Mass spectrometric identification of immunogenic SARS-CoV-2 epitopes and cognate TCRs.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections elicit both humoral and cellular immune responses. For the prevention and treatment of COVID-19, the disease caused by SARS-CoV-2, it has become increasingly apparent that T cell responses are equally if not more important than humoral responses in mediating recovery and immune protection. One major challenge in developing T cell-based therapies for infectious and malignant diseases has been the identification of immunogenic epitopes that can elicit a meaningful T cell response. Traditionally, this has been achieved using sophisticated in silico methods to predict putative epitopes deduced from binding affinities. Our studies find that, in contrast to current convention, "immunodominant" SARS-CoV-2 peptides defined by such in silico methods often fail to elicit T cell responses recognizing naturally presented SARS-CoV-2 epitopes. We postulated that immunogenic epitopes for SARS-CoV-2 are best defined empirically by directly analyzing peptides eluted from the naturally processed peptide-major histocompatibility complex (MHC) and then validating immunogenicity by determining whether such peptides can elicit T cells recognizing SARS-CoV-2 antigen-expressing cells. Using a tandem mass spectrometry approach, we identified epitopes derived from not only structural but also nonstructural genes in regions highly conserved among SARS-CoV-2 strains, including recently recognized variants. Finally, there are no reported T cell receptor-engineered T cell technology that can redirect T cell specificity to recognize and kill SARS-CoV-2 target cells. We report here several SARS-CoV-2 epitopes defined by mass spectrometric analysis of MHC-eluted peptides, provide empiric evidence for their immunogenicity, and demonstrate engineered TCR-redirected killing.

Unique potential of 4-1BB agonist antibody to promote durable regression of HPV+ tumors when combined with an E6/E7 peptide vaccine.

Antibody modulation of T-cell coinhibitory (e.g., CTLA-4) or costimulatory (e.g., 4-1BB) receptors promotes clinical responses to a variety of cancers. Therapeutic cancer vaccination, in contrast, has produced limited clinical benefit and no curative therapies. The E6 and E7 oncoproteins of human papilloma virus (HPV) drive the majority of genital cancers, and many oropharyngeal tumors. We discovered 15-19 amino acid peptides from HPV-16 E6/E7 for which induction of T-cell immunity correlates with disease-free survival in patients treated for high-grade cervical neoplasia. We report here that intranasal vaccination with these peptides and the adjuvant alpha-galactosylceramide elicits systemic and mucosal T-cell responses leading to reduced HPV(+) TC-1 tumor growth and prolonged survival in mice. We hypothesized that the inability of these T cells to fully reject established tumors resulted from suppression in the tumor microenvironment which could be ameliorated through checkpoint modulation. Combining this E6/E7 peptide vaccine with checkpoint blockade produced only modest benefit; however, coadministration with a 4-1BB agonist antibody promoted durable regression of established genital TC-1 tumors. Relative to other therapies tested, this combination of vaccine and α4-1BB promoted the highest CD8(+) versus regulatory FoxP3(+) T-cell ratios, elicited 2- to 5-fold higher infiltration by E7-specific CTL, and evoked higher densities of highly cytotoxic TcEO (T cytotoxic Eomesodermin) CD8 (>70-fold) and ThEO (T helper Eomesodermin) CD4 (>17-fold) T cells. These findings have immediate clinical relevance both in terms of the direct clinical utility of the vaccine studied and in illustrating the potential of 4-1BB antibody to convert therapeutic E6/E7 vaccines already in clinical trials into curative therapies.

HIV-1 Vpr protein inhibits telomerase activity via the EDD-DDB1-VPRBP E3 ligase complex.

Viral pathogens utilize host cell machinery for their benefits. Herein, we identify that HIV-1 Vpr (viral protein R) negatively modulates telomerase activity. Telomerase enables stem and cancer cells to evade cell senescence by adding telomeric sequences to the ends of chromosomes. We found that Vpr inhibited telomerase activity by down-regulating TERT protein, a catalytic subunit of telomerase. As a molecular adaptor, Vpr enhanced the interaction between TERT and the VPRBP substrate receptor of the DYRK2-associated EDD-DDB1-VPRBP E3 ligase complex, resulting in increased ubiquitination of TERT. In contrast, the Vpr mutant identified in HIV-1-infected long-term nonprogressors failed to promote TERT destabilization. Our results suggest that Vpr inhibits telomerase activity by hijacking the host E3 ligase complex, and we propose the novel molecular mechanism of telomerase deregulation in possibly HIV-1 pathogenesis.

Nanofibrous MultiDomain Peptide Hydrogels Provide T Cells a 3D, Cytocompatible Environment for Cell Expansion and Antigen-Specific Killing.

T cells have the ability to recognize and kill specific target cells, giving therapies based on their potential for treating infection, diabetes, cancer, and other diseases. However, the advancement of T cell-based treatments has been hindered by difficulties in their ex vivo activation and expansion, the number of cells required for sustained in vivo levels, and preferential localization following systemic delivery. Biomaterials may help to overcome many of these challenges by providing a combined means of proliferation, antigen presentation, and cell localization upon delivery. In this work, we studied self-assembling Multidomain Peptides (MDPs) as scaffolds for T cell culture, activation, and expansion. We evaluated the effect of different MDP chemistries on their biocompatibility with T cells and the maintenance of antigen specificity for T cells cultured in the hydrogels. We also examined the potential application of MDPs as scaffolds for T cell activation and expansion and the effect of MDP encapsulation on T cell phenotype. We found high cell viability when T cells were encapsulated in noncationic MDPs, O5 and D2, and superior retention of antigen specificity and tumor-reactivity were preserved in the anionic MDP, D2. Maintenance of antigen recognition by T cells in D2 hydrogels was confirmed by quantifying immune synapses of T Cells engaged with antigen-presenting cancer cells. When 3D cultured in anionic MDP D2 coloaded with anti-CD3, anti-CD28, IL2, IL7, and IL15, we observed successful T cell proliferation evidenced by upregulation of CD27 and CD107a. This study is the first to investigate the potential of self-assembling peptide-based hydrogels as 3D scaffolds for human T cell applications and demonstrates that MDP hydrogels are a viable platform for enabling T cell in vitro activation, expansion, and maintenance of antigen specificity and therefore a promising tool for future T cell-based therapies.

Intranasal but not intravenous delivery of the adjuvant α-galactosylceramide permits repeated stimulation of natural killer T cells in the lung.

Efficient induction of antigen-specific immunity is achieved by delivering multiple doses of vaccine formulated with appropriate adjuvants that can harness the benefits of innate immune mediators. The synthetic glycolipid α-galactosylceramide (α-GalCer) is a potent activator of NKT cells, a major innate immune mediator cell type effective in inducing maturation of DCs for efficient presentation of co-administered antigens. However, systemic administration of α-GalCer results in NKT cell anergy in which the cells are unresponsive to subsequent doses of α-GalCer. We show here that α-GalCer delivered as an adjuvant by the intranasal route, as opposed to the intravenous route, enables repeated activation of NKT cells and DCs, resulting in efficient induction of cellular immune responses to co-administered antigens. We show evidence that after intranasal delivery,α-GalCer is selectively presented by DCs for the activation of NKT cells, not B cells. Furthermore, higher levels of PD-1 expression, a potential marker for functional exhaustion of the NKT cells when α-GalCer is delivered by the intravenous route, are not observed after intranasal delivery. These results support a mucosal route of delivery for the utility of α-GalCer as an adjuvant for vaccines, which often requires repeated dosing to achieve durable protective immunity.

Tertiary lymphoid structure signatures are associated with immune checkpoint inhibitor related acute interstitial nephritis.

Tertiary lymphoid structures (TLSs) are associated with anti-tumor response following immune checkpoint inhibitor (ICI) therapy, but a commensurate observation of TLS is absent for immune related adverse events (irAEs) i.e. acute interstitial nephritis (AIN). We hypothesized that TLS-associated inflammatory gene signatures are present in AIN and performed NanoString-based gene expression and multiplex 12-chemokine profiling on paired kidney tissue, urine and plasma specimens of 36 participants who developed acute kidney injury (AKI) on ICI therapy: AIN (18), acute tubular necrosis (9), or HTN nephrosclerosis (9). Increased T and B cell scores, a Th1-CD8+ T cell axis accompanied by interferon-g and TNF superfamily signatures were detected in the ICI-AIN group. TLS signatures were significantly increased in AIN cases and supported by histopathological identification. Furthermore, urinary TLS signature scores correlated with ICI-AIN diagnosis but not paired plasma. Urinary CXCL9 correlated best to tissue CXCL9 expression (rho 0.75, p < 0.001) and the ability to discriminate AIN vs. non-AIN (AUC 0.781, p-value 0.003). For the first time, we report the presence of TLS signatures in irAEs, define distinctive immune signatures, identify chemokine markers distinguishing ICI-AIN from common AKI etiologies and demonstrate that urine chemokine markers may be used as a surrogate for ICI-AIN diagnoses.

Urinary T cells are detected in patients with immune checkpoint inhibitor-associated immune nephritis that are clonotypically identical to kidney T cell infiltrates.

Acute kidney injury (AKI) occurs in ~20% of patients receiving immune checkpoint inhibitor (ICI) therapy; however, only 2-5% will develop ICI-mediated immune nephritis. Conventional tests are nonspecific in diagnosing disease pathology and invasive procedures (i.e. kidney biopsy) may not be feasible. In other autoimmune renal diseases, urinary immune cells correlated with the pathology or were predictive of disease activity. Corresponding evidence and analysis are absent for ICI-mediated immune nephritis. We report the first investigation analyzing immune cell profiles of matched kidney biopsies and urine of patients with ICI-AKI. We demonstrated the presence of urinary T cells in patients with immune nephritis by flow cytometry analysis. Clonotype analysis of T cell receptor (TCR) sequences confirmed enrichment of kidney TCRs in urine. As ICI therapies become standard of care for more cancers, noninvasively assessing urinary immune cells of ICI therapy recipients can facilitate clinical management and an opportunity to tailor ICI-nephritis treatment.

Mucoadhesive wafers composed of binary polymer blends for sublingual delivery and preservation of protein vaccines.

The objective of this study is to develop a simple biopolymer platform of mucoadhesive wafers that enables effective sublingual delivery and preservation of protein vaccines. The wafers were composed of a series of binary polymer blends of carboxymethylcellulose (CMC) and alginate (ALG). Varying the ratio between CMC and ALG resulted in wafers with different microstructure, mechanical properties, disintegration time, and release kinetics of model compounds. Wafers with high CMC content were highly mucoadhesive to sublingual mucosal tissue and could withstand extensive washing, leading to improved protein permeation into the tissue. On the other hand, wafers with high ALG content were not only mechanically robust, but also able to protect a model enzyme (ß-galactosidase) against lyophilization and heat challenge. HIV gp140 protein loaded in wafers of the optimal composition could be stored and transported without cold chain, while maintaining antigen-specific immunogenicity after sublingual vaccination in mice. These findings established that the CMC/ALG binary blend polymer wafers have the potential to improve the sublingual delivery and storage stability of protein-based vaccines.

In Silico Defined SARS-CoV2 Epitopes May Not Predict Immunogenicity to COVID19.

SARS-CoV-2 infections elicit both humoral and cellular immune responses. For the prevention and treatment of COVID19, the disease caused by SARS-CoV-2, it has become increasingly apparent that T cell responses are equally, if not more important than humoral responses in mediating recovery and immune-protection. One of the major challenges in developing T cell-based therapies for infectious and malignant diseases has been the identification of immunogenic epitopes that can elicit a meaningful T cell response. Traditionally, this has been achieved using sophisticated in silico methods to predict putative epitopes deduced from binding affinities and consensus data. Our studies find that, in contrast to current dogma, 'immunodominant' SARS-CoV-2 peptides defined by such in silico methods often fail to elicit T cell responses recognizing naturally presented SARS-CoV-2 epitopes.

Immunogenic SARS-CoV2 Epitopes Defined by Mass Spectrometry.

SARS-CoV-2 infections elicit both humoral and cellular immune responses. For the prevention and treatment of COVID19, the disease caused by SARS-CoV-2, T cell responses are important in mediating recovery and immune-protection. The identification of immunogenic epitopes that can elicit a meaningful T cell response can be elusive. Traditionally, this has been achieved using sophisticated in silico methods to predict putative epitopes; however, our previous studies find that 'immunodominant' SARS-CoV-2 peptides defined by such in silico methods often fail to elicit T cell responses recognizing SARS-CoV-2. We postulated that immunogenic epitopes for SARS-CoV-2 are best defined by directly analyzing peptides eluted from the peptide-MHC complex and then validating immunogenicity empirically by determining if such peptides can elicit T cells recognizing SARS-CoV-2 antigen-expressing cells. Using a tandem mass spectrometry approach, we identified epitopes of SARS-CoV-2 derived not only from structural but also non-structural genes in regions highly conserved among SARS-CoV-2 strains including recently recognized variants. We report here, for the first time, several novel SARS-CoV-2 epitopes from membrane glycol-protein (MGP) and non-structure protein-13 (NSP13) defined by mass-spectrometric analysis of MHC-eluted peptides, provide empiric evidence for their immunogenicity to induce T cell response. SIGNIFICANCE STATEMENT: Current state of the art uses putative epitope peptides based on in silico prediction algorithms to evaluate the T cell response among COVID-19 patients. However, none of these peptides have been tested for immunogenicity, i.e. the ability to elicit a T cell response capable of recognizing endogenously presented peptide. In this study, we used MHC immune-precipitation, acid elution and tandem mass spectrometry to define the SARS-CoV-2 immunopeptidome for membrane glycol-protein and the non-structural protein. Furthermore, taking advantage of a highly robust endogenous T cell (ETC) workflow, we verify the immunogenicity of these MS-defined peptides by in vitro generation of MGP and NSP13 peptide-specific T cells and confirm T cell recognition of MGP or NSP13 endogenously expressing cell lines.

Single-dose intranasal vaccination elicits systemic and mucosal immunity against SARS-CoV-2.

Despite remarkable progress in the development and authorization of vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), there is a need to validate vaccine platforms for broader application. The current intramuscular vaccines are designed to elicit systemic immunity without conferring mucosal immunity in the nasal compartment, which is the first barrier that SARS-CoV-2 virus breaches before dissemination to the lung. We report the development of an intranasal subunit vaccine that uses lyophilized spike protein and liposomal STING agonist as an adjuvant. This vaccine induces systemic neutralizing antibodies, IgA in the lung and nasal compartments, and T-cell responses in the lung of mice. Single-cell RNA sequencing confirmed the coordinated activation of T/B-cell responses in a germinal center-like manner within the nasal-associated lymphoid tissues, confirming its role as an inductive site to enable durable immunity. The ability to elicit immunity in the respiratory tract can prevent the establishment of infection in individuals and prevent disease transmission.

Single-dose intranasal vaccination elicits systemic and mucosal immunity against SARS-CoV-2.

A safe and durable vaccine is urgently needed to tackle the COVID19 pandemic that has infected >15 million people and caused >620,000 deaths worldwide. As with other respiratory pathogens, the nasal compartment is the first barrier that needs to be breached by the SARS-CoV-2 virus before dissemination to the lung. Despite progress at remarkable speed, current intramuscular vaccines are designed to elicit systemic immunity without conferring mucosal immunity. We report the development of an intranasal subunit vaccine that contains the trimeric or monomeric spike protein and liposomal STING agonist as adjuvant. This vaccine induces systemic neutralizing antibodies, mucosal IgA responses in the lung and nasal compartments, and T-cell responses in the lung of mice. Single-cell RNA-sequencing confirmed the concomitant activation of T and B cell responses in a germinal center-like manner within the nasal-associated lymphoid tissues (NALT), confirming its role as an inductive site that can lead to long-lasting immunity. The ability to elicit immunity in the respiratory tract has can prevent the initial establishment of infection in individuals and prevent disease transmission across humans.

Selective Inhibition of HDAC3 Targets Synthetic Vulnerabilities and Activates Immune Surveillance in Lymphoma.

CREBBP mutations are highly recurrent in B-cell lymphomas and either inactivate its histone acetyltransferase (HAT) domain or truncate the protein. Herein, we show that these two classes of mutations yield different degrees of disruption of the epigenome, with HAT mutations being more severe and associated with inferior clinical outcome. Genes perturbed by CREBBP mutation are direct targets of the BCL6-HDAC3 onco-repressor complex. Accordingly, we show that HDAC3-selective inhibitors reverse CREBBP-mutant aberrant epigenetic programming, resulting in: (i) growth inhibition of lymphoma cells through induction of BCL6 target genes such as CDKN1A and (ii) restoration of immune surveillance due to induction of BCL6-repressed IFN pathway and antigen-presenting genes. By reactivating these genes, exposure to HDAC3 inhibitors restored the ability of tumor-infiltrating lymphocytes to kill DLBCL cells in an MHC class I and II-dependent manner, and synergized with PD-L1 blockade in a syngeneic model in vivo. Hence, HDAC3 inhibition represents a novel mechanism-based immune epigenetic therapy for CREBBP-mutant lymphomas. SIGNIFICANCE: We have leveraged the molecular characterization of different types of CREBBP mutations to define a rational approach for targeting these mutations through selective inhibition of HDAC3. This represents an attractive therapeutic avenue for targeting synthetic vulnerabilities in CREBBP-mutant cells in tandem with promoting antitumor immunity.This article is highlighted in the In This Issue feature, p. 327.

Spatially resolved analyses link genomic and immune diversity and reveal unfavorable neutrophil activation in melanoma.

Complex tumor microenvironmental (TME) features influence the outcome of cancer immunotherapy (IO). Here we perform immunogenomic analyses on 67 intratumor sub-regions of a PD-1 inhibitor-resistant melanoma tumor and 2 additional metastases arising over 8 years, to characterize TME interactions. We identify spatially distinct evolution of copy number alterations influencing local immune composition. Sub-regions with chromosome 7 gain display a relative lack of leukocyte infiltrate but evidence of neutrophil activation, recapitulated in The Cancer Genome Atlas (TCGA) samples, and associated with lack of response to IO across three clinical cohorts. Whether neutrophil activation represents cause or consequence of local tumor necrosis requires further study. Analyses of T-cell clonotypes reveal the presence of recurrent priming events manifesting in a dominant T-cell clonotype over many years. Our findings highlight the links between marked levels of genomic and immune heterogeneity within the physical space of a tumor, with implications for biomarker evaluation and immunotherapy response.

Nanofluidic drug-eluting seed for sustained intratumoral immunotherapy in triple negative breast cancer.

Conventional systemic immunotherapy administration often results in insufficient anti-tumor immune response and adverse side effects. Delivering immunotherapeutics intratumorally could maximize tumor exposure, elicit efficient anti-tumor immune response, and minimize toxicity. To fulfill the unmet clinical need for sustained local drug delivery and to avoid repeated intratumoral injections, we developed a nanofluidic-based device for intratumoral drug delivery called the nanofluidic drug-eluting seed (NDES). The NDES is inserted intratumorally using a minimally invasive trocar method similar to brachytherapy seed insertion and offers a clinical advantage of drug elution. Drug diffusion from the NDES is regulated by physical and electrostatic nanoconfinement, thereby resulting in constant and sustained immunotherapeutic delivery without the need for injections or clinician intervention. In this study, the NDES was used to deliver immunotherapeutics intratumorally in the 4 T1 orthotopic murine mammary carcinoma model, which recapitulates triple negative breast cancer. We demonstrated that NDES-mediated intratumoral release of agonist monoclonal antibodies, OX40 and CD40, resulted in potentiation of local and systemic anti-tumor immune response and inhibition of tumor growth compared to control mice. Further, mice treated with NDES-CD40 demonstrated minimal liver damage compared to systemically treated mice. Collectively, our study highlights the NDES as an effective platform for sustained intratumoral immunotherapeutic delivery. The potential clinical impact is tremendous given that the NDES is applicable to a broad spectrum of drugs and solid tumors.

Prophylactic Sublingual Immunization with Mycobacterium tuberculosis Subunit Vaccine Incorporating the Natural Killer T Cell Agonist Alpha-Galactosylceramide Enhances Protective Immunity to Limit Pulmonary and Extra-Pulmonary Bacterial Burden in Mice.

Infection by Mycobacterium tuberculosis (Mtb) remains a major global concern and the available Bacillus Calmette-Guerin (BCG) vaccine is poorly efficacious in adults. Therefore, alternative vaccines and delivery strategies focusing on Mtb antigens and appropriate immune stimulating adjuvants are needed to induce protective immunity targeted to the lungs, the primary sites of infections and pathology. We present here evidence in support of mucosal vaccination by the sublingual route in mice using the subunit Mtb antigens Ag85B and ESAT-6 adjuvanted with the glycolipid alpha-galactosylceramide (α-GalCer), a potent natural killer T (NKT) cell agonist. Vaccinated animals exhibited strong antigen-specific CD4 and CD8 T cells responses in the spleen, cervical lymph nodes and lungs. In general, inclusion of the α-GalCer adjuvant significantly enhanced these responses that persisted over 50 days. Furthermore, aerosolized Mtb infection of vaccinated mice resulted in a significant reduction of bacterial load of the lungs and spleens as compared to levels seen in naïve controls or those vaccinated with subunit proteins, adjuvant , or BCG alone. The protection induced by the Mtb antigens and-GalCer vaccine through sublingual route correlated with a TH1-type immunity mediated by antigen-specific IFN-γ and IL-2 producing T cells.

Intranasal Vaccination Affords Localization and Persistence of Antigen-Specific CD8⁺ T Lymphocytes in the Female Reproductive Tract.

Immunization strategies generating large numbers of antigen-specific T cells in the female reproductive tract (FRT) can provide barrier protection against sexually-transmitted pathogens, such as the human immunodeficiency virus (HIV) and human papillomaviruses (HPV). The kinetics and mechanisms of regulation of vaccine-induced adaptive T cell-mediated immune responses in FRT are less well defined. We present here evidence for intranasal delivery of the model antigen ovalbumin (OVA) along with alpha-galactosylceramide adjuvant as a protein vaccine to induce significantly higher levels of antigen-specific effector and memory CD8⁺ T cells in the FRT, relative to other systemic and mucosal tissues. Antibody blocking of the CXCR3 receptor significantly reduced antigen-specific CD8⁺ T cells subsequent to intranasal delivery of the protein vaccine suggesting an important role for the CXCR3 chemokine-receptor signaling for T cell trafficking. Further, intranasal vaccination with an adenoviral vector expressing OVA or HIV-1 envelope was as effective as intramuscular vaccination for generating OVA- or ENV-specific immunity in the FRT. These results support the application of the needle-free intranasal route as a practical approach to delivering protein as well as DNA/virus vector-based vaccines for efficient induction of effector and memory T cell immunity in the FRT.

Sublingual injection of microparticles containing glycolipid ligands for NKT cells and subunit vaccines induces antibody responses in oral cavity.

Natural Killer T (NKT) cells are a unique type of innate immune cells which exert paradoxical roles in animal models through producing either Th1 or Th2 cytokines and activating dendritic cells. Alpha-galactosylceramide (αGalCer), a synthetic antigen for NKT cells, was found to be safe and immune stimulatory in cancer and hepatitis patients. We recently developed microparticle-formulated αGalCer, which is selectively presented by dendritic cells and macrophages, but not B cells, and thus can avoid the anergy of NKT cells. In this study, we have examined the immunogenicity of microparticles containing αGalCer and protein vaccine components through sublingual injection in mice. The results showed that sublingual injection of microparticles containing αGalCer and ovalbumin triggered IgG responses in serum (titer >1:100,000), which persisted for more than 3months. Microparticles containing ovalbumin alone also induced comparable level of IgG responses. However, immunoglobulin subclass analysis showed that sublingually injected microparticles containing αGalCer and ovalbumin induced 20 fold higher Th1 biased antibody (IgG2c) than microparticles containing OVA alone (1:20,000 as compared to 1:1000 titer). Sublingual injection of microparticles containing αGalCer and ovalbumin induced secretion of both IgG (titer >1:1000) and IgA (titer=1:80) in saliva secretion, while microparticles containing ovalbumin alone only induced secretion of IgG in saliva. Our results suggest that sublingual injection of microparticles and their subsequent trafficking to draining lymph nodes may induce adaptive immune responses in mucosal compartments. Ongoing studies are focused on the mechanism of antigen presentation and lymphocyte biology in the oral cavity, as well as the toxicity and efficacy of these candidate microparticles for future applications.

Natural killer T cell and TLR9 agonists as mucosal adjuvants for sublingual vaccination with clade C HIV-1 envelope protein.

The vast majority of HIV-1 infections occur at mucosa during sexual contact. It may therefore be advantageous to provide mucosal barrier protection against this entry by mucosal vaccination. While a number of mucosal routes of vaccination are possible, many like enteric oral vaccines or intranasal vaccines have significant impediments that limit vaccine efficacy or pose safety risks. In contrast, immunogens applied to the sublingual region of the mouth could provide a simple route for mucosal vaccination. While sublingual immunization is appealing, this site does not always drive strong immune responses, particularly when using protein antigens. To address this issue, we have tested the ability of two mucosal adjuvants: alpha-galactosylceramide (αGalCer) that is a potent stimulator of natural killer T cells and CpG-oligodeoxynucleotide (CpG-ODN) a TLR9 agonist for their ability to amplify immune responses against clade C gp140 HIV-1 envelope protein antigen. Immunization with envelope protein alone resulted in a weak T cell and antibody responses. In contrast, CD4(+) and CD8(+) T cells responses in systemic and mucosal tissues were significantly higher in mice immunized with gp140 in the presence of either αGalCer or CpG-ODN and these responses were further augmented when the two adjuvants were used together. While both the adjuvants effectively increased gp140-specific serum IgG and vaginal IgA antibody levels, combining both significantly improved these responses. Memory T cell responses 60 days after immunization revealed αGalCer to be more potent than CpG-ODN and the combination of the αGalCer and CpG-ODN adjuvants was more effective than either alone. Serum and vaginal washes collected 60 days after immunization with gp140 with both αGalCer and CpG-ODN adjuvants had significant neutralization activity against Tier 1 and Tier 2 SHIVs. These data support the utility of the sublingual route for mucosal vaccination particularly in combination with αGalCer and CpG-ODN adjuvants.

Sublingual vaccination induces mucosal and systemic adaptive immunity for protection against lung tumor challenge.

Sublingual route offers a safer and more practical approach for delivering vaccines relative to other systemic and mucosal immunization strategies. Here we present evidence demonstrating protection against ovalbumin expressing B16 (B16-OVA) metastatic melanoma lung tumor formation by sublingual vaccination with the model tumor antigen OVA plus synthetic glycolipid alpha-galactosylceramide (aGalCer) for harnessing the adjuvant potential of natural killer T (NKT) cells, which effectively bridge innate and adaptive arms of the immune system. The protective efficacy of immunization with OVA plus aGalCer was antigen-specific as immunized mice challenged with parental B16 tumors lacking OVA expression were not protected. Multiple sublingual immunizations in the presence, but not in the absence of aGalCer, resulted in repeated activation of NKT cells in the draining lymph nodes, spleens, and lungs of immunized animals concurrent with progressively increasing OVA-specific CD8+ T cell responses as well as serum IgG and vaginal IgA levels. Furthermore, sublingual administration of the antigen only in the presence of the aGalCer adjuvant effectively boosted the OVA-specific immune responses. These results support potential clinical utility of sublingual route of vaccination with aGalCer-for prevention of pulmonary metastases.