Phenotypic signatures of circulating neoantigen-reactive CD8+ T cells in patients with metastatic cancers.

Circulating T cells from peripheral blood (PBL) can provide a rich and noninvasive source for antitumor T cells. By single-cell transcriptomic profiling of 36 neoantigen-specific T cell clones from 6 metastatic cancer patients, we report the transcriptional and cell surface signatures of antitumor PBL-derived CD8+ T cells (NeoTCRPBL). Comparison of tumor-infiltrating lymphocyte (TIL)- and PBL-neoantigen-specific T cells revealed that NeoTCRPBL T cells are low in frequency and display less-dysfunctional memory phenotypes relative to their TIL counterparts. Analysis of 100 antitumor TCR clonotypes indicates that most NeoTCRPBL populations target the same neoantigens as TILs. However, NeoTCRPBL TCR repertoire is only partially shared with TIL. Prediction and testing of NeoTCRPBL signature-derived TCRs from PBL of 6 prospective patients demonstrate high enrichment of clonotypes targeting tumor mutations, a viral oncogene, and patient-derived tumor. Thus, the NeoTCRPBL signature provides an alternative source for identifying antitumor T cells from PBL of cancer patients, enabling immune monitoring and immunotherapies.

Healthcare Professionals' Attitudes towards and Knowledge and Understanding of Paediatric Palliative Medicine (PPM) and Its Meaning within the Paediatric Intensive Care Unit (PICU): A Summative Content Analysis in a Tertiary Children's Hospital in Scotland-"An In Vitro Study".

Background: Paediatric palliative medicine (PPM) is a holistic approach to care for children and their families. Services are growing and developing worldwide but significant disparity in service provision remains. The Paediatric Supportive and Palliative Care Team (PSPCT) at the Royal Hospital for Children in Glasgow was established in 2019, but there is still no clear integrated role within the paediatric intensive care unit (PICU) at present. Through analysing the attitudes, meaning, knowledge and understanding of PPM in the PICU environment, we hoped to explore the experiences of those providing paediatric palliative care and to identify any barriers to or facilitators of integrated working to gain a better understanding of providing this care. Methods: This qualitative study used a survey composed of five open-ended and five closed questions. Sixteen out of a possible thirty-two responses (50%) were accrued from PICU healthcare professionals, including consultants (n = 19), advanced nurse practitioners (n = 4) and band-seven nurses (n = 9). The data were comprehensively studied and analysed by two coders using summative content analysis with assistance from data management software. Codes were further developed to form categories and subcategories. Results: Two categories were found: (1) the role of palliative care and (2) experiences of providing palliative care. A total of five subcategories were found, demonstrating that the PSPCT can enhance care in PICU through collaborative working. Barriers identified included staffing, funding and stigma around palliative care. Conclusions: This study shows that PICU professionals have a good understanding of the concepts of PPM and view it as an essential part of PICU work. Barriers related to resources and misperceptions of palliative care can be overcome through improved education, funding and staff retention, but this would require buy-in from policymakers. The perspective from our relatively small team increases generalizability to growing teams across the country.

Cell surface marker-based capture of neoantigen-reactive CD8+ T-cell receptors from metastatic tumor digests.

BACKGROUND: Cellular immunotherapies using autologous tumor-infiltrating lymphocytes (TIL) can induce durable regression of epithelial cancers in selected patients with treatment-refractory metastatic disease. As the genetic engineering of T cells with tumor-reactive T-cell receptors (TCRs) comes to the forefront of clinical investigation, the rapid, scalable, and cost-effective detection of patient-specific neoantigen-reactive TIL remains a top priority. METHODS: We analyzed the single-cell transcriptomic states of 31 neoantigen-specific T-cell clonotypes to identify cell surface dysfunction markers that best identified the metastatic transcriptional states enriched with antitumor TIL. We developed an efficient method to capture neoantigen-reactive TCRs directly from resected human tumors based on cell surface co-expression of CD39, programmed cell death protein-1, and TIGIT dysfunction markers (CD8+ TILTP). RESULTS: TILTP TCR isolation achieved a high degree of correlation with single-cell transcriptomic signatures that identify neoantigen-reactive TCRs, making it a cost-effective strategy using widely available resources. Reconstruction of additional TILTP TCRs from tumors identified known and novel antitumor TCRs, showing that at least 39.5% of TILTP TCRs are neoantigen-reactive or tumor-reactive. Despite their substantial enrichment for neoantigen-reactive TCR clonotypes, clonal dynamics of 24 unique antitumor TILTP clonotypes from four patients indicated that most in vitro expanded TILTP populations failed to demonstrate neoantigen reactivity, either by loss of neoantigen-reactive clones during TIL expansion, or through functional impairment during cognate neoantigen recognition. CONCLUSIONS: While direct usage of in vitro-expanded CD8+ TILTP as a source for cellular therapy might be precluded by profound TIL dysfunction, isolating TILTP represents a streamlined effective approach to rapidly identify neoantigen-reactive TCRs to design engineered cellular immunotherapies against cancer.

Adoptive Cellular Therapy with Autologous Tumor-Infiltrating Lymphocytes and T-cell Receptor-Engineered T Cells Targeting Common p53 Neoantigens in Human Solid Tumors.

Adoptive cellular therapy (ACT) targeting neoantigens can achieve durable clinical responses in patients with cancer. Most neoantigens arise from patient-specific mutations, requiring highly individualized treatments. To broaden the applicability of ACT targeting neoantigens, we focused on TP53 mutations commonly shared across different cancer types. We performed whole-exome sequencing on 163 patients with metastatic solid cancers, identified 78 who had TP53 missense mutations, and through immunologic screening, identified 21 unique T-cell reactivities. Here, we report a library of 39 T-cell receptors (TCR) targeting TP53 mutations shared among 7.3% of patients with solid tumors. These TCRs recognized tumor cells in a TP53 mutation- and human leucocyte antigen (HLA)-specific manner in vitro and in vivo. Twelve patients with chemorefractory epithelial cancers were treated with ex vivo-expanded autologous tumor-infiltrating lymphocytes (TIL) that were naturally reactive against TP53 mutations. However, limited clinical responses (2 partial responses among 12 patients) were seen. These infusions contained low frequencies of mutant p53-reactive TILs that had exhausted phenotypes and showed poor persistence. We also treated one patient who had chemorefractory breast cancer with ACT comprising autologous peripheral blood lymphocytes transduced with an allogeneic HLA-A*02-restricted TCR specific for p53R175H. The infused cells exhibited an improved immunophenotype and prolonged persistence compared with TIL ACT and the patient experienced an objective tumor regression (-55%) that lasted 6 months. Collectively, these proof-of-concept data suggest that the library of TCRs targeting shared p53 neoantigens should be further evaluated for the treatment of patients with advanced human cancers. See related Spotlight by Klebanoff, p. 919.

Breast Cancers Are Immunogenic: Immunologic Analyses and a Phase II Pilot Clinical Trial Using Mutation-Reactive Autologous Lymphocytes.

PURPOSE: Metastatic breast cancer (mBrCa) is most often an incurable disease with only modest responses to available immunotherapies. This study investigates the immunogenicity of somatic mutations in breast cancer and explores the therapeutic efficacy in a pilot trial of mutation-reactive tumor-infiltrating lymphocytes (TILs) in patients with metastatic disease. PATIENTS AND METHODS: Forty-two patients with mBrCa refractory to previous lines of treatment underwent surgical resection of a metastatic lesion(s), isolation of TIL cultures, identification of exomic nonsynonymous tumor mutations, and immunologic screening for neoantigen reactivity. Clinically eligible patients with appropriate reactivity were enrolled into one cohort of an ongoing phase II pilot trial of adoptive cell transfer of selected neoantigen-reactive TIL, with a short course of pembrolizumab (ClinicalTrials.gov identifier: NCT01174121). RESULTS: TILs were isolated and grown in culture from the resected lesions of all 42 patients with mBrCa, and a median number of 112 (range: 6-563) nonsynonymous mutations per patient were identified. Twenty-eight of 42 (67%) patients contained TIL that recognized at least one immunogenic somatic mutation (median: 3 neoantigens per patient, range: 1-11), and 13 patients demonstrated robust reactivity appropriate for adoptive transfer. Eight patients remained clinically eligible for treatment, and six patients were enrolled on a protocol of adoptive cell transfer of enriched neoantigen-specific TIL, in combination with pembrolizumab (≤ 4 doses). Objective tumor regression was noted in three patients, including one complete response (now ongoing over 5.5 years) and two partial responses (6 and 10 months). CONCLUSION: Most patients with breast cancer generated a natural immune response targeting the expressed products of their cancer mutations. Adoptive transfer of TIL is a highly personalized experimental option for patients with mBrCa shown to be capable of mediating objective responses in this pilot trial and deserves further study.

Molecular signatures of antitumor neoantigen-reactive T cells from metastatic human cancers.

The accurate identification of antitumor T cell receptors (TCRs) represents a major challenge for the engineering of cell-based cancer immunotherapies. By mapping 55 neoantigen-specific TCR clonotypes (NeoTCRs) from 10 metastatic human tumors to their single-cell transcriptomes, we identified signatures of CD8+ and CD4+ neoantigen-reactive tumor-infiltrating lymphocytes (TILs). Neoantigen-specific TILs exhibited tumor-specific expansion with dysfunctional phenotypes, distinct from blood-emigrant bystanders and regulatory TILs. Prospective prediction and testing of 73 NeoTCR signature-derived clonotypes demonstrated that half of the tested TCRs recognized tumor antigens or autologous tumors. NeoTCR signatures identified TCRs that target driver neoantigens and nonmutated viral or tumor-associated antigens, suggesting a common metastatic TIL exhaustion program. NeoTCR signatures delineate the landscape of TILs across metastatic tumors, enabling successful TCR prediction based purely on TIL transcriptomic states for use in cancer immunotherapy.

Pre-existing antibodies directed against a tetramerizing domain enhance the immune response against artificially stabilized soluble tetrameric influenza neuraminidase.

The neuraminidase (NA) is an abundant antigen at the surface of influenza virions. Recent studies have highlighted the immune-protective potential of NA against influenza and defined anti-NA antibodies as an independent correlate of protection. Even though NA head domain changes at a slightly slower pace than hemagglutinin (HA), NA is still subject to antigenic drift, and therefore an NA-based influenza vaccine antigen may have to be updated regularly and thus repeatedly administered. NA is a tetrameric type II membrane protein, which readily dissociates into dimers and monomers when expressed in a soluble form. By using a tetramerizing zipper, such as the tetrabrachion (TB) from Staphylothermus marinus, it is possible to stabilize soluble NA in its active tetrameric conformation, an imperative for the optimal induction of protective NA inhibitory antibodies. The impact of repetitive immunizations with TB-stabilized antigens on the immunogenicity of soluble TB-stabilized NA is unknown. We demonstrate that TB is immunogenic in mice. Interestingly, preexisting anti-TB antibodies enhance the anti-NA antibody response induced by immunization with TB-stabilized NA. This immune-enhancing effect was transferable by serum and operated independently of activating Fcγ receptors. We also demonstrate that priming with TB-stabilized NA antigens, enhances the NA inhibitory antibody responses against a heterosubtypic TB-stabilized NA. These findings have implications for the clinical development of oligomeric vaccine antigens that are stabilized by a heterologous oligomerizing domain.

Identification and Validation of T-cell Receptors Targeting RAS Hotspot Mutations in Human Cancers for Use in Cell-based Immunotherapy.

PURPOSE: Immunotherapies mediate the regression of human tumors through recognition of tumor antigens by immune cells that trigger an immune response. Mutations in the RAS oncogenes occur in about 30% of all patients with cancer. These mutations play an important role in both tumor establishment and survival and are commonly found in hotspots. Discovering T-cell receptors (TCR) that recognize shared mutated RAS antigens presented on MHC class I and class II molecules are thus promising reagents for "off-the-shelf" adoptive cell therapies (ACT) following insertion of the TCRs into lymphocytes. EXPERIMENTAL DESIGN: In this ongoing work, we screened for RAS antigen recognition in tumor-infiltrating lymphocytes (TIL) or by in vitro stimulation of peripheral blood lymphocytes (PBL). TCRs recognizing mutated RAS were identified from the reactive T cells. The TCRs were then reconstructed and virally transduced into PBLs and tested. RESULTS: Here, we detect and report multiple novel TCR sequences that recognize nonsynonymous mutant RAS hotspot mutations with high avidity and specificity and identify the specific class-I and -II MHC restriction elements involved in the recognition of mutant RAS. CONCLUSIONS: The TCR library directed against RAS hotspot mutations described here recognize RAS mutations found in about 45% of the Caucasian population and about 60% of the Asian population and represent promising reagents for "off-the-shelf" ACTs.

Characteristics and outcome of pediatric renal cell carcinoma patients registered in the International Society of Pediatric Oncology (SIOP) 93-01, 2001 and UK-IMPORT database: A report of the SIOP-Renal Tumor Study Group.

In children, renal cell carcinoma (RCC) is rare. This study is the first report of pediatric patients with RCC registered by the International Society of Pediatric Oncology-Renal Tumor Study Group (SIOP-RTSG). Pediatric patients with histologically confirmed RCC, registered in SIOP 93-01, 2001 and UK-IMPORT databases, were included. Event-free survival (EFS) and overall survival (OS) were analyzed using the Kaplan-Meier method. Between 1993 and 2019, 122 pediatric patients with RCC were registered. Available detailed data (n = 111) revealed 56 localized, 30 regionally advanced, 25 metastatic and no bilateral cases. Histological classification according to World Health Organization 2004, including immunohistochemical and molecular testing for transcription factor E3 (TFE3) and/or EB (TFEB) translocation, was available for 65/122 patients. In this group, the most common histological subtypes were translocation type RCC (MiT-RCC) (36/64, 56.3%), papillary type (19/64, 29.7%) and clear cell type (4/64, 6.3%). One histological subtype was not reported. In the remaining 57 patients, translocation testing could not be performed, or TFE-cytogenetics and/or immunohistochemistry results were missing. In this group, the most common RCC histological subtypes were papillary type (21/47, 44.7%) and clear cell type (11/47, 23.4%). Ten histological subtypes were not reported. Estimated 5-year (5y) EFS and 5y OS of the total group was 70.5% (95% CI = 61.7%-80.6%) and 84.5% (95% CI = 77.5%-92.2%), respectively. Estimated 5y OS for localized, regionally advanced, and metastatic disease was 96.8%, 92.3%, and 45.6%, respectively. In conclusion, the registered pediatric patients with RCC showed a reasonable outcome. Survival was substantially lower for patients with metastatic disease. This descriptive study stresses the importance of full, prospective registration including TFE-testing.

Rapid Identification and Evaluation of Neoantigen-reactive T-Cell Receptors From Single Cells.

Engineered T cells expressing tumor-specific T-cell receptors (TCRs) are emerging as a mode of personalized cancer immunotherapy that requires identification of TCRs against the products of known driver mutations and novel mutations in a timely fashion. We present a nonviral and non-next-generation sequencing platform for rapid, and efficient neoantigen-specific TCR identification and evaluation that does not require the use of recombinant cloning techniques. The platform includes an innovative method of TCRα detection using Sanger sequencing, TCR pairings and the use of TCRα/ß gene fragments for putative TCR evaluation. Using patients' samples, we validated and compared our new methods head-to-head with conventional approaches used for TCR discovery. Development of a unique demultiplexing method for identification of TCRα, adaptation of synthetic TCRs for gene transfer, and a reliable reporter system significantly shortens TCR discovery time over conventional methods and increases throughput to facilitate testing prospective personalized TCRs for adoptive cell therapy.

An Unusual Cause of Hemorrhagic Cystitis in a Teenager With Medulloblastoma.

Hemorrhagic cystitis is a life-threatening condition in which the transitional epithelium and blood vessels of the bladder necrose leading to severe hematuria, abdominal pain, and voiding lower urinary tract symptoms. Etiology includes chemotherapy (cyclophosphamide, ifosfamide, busulfan), radiotherapy, or infectious agents. We present a pediatric case of a 15-year-old boy with medulloblastoma who developed hemorrhagic cystitis following cisplatin chemotherapy. All other causes were ruled out and it is therefore likely that the agent, in this case, was cisplatin, which has never had hemorrhagic cystitis reported as a side effect. We also suggest a mechanism for urothelial injury centered around OCT-2 receptors.

Pediatric and young adult renal cell carcinoma.

Renal cell carcinoma (RCC) is rare in children but is the most common renal tumor in adults. Pediatric RCC has different clinical characteristics, histopathology, and treatment compared with adult disease. Databases were reviewed from inception to February 2020, identifying 32 publications pertaining to 350 patients under 27 years. Surgery is the cornerstone for cure in localized RCC. Lymph node dissection remains controversial. Conventional radiotherapy has no curative role in RCC; similarly, conventional chemotherapy has not proven to be effective in large cohorts. Pediatric metastatic RCC has a poor outlook. There are no published prospective studies demonstrating which adjuvant therapy could improve outcome. Sunitinib, a tyrosine kinase inhibitor, is recommended in this group despite limited evidence. This review provides an overview for pediatric RCC, including the evolving role of precision medicine.

Characteristics and Outcome of Children with Renal Cell Carcinoma: A Narrative Review.

Pediatric renal cell carcinoma (RCC) is a rare type of kidney cancer, most commonly occurring in teenagers and young adolescents. Few relatively large series of pediatric RCC have been reported. Knowledge of clinical characteristics, outcome and treatment strategies are often based on the more frequently occurring adult types of RCC. However, published pediatric data suggest that clinical, molecular and histological characteristics of pediatric RCC differ from adult RCC. This paper summarizes reported series consisting of ≥10 RCC pediatric patients in order to create an up-to-date overview of the clinical and histopathological characteristics, treatment and outcome of pediatric RCC patients.

Antigen Experienced T Cells from Peripheral Blood Recognize p53 Neoantigens.

PURPOSE: The purpose of this study was to evaluate antigen experienced T cells in peripheral blood lymphocytes (PBL) for responses to p53 neoantigens. EXPERIMENTAL DESIGN: PBLs from patients with a mutated TP53 tumor were sorted for antigen-experienced T cells and in vitro stimulation (IVS) was performed with p53 neoantigens. The IVS cultures were stimulated with antigen-presenting cells expressing p53 neoantigens, enriched for 41BB/OX40 and grown with rapid expansion protocol. RESULTS: T-cell responses were not observed in the PBLs of 4 patients who did not have tumor-infiltrating lymphocyte (TIL) responses to mutated TP53. In contrast, 5 patients with TIL responses to mutated TP53 also had similar T-cell responses in their PBLs, indicating that the PBLs and TILs were congruent in p53 neoantigen reactivity. CD4+ and CD8+ T cells were specific for p53R175H, p53Y220C, or p53R248W neoantigens, including a 78% reactive T-cell culture against p53R175H and HLA-A*02:01. Tracking TCRB clonotypes (clonality, top ranked, and TP53 mutation-specific) supported the enrichment of p53 neoantigen-reactive T cells from PBLs. The same T-cell receptor (TCR) from the TIL was found in the IVS cultures in three cases and multiple unique TCRs were found in another patient. TP53 mutation-specific T cells also recognized tumor cell lines bearing the appropriate human leukocyte antigen restriction element and TP53 mutation, indicating these T cells could recognize processed and presented p53 neoantigens. CONCLUSIONS: PBL was a noninvasive source of T cells targeting TP53 mutations for cell therapy and can provide a window into intratumoral p53 neoantigen immune responses.See related commentary by Olivera et al., p. 1203.

Recognition of human gastrointestinal cancer neoantigens by circulating PD-1+ lymphocytes.

Tumor-resident lymphocytes can mount a response against neoantigens expressed in microsatellite-stable gastrointestinal (GI) cancers, and adoptive transfer of neoantigen-specific lymphocytes has demonstrated antitumor activity in selected patients. However, whether peripheral blood could be used as an alternative minimally invasive source to identify lymphocytes targeting neoantigens in patients with GI cancer with relatively low mutation burden is unclear. We used a personalized high-throughput screening strategy to investigate whether PD-1 expression in peripheral blood could be used to identify CD8+ or CD4+ lymphocytes recognizing neoantigens identified by whole-exome sequencing in 7 patients with GI cancer. We found that neoantigen-specific lymphocytes were preferentially enriched in the CD8+PD-1+/hi or CD4+PD-1+/hi subsets, but not in the corresponding bulk or PD-1- fractions. In 6 of 7 individuals analyzed we identified circulating CD8+ and CD4+ lymphocytes targeting 6 and 4 neoantigens, respectively. Moreover, neoantigen-reactive T cells and a T cell receptor (TCR) isolated from the CD8+PD-1+ subsets recognized autologous tumor, albeit at reduced levels, in 2 patients with available cell lines. These data demonstrate the existence of circulating T cells targeting neoantigens in GI cancer patients and provide an approach to generate enriched populations of personalized neoantigen-specific lymphocytes and isolate TCRs that could be exploited therapeutically to treat cancer.

Unique Neoantigens Arise from Somatic Mutations in Patients with Gastrointestinal Cancers.

Immunotherapies can mediate regression of human tumors with high mutation rates, but responses are rarely observed in patients with common epithelial cancers. This raises the question of whether patients with these common cancers harbor T lymphocytes that recognize mutant proteins expressed by autologous tumors that may represent ideal targets for immunotherapy. Using high-throughput immunologic screening of mutant gene products identified via whole-exome sequencing, we identified neoantigen-reactive tumor-infiltrating lymphocytes (TIL) from 62 of 75 (83%) patients with common gastrointestinal cancers. In total, 124 neoantigen-reactive TIL populations were identified, and all but one of the neoantigenic determinants were unique. The results of in vitro T-cell recognition assays demonstrated that 1.6% of the gene products encoded by somatic nonsynonymous mutations were immunogenic. These findings demonstrate that the majority of common epithelial cancers elicit immune recognition and open possibilities for cell-based immunotherapies for patients bearing these cancers. SIGNIFICANCE: TILs cultured from 62 of 75 (83%) patients with gastrointestinal cancers recognized neoantigens encoded by 1.6% of somatic mutations expressed by autologous tumor cells, and 99% of the neoantigenic determinants appeared to be unique and not shared between patients.This article is highlighted in the In This Issue feature, p. 983.

Immunologic Recognition of a Shared p53 Mutated Neoantigen in a Patient with Metastatic Colorectal Cancer.

Adoptive cell therapy (ACT) with T cells targeting neoantigens can mediate durable responses in patients with metastatic cancer. Cell therapies targeting common shared antigens for epithelial cancers are not yet broadly available. Here, we report the identification and characterization in one patient of T-cell receptors (TCRs) recognizing mutated p53 p.R175H, which is shared among a subset of patients with cancer. Tumor-infiltrating lymphocytes were screened for recognition of mutated neoantigens in a patient with metastatic colorectal cancer. HLA-A*0201-restricted recognition of mutated p53 p.R175H was identified, and the minimal peptide epitope was HMTEVVRHC. Reactive T cells were isolated by tetramer sorting, and three TCRs were identified. These TCRs mediated recognition of commercially available ovarian cancer, uterine carcinoma, and myeloma cell lines, as well as an NIH patient-derived esophageal adenocarcinoma line that endogenously expressed p53 p.R175H and HLA-A*0201. They also mediated recognition of p53 p.R175H+ colon, breast, and leukemia cell lines after transduction with a retrovirus encoding HLA-A*0201. This work demonstrates that common shared mutated epitopes such as those found in p53 can elicit immunogenic responses and that the application of ACT may be extended to patients with any cancer histology that expresses both HLA-A*0201 and the p53 p.R175H mutation.

Memory T cells targeting oncogenic mutations detected in peripheral blood of epithelial cancer patients.

T cells targeting shared oncogenic mutations can induce durable tumor regression in epithelial cancer patients. Such T cells can be detected in tumor infiltrating lymphocytes, but whether such cells can be detected in the peripheral blood of patients with the common metastatic epithelial cancer patients is unknown. Using a highly sensitive in vitro stimulation and cell enrichment of peripheral memory T cells from six metastatic cancer patients, we identified and isolated CD4+, and CD8+ memory T cells targeting the mutated KRASG12D and KRASG12V variants, respectively, in three patients. In an additional two metastatic colon cancer patients, we detected CD8+ neoantigen-specific cells targeting the mutated SMAD5 and MUC4 proteins. Therefore, memory T cells targeting unique as well as shared somatic mutations can be detected in the peripheral blood of epithelial cancer patients and can potentially be used for the development of effective personalized T cell-based cancer immunotherapy across multiple patients.

Correction: Split inactivated COBRA vaccine elicits protective antibodies against H1N1 and H3N2 influenza viruses.

[This corrects the article DOI: 10.1371/journal.pone.0204284.].

Split inactivated COBRA vaccine elicits protective antibodies against H1N1 and H3N2 influenza viruses.

Development of broadly reactive or universal influenza vaccines will be a paradigm shifting event for the influenza vaccine field. These next generation vaccines could replace the current standard of care with vaccines that elicit broadly cross-protective immune responses. However, a variety of in vitro and in vivo models are necessary to make the best assessments of these vaccine formulations to determine their mechanisms of action, and allow for downselection of candidates prior to human clinical trials. Our group has developed the computationally optimized broadly reactive antigen (COBRA) technology to develop HA head-based strategies to elicit antibodies against H1, H3, and H5 influenza strains. These vaccines elicit broadly reactive antibody responses that neutralize not only historical and contemporary vaccine strains, but also co-circulating variants in mice. In this study, we used H1 and H3 HA antigens in a split, inactivated vaccine (IIV) formulation in combination with the AF03 squalene-in-water emulsion adjuvant in ferrets immunologically naïve to influenza virus. The H3 COBRA IIV vaccine T11 elicited antibodies with HAI activity against more H3N2 influenza strains compared to IIV expressing wild-type H3 HA antigens, except for IIV vaccines expressing the HA from A/Texas/50/2012 (Tx/12) virus. H1 COBRA IIV vaccines, P1 and X6, elicited antibodies that recognized a similar number of H1N1 viruses as those antibodies elicited by IIV expressing the A/California/07/2009 (CA/09) HA. Ferrets vaccinated with the P1 or X6 COBRA IIV were protected against CA/09 challege and cleared virus from the lungs of the ferrets, similar to ferrets vaccinated with the CA/09 IIV.