Cyclic Dinucleotide–Adjuvanted Dengue Virus Nonstructural Protein 1 Induces Protective Antibody and T Cell Responses

Endothelial dysfunction and vascular leak, pathogenic hallmarks of severe dengue disease, are directly triggered by dengue virus (DENV) nonstructural protein 1 (NS1). Previous studies have shown that immunization with NS1, as well as passive transfer of NS1-immune serum or anti-NS1 mAb, prevent NS1-mediated lethality in vivo. In this study, we evaluated the immunogenicity and protective capacity of recombinant DENV NS1 administered with cyclic dinucleotides (CDNs), potent activators of innate immune pathways and highly immunogenic adjuvants. Using both wild-type C57BL/6 mice and IFN-α/β receptor-deficient mice, we show that NS1-CDN immunizations elicit serotype-specific and cross-reactive Ab and T cell responses. Furthermore, NS1-CDN vaccinations conferred significant homotypic and heterotypic protection from DENV2-induced morbidity and mortality. In addition, we demonstrate that high anti-NS1 Ab titers are associated with protection, supporting the role of humoral responses against DENV NS1 as correlates of protection. These findings highlight the potential of CDN-based adjuvants for inducing Ab and T cell responses and validate NS1 as an important candidate for dengue vaccine development

NSG Mice Provide a Better Spontaneous Model of Breast Cancer Metastasis than Athymic (Nude) Mice.

Metastasis is the most common cause of mortality in breast cancer patients worldwide. To identify improved mouse models for breast cancer growth and spontaneous metastasis, we examined growth and metastasis of both estrogen receptor positive (T47D) and negative (MDA-MB-231, SUM1315, and CN34BrM) human breast cancer cells in nude and NSG mice. Both primary tumor growth and spontaneous metastases were increased in NSG mice compared to nude mice. In addition, a pattern of metastasis similar to that observed in human breast cancer patients (metastases to the lungs, liver, bones, brain, and lymph nodes) was found in NSG mice. Furthermore, there was an increase in the metastatic burden in NSG compared to nude mice that were injected with MDA-MB-231 breast cancer cells in an intracardiac experimental metastasis model. This data demonstrates that NSG mice provide a better model for studying human breast cancer metastasis compared to the current nude mouse model

Magnitude of Therapeutic STING Activation Determines CD8⁺ T Cell-Mediated Anti-tumor Immunity

STING pathway activation by intratumoral administration of cyclic dinucleotides (CDNs) results in stable tumor regression, yet the underlying innate and adaptive immune mechanisms are not fully established. ADU-S100, CDN under clinical evaluation, was used with an optimized dosing regimen to uncover key immune requirements for tumor regression. An immunogenic dose induces local expansion of tumor-specific CD8+ T cells, which are both necessary and sufficient for durable anti-tumor immunity and correlated with STING activation in hematopoietic but not tumor or stromal cells. Type I IFN, but not TNFα, drives optimal anti-tumor immune responses. The function of ADU-S100-induced CD8+ T cells was enhanced by combination with immune checkpoint modulators anti-PD-1 and anti-CTLA4. These results provide fundamental mechanistic insights into STING IT therapy

Human breast cancer cells grow better in NSG mice than in nude mice.

<p>Human breast cancer cells were injected into the lactiferous duct of each of the fourth mammary glands in female nude or NSG mice. A. Tumor volume was measured starting at 21 days after injection of the cells. Breast cancer cells grew significantly faster in NSG mice (black squares) versus those growing in nude mice (open circles). Data are mean ± SEM. B. Tumors were weighed at the endpoint. The weight of tumors that arose in NSG mice were significantly larger than those grown in nude mice for all breast cancer cells examined. The increase in end tumor weight that arose from these cell lines grown in NSG mice was as follows: CN34BrM, 58%; MDA-MB-231, 65%; and SUM1315, 95%. The line indicates mean tumor weight. The mean is reported as the average of each tumor. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163521#pone.0163521.t002" target="_blank">Table 2</a> for the number of mice per group. **p<0.01, ***p<0.001. Please see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163521#sec002" target="_blank">Methods</a> for the statistical analysis performed.</p

Human breast cancer cells are more metastatic in NSG mice than in nude mice in an experimental metastasis model.

<p>MDA-MB-231 cells were injected into the heart of both nude (n = 7) and NSG (n = 8) mice and metastases were analyzed. A. Fluorescence images (white area is fluorescent tumors) were taken using a Zeiss SteroDiscovery.V12 fluorescence dissecting microscope with an AxioCam MRm digital camera. Representative images are shown from several different organs. Arrows, examples of numerous micrometastases in the lungs and brain of NSG mice (≤10 mm²). Bar, 2 mm. B. The metastases in the lungs, liver, brain (cranial and caudal) and bones (spine, ribs, scapula, radius, humerus, pelvis, femur and tibia) were enumerated for each mouse using Image J.</p

NSG mice have a higher metastatic burden than nude mice in a spontaneous metastasis model of breast cancer.

<p>Metastases were analyzed from the mice described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163521#pone.0163521.g001" target="_blank">Fig 1</a>. Fluorescent images as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163521#pone.0163521.g002" target="_blank">Fig 2</a> were counted from each organ for each mouse in Image J. Metastases were counted in both the cranial and caudal side of the brain. Bone metastases were found in the spine, ribs, scapula, radius, humerus, pelvis, femur and tibia of NSG mice injected with MDA-MB-231 cells and only in the tibia and femur of NSG mice injected with SUM1315 cells. Statistics determined by an unpaired t-test. *p<0.05, **p<0.01, ****p<0.0001</p

Comparison of immunodeficient mouse strains used in breast cancer research.

<p>Comparison of immunodeficient mouse strains used in breast cancer research.</p

STING agonist formulated cancer vaccines can cure established tumors resistant to PD-1 blockade.

Stimulator of interferon genes (STING) is a cytosolic receptor that senses both exogenous and endogenous cytosolic cyclic dinucleotides (CDNs), activating TBK1/IRF3 (interferon regulatory factor 3), NF-κB (nuclear factor κB), and STAT6 (signal transducer and activator of transcription 6) signaling pathways to induce robust type I interferon and proinflammatory cytokine responses. CDN ligands were formulated with granulocyte-macrophage colony-stimulating factor (GM-CSF)-producing cellular cancer vaccines--termed STINGVAX--that demonstrated potent in vivo antitumor efficacy in multiple therapeutic models of established cancer. We found that rationally designed synthetic CDN derivative molecules, including one with an Rp,Rp dithio diastereomer and noncanonical c[A(2',5')pA(3',5')p] phosphate bridge structure, enhanced antitumor efficacy of STINGVAX in multiple aggressive therapeutic models of established cancer in mice. Antitumor activity was STING-dependent and correlated with increased activation of dendritic cells and tumor antigen-specific CD8(+) T cells. Tumors from STINGVAX-treated mice demonstrated marked PD-L1 (programmed death ligand 1) up-regulation, which was associated with tumor-infiltrating CD8(+)IFNγ(+) T cells. When combined with PD-1 (programmed death 1) blockade, STINGVAX induced regression of palpable, poorly immunogenic tumors that did not respond to PD-1 blockade alone