The Predictive Utility of MammaPrint and BluePrint in Identifying Patients with Locally Advanced Breast Cancer Who are Most Likely to Have Nodal Downstaging and a Pathologic Complete Response After Neoadjuvant Chemotherapy.

BACKGROUND: Neoadjuvant chemotherapy (NCT) increases the feasibility of surgical resection by downstaging large primary breast tumors and nodal involvement, which may result in surgical de-escalation and improved outcomes. This subanalysis from the Multi-Institutional Neo-adjuvant Therapy MammaPrint Project I (MINT) trial evaluated the association between MammaPrint and BluePrint with nodal downstaging. PATIENTS AND METHODS: The prospective MINT trial (NCT01501487) enrolled 387 patients between 2011 and 2016 aged ≥ 18 years with invasive breast cancer (T2-T4). This subanalysis includes 146 patients with stage II-III, lymph node positive, who received NCT. MammaPrint stratifies tumors as having a Low Risk or High Risk of distant metastasis. Together with MammaPrint, BluePrint genomically (g) categorizes tumors as gLuminal A, gLuminal B, gHER2, or gBasal. RESULTS: Overall, 45.2% (n = 66/146) of patients had complete nodal downstaging, of whom 60.6% (n = 40/66) achieved a pathologic complete response. MammaPrint and combined MammaPrint and BluePrint were significantly associated with nodal downstaging (p = 0.007 and p < 0.001, respectively). A greater proportion of patients with MammaPrint High Risk tumors had nodal downstaging compared with Low Risk (p = 0.007). When classified with MammaPrint and BluePrint, more patients with gLuminal B, gHER2, and gBasal tumors had nodal downstaging compared with HR+HER2-, gLuminal A tumors (p = 0.538, p < 0.001, and p = 0.013, respectively). CONCLUSIONS: Patients with genomically High Risk tumors, defined by MammaPrint with or without BluePrint, respond better to NCT and have a higher likelihood of nodal downstaging compared with patients with gLuminal A tumors. These genomic signatures can be used to select node-positive patients who are more likely to have nodal downstaging and avoid invasive surgical procedures.

BluePrint breast cancer molecular subtyping recognizes single and dual subtype tumors with implications for therapeutic guidance.

PURPOSE: BluePrint (BP) is an 80-gene molecular subtyping test that classifies early-stage breast cancer (EBC) into Basal, Luminal, and HER2 subtypes. In most cases, breast tumors have one dominant subtype, representative of a single activated pathway. However, some tumors show a statistically equal representation of more than one subtype, referred to as dual subtype. This study aims to identify and examine dual subtype tumors by BP to understand their biology and possible implications for treatment guidance. METHODS: The BP scores of over 15,000 tumor samples from EBC patients were analyzed, and the differences between the highest and the lowest scoring subtypes were calculated. Based upon the distribution of the differences between BP scores, a threshold was determined for each subtype to identify dual versus single subtypes. RESULTS: Approximately 97% of samples had one single activated BluePrint molecular subtype, whereas ~ 3% of samples were classified as BP dual subtype. The most frequently occurring dual subtypes were the Luminal-Basal-type and Luminal-HER2-type. Luminal-Basal-type displays a distinct biology from the Luminal single type and Basal single type. Burstein's classification of the single and dual Basal samples showed that the Luminal-Basal-type is mostly classified as 'luminal androgen receptor' and 'mesenchymal' subtypes, supporting molecular evidence of AR activation in the Luminal-Basal-type tumors. Tumors classified as Luminal-HER2-type resemble features of both Luminal-single-type and HER2-single-type. However, patients with dual Luminal-HER2-type have a lower pathological complete response after receiving HER2-targeted therapies in addition to chemotherapy in comparison with patients with a HER2-single-type. CONCLUSION: This study demonstrates that BP identifies tumors with two active functional pathways (dual subtype) with specific transcriptional characteristics and highlights the added value of distinguishing BP dual from single subtypes as evidenced by distinct treatment response rates.

Age-Independent Preoperative Chemosensitivity and 5-Year Outcome Determined by Combined 70- and 80-Gene Signature in a Prospective Trial in Early-Stage Breast Cancer.

BACKGROUND: The Neoadjuvant Breast Symphony Trial (NBRST) demonstrated the 70-gene risk of distant recurrence signature, MammaPrint, and the 80-gene molecular subtyping signature, BluePrint, precisely determined preoperative pathological complete response (pCR) in breast cancer patients. We report 5-year follow-up results in addition to an exploratory analysis by age and menopausal status. METHODS: The observational, prospective NBRST (NCT01479101) included 954 early-stage breast cancer patients aged 18-90 years who received neoadjuvant chemotherapy and had clinical and genomic data available. Chemosensitivity and 5-year distant metastasis-free survival (DMFS) and overall survival (OS) were assessed. In a post hoc subanalysis, results were stratified by age (≤ 50 vs. > 50 years) and menopausal status in patients with hormone receptor-positive/human epidermal growth factor receptor 2-negative (HR+/HER2-) tumors. RESULTS: MammaPrint and BluePrint further classified 23% of tumors to a different subtype compared with immunohistochemistry, with more precise correspondence to pCR rates. Five-year DMFS and OS were highest in MammaPrint Low Risk, Luminal A-type and HER2-type tumors, and lowest in MammaPrint High Risk, Luminal B-type and Basal-type tumors. There was no significant difference in chemosensitivity between younger and older patients with Low-Risk (2.2% vs. 3.8%; p = 0.64) or High-Risk tumors (14.5% vs. 11.5%; p = 0.42), or within each BluePrint subtype; this was similar when stratifying by menopausal status. The 5-year outcomes were comparable by age or menopausal status for each molecular subtype. CONCLUSION: Intrinsic preoperative chemosensitivity and long-term outcomes were precisely determined by BluePrint and MammaPrint regardless of patient age, supporting the utility of these assays to inform treatment and surgical decisions in early-stage breast cancer.

Phase II Trial of Neoadjuvant Carboplatin and Nab-Paclitaxel in Patients with Triple-Negative Breast Cancer.

BACKGROUND: In this phase II clinical trial, we evaluated the efficacy of the nonanthracycline combination of carboplatin and nab-paclitaxel in early stage triple-negative breast cancer (TNBC). PATIENTS AND METHODS: Patients with newly diagnosed stage II-III TNBC (n = 69) were treated with neoadjuvant carboplatin (area under the curve 6) every 28 days for four cycles plus nab-paclitaxel (100 mg/m2 ) weekly for 16 weeks. Pathological complete response (pCR) and residual cancer burden (RCB) were analyzed with germline mutation status, tumor-infiltrating lymphocytes (TILs), TNBC molecular subtype, and GeparSixto immune signature (GSIS). RESULTS: Sixty-seven patients were evaluable for safety and response. Fifty-three (79%) patients experienced grade 3/4 adverse events, including grade 3 anemia (43%), neutropenia (39%), leukopenia (15%), thrombocytopenia (12%), fatigue (7%), peripheral neuropathy (7%), neutropenia (16%), and leukopenia (1%). Twenty-four patients (35%) had at least one dose delay, and 50 patients (72%) required dose reduction. Sixty-three (94%) patients completed scheduled treatment. The responses were as follows: 32 of 67 patients (48%) had pCR (RCB 0), 10 of 67 (15%) had RCB I, 19 of 67 (28%) had RCB II, 5 of 67 (7%) had RCB III, and 1 of 67 (2%) progressed and had no surgery. Univariate analysis showed that immune-hot GSIS and DNA repair defect (DRD) were associated with higher pCR with odds ratios of 4.62 (p = .005) and 4.76 (p = .03), respectively, and with RCB 0/I versus RCB II/III with odds ratio 4.80 (p = .01). Immune-hot GSIS was highly correlated with DRD status (p = .03), TIL level (p < .001), and TNBC molecular subtype (p < .001). After adjusting for age, race, stage, and grade, GSIS remained associated with higher pCR and RCB class 0/I versus II/III with odds ratios 7.19 (95% confidence interval [CI], 2.01-25.68; p = .002) and 8.95 (95% CI, 2.09-38.23; p = .003), respectively. CONCLUSION: The combination of carboplatin and nab-paclitaxel for early stage high-risk TNBC showed manageable toxicity and encouraging antitumor activity. Immune-hot GSIS is associated with higher pCR rate and RCB class 0/1. This study provides an additional rationale for using nonanthracycline platinum-based therapy for future neoadjuvant trials in early stage TNBCs. Clinical trial identification number: NCT01525966 IMPLICATIONS FOR PRACTICE: Platinum is an important neoadjuvant chemotherapy agent for treatment of early stage triple-negative breast cancer (TNBC). In this study, carboplatin and nab-paclitaxel were well tolerated and highly effective in TNBC, resulting in pathological complete response of 48%. In univariate and multivariate analyses adjusting for age, race, tumor stage and grade, "immune-hot" GeparSixto immune signature (GSIS) and DNA repair defect (DRD) were associated with higher pathological complete response (pCR) and residual cancer burden class 0/1. The association of immune-hot GSIS with higher pCR holds promise for de-escalating neoadjuvant chemotherapy for patients with early stage TNBC. Although GSIS is not routinely used in clinic, further development of this immune signature into a clinically applicable assay is indicated.

Henipavirus W Proteins Interact with 14-3-3 To Modulate Host Gene Expression.

Nipah virus (NiV) and Hendra virus (HeV), members of the Henipavirus genus in the Paramyxoviridae family, are recently emerged, highly lethal zoonotic pathogens. The NiV and HeV nonsegmented, negative-sense RNA genomes encode nine proteins, including the W protein. Expressed from the P gene through mRNA editing, W shares a common N-terminus with P and V but has a unique C-terminus. Expressed alone, W modulates innate immune responses by several mechanisms, and elimination of W from NiV alters the course of infection in experimentally infected ferrets. However, the specific host interactions that allow W to modulate innate immunity are incompletely understood. This study demonstrates that the NiV and HeV W proteins interact with all seven isoforms of the 14-3-3 family, regulatory molecules that preferentially bind phosphorylated target proteins to regulate a wide range of cellular functions. The interaction is dependent on the penultimate amino acid residue in the W sequence, a conserved, phosphorylated serine. The cocrystal structure of the W C-terminal binding motif with 14-3-3 provides only the second structure of a complex containing a mode III interactor, which is defined as a 14-3-3 interaction with a phosphoserine/phosphothreonine at the C-termini of the target protein. Transcriptomic analysis of inducible cell lines infected with an RNA virus and expressing either wild-type W or W lacking 14-3-3 binding, identifies new functions for W. These include the regulation of cellular metabolic processes, extracellular matrix organization, and apoptosis.IMPORTANCE Nipah virus (NiV) and Hendra virus (HeV), members of the Henipavirus genus, are recently emerged, highly lethal zoonotic pathogens that cause yearly outbreaks. NiV and HeV each encode a W protein that has roles in regulating host signaling pathways, including antagonism of the innate immune response. However, the mechanisms used by W to regulate these host responses are not clear. Here, characterization of the interaction of NiV and HeV W with 14-3-3 identifies modulation of 14-3-3-regulated host signaling pathways not previously associated with W, suggesting new avenues of research. The cocrystal structure of the NiV W:14-3-3 complex, as only the second structure of a 14-3-3 mode III interactor, provides further insight into this less-well-understood 14-3-3 binding motif.

Early Transcriptional Changes within Liver, Adrenal Gland, and Lymphoid Tissues Significantly Contribute to Ebola Virus Pathogenesis in Cynomolgus Macaques.

Ebola virus (EBOV) continues to pose a significant threat to human health, as evidenced by the 2013-2016 epidemic in West Africa and the ongoing outbreak in the Democratic Republic of the Congo. EBOV causes hemorrhagic fever, organ damage, and shock culminating in death, with case fatality rates as high as 90%. This high lethality combined with the paucity of licensed medical countermeasures makes EBOV a critical human pathogen. Although EBOV infection results in significant damage to the liver and the adrenal glands, little is known about the molecular signatures of injury in these organs. Moreover, while changes in peripheral blood cells are becoming increasingly understood, the host responses within organs and lymphoid tissues remain poorly characterized. To address this knowledge gap, we tracked longitudinal transcriptional changes in tissues collected from EBOV-Makona-infected cynomolgus macaques. Following infection, both liver and adrenal glands exhibited significant and early downregulation of genes involved in metabolism, coagulation, hormone synthesis, and angiogenesis; upregulated genes were associated with inflammation. Analysis of lymphoid tissues showed early upregulation of genes that play a role in innate immunity and inflammation and downregulation of genes associated with cell cycle and adaptive immunity. Moreover, transient activation of innate immune responses and downregulation of humoral immune responses in lymphoid tissues were confirmed with flow cytometry. Together, these data suggest that the liver, adrenal gland, and lymphatic organs are important sites of EBOV infection and that dysregulating the function of these vital organs contributes to the development of Ebola virus disease.IMPORTANCE Ebola virus (EBOV) remains a high-priority pathogen since it continues to cause outbreaks with high case fatality rates. Although it is well established that EBOV results in severe organ damage, our understanding of tissue injury in the liver, adrenal glands, and lymphoid tissues remains limited. We begin to address this knowledge gap by conducting longitudinal gene expression studies in these tissues, which were collected from EBOV-infected cynomolgus macaques. We report robust and early gene expression changes within these tissues, indicating they are primary sites of EBOV infection. Furthermore, genes involved in metabolism, coagulation, and adaptive immunity were downregulated, while inflammation-related genes were upregulated. These results indicate significant tissue damage consistent with the development of hemorrhagic fever and lymphopenia. Our study provides novel insight into EBOV-host interactions and elucidates how host responses within the liver, adrenal glands, and lymphoid tissues contribute to EBOV pathogenesis.

Prediction of Benefit From Adjuvant Pertuzumab by 80-Gene Signature in the APHINITY (BIG 4-11) Trial.

PURPOSE: At the primary analysis, the APHINITY trial reported a statistically significant but modest benefit of adding pertuzumab to standard adjuvant chemotherapy plus trastuzumab in patients with histologically confirmed human epidermal growth factor receptor 2 (HER2)-positive early-stage breast cancer. This study evaluated whether the 80-gene molecular subtyping signature (80-GS) could identify patients within the APHINITY population who derive the most benefit from dual anti-HER2 therapy. METHODS: In a nested case-control study design of 1,023 patients (matched event to control ratio of 3:1), the 80-GS classified breast tumors into functional luminal type, HER2 type, or basal type. Additionally, 80-GS distinguished tumor subtypes that exhibited a single-dominant functional pathway versus tumors with multiple activated pathways. The primary end point was invasive disease-free survival (IDFS). Hazard ratios (HRs) were evaluated by Cox regression. After excluding patients without appropriate consent and those with missing data, 964 patients were included. RESULTS: The 80-GS classified 50% (n = 479) of tumors as luminal type, 28% (n = 275) as HER2 type, and 22% (n = 209) as basal type. Most luminal-type tumors (86%) displayed a single-activated pathway, whereas 49% of HER2-type and 42% of basal-type tumors were dual activated. There was no significant difference in IDFS among different conventional 80-GS subtypes (single- and dual-activated subtypes combined). However, basal single-subtype tumors were significantly more likely to have an IDFS event (hazard ratio, 1.69 [95% CI, 1.12 to 2.54]) compared with other subtypes. HER2 single-subtype tumors displayed a trend toward greater beneficial effect on the addition of pertuzumab (hazard ratio, 0.56 [95% CI, 0.27 to 1.16]) compared with all other subtypes. CONCLUSION: The 80-GS identified subgroups of histologically confirmed HER2-positive tumors with distinct biological characteristics. Basal single-subtype tumors exhibit an inferior prognosis compared with other subgroups and may be candidates for additional therapeutic strategies. Preliminary results suggest patients with HER2-positive, genomically HER2 single-subtype tumors may particularly benefit from added pertuzumab, which warrants further investigation.

CX3CR1-Expressing Myeloid Cells Regulate Host-Helminth Interaction and Lung Inflammation.

Many helminth life cycles, including hookworm, involve a mandatory lung phase, where myeloid and granulocyte subsets interact with the helminth and respond to infection-induced lung injury. To evaluate these innate subsets in Nippostrongylus brasiliensis infection, reporter mice for myeloid cells (CX3CR1GFP ) and granulocytes (PGRPdsRED ) are employed. Nippostrongylus infection induces lung infiltration of reporter cells, including CX3CR1+ myeloid cells and PGRP+ eosinophils. Strikingly, CX3CR1GFP/GFP mice, which are deficient in CX3CR1, are protected from Nippostrongylus infection with reduced weight loss, lung leukocyte infiltration, and worm burden compared to CX3CR1+/+ mice. This protective effect is specific for CX3CR1 as CCR2-deficient mice do not exhibit reduced worm burdens. Nippostrongylus co-culture with lung Ly6C+ monocytes or CD11c+ cells demonstrates that CX3CR1GFP/GFP monocytes secrete more pro-inflammatory cytokines and actively bind the parasites causing reduced motility. RNA sequencing of Ly6C+ or CD11c+ cells shows Nippostrongylus-induced gene expression changes, particularly in monocytes, associated with inflammation, chemotaxis, and extracellular matrix remodeling pathways. Analysis reveals cytotoxic and adhesion molecules as potential effectors against the parasite, such as Gzma and Gzmb, which are elevated in CX3CR1GFP/GFP monocytes. These studies validate a dual innate cell reporter for lung helminth infection and demonstrate that CX3CR1 impairs monocyte-helminth interaction.

Combined 70- and 80-gene signatures identify tumors with genomically luminal biology responsive to neoadjuvant endocrine therapy and are prognostic of 5-year outcome in early-stage breast cancer.

BACKGROUND: As more patients with early-stage breast cancer receive neoadjuvant endocrine therapy (NET), there is a need for reliable biomarkers that can identify patients with HR+ HER2- tumors who are likely to benefit from NET. NBRST (NCT01479101) compared the prognostic value of the 70-gene risk classification and 80-gene molecular subtyping signatures with conventional pathological classification methods in response to neoadjuvant therapy. We evaluated the association of these signatures with clinical response and 5-year outcome of patients treated with NET. METHODS: 1091 patients with early-stage breast cancer scheduled to receive neoadjuvant therapy were prospectively enrolled into NBRST, and a sub-analysis of 67 patients treated with NET was performed. Patients received standard of care genomic testing using the 70-gene and 80-gene signatures and were treated with NET, per physician's discretion. The primary endpoint was pathologic partial response (pPR) and secondary endpoints were distant metastasis-free survival (DMFS) and overall survival (OS). Clinical benefit was defined as having a pPR or stable disease (SD) with NET. RESULTS: Overall, 94.4% of patients with genomically (g) Luminal A-Type (50.0% pPR and 44.4% SD) and 95.0% with Luminal B-Type tumors (55.0% pPR and 40.0% SD) exhibited clinical benefit. At 5 years, patients with gLuminal B tumors had significantly worse DMFS (75.6%, 95% CI 50.8-89.1) than patients with gLuminal A (91.1%; 95% CI 74.8-97.1; p = 0.047), with a similar trend for OS, albeit not significant (81.0%, 95% CI 56.9-92.4 and 91.1%, 95% CI 74.8-97.1, respectively; p = 0.13). CONCLUSIONS: Genomic assays offer a broader understanding of the underlying tumor biology, which adds precision to pathology as a preoperative risk classifier. Patients with 70-gene signature Low Risk, gLuminal A tumors treated with endocrine therapy alone have excellent 5-year outcomes. Most patients with genomically-defined Luminal A- and B-Type tumors respond well to NET, suggesting these patients may be safely treated with NET, while those with gLuminal B tumors will also require post-operative chemotherapy or CDK4/6 inhibitors to improve long-term outcomes. Overall, these findings demonstrate that genomic classification, defined by the combined 70- and 80-gene signatures, is associated with tumor response and prognostic of long-term outcomes.

Genomic Classification of HER2-Positive Patients With 80-Gene and 70-Gene Signatures Identifies Diversity in Clinical Outcomes With HER2-Targeted Neoadjuvant Therapy.

PURPOSE: The prospective Neoadjuvant Breast Registry Symphony Trial compared the 80-gene molecular subtyping signature with clinical assessment by immunohistochemistry and/or fluorescence in situ hybridization in predicting pathologic complete response (pCR) and 5-year outcomes in patients with early-stage breast cancer. METHODS: Standard-of-care neoadjuvant chemotherapy combined with trastuzumab or trastuzumab plus pertuzumab was given to patients with human epidermal growth factor receptor 2 (HER2)-positive tumors (n = 295). pCR was the primary end point, with secondary end points of distant metastasis-free survival and overall survival at 5 years. RESULTS: Among clinically defined HER2-positive (cHER2) tumors, the 80-gene assay identified 29.5% (87 of 295) as Luminal-Type (cHER2/gLuminal), 14.9% (44 of 295) as Basal-Type (cHER2/gBasal), and 55.6% (164 of 295) as HER2-Type (cHER2/genomically classified as HER2 [gHER2]). Patients with cHER2/gHER2 tumors had a higher pCR rate (61.6%) compared with non-gHER2 tumors (26.7%; P < .001). Dual targeting for cHER2/gHER2 tumors yielded a higher pCR rate (75%) compared with those treated with single HER2-targeted therapy (54%; P = .006). For cHER2/gBasal tumors, the 42.9% pCR rate observed with dual targeting was not different from that with trastuzumab alone (46.4%; P = .830). Among those with cHER2/gBasal tumors, 5-year distant metastasis-free survival (68.6%; 95% CI, 49.1 to 81.9) was significantly worse than in patients with cHER2/gLuminal tumors (88.9%; 95% CI, 78.0 to 94.6) and cHER2/gHER2 tumors (87.4%; 95% CI, 80.2 to 92.2; P = .010), with similar corresponding overall survival differences. CONCLUSION: The 80-gene assay identified meaningful genomic diversity in patients with cHER2 disease. Patients with cHER2/gHER2 tumors, who benefitted most from dual HER2-targeted therapy, accounted for approximately half of the cHER2 cohort. Genomically Luminal tumors had low pCR rates but good 5-year outcomes. cHER2/gBasal tumors derived no benefit from dual therapy and had significantly worse 5-year prognosis; these patients merit special consideration in future trials.

Distinct Neoadjuvant Chemotherapy Response and 5-Year Outcome in Patients With Estrogen Receptor-Positive, Human Epidermal Growth Factor Receptor 2-Negative Breast Tumors That Reclassify as Basal-Type by the 80-Gene Signature.

PURPOSE: The 80-gene molecular subtyping signature (80-GS) reclassifies a proportion of immunohistochemistry (IHC)-defined luminal breast cancers (estrogen receptor-positive [ER+], human epidermal growth factor receptor 2-negative [HER2-]) as Basal-Type. We report the association of 80-GS reclassification with neoadjuvant treatment response and 5-year outcome in patients with breast cancer. METHODS: Neoadjuvant Breast Registry Symphony Trial (NBRST; NCT01479101) is an observational, prospective study that included 1,069 patients with early-stage breast cancer age 18-90 years who received neoadjuvant therapy. Pathologic complete response (pCR) and 5-year distant metastasis-free survival (DMFS) and overall survival (OS) were assessed in 477 patients with IHC-defined ER+, HER2- tumors and in a reference group of 229 patients with IHC-defined triple-negative breast cancer (TNBC). RESULTS: 80-GS reclassified 15% of ER+, HER2- tumors (n = 73) as Basal-Type (ER+/Basal), which had similar pCR compared with TNBC/Basal tumors (34% v 38%; P = .52), and significantly higher pCR than ER+/Luminal A (2%; P < .001) and ER+/Luminal B (6%; P < .001) tumors. The 5-year DMFS (%, [95% CI]) was significantly lower for patients with ER+/Basal tumors (66% [52.6 to 77.3]), compared with those with ER+/Luminal A tumors (92.3% [85.2 to 96.1]) and ER+/Luminal B tumors (73.5% [44.5 to 79.3]). Importantly, patients with ER+/Basal or TNBC/Basal tumors that had a pCR exhibited significantly improved DMFS and OS compared with those with residual disease. By contrast, patients with ER+/Luminal B tumors had comparable 5-year DMFS and OS whether or not they achieved pCR. CONCLUSION: Significant differences in chemosensitivity and 5-year outcome suggest patients with ER+/Basal molecular subtype may benefit from neoadjuvant regimens optimized for patients with TNBC/Basal tumors compared with patients with ER+/Luminal subtype. These data highlight the importance of identifying this subset of patients to improve treatment planning and long-term survival.

Single Dose of a VSV-Based Vaccine Rapidly Protects Macaques From Marburg Virus Disease.

Marburg virus (MARV) is a member of the filovirus family that causes hemorrhagic disease with high case fatality rates. MARV is on the priority list of the World Health Organization for countermeasure development highlighting its potential impact on global public health. We developed a vesicular stomatitis virus (VSV)-based vaccine expressing the MARV glycoprotein (VSV-MARV) and previously demonstrated uniform protection of nonhuman primates (NHPs) with a single dose. Here, we investigated the fast-acting potential of this vaccine by challenging NHPs with MARV 14, 7 or 3 days after a single dose vaccination with VSV-MARV. We found that 100% of the animals survived when vaccinated 7 or 14 days and 75% of the animal survived when vaccinated 3 days prior to lethal MARV challenge. Transcriptional analysis of whole blood samples indicated activation of B cells and antiviral defense after VSV-MARV vaccination. In the day -14 and -7 groups, limited transcriptional changes after challenge were observed with the exception of day 9 post-challenge in the day -7 group where we detected gene expression profiles indicative of a recall response. In the day -3 group, transcriptional analysis of samples from surviving NHPs revealed strong innate immune activation. In contrast, the animal that succumbed to disease in this group lacked signatures of antiviral immunity. In summary, our data demonstrate that the VSV-MARV is a fast-acting vaccine suitable for the use in emergency situations like disease outbreaks in Africa.

Antiviral Innate Responses Induced by VSV-EBOV Vaccination Contribute to Rapid Protection.

Ebola virus (EBOV) is a single-stranded RNA virus that causes Ebola virus disease (EVD), characterized by excessive inflammation, lymphocyte apoptosis, hemorrhage, and coagulation defects leading to multiorgan failure and shock. Recombinant vesicular stomatitis virus expressing the EBOV glycoprotein (VSV-EBOV), which is highly efficacious against lethal challenge in nonhuman primates, is the only vaccine that successfully completed a phase III clinical trial. Additional studies showed VSV-EBOV provides complete and partial protection to macaques immunized 7 and 3 days before EBOV challenge, respectively. However, the mechanisms by which this live-attenuated vaccine elicits rapid protection are only partially understood. To address this, we carried out a longitudinal transcriptome analysis of host responses in whole-blood samples collected from cynomolgus macaques vaccinated with VSV-EBOV 28, 21, 14, 7, and 3 days before EBOV challenge. Our findings indicate the transcriptional response to the vaccine peaks 7 days following vaccination and contains signatures of both innate antiviral immunity as well as B-cell activation. EBOV challenge 1 week after vaccination resulted in large gene expression changes suggestive of a recall adaptive immune response 14 days postchallenge. Lastly, the timing and magnitude of innate immunity and interferon-stimulated gene expression correlated with viral burden and disease outcome in animals vaccinated 3 days before challenge.IMPORTANCE Ebola virus (EBOV) is the causative agent of Ebola virus disease (EVD), a deadly disease and major public health threat worldwide. A safe and highly efficacious vesicular stomatitis virus-based vaccine against EBOV is the only platform that has successfully completed phase III clinical trials and has been used in recent and ongoing outbreaks. Earlier studies showed that antibodies are the main mode of protection when this vaccine is administered 28 days before EBOV challenge. Recently, we showed this vaccine can provide protection when administered as early as 3 days before challenge and before antibodies are detected. This study seeks to identify the mechanisms of rapid protection, which in turn will pave the way for improved vaccines and therapeutics. Additionally, this study provides insight into host gene expression signatures that could provide early biomarkers to identify infected individuals who are at highest risk of poor outcomes.

Author Correction: Infection with the Makona variant results in a delayed and distinct host immune response compared to previous Ebola virus variants.

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A VP35 Mutant Ebola Virus Lacks Virulence but Can Elicit Protective Immunity to Wild-Type Virus Challenge.

Zaire ebolavirus (EBOV) VP35 protein is a suppressor of type I interferon (IFN) production, an inhibitor of dendritic cell maturation, and a putative virulence determinant. Here, a recombinant EBOV encoding a mutant VP35 virus (VP35m) is demonstrated to activate RIG-I-like receptor signaling and innate antiviral pathways. When inoculated into cynomolgus macaques, VP35m exhibits dramatic attenuation as compared to wild-type EBOV (wtEBOV), with 20 or 300 times the standard 100% lethal challenge dose not causing EBOV disease (EVD). Further, VP35m infection, despite limited replication in vivo, activates antigen presentation and innate immunity pathways and elicits increased frequencies of proliferating memory T cells and B cells and production of anti-EBOV antibodies. Upon wtEBOV challenge, VP35m-immunized animals survive, exhibiting host responses consistent with an orderly immune response and the absence of excessive inflammation. These data demonstrate that VP35 is a critical EBOV immune evasion factor and provide insights into immune mechanisms of EBOV control.

Protection Against Marburg Virus Using a Recombinant VSV-Vaccine Depends on T and B Cell Activation.

Marburg virus (MARV) is the causative agent of hemorrhagic fever outbreaks with high case fatality rates. Closely related to Ebola virus, MARV is a filamentous virus with a negative-sense, single-stranded RNA genome. Although extensive studies on filovirus countermeasures have been conducted, there are no licensed treatments against MARV infections. An experimental vaccine based on the recombinant vesicular stomatitis virus (VSV) expressing the MARV-Musoke glycoprotein demonstrated complete protection when a single dose was administered 28 days and up to 14 months prior to MARV challenge. Here, we analyzed the protective efficacy of an updated vaccine expressing the MARV-Angola glycoprotein (VSV-MARV). A single dose of VSV-MARV given 5 weeks before challenge provided uniform protection with no detectable viremia. The vaccine induced B and T cell proliferation and, importantly, antigen-specific IgG production. Transcriptomic signatures confirm these findings and suggest innate immunity engendered by VSV-MARV may direct the development of protective humoral immunity.

Transcriptomic analysis reveals a previously unknown role for CD8+ T-cells in rVSV-EBOV mediated protection.

Ebola virus (EBOV) poses a significant threat to human health as highlighted by the recent epidemic in West Africa. Data from animal studies and a ring vaccination clinical trial conducted in Guinea during the recent epidemic demonstrated that a recombinant VSV where G protein is replaced with EBOV GP (rVSV-EBOV) is safe and highly efficacious. We previously established that antibodies are essential for rVSV-EBOV mediated protection against EBOV; however, the mechanisms by which this vaccine induces a humoral response and the role of T-cells in rVSV-EBOV mediated protection remain poorly understood. Since this is the only vaccine platform that has completed Phase III clinical studies, it is imperative to gain a better understanding of its mechanisms of protection. Therefore, we performed a longitudinal gene expression analysis of samples collected from controls and T-cell-depleted macaques after rVSV-EBOV vaccination and EBOV challenge. We show that rVSV-EBOV vaccination induces gene expression changes consistent with anti-viral immunity and B-cell proliferation. We also report a previously unappreciated role for CD8+ T-cells in mediating rVSV-EBOV protection. Finally, limited viral transcription in surviving animals may boost protective responses after EBOV challenge by maintaining transcriptional changes. This study presents a novel approach in determining mechanisms of vaccine efficacy.

Infection with the Makona variant results in a delayed and distinct host immune response compared to previous Ebola virus variants.

Zaire Ebolavirus (ZEBOV) continues to pose a significant threat to human health as highlighted by the recent epidemic that originated in West Africa and the ongoing outbreak in the Democratic Republic of the Congo. Although the ZEBOV variant responsible for this epidemic (Makona) shares significant genetic similarity with previously identified variants (Kikwit and Mayinga), recent reports suggest slower disease progression in nonhuman primates. However, the pathogenesis caused by the new variant is not fully understood. We present the first comprehensive approach in understanding ZEBOV-Makona pathogenesis in cynomolgus macaques by measuring changes in immune cell frequencies, plasma levels of immune mediators, and differentially expressed genes (DEGs) within whole blood (WB) and peripheral blood mononuclear cells (PBMC). Our combined approach revealed a link between: 1) increased interferon-stimulated gene expression, IFNα levels, and activated plasmacytoid dendritic cells; 2) higher proinflammatory gene expression, cytokine and chemokine levels, and non-classical monocytes; 3) gene signature of leukocyte activation and increased granulocytes; and 4) decreased expression of lymphocyte related genes and lymphopenia. In addition, our data strongly indicate delayed disease progression as well as limited overlap (~30%) in host transcriptome changes following ZEBOV-Makona infection compared to ZEBOV-Kikwit. These observations provide novel insight into the molecular mechanisms of ZEBOV-Makona pathogenesis.

Transcriptome Analysis of Circulating Immune Cell Subsets Highlight the Role of Monocytes in Zaire Ebola Virus Makona Pathogenesis.

Existing models of Ebola virus disease (EVD) suggest antigen-presenting cells are initial targets of Zaire ebolavirus (ZEBOV). In vitro studies have shown that ZEBOV infection of monocytes and macrophages results in the production of inflammatory mediators, which may cause lymphocyte apoptosis. However, these findings have not been corroborated by in vivo studies. In this study, we report the first longitudinal analysis of transcriptional changes in purified monocytes, T-cells, and B-cells isolated from cynomolgus macaques following infection with ZEBOV-Makona. Our data reveal monocytes as one of the major immune cell subsets that supports ZEBOV replication in vivo. In addition, we report a marked increase in the transcription of genes involved in inflammation, coagulation, and vascular disease within monocytes, suggesting that monocytes contribute to EVD manifestations. Further, genes important for antigen presentation and regulation of immunity were downregulated, potentially subverting development of adaptive immunity. In contrast, lymphocytes, which do not support ZEBOV replication, showed transcriptional changes limited to a small number of interferon-stimulated genes (ISGs) and a failure to upregulate genes associated with an antiviral effector immune response. Collectively, these data suggest that ZEBOV-infected monocytes play a significant role in ZEBOV-Makona pathogenesis and strategies to suppress virus replication or modify innate responses to infection in these cells should be a priority for therapeutic intervention.