Mucosal vaccines: wisdom from now and then.

The oral and nasal cavities are covered by the mucosal epithelium that starts at the beginning of the aero-digestive tract. These mucosal surfaces are continuously exposed to environmental antigens including pathogens and allergens and are thus equipped with a mucosal immune system that mediates initial recognition of pathogenicity and initiates pathogen-specific immune responses. At the dawn of our scientific effort to explore the mucosal immune system, dental science was one of the major driving forces as it provided insights into the importance of mucosal immunity and its application for the control of oral infectious diseases. The development of mucosal vaccines for the prevention of dental caries was thus part of a novel approach that contributed to building the scientific foundations of the mucosal immune system. Since then, mucosal immunology and vaccines have gone on a scientific journey to become one of the major entities within the discipline of immunology. Here, we introduce our past and current efforts and future directions for the development of mucosal vaccines, specifically a rice-based oral vaccine (MucoRice) and a nanogel-based nasal vaccine, with the aim of preventing and controlling gastrointestinal and respiratory infectious diseases using the interdisciplinary fusion of mucosal immunology with agricultural science and biomaterial engineering, respectively.

Distinct roles of BCNP1 in B-cell development and activation.

B-cell novel protein 1 (BCNP1) has recently been identified as a new B-cell receptor (BCR) signaling molecule but its physiological function remains unknown. Here, we demonstrate that mice deficient in BCNP1 exhibit impaired B-cell maturation and a reduction of B-1a cells. BCNP1-deficient spleen B cells show enhanced survival, proliferation and Ca2+ influx in response to BCR cross-linking as compared with wild-type spleen B cells. Consistently, mutant B cells show elevated phosphorylation of SYK, B-cell linker protein (BLNK) and PLCγ2 upon BCR cross-linking. In vivo, BCNP1-deficient mice exhibit enhanced humoral immune responses to T-independent and T-dependent antigens. Moreover, aged mutant mice contain elevated levels of serum IgM and IgG3 antibodies and exhibit polyclonal and monoclonal B-cell expansion in lymphoid organs. These results reveal distinct roles for BCNP1 in B-cell development, activation and homeostasis.

Characterization and Specification of a Trivalent Protein-Based Pneumococcal Vaccine Formulation Using an Adjuvant-Free Nanogel Nasal Delivery System.

We previously developed a safe and effective nasal vaccine delivery system using a self-assembled nanosized hydrogel (nanogel) made from a cationic cholesteryl pullulan. Here, we generated three pneumococcal surface protein A (PspA) fusion antigens as a universal pneumococcal nasal vaccine and then encapsulated each PspA into a nanogel and mixed the three resulting monovalent formulations into a trivalent nanogel-PspA formulation. First, to characterize the nanogel-PspA formulations, we used native polyacrylamide gel electrophoresis (PAGE) to determine the average number of PspA molecules encapsulated per nanogel molecule. Second, we adopted two methods-a densitometric method based on lithium dodecyl sulfate (LDS)-PAGE and a biologic method involving sandwich enzyme-linked immunosorbent assay (ELISA)-to determine the PspA content in the nanogel formulations. Third, treatment of nanogel-PspA formulations by adding methyl-ß-cyclodextrin released each PspA in its native form, as confirmed through circular dichroism (CD) spectroscopy. However, when nanogel-PspA formulations were heat-treated at 80 °C for 16 h, CD spectroscopy showed that each PspA was released in a denatured form. Fourth, we confirmed that the nanogel-PspA formulations were internalized into nasal mucosa effectively and that each PspA was gradually released from the nanogel in epithelial cells in mice. Fifth, LDS-PAGE densitometry and ELISA both indicated that the amount of trivalent PspA was dramatically decreased in the heat-treated nanogel compared with that before heating. When mice were immunized nasally using the heat-treated formulation, the immunologic activity of each PspA was dramatically reduced compared with that of the untreated formulation; in both cases, the immunologic activity correlated well with the content of each PspA as determined by LDS-PAGE densitometry and ELISA. Finally, we confirmed that the trivalent nanogel-PspA formulation induced equivalent titers of PspA-specific serum IgG and mucosal IgA Abs in immunized mice. These results show that the specification methods we developed effectively characterized our nanogel-based trivalent PspA nasal vaccine formulation.

The B cell novel protein 1 (BCNP1) regulates BCR signaling and B cell apoptosis.

The BCR plays a central role in B cell development, survival, activation, and differentiation. We have identified the B cell novel protein 1 (BCNP1) as a new regulator of BCR signaling. BCNP1 contains a pleckstrin homology domain, three proline-rich motifs, and a potential SH2 binding site, and is predominantly expressed by B cells. We found that BCNP1 overexpression in WEHI231 immature B cells potentiated α-IgM-induced apoptosis. Conversely, BCNP1-deficient WEHI231 cells, generated by CRISPR-Cas9-mediated genome editing, exhibited reduced apoptosis after BCR crosslinking. Biochemical analyses revealed that BCNP1 physically interacted with the B cell linker protein (BLNK), Grb2, and PLCγ2. Moreover, absence of BCNP1 resulted in accelerated dephosphorylation of BLNK, reduced phosphorylation of SYK and PLCγ2, and decreased Ca2+ influx after BCR crosslinking. These results demonstrate that BCNP1 promotes BCR signaling by modulating the phosphorylation of BLNK, SYK, and PLCγ2.

A lysosomal protein negatively regulates surface T cell antigen receptor expression by promoting CD3zeta-chain degradation.

Modulation of surface T cell antigen receptor (TCR) expression is an important mechanism for the regulation of immune responses and the prevention of T cell hyperactivation and autoimmunity. The TCR is rapidly internalized after antigen stimulation and then degraded in lysosomes. However, few of the molecules involved in this process have been identified. We demonstrate that the lysosomal protein LAPTM5 negatively regulated surface TCR expression by specifically interacting with the invariant signal-transducing CD3zeta chain and promoting its degradation without affecting other CD3 proteins, CD3epsilon, CD3delta, or CD3gamma. TCR downmodulation required the polyproline-tyrosine motifs and the ubiquitin-interacting motif of LAPTM5. LAPTM5 deficiency resulted in elevated TCR expression on both CD4(+)CD8(+) thymocytes and spleen T cells after CD3 stimulation, as well as enhanced T cell responses in vitro and in vivo. These results identify a lysosomal protein important for CD3zeta degradation and illustrate a unique mechanism for the control of surface TCR expression and T cell activation.

FcµR interacts and cooperates with the B cell receptor To promote B cell survival.

The IgM FcR (FcµR) promotes B cell survival, but the molecular mechanism remains largely unknown. We show using FcµR(-/-) and wild-type mice that FcµR specifically enhanced B cell survival induced by BCR cross-linking with F(ab')2-anti-IgM Abs while having no effect on survival when the B cells were activated by CD40 ligation or LPS stimulation. FcµR expression was markedly upregulated by anti-IgM stimulation, which may promote enhanced FcµR signaling in these cells. Immunofluorescence and confocal microscopy analyses demonstrated that FcµR colocalized with the BCR on the plasma membrane of primary B cells. Coimmunoprecipitation analysis further revealed that FcµR physically interacted with the BCR complex. Because NF-κB plays a prominent role in B cell survival, we analyzed whether FcµR was involved in BCR-triggered NF-κB activation. FcµR did not affect BCR-triggered IκBα phosphorylation characteristic of the canonical NF-κB activation pathway but promoted the production of the noncanonical NF-κB pathway component p52. Consistent with the elevated p52 levels, FcµR enhanced BCR-triggered expression of the antiapoptotic protein BCL-xL. Importantly, FcµR stimulation alone in the absence of BCR signaling had no effect on either IκBα phosphorylation or the expression of p52 and BCL-xL. Therefore, FcµR relied on the BCR signal to activate the noncanonical NF-κB pathway and enhance B cell survival. These results reveal a cross-talk downstream of FcµR and BCR signaling and provide mechanistic insight into FcµR-mediated enhancement of B cell survival after BCR stimulation.

Critical role of the IgM Fc receptor in IgM homeostasis, B-cell survival, and humoral immune responses.

IgM antibodies have been known for decades to enhance humoral immune responses in an antigen-specific fashion. This enhancement has been thought to be dependent on complement activation by IgM-antigen complexes; however, recent genetic studies render this mechanism unlikely. Here, we describe a likely alternative explanation; mice lacking the recently identified Fc receptor for IgM (FcµR) on B cells produced significantly less antibody to protein antigen during both primary and memory responses. This immune deficiency was accompanied by impaired germinal center formation and decreased plasma and memory B-cell generation. FcµR did not affect steady-state B-cell survival but specifically enhanced the survival and proliferation induced by B-cell receptor cross-linking. Moreover, FcµR-deficient mice produced far more autoantibodies than control mice as they aged, suggesting that FcµR is also required for maintaining tolerance to self-antigens. Our results thus define a unique pathway mediated by the FcµR for regulating immunity and tolerance and suggest that IgM antibodies promote humoral immune responses to foreign antigen yet suppress autoantibody production through at least two pathways: complement activation and FcµR.

LAPTM5 promotes lysosomal degradation of intracellular CD3ζ but not of cell surface CD3ζ.

The lysosomal protein LAPTM5 has been shown to negatively regulate cell surface T cell receptor (TCR) expression and T-cell activation by promoting CD3ζ degradation in lysosomes, but the mechanism remains largely unknown. Here we show that LAPTM5 promotes lysosomal translocation of intracellular CD3ζ but not of the cell surface CD3ζ associated with the mature TCR complex. Kinetic analysis of the subcellular localization of the newly synthesized CD3ζ suggests that LAPTM5 targets CD3ζ in the Golgi apparatus and promotes its lysosomal translocation. Consistently, a Golgi-localizing mutant CD3ζ can be transported to and degraded in the lysosome by LAPTM5. A CD3ζ YF mutant in which all six tyrosine residues in the immunoreceptor tyrosine-based activation motif are mutated to phenylalanines is degraded as efficiently as is wild type CD3ζ, further suggesting that TCR signaling-triggered tyrosine phosphorylation of CD3ζ is dispensable for LAPTM5-mediated degradation. Previously, Src-like adapter protein (SLAP) and E3 ubiquitin ligase c-Cbl have been shown to mediate the ubiquitination of CD3ζ in the internalized TCR complex and its subsequent lysosomal degradation. We show that LAPTM5 and SLAP/c-Cbl function in distinct genetic pathways to negatively regulate TCR expression. Collectively, these results suggest that CD3ζ can be degraded by two pathways: SLAP/c-Cbl, which targets internalized cell surface CD3ζ dependent on TCR signaling, and LAPTM5, which targets intracellular CD3ζ independent of TCR signaling.

Glycoprotein 2 as a gut gate keeper for mucosal equilibrium between inflammation and immunity.

Glycoprotein 2 (GP2) is a widely distributed protein in the digestive tract, contributing to mucosal barrier maintenance, immune homeostasis, and antigen-specific immune response, while also being linked to inflammatory bowel disease (IBD) pathogenesis. This review sheds light on the extensive distribution of GP2 within the gastrointestinal tract and its intricate interplay with the immune system. Furthermore, the significance of GP2 autoantibodies in diagnosing and categorizing IBD is underscored, alongside the promising therapeutic avenues for modulating GP2 to regulate immunity and maintain mucosal balance.

MucoRice-CTB line 19A, a new marker-free transgenic rice-based cholera vaccine produced in an LED-based hydroponic system.

We previously established the selection-marker-free rice-based oral cholera vaccine (MucoRice-CTB) line 51A for human use by Agrobacterium-mediated co-transformation and conducted a double-blind, randomized, placebo-controlled phase I trial in Japan and the United States. Although MucoRice-CTB 51A was acceptably safe and well tolerated by healthy Japanese and U.S. subjects and induced CTB-specific antibodies neutralizing cholera toxin secreted by Vibrio cholerae, we were limited to a 6-g cohort in the U.S. trial because of insufficient production of MucoRice-CTB. Since MucoRice-CTB 51A did not grow in sunlight, we re-examined the previously established marker-free lines and selected MucoRice-CTB line 19A. Southern blot analysis of line 19A showed a single copy of the CTB gene. We resequenced the whole genome and detected the transgene in an intergenic region in chromosome 1. After establishing a master seed bank of MucoRice-CTB line 19A, we established a hydroponic production facility with LED lighting to reduce electricity consumption and to increase production capacity for clinical trials. Shotgun MS/MS proteomics analysis of MucoRice-CTB 19A showed low levels of α-amylase/trypsin inhibitor-like proteins (major rice allergens), which was consistent with the data for line 51A. We also demonstrated that MucoRice-CTB 19A had high oral immunogenicity and induced protective immunity against cholera toxin challenge in mice. These results indicate that MucoRice-CTB 19A is a suitable oral cholera vaccine candidate for Phase I and II clinical trials in humans, including a V. cholerae challenge study.

LAPTM5 protein is a positive regulator of proinflammatory signaling pathways in macrophages.

LAPTM5 (lysosomal-associated protein transmembrane 5) is a protein that is preferentially expressed in immune cells, and it interacts with the Nedd4 family of ubiquitin ligases. Recent studies in T and B cells identified LAPTM5 as a negative regulator of T and B cell receptor levels at the plasma membrane. Here we investigated the function of LAPTM5 in macrophages. We demonstrate that expression of LAPTM5 is required for the secretion of proinflammatory cytokines in response to Toll-like receptor ligands. We also show that RAW264.7 cells knocked down for LAPTM5 or macrophages from LAPTM5(-/-) mice exhibit reduced activation of NF-κB and MAPK signaling pathways mediated by the TNF receptor, as well as multiple pattern recognition receptors in various cellular compartments. TNF stimulation of LAPTM5-deficient macrophages leads to reduced ubiquitination of RIP1 (receptor-interacting protein 1), suggesting a role for LAPTM5 at the receptor-proximate level. Interestingly, we find that macrophages from LAPTM5(-/-) mice display up-regulated levels of A20, a ubiquitin-editing enzyme responsible for deubiquitination of RIP1 and subsequent termination of NF-κB activation. Our studies thus indicate that, in contrast to its negative role in T and B cell activation, LAPTM5 acts as a positive modulator of inflammatory signaling pathways and hence cytokine secretion in macrophages. They also highlight a role for the endosomal/lysosomal system in regulating signaling via cytokine and pattern recognition receptors.

Nasal vaccines: solutions for respiratory infectious diseases.

Nasal vaccines induce pathogen-specific dual protective immunity at mucosal surfaces and systemically throughout the body. Consequently, nasal vaccines both prevent pathogen invasion and reduce disease severity. Because of these features, nasal vaccines are considered to be a next-generation tool for preventing respiratory infectious diseases, including COVID-19. However, nasal vaccines must overcome key safety concerns given the anatomic proximity of the central nervous system (CNS) via the olfactory bulbs which lie next to the nasal cavity. This review summarizes current efforts to develop safe and effective nasal vaccines and delivery systems, as well as their clinical applications for the prevention of respiratory infections. We also discuss various concerns regarding the safety of nasal vaccines and introduce a system for evaluating them.

Biodistribution assessment of cationic pullulan nanogel, a nasal vaccine delivery system, in mice and non-human primates.

Cationic cholesteryl-group-bearing pullulan nanogel (cCHP-nanogel) is an effective drug-delivery system for nasal vaccines. However, cCHP-nanogel-based nasal vaccines might access the central nervous system due to its close proximity via the olfactory bulb in the nasal cavity. Using real-time quantitative tracking of the nanogel-based nasal botulinum neurotoxin and pneumococcal vaccines, we previously confirmed the lack of deposition of vaccine antigen in the cerebrum or olfactory bulbs of mice and non-human primates (NHPs), rhesus macaques. Here, we used positron emission tomography to investigate the biodistribution of the drug-delivery system itself, cCHP-nanogel after mice and NHPs were nasally administered with 18F-labeled cCHP nanogel. The results generated by the PET analysis of rhesus macaques were consistent with the direct counting of radioactivity due to 18F or 111In in dissected mouse tissues. Thus, no depositions of cCHP-nanogel were noted in the cerebrum, olfactory bulbs, or eyes of both species after nasal administration of the radiolabeled cCHP-nanogel compound. Our findings confirm the safe biodistribution of the cCHP-nanogel-based nasal vaccine delivery system in mice and NHPs.

Cationic-nanogel nasal vaccine containing the ectodomain of RSV-small hydrophobic protein induces protective immunity in rodents.

Respiratory syncytial virus (RSV) is a leading cause of upper and lower respiratory tract infection, especially in children and the elderly. Various vaccines containing the major transmembrane surface proteins of RSV (proteins F and G) have been tested; however, they have either afforded inadequate protection or are associated with the risk of vaccine-enhanced disease (VED). Recently, F protein-based maternal immunization and vaccines for elderly patients have shown promising results in phase III clinical trials, however, these vaccines have been administered by injection. Here, we examined the potential of using the ectodomain of small hydrophobic protein (SHe), also an RSV transmembrane surface protein, as a nasal vaccine antigen. A vaccine was formulated using our previously developed cationic cholesteryl-group-bearing pullulan nanogel as the delivery system, and SHe was linked in triplicate to pneumococcal surface protein A as a carrier protein. Nasal immunization of mice and cotton rats induced both SHe-specific serum IgG and mucosal IgA antibodies, preventing viral invasion in both the upper and lower respiratory tracts without inducing VED. Moreover, nasal immunization induced greater protective immunity against RSV in the upper respiratory tract than did systemic immunization, suggesting a critical role for mucosal RSV-specific IgA responses in viral elimination at the airway epithelium. Thus, our nasal vaccine induced effective protection against RSV infection in the airway mucosa and is therefore a promising vaccine candidate for further development.

A role for lysosomal-associated protein transmembrane 5 in the negative regulation of surface B cell receptor levels and B cell activation.

Mechanisms by which cell surface levels of the BCR are regulated remain largely unknown. We found that B cells lacking the lysosomal-associated protein transmembrane 5 (LAPTM5) expressed higher levels of cell surface BCR than did wild-type (WT) B cells after Ag stimulation in vitro and in vivo. In addition, LAPTM5-deficient mice contained an increased frequency of Ag-specific B cells and produced greater amounts of Abs than did WT mice after immunization with a T-dependent Ag. Adoptive transfer of LAPTM5-deficient B cells with WT T cells into RAG1-deficient mice revealed that the increased surface BCR levels and the enhanced B cell activation and Ab production were due to a B cell intrinsic defect. As they aged, the LAPTM5-deficient mice had increased titers of serum IgM and autoantibodies and immune complex deposition in the kidney. Immunofluorescent and biochemical analysis revealed that LAPTM5 physically interacted with the BCR complex and promoted its degradation in the lysosomal compartment in mouse B cells. These results demonstrate a role for LAPTM5 in the negative regulation of cell surface BCR levels and B cell activation.

Induction of Mucosal IgA-Mediated Protective Immunity Against Nontypeable Haemophilus influenzae Infection by a Cationic Nanogel-Based P6 Nasal Vaccine.

Nontypeable Haemophilus influenzae (NTHi) strains form a major group of pathogenic bacteria that colonizes the nasopharynx and causes otitis media in young children. At present, there is no licensed vaccine for NTHi. Because NTHi colonizes the upper respiratory tract and forms biofilms that cause subsequent infectious events, a nasal vaccine that induces NTHi-specific secretory IgA capable of preventing biofilm formation in the respiratory tract is desirable. Here, we developed a cationic cholesteryl pullulan-based (cCHP nanogel) nasal vaccine containing the NTHi surface antigen P6 (cCHP-P6) as a universal vaccine antigen, because P6 expression is conserved among 90% of NTHi strains. Nasal immunization of mice with cCHP-P6 effectively induced P6-specific IgA in mucosal fluids, including nasal and middle ear washes. The vaccine-induced P6-specific IgA showed direct binding to the NTHi via the surface P6 proteins, resulting in the inhibition of NTHi biofilm formation. cCHP-P6 nasal vaccine thus protected mice from intranasal NTHi challenge by reducing NTHi colonization of nasal tissues and eventually eliminated the bacteria. In addition, the vaccine-induced IgA bound to different NTHi clinical isolates from patients with otitis media and inhibited NTHi attachment in a three-dimensional in vitro model of the human nasal epithelial surface. Therefore, the cCHP-P6 nanogel nasal vaccine induced effective protection in the airway mucosa, making it a strong vaccine candidate for preventing NTHi-induced infectious diseases, such as otitis media, sinusitis, and pneumonia.

Oral MucoRice-CTB vaccine is safe and immunogenic in healthy US adults.

MucoRice-CTB is a promising cold-chain-free oral cholera vaccine candidate. Here, we report a double-blind, randomized, placebo-controlled, phase I study conducted in the USA in which vaccination with the 6-g dose of MucoRice-CTB induced cross-reactive antigen-specific antibodies against the B subunit of cholera toxin (CTB) and enterotoxigenic Escherichia coli heat-labile enterotoxin without inducing serious adverse events. This dosage was acceptably safe and tolerable in healthy men and women. In addition, it induced a CTB-specific IgA response in the saliva of two of the nine treated subjects; in one subject, the immunological kinetics of the salivary IgA were similar to those of the serum CTB-specific IgA. Antibodies from three of the five responders to the vaccine prevented CTB from binding its GM1 ganglioside receptor. These results are consistent with those of the phase I study in Japan, suggesting that oral MucoRice-CTB induces neutralizing antibodies against diarrheal toxins regardless of ethnicity.

A critical role for REV1 in regulating the induction of C:G transitions and A:T mutations during Ig gene hypermutation.

REV1 is a deoxycytidyl transferase that catalyzes the incorporation of deoxycytidines opposite deoxyguanines and abasic sites. To explore the role of its catalytic activity in Ig gene hypermutation in mammalian cells, we have generated mice expressing a catalytically inactive REV1 (REV1AA). REV1AA mice developed normally and were fertile on a pure C57BL/6 genetic background. B and T cell development and maturation were not affected, and REV1AA B cells underwent normal activation and class switch recombination. Analysis of Ig gene hypermutation in REV1AA mice revealed a great decrease of C to G and G to C transversions, consistent with the disruption of its deoxycytidyl transferase activity. Intriguingly, REV1AA mice also exhibited a significant reduction of C to T and G to A transitions. Moreover, each type of nucleotide substitutions at A:T base pairs was uniformly reduced in REV1AA mice, a phenotype similar to that observed in mice haploinsufficient for Polh. These results reveal an unexpected role for REV1 in the generation of C:G transitions and A:T mutations and suggest that REV1 is involved in multiple mutagenic pathways through functional interaction with other polymerases during the hypermutation process.

Development of Antibody-Fragment-Producing Rice for Neutralization of Human Norovirus.

Human norovirus is the leading cause of acute nonbacterial gastroenteritis in people of all ages worldwide. Currently, no licensed norovirus vaccine, pharmaceutical drug, or therapy is available for the control of norovirus infection. Here, we used a rice transgenic system, MucoRice, to produce a variable domain of a llama heavy-chain antibody fragment (VHH) specific for human norovirus (MucoRice-VHH). VHH is a small heat- and acid-stable protein that resembles a monoclonal antibody. Consequently, VHHs have become attractive and useful antibodies (Abs) for oral immunotherapy against intestinal infectious diseases. MucoRice-VHH constructs were generated at high yields in rice seeds by using an overexpression system with RNA interference to suppress the production of the major rice endogenous storage proteins. The average production levels of monomeric VHH (7C6) to GII.4 norovirus and heterodimeric VHH (7C6-1E4) to GII.4 and GII.17 noroviruses in rice seed were 0.54 and 0.28% (w/w), respectively, as phosphate buffered saline (PBS)-soluble VHHs. By using a human norovirus propagation system in human induced pluripotent stem-cell-derived intestinal epithelial cells (IECs), we demonstrated the high neutralizing activity of MucoRice expressing monomeric VHH (7C6) against GII.4 norovirus and of heterodimeric VHH (7C6-1E4) against both GII.4 and GII.17 noroviruses. In addition, MucoRice-VHH (7C6-1E4) retained neutralizing activity even after heat treatment at 90°C for 20 min. These results build a fundamental platform for the continued development of MucoRice-VHH heterodimer as a candidate for oral immunotherapy and for prophylaxis against GII.4 and GII.17 noroviruses in not only healthy adults and children but also immunocompromised patients and the elderly.

A nanogel-based trivalent PspA nasal vaccine protects macaques from intratracheal challenge with pneumococci.

Current polysaccharide-based pneumococcal vaccines are effective but not compatible with all serotypes of Streptococcus pneumoniae. We previously developed an adjuvant-free cationic nanogel nasal vaccine containing pneumococcal surface protein A (PspA), which is expressed on the surfaces of all pneumococcal serotypes. Here, to address the sequence diversity of PspA proteins, we formulated a cationic nanogel-based trivalent pneumococcal nasal vaccine and demonstrated the vaccine's immunogenicity and protective efficacy in macaques by using a newly developed nasal spray device applicable to humans. Nasal vaccination of macaques with cationic cholesteryl pullulan nanogel (cCHP)-trivalent PspA vaccine effectively induced PspA-specific IgGs that bound to pneumococcal surfaces and triggered complement C3 deposition. The immunized macaques were protected from pneumococcal intratracheal challenge through both inhibition of lung inflammation and a dramatic reduction in the numbers of bacteria in the lungs. These results demonstrated that the cCHP-trivalent PspA vaccine is an effective candidate vaccine against pneumococcal infections.