer patients treated with Fce anti-TIGIT. In contrast, Fcs anti-TIGIT did not deplete human Treg in vitro and was associated with anecdotal objective clinical responses in two Phase I solid tumor cancer patients in whom peripheral Treg frequencies remained stable on treatment. Collectively, these data provide evidence of pharmacological activity and anti-tumor efficacy of anti-TIGIT antibodies lacking the ability to engage FcγR.

mRNA Vaccine Nanoplatforms and Innate Immunity.

mRNA-based vaccine technology has been significantly developed and enhanced, particularly highlighted by the authorization of mRNA vaccines for addressing the COVID-19 pandemic. Various biomaterials are developed in nano-scales and applied as mRNA vaccine delivery platforms. However, how these mRNA nanoplatforms influence immune responses has not been thoroughly studied. Hence, we have reviewed the current understanding of various mRNA vaccine platforms. We discussed the possible pathways through which these platforms moderate the host's innate immunity and contribute to the development of adaptive immunity. We shed light on their development in reducing biotoxicity and enhancing antigen delivery efficiency. Beyond the built-in adjuvanticity of mRNA vaccines, we propose that supplementary adjuvants may be required to fine-tune and precisely control innate immunity and subsequent adaptive immune responses.

Perturbational phenotyping of human blood cells reveals genetically determined latent traits associated with subsets of common diseases.

Although genome-wide association studies (GWAS) have successfully linked genetic risk loci to various disorders, identifying underlying cellular biological mechanisms remains challenging due to the complex nature of common diseases. We established a framework using human peripheral blood cells, physical, chemical and pharmacological perturbations, and flow cytometry-based functional readouts to reveal latent cellular processes and performed GWAS based on these evoked traits in up to 2,600 individuals. We identified 119 genomic loci implicating 96 genes associated with these cellular responses and discovered associations between evoked blood phenotypes and subsets of common diseases. We found a population of pro-inflammatory anti-apoptotic neutrophils prevalent in individuals with specific subsets of cardiometabolic disease. Multigenic models based on this trait predicted the risk of developing chronic kidney disease in type 2 diabetes patients. By expanding the phenotypic space for human genetic studies, we could identify variants associated with large effect response differences, stratify patients and efficiently characterize the underlying biology.

Influenza immune imprinting synergizes PEI-HA/CpG nanoparticle vaccine protection against heterosubtypic infection in mice.

The first influenza virus infection (imprinting) can lead to long-term immune memory and influence subsequent vaccinations and infections. Previously, we reported a polyethyleneimine (PEI)-Aichi hemagglutinin (HA)/CpG (PHC) nanoparticle with cross-protective potential against homologous and heterologous influenza strains. Here we studied how influenza immune imprinting influences the antibody responses to the PHC vaccination and the protection against heterosubtypic virus challenges. We found that pre-existing virus immunity can maintain or synergize the vaccine-induced antibody titers, depending on the imprinting virus HA phylogenetic group. The HA group 1 virus (PR8, H1N1)-imprinted mice displayed comparable antigen-specific antibody responses to those without imprinting post-PHC vaccination. In contrast, the group 2 virus (Phi, H3N2)-imprinted mice showed significantly more robust and balanced antibodies post-vaccination, conferring complete protection against body weight loss and lung inflammation upon heterosubtypic reassortant A/Shanghai/2/2013 (rSH, H7N9) virus challenge. Our findings suggest that influenza imprinting from the same HA phylogenetic group can synergize subsequent vaccination, conferring heterosubtypic protection.

GCLiPP: global crosslinking and protein purification method for constructing high-resolution occupancy maps for RNA binding proteins.

GCLiPP is a global RNA interactome capture method that detects RNA-binding protein (RBP) occupancy transcriptome-wide. GCLiPP maps RBP-occupied sites at a higher resolution than phase separation-based techniques. GCLiPP sequence tags correspond with known RBP binding sites and are enriched for sites detected by RBP-specific crosslinking immunoprecipitation (CLIP) for abundant cytosolic RBPs. Comparison of human Jurkat T cells and mouse primary T cells uncovers shared peaks of GCLiPP signal across homologous regions of human and mouse 3' UTRs, including a conserved mRNA-destabilizing cis-regulatory element. GCLiPP signal overlapping with immune-related SNPs uncovers stabilizing cis-regulatory regions in CD5, STAT6, and IKZF1.

The lncRNA Malat1 inhibits miR-15/16 to enhance cytotoxic T cell activation and memory cell formation.

Proper activation of cytotoxic T cells via the T cell receptor and the costimulatory receptor CD28 is essential for adaptive immunity against viruses, intracellular bacteria, and cancers. Through biochemical analysis of RNA:protein interactions, we uncovered a non-coding RNA circuit regulating activation and differentiation of cytotoxic T cells composed of the long non-coding RNA Malat1 (Metastasis Associated Lung Adenocarcinoma Transcript 1) and the microRNA family miR-15/16. miR-15/16 is a widely and highly expressed tumor suppressor miRNA family important for cell proliferation and survival. miR-15/16 play important roles in T cell responses to viral infection, including the regulation of antigen-specific T cell expansion and memory. Comparative Argonaute-2 high-throughput sequencing of crosslinking immunoprecipitation (AHC) combined with gene expression profiling in normal and miR-15/16-deficient mouse T cells revealed a large network of hundreds of direct miR-15/16 target mRNAs, many with functional relevance for T cell activation, survival and memory formation. Among these targets, Malat1 contained the largest absolute magnitude miR-15/16-dependent AHC peak. This binding site was among the strongest lncRNA:miRNA interactions detected in the T cell transcriptome. We used CRISPR targeting with homology directed repair to generate mice with a 5-nucleotide mutation in the miR-15/16-binding site in Malat1. This mutation interrupted Malat1:miR-15/16 interaction, and enhanced the repression of other miR-15/16 target genes, including CD28. Interrupting Malat1 interaction with miR-15/16 decreased cytotoxic T cell activation, including the expression of interleukin 2 (IL-2) and a broader CD28-responsive gene program. Accordingly, Malat1 mutation diminished memory cell persistence in mice following LCMV Armstrong and Listeria monocytogenes infection. This study marks a significant advance in the study of long non-coding RNAs in the immune system by ascribing cell-intrinsic, sequence-specific in vivo function to Malat1. These findings have implications for T cell-mediated autoimmune diseases, antiviral and anti-tumor immunity, as well as lung adenocarcinoma and other malignancies where Malat1 is overexpressed.

Modeling of large-scale hoxbb cluster deletions in zebrafish uncovers a role for segmentation pathways in atrioventricular boundary specification.

Hox genes orchestrate the segmental specification of the muscular circulatory system in invertebrates but it has not proven straightforward to decipher segmental parallels in the vertebrate heart. Recently, patients with HOXB gene cluster deletion were found to exhibit abnormalities including atrioventricular canal defects. Using CRISPR, we established a mutant with the orthologous hoxbb cluster deletion in zebrafish. The mutant exhibited heart failure and atrioventricular regurgitation at 5 days. Analyzing the four genes in the hoxbb cluster, isolated deletion of hoxb1b-/- recapitulated the cardiac abnormalities, supporting hoxb1b as the causal gene. Both in situ and in vitro data indicated that hoxb1b regulates gata5 to inhibit hand2 expression and ultimately is required to pattern the vertebrate atrioventricular boundary. Together, these data reveal a role for segmental specification in vertebrate cardiac development and highlight the utility of CRISPR techniques for efficiently exploring the function of large structural genomic lesions.

RNA circuits and RNA-binding proteins in T cells.

RNA is integral to the regulatory circuits that control cell identity and behavior. Cis-regulatory elements in mRNAs interact with RNA-binding proteins (RBPs) that can alter RNA sequence, stability, and translation into protein. Similarly, long noncoding RNAs (lncRNAs) scaffold ribonucleoprotein complexes that mediate transcriptional and post-transcriptional regulation of gene expression. Indeed, cell programming is fundamental to multicellular life and, in this era of cellular therapies, it is of particular interest in T cells. Here, we review key concepts and recent advances in our understanding of the RNA circuits and RBPs that govern mammalian T cell differentiation and immune function.

The lncRNA Malat1 Inhibits miR-15/16 to Enhance Cytotoxic T Cell Activation and Memory Cell Formation.

Proper activation of cytotoxic T cells via the T cell receptor and the costimulatory receptor CD28 is essential for adaptive immunity against viruses, many intracellular bacteria and cancers. Through biochemical analysis of RNA:protein interactions, we uncovered a non-coding RNA circuit regulating activation and differentiation of cytotoxic T cells composed of the long non-coding RNA Malat1 (Metastasis Associated Lung Adenocarcinoma Transcript 1) and the microRNA family miR-15/16. miR-15/16 is a widely and highly expressed tumor suppressor miRNA family important for cell proliferation and survival. miR-15/16 also play important roles in T cell responses to viral infection, including the regulation of antigen-specific T cell expansion and T cell memory. Comparative Argonaute-2 high throughput sequencing of crosslinking immunoprecipitation (Ago2 HITS-CLIP, or AHC) combined with gene expression profiling in normal and miR-15/16-deficient T cells revealed a large network of several hundred direct miR-15/16 target mRNAs, many with functional relevance for T cell activation, survival and memory formation. Among these targets, the long non-coding RNA Malat1 contained the largest absolute magnitude miR-15/16-dependent AHC peak in T cells. This binding site was also among the strongest lncRNA:miRNA interactions detected in the T cell transcriptome. We used CRISPR targeting with homology directed repair to generate mice with a 5-nucleotide mutation in the miR-15/16 binding site in Malat1. This mutation interrupted Malat1:miR-15/16 interaction, and enhanced the repression of other miR-15/16 target genes, including CD28. Interrupting Malat1 interaction with miR-15/16 decreased cytotoxic T cell activation, including the expression of IL-2 and a broader CD28-responsive gene program. Accordingly, Malat1 mutation diminished memory cell persistence following LCMV Armstrong and Listeria monocytogenes infection. This study marks a significant advance in the study of long noncoding RNAs in the immune system by ascribing cell-intrinsic, sequence-specific in vivo function to Malat1. These findings have implications for T cell-mediated autoimmune diseases, antiviral and anti-tumor immunity, as well as lung adenocarcinoma and other malignancies where Malat1 is overexpressed.

Stromal STAT5-Mediated Trophic Activity Regulates Hematopoietic Niche Factors.

Signal transducer and activator of transcription 5 (STAT5a and STAT5b) are intrinsically critical for normal hematopoiesis but are also expressed in stromal cells. Here, STAT5ab knockout (KO) was generated with a variety of bone marrow hematopoietic and stromal Cre transgenic mouse strains. Vav1-Cre/+STAT5abfl/fl, the positive control for loss of multipotent hematopoietic function, surprisingly dysregulated niche factor mRNA expression, and deleted STAT5ab in CD45neg cells. Single-cell transcriptome analysis of bone marrow from Vav1-Cre/+ wild-type or Vav1-Cre/+STAT5abfl/fl mice showed hematopoietic stem cell (HSC) myeloid commitment priming. Nes+ cells were detected in both CD45neg and CD45+ clusters and deletion of STAT5ab with Nes-Cre caused hematopoietic repopulating defects. To follow up on these promiscuous Cre promoter deletions in CD45neg and CD45+ bone marrow cell populations, more stroma-specific Cre strains were generated and demonstrated a reduction in multipotent hematopoietic progenitors. Functional support for niche-supporting activity was assessed using STAT5-deficient mesenchymal stem cells (MSCs). With Lepr-Cre/+STAT5abfl/fl, niche factor mRNAs were downregulated with validation of reduced IGF-1 and CXCL12 proteins. Furthermore, advanced computational analyses revealed a key role for STAT5ab/Cish balance with Cish strongly co-expressed in MSCs and HSCs primed for differentiation. Therefore, STAT5ab-associated gene regulation supports the bone marrow microenvironment.

Stimulation of ectopically expressed muscarinic receptors induces IFN-γ but suppresses IL-2 production by inhibiting activation of pAKT pathways in primary T cells.

T cell antigen receptor stimulation induces tyrosine phosphorylation of downstream signaling molecules and the phosphatidylinositol, Ras, MAPK, and PI3 kinase pathways, leading to T cell activation. Previously, we reported that the G-protein-coupled human muscarinic receptor could bypass tyrosine kinases to activate the phosphatidylinositol pathway and induce interleukin-2 production in Jurkat leukemic T cells. Here, we demonstrate that stimulating G-protein-coupled muscarinic receptors (M1 and synthetic hM3Dq) can activate primary mouse T cells if PLCß1 is coexpressed. Resting peripheral hM3Dq+PLCß1 (hM3Dq/ß1) T cells did not respond to clozapine, an hM3Dq agonist, unless they were preactivated by TCR and CD28 stimulation which increased hM3Dq and PLCß1 expression. This permitted large calcium and phosphorylated ERK responses to clozapine. Clozapine treatment induced high IFN-γ, CD69, and CD25 expression, but surprisingly did not induce substantial IL-2 in hM3Dq/ß1 T cells. Importantly, costimulation of both muscarinic receptors plus the TCR even led to reduced IL-2 expression, suggesting a selective inhibitory effect of muscarinic receptor costimulation. Stimulation of muscarinic receptors induced strong nuclear translocation of NFAT and NFκB and activated AP-1. However, stimulation of hM3Dq led to reduced IL-2 mRNA stability which correlated with an effect on the IL-2 3'UTR activity. Interestingly, stimulation of hM3Dq resulted in reduced pAKT and its downstream pathway. This may explain the inhibitory impact on IL-2 production in hM3Dq/ß1T cells. Moreover, an inhibitor of PI3K reduced IL-2 production in TCR-stimulated hM3Dq/ß1 CD4 T cells, suggesting that activating the pAKT pathway is critical for IL-2 production in T cells.

ISCOMs/MPLA-Adjuvanted SDAD Protein Nanoparticles Induce Improved Mucosal Immune Responses and Cross-Protection in Mice.

The epidemics caused by the influenza virus are a serious threat to public health and the economy. Adding appropriate adjuvants to improve immunogenicity and finding effective mucosal vaccines to combat respiratory infection at the portal of virus entry are important strategies to boost protection. In this study, a novel type of core/shell protein nanoparticle consisting of influenza nucleoprotein (NP) as the core and NA1-M2e or NA2-M2e fusion proteins as the coating antigens by SDAD hetero-bifunctional crosslinking is exploited. Immune-stimulating complexes (ISCOMs)/monophosphoryl lipid A (MPLA) adjuvants further boost the NP/NA-M2e SDAD protein nanoparticle-induced immune responses when administered intramuscularly. The ISCOMs/MPLA-adjuvanted protein nanoparticles are delivered through the intranasal route to validate the application as mucosal vaccines. ISCOMs/MPLA-adjuvanted nanoparticles induce significantly strengthened antigen-specific antibody responses, cytokine-secreting splenocytes in the systemic compartment, and higher levels of antigen-specific IgA and IgG in the local mucosa. Meanwhile, significantly expanded lung resident memory (RM) T and B cells (TRM /BRM ) and alveolar macrophages population are observed in ISCOMs/MPLA-adjuvanted nanoparticle-immunized mice with a 100% survival rate after homogeneous and heterogeneous H3N2 viral challenges. Taken together, ISCOMs/MPLA-adjuvanted protein nanoparticles could improve strong systemic and mucosal immune responses conferring protection in different immunization routes.

Chinese herbal medicine for the treatment of cardiovascular diseases ─ Targeting cardiac ion channels.

Cardiovascular disease (CVD) remains the leading cause of morbidity and mortality, imposing an increasing global health burden. Cardiac ion channels (voltage-gated NaV, CaV, KVs, and others) synergistically shape the cardiac action potential (AP) and control the heartbeat. Dysfunction of these channels, due to genetic mutations, transcriptional or post-translational modifications, may disturb the AP and lead to arrhythmia, a major risk for CVD patients. Although there are five classes of anti-arrhythmic drugs available, they can have varying levels of efficacies and side effects on patients, possibly due to the complex pathogenesis of arrhythmias. As an alternative treatment option, Chinese herbal remedies have shown promise in regulating cardiac ion channels and providing anti-arrhythmic effects. In this review, we first discuss the role of cardiac ion channels in maintaining normal heart function and the pathogenesis of CVD, then summarize the classification of Chinese herbal compounds, and elaborate detailed mechanisms of their efficacy in regulating cardiac ion channels and in alleviating arrhythmia and CVD. We also address current limitations and opportunities for developing new anti-CVD drugs based on Chinese herbal medicines.

Monophosphoryl lipid A-adjuvanted nucleoprotein-neuraminidase nanoparticles improve immune protection against divergent influenza viruses.

Universal influenza vaccines are urgently needed to prevent recurrent influenza epidemics and inevitable pandemics. We generated double-layered protein nanoparticles incorporating two conserved influenza antigens-nucleoprotein and neuraminidase-through a two-step desolvation-crosslinking method. These protein nanoparticles displayed immunostimulatory properties to antigen-presenting cells by promoting inflammatory cytokine (IL-6 and TNF-α) secretion from JAWS II dendric cells. The nanoparticle immunization induced significant antigen-specific humoral and cellular responses, including antigen-binding and neutralizing antibodies, antibody- and cytokine (IFN-γ and IL-4)-secreting cells, and NP147-155 tetramer-specific cytotoxic T lymphocyte (CTL) responses. Co-administration of monophosphoryl lipid A (MPLA, a toll-like receptor 4 agonist) with the protein nanoparticles further improved immune responses and conferred heterologous and heterosubtypic influenza protection. The MPLA-adjuvanted nanoparticles reduced lung inflammation post-infection. The results demonstrated that the combination of MPLA and conserved protein nanoparticles could be developed into an improved universal influenza vaccine strategy.

cGAMP-adjuvanted multivalent influenza mRNA vaccines induce broadly protective immunity through cutaneous vaccination in mice.

Increasing preclinical and clinical results have demonstrated that mRNA vaccines efficiently prevent infectious diseases and are safe in animal models and humans. In this study, we fabricated a multivalent influenza mRNA lipid nanoparticle (LNP) vaccine with mRNAs of hemagglutinins from influenza H1N1 and H3N2 viruses, matrix protein 1, and nucleoprotein. We found that cutaneous immunization with mRNA LNPs induced strong Th1 and Th2 cellular immunity with robust antigen-specific antibody titers and increased cytokine-secreting splenocytes and antibody-secreting cells. The supplement of cGAMP improved the immunogenicity of mRNA LNPs. Compared with αGC or cGAMP/αGC adjuvanted mRNA LNP formulations in our study, cGAMP mRNA LNPs induced more robust antibody responses. Enhanced cellular immunity with more IL-4 and IFN-γ secreting cells and effector memory T cell populations in spleens, as well as increased CD4+ resident memory (TRM) T cells in lungs were observed in cGAMP mRNA LNPs immunized group. These results demonstrated that cGAMP is an effective adjuvant for cutaneous vaccination of multivalent mRNA LNP vaccines in mice to induce stronger immune responses in the spleen and lung, and the cGAMP-adjuvanted mRNA LNPs protected against homologous and heterologous viral infection.

Layered protein nanoparticles containing influenza B HA stalk induced sustained cross-protection against viruses spanning both viral lineages.

The influenza epidemics pose a significant threat to public health. Of them, type B influenza coincided with several severe flu outbreaks. The efficacy of the current seasonal flu vaccine is limited due to the antigenicity changes of circulating strains. In this study, we generated structure-stabilized HA stalk antigens from influenza B and fabricated double-layered protein nanoparticles as universal influenza B vaccine candidates. In vitro studies found that the resulting protein nanoparticles were effectively taken up to activate dendritic cells. Nanoparticle immunization induced broadly reactive immune responses conferring robust and sustained cross-immune protection against influenza B virus strains of both lineages. The results reveal the potential of layered protein nanoparticles incorporated with structure-stabilized constant antigens as a universal influenza vaccine with improved immune protective potency and breadth.

Single Cell Biology: Exploring Somatic Cell Behaviors, Competition and Selection in Chronic Disease.

The full range of cell functions is under-determined in most human diseases. The evidence that somatic cell competition and clonal imbalance play a role in non-neoplastic chronic disease reveal a need for a dedicated effort to explore single cell function if we are to understand the mechanisms by which cell population behaviors influence disease. It will be vital to document not only the prevalent pathologic behaviors but also those beneficial functions eliminated or suppressed by competition. An improved mechanistic understanding of the role of somatic cell biology will help to stratify chronic disease, define more precisely at an individual level the role of environmental factors and establish principles for prevention and potential intervention throughout the life course and across the trajectory from wellness to disease.

SARS-CoV-2 Spike Stem Protein Nanoparticles Elicited Broad ADCC and Robust Neutralization against Variants in Mice.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused the global pandemic. The virus is rapidly evolving, characterized by the emergence of several major variants. Stable prefusion spike protein (Pre) is the immunogen in current vaccines but is limited in protecting against different variants. Here, the immune responses induced by the relatively conserved stem subunit (S2) of spike protein versus Pre are investigated. Pre generates the most robust neutralization responses against SARS-CoV-2 variants in vesicular stomatitis virus pseudovirus-based assessment but elicits less antibody-dependent cellular cytotoxicity (ADCC) activity than S2. By contrast, S2 induces the most balanced immunoglobulin G (IgG) antibodies with potent and broad ADCC activity although produces weaker neutralization. The immunogenicity of S2 and Pre improves by incorporating the two proteins into double-layered protein nanoparticles. The resulting protein nanoparticles Pre/S2 elicit higher neutralizing antibodies than Pre alone, and stronger ADCC than S2 alone. Moreover, nanoparticles produce more potent and balanced serum IgG antibodies than the corresponding soluble protein mixture, and the immune responses are sustained for at least four months after the immunization. Thus, the double-layered protein nanoparticles have the potential to be developed into broader SARS-CoV-2 vaccines with excellent safety profiles.

Obesity alters pathology and treatment response in inflammatory disease.

Decades of work have elucidated cytokine signalling and transcriptional pathways that control T cell differentiation and have led the way to targeted biologic therapies that are effective in a range of autoimmune, allergic and inflammatory diseases. Recent evidence indicates that obesity and metabolic disease can also influence the immune system1-7, although the mechanisms and effects on immunotherapy outcomes remain largely unknown. Here, using two models of atopic dermatitis, we show that lean and obese mice mount markedly different immune responses. Obesity converted the classical type 2 T helper (TH2)-predominant disease associated with atopic dermatitis to a more severe disease with prominent TH17 inflammation. We also observed divergent responses to biologic therapies targeting TH2 cytokines, which robustly protected lean mice but exacerbated disease in obese mice. Single-cell RNA sequencing coupled with genome-wide binding analyses revealed decreased activity of nuclear receptor peroxisome proliferator-activated receptor-γ (PPARγ) in TH2 cells from obese mice relative to lean mice. Conditional ablation of PPARγ in T cells revealed that PPARγ is required to focus the in vivo TH response towards a TH2-predominant state and prevent aberrant non-TH2 inflammation. Treatment of obese mice with a small-molecule PPARγ agonist limited development of TH17 pathology and unlocked therapeutic responsiveness to targeted anti-TH2 biologic therapies. These studies reveal the effects of obesity on immunological disease and suggest a precision medicine approach to target the immune dysregulation caused by obesity.

Polycationic HA/CpG Nanoparticles Induce Cross-Protective Influenza Immunity in Mice.

The intranasal (i.n.) route is an ideal vaccination approach for infectious respiratory diseases like influenza. Polycationic polyethylenimine (PEI) could form nanoscale complexes with negatively charged viral glycoproteins. Here we fabricated PEI-hemagglutinin (HA) and PEI-HA/CpG nanoparticles and investigated their immune responses and protective efficacies with an i.n. vaccination regimen in mice. Our results revealed that the nanoparticles significantly enhanced HA immunogenicity, providing heterologous cross-protection. The conserved HA stalk region induced substantial antibodies in the nanoparticle immunization groups. In contrast to the Th2-biased, IgG1-dominant antibody response generated by PEI-HA nanoparticles, PEI-HA/CpG nanoparticles generated more robust and balanced IgG1/IgG2a antibody responses with augmented neutralization activity and Fc-mediated antibody-dependent cellular cytotoxicity (ADCC). PEI-HA/CpG nanoparticles also induced enhanced local and systemic cellular immune responses. These immune responses did not decay over six months of observation postimmunization. PEI and CpG synergized these comprehensive immune responses. Thus, the PEI-HA/CpG nanoparticle is a potential cross-protective influenza vaccine candidate. Polycationic PEI nanoplatforms merit future development into mucosal vaccine systems.