Mutation-guided vaccine design: A process for developing boosting immunogens for HIV broadly neutralizing antibody induction.

A major goal of HIV-1 vaccine development is the induction of broadly neutralizing antibodies (bnAbs). Although success has been achieved in initiating bnAb B cell lineages, design of boosting immunogens that select for bnAb B cell receptors with improbable mutations required for bnAb affinity maturation remains difficult. Here, we demonstrate a process for designing boosting immunogens for a V3-glycan bnAb B cell lineage. The immunogens induced affinity-matured antibodies by selecting for functional improbable mutations in bnAb precursor knockin mice. Moreover, we show similar success in prime and boosting with nucleoside-modified mRNA-encoded HIV-1 envelope trimer immunogens, with improved selection by mRNA immunogens of improbable mutations required for bnAb binding to key envelope glycans. These results demonstrate the ability of both protein and mRNA prime-boost immunogens for selection of rare B cell lineage intermediates with neutralizing breadth after bnAb precursor expansion, a key proof of concept and milestone toward development of an HIV-1 vaccine.

Breadth of SARS-CoV-2 neutralization and protection induced by a nanoparticle vaccine.

Coronavirus vaccines that are highly effective against current and anticipated SARS-CoV-2 variants are needed to control COVID-19. We previously reported a receptor-binding domain (RBD)-sortase A-conjugated ferritin nanoparticle (scNP) vaccine that induced neutralizing antibodies against SARS-CoV-2 and pre-emergent sarbecoviruses and protected non-human primates (NHPs) from SARS-CoV-2 WA-1 infection. Here, we find the RBD-scNP induced neutralizing antibodies in NHPs against pseudoviruses of SARS-CoV and SARS-CoV-2 variants including 614G, Beta, Delta, Omicron BA.1, BA.2, BA.2.12.1, and BA.4/BA.5, and a designed variant with escape mutations, PMS20. Adjuvant studies demonstrate variant neutralization titers are highest with 3M-052-aqueous formulation (AF). Immunization twice with RBD-scNPs protect NHPs from SARS-CoV-2 WA-1, Beta, and Delta variant challenge, and protect mice from challenges of SARS-CoV-2 Beta variant and two other heterologous sarbecoviruses. These results demonstrate the ability of RBD-scNPs to induce broad neutralization of SARS-CoV-2 variants and to protect animals from multiple different SARS-related viruses. Such a vaccine could provide broad immunity to SARS-CoV-2 variants.

Stabilized HIV-1 envelope immunization induces neutralizing antibodies to the CD4bs and protects macaques against mucosal infection.

A successful HIV-1 vaccine will require induction of a polyclonal neutralizing antibody (nAb) response, yet vaccine-mediated induction of such a response in primates remains a challenge. We found that a stabilized HIV-1 CH505 envelope (Env) trimer formulated with a Toll-like receptor 7/8 agonist induced potent HIV-1 polyclonal nAbs that correlated with protection from homologous simian-human immunodeficiency virus (SHIV) infection. The serum dilution that neutralized 50% of virus replication (ID50 titer) required to protect 90% of macaques was 1:364 against the challenge virus grown in primary rhesus CD4+ T cells. Structural analyses of vaccine-induced nAbs demonstrated targeting of the Env CD4 binding site or the N156 glycan and the third variable loop base. Autologous nAb specificities similar to those elicited in macaques by vaccination were isolated from the human living with HIV from which the CH505 Env immunogen was derived. CH505 viral isolates were isolated that mutated the V1 to escape both the infection-induced and vaccine-induced antibodies. These results define the specificities of a vaccine-induced nAb response and the protective titers of HIV-1 vaccine-induced nAbs required to protect nonhuman primates from low-dose mucosal challenge by SHIVs bearing a primary transmitted/founder Env.

Breadth of SARS-CoV-2 Neutralization and Protection Induced by a Nanoparticle Vaccine.

Coronavirus vaccines that are highly effective against SARS-CoV-2 variants are needed to control the current pandemic. We previously reported a receptor-binding domain (RBD) sortase A-conjugated ferritin nanoparticle (RBD-scNP) vaccine that induced neutralizing antibodies against SARS-CoV-2 and pre-emergent sarbecoviruses and protected monkeys from SARS-CoV-2 WA-1 infection. Here, we demonstrate SARS-CoV-2 RBD-scNP immunization induces potent neutralizing antibodies in non-human primates (NHPs) against all eight SARS-CoV-2 variants tested including the Beta, Delta, and Omicron variants. The Omicron variant was neutralized by RBD-scNP-induced serum antibodies with a mean of 10.6-fold reduction of ID50 titers compared to SARS-CoV-2 D614G. Immunization with RBD-scNPs protected NHPs from SARS-CoV-2 WA-1, Beta, and Delta variant challenge, and protected mice from challenges of SARS-CoV-2 Beta variant and two other heterologous sarbecoviruses. These results demonstrate the ability of RBD-scNPs to induce broad neutralization of SARS-CoV-2 variants and to protect NHPs and mice from multiple different SARS-related viruses. Such a vaccine could provide the needed immunity to slow the spread of and reduce disease caused by SARS-CoV-2 variants such as Delta and Omicron.

Different adjuvanted pediatric HIV envelope vaccines induced distinct plasma antibody responses despite similar B cell receptor repertoires in infant rhesus macaques.

Different HIV vaccine regimens elicit distinct plasma antibody responses in both human and nonhuman primate models. Previous studies in human and non-human primate infants showed that adjuvants influenced the quality of plasma antibody responses induced by pediatric HIV envelope vaccine regimens. We recently reported that use of the 3M052-SE adjuvant and longer intervals between vaccinations are associated with higher magnitude of antibody responses in infant rhesus macaques. However, the impact of different adjuvants in HIV vaccine regimens on the developing infant B cell receptor (BCR) repertoire has not been studied. This study evaluated whether pediatric HIV envelope vaccine regimens with different adjuvants induced distinct antigen-specific memory B cell repertoires and whether specific immunoglobulin (Ig) immunogenetic characteristics are associated with higher magnitude of plasma antibody responses in vaccinated infant rhesus macaques. We utilized archived preclinical pediatric HIV vaccine studies PBMCs and tissue samples from 19 infant rhesus macaques immunized either with (i) HIV Env protein with a squalene adjuvant, (ii) MVA-HIV and Env protein co-administered using a 3-week interval, (iii) MVA-HIV prime/ protein boost with an extended 6-week interval between immunizations, or (iv) with HIV Env administered with 3M-052-SE adjuvant. Frequencies of vaccine-elicited HIV Env-specific memory B cells from PBMCs and tissues were similar across vaccination groups (frequency range of 0.06-1.72%). There was no association between vaccine-elicited antigen-specific memory B cell frequencies and plasma antibody titer or avidity. Moreover, the epitope specificity and Ig immunogenetic features of vaccine-elicited monoclonal antibodies did not differ between the different vaccine regimens. These data suggest that pediatric HIV envelope vaccine candidates with different adjuvants that previously induced higher magnitude and quality of plasma antibody responses in infant rhesus macaques were not driven by distinct antigen-specific memory BCR repertoires.

Development of COVID-19 vaccine using a dual Toll-like receptor ligand liposome adjuvant.

We developed a SARS-CoV-2 spike subunit vaccine formulation containing dual TLR ligand liposome adjuvant. The vaccine-induced robust systemic neutralizing antibodies and completely protected mice from a lethal challenge. Two immunizations protected against lung injury and cleared the virus from lungs upon challenge. The adjuvanted vaccine also elicited systemic and local anti-Spike IgA which can be an important feature for a COVID-19 vaccine.

A yeast expressed RBD-based SARS-CoV-2 vaccine formulated with 3M-052-alum adjuvant promotes protective efficacy in non-human primates.

Ongoing SARS-CoV-2 vaccine development is focused on identifying stable, cost-effective, and accessible candidates for global use, specifically in low and middle-income countries. Here, we report the efficacy of a rapidly scalable, novel yeast expressed SARS-CoV-2 specific receptor-binding domain (RBD) based vaccine in rhesus macaques. We formulated the RBD immunogen in alum, a licensed and an emerging alum adsorbed TLR-7/8 targeted, 3M-052-alum adjuvants. The RBD+3M-052-alum adjuvanted vaccine promoted better RBD binding and effector antibodies, higher CoV-2 neutralizing antibodies, improved Th1 biased CD4+T cell reactions, and increased CD8+ T cell responses when compared to the alum-alone adjuvanted vaccine. RBD+3M-052-alum induced a significant reduction of SARS-CoV-2 virus in respiratory tract upon challenge, accompanied by reduced lung inflammation when compared with unvaccinated controls. Anti-RBD antibody responses in vaccinated animals inversely correlated with viral load in nasal secretions and BAL. RBD+3M-052-alum blocked a post SARS-CoV-2 challenge increase in CD14+CD16++ intermediate blood monocytes, and Fractalkine, MCP-1, and TRAIL in the plasma. Decreased plasma analytes and intermediate monocyte frequencies correlated with reduced nasal and BAL viral loads. Lastly, RBD-specific plasma cells accumulated in the draining lymph nodes and not in the bone marrow, contrary to previous findings. Together, these data show that a yeast expressed, RBD-based vaccine+3M-052-alum provides robust immune responses and protection against SARS-CoV-2, making it a strong and scalable vaccine candidate.

Optimizing a Multi-Component Intranasal Entamoeba Histolytica Vaccine Formulation Using a Design of Experiments Strategy.

Amebiasis is a neglected tropical disease caused by Entamoeba histolytica. Although the disease burden varies geographically, amebiasis is estimated to account for some 55,000 deaths and millions of infections globally per year. Children and travelers are among the groups with the greatest risk of infection. There are currently no licensed vaccines for prevention of amebiasis, although key immune correlates for protection have been proposed from observational studies in humans. We previously described the development of a liposomal adjuvant formulation containing two synthetic TLR ligands (GLA and 3M-052) that enhanced antigen-specific fecal IgA, serum IgG2a, a mixed IFNγ and IL-17A cytokine profile from splenocytes, and protective efficacy following intranasal administration with the LecA antigen. By applying a statistical design of experiments (DOE) and desirability function approach, we now describe the optimization of the dose of each vaccine formulation component (LecA, GLA, 3M-052, and liposome) as well as the excipient composition (acyl chain length and saturation; PEGylated lipid:phospholipid ratio; and presence of antioxidant, tonicity, or viscosity agents) to maximize desired immunogenicity characteristics while maintaining physicochemical stability. This DOE/desirability index approach led to the identification of a lead candidate composition that demonstrated immune response durability and protective efficacy in the mouse model, as well as an assessment of the impact of each active vaccine formulation component on protection. Thus, we demonstrate that both GLA and 3M-052 are required for statistically significant protective efficacy. We also show that immunogenicity and efficacy results differ in female vs male mice, and the differences appear to be at least partly associated with adjuvant formulation composition.

Neutralizing antibody vaccine for pandemic and pre-emergent coronaviruses.

Betacoronaviruses caused the outbreaks of severe acute respiratory syndrome (SARS) and Middle East respiratory syndrome, as well as the current pandemic of SARS coronavirus 2 (SARS-CoV-2)1-4. Vaccines that elicit protective immunity against SARS-CoV-2 and betacoronaviruses that circulate in animals have the potential to prevent future pandemics. Here we show that the immunization of macaques with nanoparticles conjugated with the receptor-binding domain of SARS-CoV-2, and adjuvanted with 3M-052 and alum, elicits cross-neutralizing antibody responses against bat coronaviruses, SARS-CoV and SARS-CoV-2 (including the B.1.1.7, P.1 and B.1.351 variants). Vaccination of macaques with these nanoparticles resulted in a 50% inhibitory reciprocal serum dilution (ID50) neutralization titre of 47,216 (geometric mean) for SARS-CoV-2, as well as in protection against SARS-CoV-2 in the upper and lower respiratory tracts. Nucleoside-modified mRNAs that encode a stabilized transmembrane spike or monomeric receptor-binding domain also induced cross-neutralizing antibody responses against SARS-CoV and bat coronaviruses, albeit at lower titres than achieved with the nanoparticles. These results demonstrate that current mRNA-based vaccines may provide some protection from future outbreaks of zoonotic betacoronaviruses, and provide a multimeric protein platform for the further development of vaccines against multiple (or all) betacoronaviruses.

SARS-CoV-2 vaccination induces neutralizing antibodies against pandemic and pre-emergent SARS-related coronaviruses in monkeys.

Betacoronaviruses (betaCoVs) caused the severe acute respiratory syndrome (SARS) and Middle East Respiratory Syndrome (MERS) outbreaks, and now the SARS-CoV-2 pandemic. Vaccines that elicit protective immune responses against SARS-CoV-2 and betaCoVs circulating in animals have the potential to prevent future betaCoV pandemics. Here, we show that immunization of macaques with a multimeric SARS-CoV-2 receptor binding domain (RBD) nanoparticle adjuvanted with 3M-052-Alum elicited cross-neutralizing antibody responses against SARS-CoV-1, SARS-CoV-2, batCoVs and the UK B.1.1.7 SARS-CoV-2 mutant virus. Nanoparticle vaccination resulted in a SARS-CoV-2 reciprocal geometric mean neutralization titer of 47,216, and robust protection against SARS-CoV-2 in macaque upper and lower respiratory tracts. Importantly, nucleoside-modified mRNA encoding a stabilized transmembrane spike or monomeric RBD protein also induced SARS-CoV-1 and batCoV cross-neutralizing antibodies, albeit at lower titers. These results demonstrate current mRNA vaccines may provide some protection from future zoonotic betaCoV outbreaks, and provide a platform for further development of pan-betaCoV nanoparticle vaccines.

Intratumoral immunotherapy with TLR7/8 agonist MEDI9197 modulates the tumor microenvironment leading to enhanced activity when combined with other immunotherapies.

BACKGROUND: Immune checkpoint blockade (ICB) promotes adaptive immunity and tumor regression in some cancer patients. However, in patients with immunologically "cold" tumors, tumor-resident innate immune cell activation may be required to prime an adaptive immune response and so exploit the full potential of ICB. Whilst Toll-like receptor (TLR) agonists have been used topically to successfully treat some superficial skin tumors, systemic TLR agonists have not been well-tolerated. METHODS: The response of human immune cells to TLR7 and 8 agonism was measured in primary human immune cell assays. MEDI9197 (3M-052) was designed as a novel lipophilic TLR7/8 agonist that is retained at the injection site, limiting systemic exposure. Retention of the TLR7/8 agonist at the site of injection was demonstrated using quantitative whole-body autoradiography, HPLC-UV, and MALDI mass spectrometry imaging. Pharmacodynamic changes on T cells from TLR7/8 agonist treated B16-OVA tumors was assessed by histology, quantitative real time PCR, and flow cytometry. Combination activity of TLR7/8 agonism with immunotherapies was assessed in vitro by human DC-T cell MLR assay, and in vivo using multiple syngeneic mouse tumor models. RESULTS: Targeting both TLR7 and 8 triggers an innate and adaptive immune response in primary human immune cells, exemplified by secretion of IFNα, IL-12 and IFNγ. In contrast, a STING or a TLR9 agonist primarily induces release of IFNα. We demonstrate that the TLR7/8 agonist, MEDI9197, is retained at the sight of injection with limited systemic exposure. This localized TLR7/8 agonism leads to Th1 polarization, enrichment and activation of natural killer (NK) and CD8+ T cells, and inhibition of tumor growth in multiple syngeneic models. The anti-tumor activity of this TLR7/8 agonist is enhanced when combined with T cell-targeted immunotherapies in pre-clinical models. CONCLUSION: Localized TLR7/8 agonism can enhance recruitment and activation of immune cells in tumors and polarize anti-tumor immunity towards a Th1 response. Moreover, we demonstrate that the anti-tumor effects of this TLR7/8 agonist can be enhanced through combination with checkpoint inhibitors and co-stimulatory agonists.

Adjuvant composition and delivery route shape immune response quality and protective efficacy of a recombinant vaccine for Entamoeba histolytica.

Amebiasis caused by Entamoeba histolytica is the third leading cause of parasitic mortality globally, with some 100,000 deaths annually, primarily among young children. Protective immunity to amebiasis is associated with fecal IgA and IFN-γ in humans; however, no vaccine exists. We have previously identified recombinant LecA as a potential protective vaccine antigen. Here we describe the development of a stable, manufacturable PEGylated liposomal adjuvant formulation containing two synthetic Toll-like receptor (TLR) ligands: GLA (TLR4) and 3M-052 (TLR7/8). The liposomes stimulated production of monocyte/macrophage chemoattractants MCP-1 and Mip-1ß, and Th1-associated cytokines IL-12p70 and IFN-γ from human whole blood dependent on TLR ligand composition and dose. The liposomes also demonstrated acceptable physicochemical compatibility with the recombinant LecA antigen. Whereas mice immunized with LecA and GLA-liposomes demonstrated enhanced antigen-specific fecal IgA titers, mice immunized with LecA and 3M-052-liposomes showed a stronger Th1 immune profile. Liposomes containing GLA and 3M-052 together elicited both LecA-specific fecal IgA and Th1 immune responses. Furthermore, the quality of the immune response could be modulated with modifications to the liposomal formulation based on PEG length. Compared to subcutaneous administration, the optimized liposome adjuvant composition with LecA antigen administered intranasally resulted in significantly enhanced fecal IgA, serum IgG2a, as well as systemic IFN-γ and IL-17A levels in mice. The optimized intranasal regimen provided greater than 80% protection from disease as measured by parasite antigen in the colon. This work demonstrates the physicochemical and immunological characterization of an optimized mucosal adjuvant system containing a combination of TLR ligands with complementary activities and illustrates the importance of adjuvant composition and route of delivery to enhance a multifaceted and protective immune response to amebiasis.

TLR7/8 adjuvant overcomes newborn hyporesponsiveness to pneumococcal conjugate vaccine at birth.

Infection is the most common cause of mortality in early life, and immunization is the most promising biomedical intervention to reduce this burden. However, newborns fail to respond optimally to most vaccines. Adjuvantation is a key approach to enhancing vaccine immunogenicity, but responses of human newborn leukocytes to most candidate adjuvants, including most TLR agonists, are functionally distinct. Herein, we demonstrate that 3M-052 is a locally acting lipidated imidazoquinoline TLR7/8 agonist adjuvant in mice, which, when properly formulated, can induce robust Th1 cytokine production by human newborn leukocytes in vitro, both alone and in synergy with the alum-adjuvanted pneumococcal conjugate vaccine 13 (PCV13). When admixed with PCV13 and administered i.m. on the first day of life to rhesus macaques, 3M-052 dramatically enhanced generation of Th1 CRM-197-specific neonatal CD4+ cells, activation of newborn and infant Streptococcus pneumoniae polysaccharide-specific (PnPS-specific) B cells as well as serotype-specific antibody titers, and opsonophagocytic killing. Remarkably, a single dose at birth of PCV13 plus 0.1 mg/kg 3M-052 induced PnPS-specific IgG responses that were approximately 10-100 times greater than a single birth dose of PCV13 alone, rapidly exceeding the serologic correlate of protection, as early as 28 days of life. This potent immunization strategy, potentially effective with one birth dose, could represent a new paradigm in early life vaccine development.

Nanoformulation of synergistic TLR ligands to enhance vaccination against Entamoeba histolytica.

Diarrheal infectious diseases represent a major cause of global morbidity and mortality. There is an urgent need for vaccines against diarrheal pathogens, especially parasites. Modern subunit vaccines rely on combining a highly purified antigen with an adjuvant to increase their efficacy. In the present study, we evaluated the ability of a nanoliposome adjuvant system to trigger a strong mucosal immune response to the Entamoeba histolytica Gal/GalNAc lectin LecA antigen. CBA/J mice were immunized with alum, emulsion or liposome based formulations containing synthetic TLR agonists. A liposome formulation containing TLR4 and TLR7/8 agonists was selected based on its ability to generate intestinal IgA, plasma IgG2a/IgG1, IFN-γ and IL-17A. Immunization with a mucosal prime followed by a parenteral boost generated a high mucosal IgA response that inhibited adherence of parasites to mammalian cells. Inclusion of the immune potentiator all-trans retinoic acid in the regimen further improved the mucosal IgA response. Immunization protected from infection with up to 55% efficacy. Our results show that a nanoliposome delivery system containing TLR agonists is a promising prospect for the development of vaccines against enteric pathogens, especially when a multifaceted immune response is desired.

Adsorption of a synthetic TLR7/8 ligand to aluminum oxyhydroxide for enhanced vaccine adjuvant activity: A formulation approach.

For nearly a century, aluminum salts have been the most widely used vaccine adjuvant formulation, and have thus established a history of safety and efficacy. Nevertheless, for extremely challenging disease targets such as tuberculosis or HIV, the adjuvant activity of aluminum salts may not be potent enough to achieve protective efficacy. Adsorption of TLR ligands to aluminum salts facilitates enhanced adjuvant activity, such as in the human papilloma virus vaccine Cervarix®. However, some TLR ligands such as TLR7/8 agonist imidazoquinolines do not efficiently adsorb to aluminum salts. The present report describes a formulation approach to solving this challenge by developing a lipid-based nanosuspension of a synthetic TLR7/8 ligand (3M-052) that facilitates adsorption to aluminum oxyhydroxide via the structural properties of the helper lipid employed. In immunized mice, the aluminum oxyhydroxide-adsorbed formulation of 3M-052 enhanced antibody and TH1-type cellular immune responses to vaccine antigens for tuberculosis and HIV.

Radiotherapy combined with TLR7/8 activation induces strong immune responses against gastrointestinal tumors.

In addition to local cytotoxic activity, radiotherapy may also elicit local and systemic antitumor immunity, which may be augmented by immunotherapeutic agents including Toll-like receptor (TLR) 7/8 agonists. Here, we investigated the ability of 3M-011 (854A), a TLR7/8 agonist, to boost the antigen-presenting activity of dendritic cells (DC) as an adjuvant to radiotherapy. The combined treatment induced marked local and systemic responses in subcutaneous and orthotopic mouse models of colorectal and pancreatic cancer. In vitro cytotoxicity assays as well as in vivo depletion experiments with monoclonal antibodies identified NK and CD8 T cells as the cell populations mediating the cytotoxic effects of the treatment, while in vivo depletion of CD11c+ dendritic cells (DC) in CD11c-DTR transgenic mice revealed DC as the pivotal immune hub in this setting. The specificity of the immune reaction was confirmed by ELISPOT assays. TLR7/8 agonists therefore seem to be potent adjuvants to radiotherapy, inducing strong local and profound systemic immune responses to tumor antigens released by conventional therapy.

The use of Toll-like receptor 7/8 agonists as vaccine adjuvants.

Small molecule Toll-like receptor (TLR) 7/8 agonists have demonstrated potential as vaccine adjuvants, since they directly activate APCs and can enhance both humoral and cellular immune responses, especially Th1 responses. Although the natural ligands for TLR7 and TLR8 are ssRNA, the vast majority of vaccine studies performed thus far have been performed with synthetic small molecule imidazoquinolines, such as imiquimod and resiquimod. Despite the approved clinical use of the topical TLR7 agonist, imiquimod (Aldara(®) Imiquimod 5% cream; 3M, MN, USA), for external genital warts, superficial basal cell carcinoma and actinic keratosis, no vaccines using TLR7, TLR8 or TLR7/8 agonists have progressed beyond early-phase clinical studies thus far. This review will highlight the nonclinical and clinical studies that indicate promise for TLR7/8 ligands as vaccine adjuvants, reasons for inconsistent results thus far, problems with current technology and potential paths forward for TLR7/8 agonists as vaccine adjuvants.

Potentiation of the anti-tumor effects of imidazoquinoline immune response modifiers by cyclophosphamide.

The aim of the current study was to evaluate the influence of chemotherapeutic drugs on immunotherapy with Imidazoquinoline Toll-like Receptor (TLR) agonists in cancer. First, the previously described antitumor efficacy of TLR agonists [i.e. a TLR7 agonist (852A) and a dual TLR7/8 agonist (3M-011)] was confirmed in additional cancer models, and second the therapeutic potential of TLR agonists in combination with cyclophosphamide was investigated. The antitumor potential was evaluated against a panel of syngeneic tumor models; namely B16-F10 melanoma, M3 melanoma and MC-26 colon carcinoma. Systemic administration of either 3M-011 or 852A in these various syngeneic models induced significant antitumor activity as evidenced by delays in tumor growth curves. Combination of cyclophosphamide with either 3M-011 or 852A demonstrated that cyclophosphamide does not negatively interfere with the TLR agonist's antitumor effects, but may, depending on the dosing schedule, to actually potentiate the effect. These findings suggest that the immunomodulatory TLR agonists may be used in combination with cytotoxic agents in the treatment of cancer.

NK1.1+ cells mediate the antitumor effects of a dual Toll-like receptor 7/8 agonist in the disseminated B16-F10 melanoma model.

Innate immune stimulation with Toll-like receptor (TLR) agonists is a proposed modality for immunotherapy of melanoma. Here, a TLR7/8 agonist, 3M-011, was used effectively as a single systemic agent against disseminated mouse B16-F10 melanoma. The investigation of the mechanism of antitumor action revealed that the agonist had no direct cytotoxic effects on tumor cells tested in vitro. In addition, 3M-011 retained its effectiveness in scid/B6 mice and scid/NOD mice, eliminating the requirement for T and B cells, but lost its activity in beige (bg/bg) and NK1.1-immunodepleted mice, suggesting a critical role for natural killer (NK) cells in the antitumor response. NK cytotoxicity was enhanced in vivo by the TLR7/8 agonist; this activation was long lasting, as determined by sustained expression of the activation marker CD69. Also, in human in vitro studies, 3M-011 potentiated NK cytotoxicity. TLR7/8-mediated NK-dependent antitumor activity was retained in IFN-alpha/beta receptor-deficient as well as perforin-deficient mice, while depletion of IFN-gamma significantly decreased the ability of 3M-011 to delay tumor growth. Thus, IFN-gamma-dependent functions of NK cell populations appear essential for cancer immunotherapy with TLR7/8 agonists.