Syndromic immune disorder caused by a viable hypomorphic allele of spliceosome component Snrnp40.

We report a new immunodeficiency disorder in mice caused by a viable hypomorphic mutation of Snrnp40, an essential gene encoding a subunit of the U5 small nuclear ribonucleoprotein (snRNP) complex of the spliceosome. Snrnp40 is ubiquitous but strongly expressed in lymphoid tissue. Homozygous mutant mice showed hypersusceptibility to infection by murine cytomegalovirus and multiple defects of lymphoid development, stability and function. Cell-intrinsic defects of hematopoietic stem cell differentiation also affected homozygous mutants. SNRNP40 deficiency in primary hematopoietic stem cells or T cells or the EL4 cell line increased the frequency of splicing errors, mostly intron retention, in several hundred messenger RNAs. Altered expression of proteins associated with immune cell function was also observed in Snrnp40-mutant cells. The immunological consequences of SNRNP40 deficiency presumably result from cumulative, moderate effects on processing of many different mRNA molecules and secondary reductions in the expression of critical immune proteins, yielding a syndromic immune disorder.

Disruption of the ZFP574-THAP12 complex suppresses B cell malignancies in mice.

Despite the availability of life-extending treatments for B cell leukemias and lymphomas, many of these cancers remain incurable. Thus, the development of new molecular targets and therapeutics is needed to expand treatment options. To identify new molecular targets, we used a forward genetic screen in mice to identify genes required for development or survival of lymphocytes. Here, we describe Zfp574, an essential gene encoding a zinc finger protein necessary for normal and malignant lymphocyte survival. We show that ZFP574 interacts with zinc finger protein THAP12 and promotes the G1-to-S-phase transition during cell cycle progression. Mutation of ZFP574 impairs nuclear localization of the ZFP574-THAP12 complex. ZFP574 or THAP12 deficiency results in cell cycle arrest and impaired lymphoproliferation. Germline mutation, acute gene deletion, or targeted degradation of ZFP574 suppressed Myc-driven B cell leukemia in mice, but normal B cells were largely spared, permitting long-term survival, whereas complete lethality was observed in control animals. Our findings support the identification of drugs targeting ZFP574-THAP12 as a unique strategy to treat B cell malignancies.

Essential requirement for IER3IP1 in B cell development.

In a forward genetic screen of mice with N-ethyl-N-nitrosourea-induced mutations for aberrant immune function, we identified animals with low percentages of B220+ cells in the peripheral blood. The causative mutation was in Ier3ip1, encoding immediate early response 3 interacting protein 1 (IER3IP1), an endoplasmic reticulum membrane protein mutated in an autosomal recessive neurodevelopmental disorder termed Microcephaly with simplified gyration, Epilepsy and permanent neonatal Diabetes Syndrome (MEDS) in humans. However, no immune function for IER3IP1 had previously been reported. The viable hypomorphic Ier3ip1 allele uncovered in this study, identical to a reported IER3IP1 variant in a MEDS patient, reveals an essential hematopoietic-intrinsic role for IER3IP1 in B cell development and function. We show that IER3IP1 forms a complex with the Golgi transmembrane protein 167A and limits activation of the unfolded protein response mediated by inositol-requiring enzyme-1α and X-box binding protein 1 in B cells. Our findings suggest that B cell deficiency may be a feature of MEDS.

Essential role of MFSD1-GLMP-GIMAP5 in lymphocyte survival and liver homeostasis.

We detected ENU-induced alleles of Mfsd1 (encoding the major facilitator superfamily domain containing 1 protein) that caused lymphopenia, splenomegaly, progressive liver pathology, and extramedullary hematopoiesis (EMH). MFSD1 is a lysosomal membrane-bound solute carrier protein with no previously described function in immunity. By proteomic analysis, we identified association between MFSD1 and both GLMP (glycosylated lysosomal membrane protein) and GIMAP5 (GTPase of immunity-associated protein 5). Germline knockout alleles of Mfsd1, Glmp, and Gimap5 each caused lymphopenia, liver pathology, EMH, and lipid deposition in the bone marrow and liver. We found that the interactions of MFSD1 and GLMP with GIMAP5 are essential to maintain normal GIMAP5 expression, which in turn is critical to support lymphocyte development and liver homeostasis that suppresses EMH. These findings identify the protein complex MFSD1-GLMP-GIMAP5 operating in hematopoietic and extrahematopoietic tissues to regulate immunity and liver homeostasis.

RNPS1 inhibits excessive tumor necrosis factor/tumor necrosis factor receptor signaling to support hematopoiesis in mice.

Null mutations of spliceosome components or cofactors are homozygous lethal in eukaryotes, but viable hypomorphic mutations provide an opportunity to understand the physiological impact of individual splicing proteins. We describe a viable missense allele (F181I) of Rnps1 encoding an essential regulator of splicing and nonsense-mediated decay (NMD), identified in a mouse genetic screen for altered immune cell development. Homozygous mice displayed a stem cell­intrinsic defect in hematopoiesis of all lineages due to excessive apoptosis induced by tumor necrosis factor (TNF)­dependent death signaling. Numerous transcript splice variants containing retained introns and skipped exons were detected at elevated frequencies in Rnps1F181I/F181I splenic CD8+ T cells and hematopoietic stem cells (HSCs), but NMD appeared normal. Strikingly, Tnf knockout rescued all hematopoietic cells to normal or near-normal levels in Rnps1F181I/F181I mice and dramatically reduced intron retention in Rnps1F181I/F181I CD8+ T cells and HSCs. Thus, RNPS1 is necessary for accurate splicing, without which disinhibited TNF signaling triggers hematopoietic cell death.

Thousands of induced germline mutations affecting immune cells identified by automated meiotic mapping coupled with machine learning.

Forward genetic studies use meiotic mapping to adduce evidence that a particular mutation, normally induced by a germline mutagen, is causative of a particular phenotype. Particularly in small pedigrees, cosegregation of multiple mutations, occasional unawareness of mutations, and paucity of homozygotes may lead to erroneous declarations of cause and effect. We sought to improve the identification of mutations causing immune phenotypes in mice by creating Candidate Explorer (CE), a machine-learning software program that integrates 67 features of genetic mapping data into a single numeric score, mathematically convertible to the probability of verification of any putative mutation-phenotype association. At this time, CE has evaluated putative mutation-phenotype associations arising from screening damaging mutations in ∼55% of mouse genes for effects on flow cytometry measurements of immune cells in the blood. CE has therefore identified more than half of genes within which mutations can be causative of flow cytometric phenovariation in Mus musculus The majority of these genes were not previously known to support immune function or homeostasis. Mouse geneticists will find CE data informative in identifying causative mutations within quantitative trait loci, while clinical geneticists may use CE to help connect causative variants with rare heritable diseases of immunity, even in the absence of linkage information. CE displays integrated mutation, phenotype, and linkage data, and is freely available for query online.

Essential requirement for nicastrin in marginal zone and B-1 B cell development.

γ-secretase is an intramembrane protease complex that catalyzes the proteolytic cleavage of amyloid precursor protein and Notch. Impaired γ-secretase function is associated with the development of Alzheimer's disease and familial acne inversa in humans. In a forward genetic screen of mice with N-ethyl-N-nitrosourea-induced mutations for defects in adaptive immunity, we identified animals within a single pedigree exhibiting both hypopigmentation of the fur and diminished T cell-independent (TI) antibody responses. The causative mutation was in Ncstn, an essential gene encoding the protein nicastrin (NCSTN), a member of the γ-secretase complex that functions to recruit substrates for proteolysis. The missense mutation severely limits the glycosylation of NCSTN to its mature form and impairs the integrity of the γ-secretase complex as well as its catalytic activity toward its substrate Notch, a critical regulator of B cell and T cell development. Strikingly, however, this missense mutation affects B cell development but not thymocyte or T cell development. The Ncstn allele uncovered in these studies reveals an essential requirement for NCSTN during the type 2 transitional-marginal zone precursor stage and peritoneal B-1 B cell development, the TI antibody response, fur pigmentation, and intestinal homeostasis in mice.

Genetic and structural studies of RABL3 reveal an essential role in lymphoid development and function.

The small GTPase RABL3 is an oncogene of unknown physiological function. Homozygous knockout alleles of mouse Rabl3 were embryonic lethal, but a viable hypomorphic allele (xiamen [xm]) causing in-frame deletion of four amino acids from the interswitch region resulted in profound defects in lymphopoiesis. Impaired lymphoid progenitor development led to deficiencies of B cells, T cells, and natural killer (NK) cells in Rabl3xm/xm mice. T cells and NK cells exhibited impaired cytolytic activity, and mice infected with mouse cytomegalovirus (MCMV) displayed elevated titers in the spleen. Myeloid cells were normal in number and function. Biophysical and crystallographic studies demonstrated that RABL3 formed a homodimer in solution via interactions between the effector binding surfaces on each subunit; monomers adopted a typical small G protein fold. RABL3xm displayed a large compensatory alteration in switch I, which adopted a ß-strand configuration normally provided by the deleted interswitch residues, thereby permitting homodimer formation. Dysregulated effector binding due to conformational changes in the switch I-interswitch-switch II module likely underlies the xm phenotype. One such effector may be GPR89, putatively an ion channel or G protein-coupled receptor (GPCR). RABL3, but not RABL3xm, strongly associated with and stabilized GPR89, and an N-ethyl-N-nitrosourea (ENU)-induced mutation (explorer) in Gpr89 phenocopied Rabl3xm.

Enhanced susceptibility to chemically induced colitis caused by excessive endosomal TLR signaling in LRBA-deficient mice.

LPS-responsive beige-like anchor (LRBA) protein deficiency in humans causes immune dysregulation resulting in autoimmunity, inflammatory bowel disease (IBD), hypogammaglobulinemia, regulatory T (Treg) cell defects, and B cell functional defects, but the cellular and molecular mechanisms responsible are incompletely understood. In an ongoing forward genetic screen for N-ethyl-N-nitrosourea (ENU)-induced mutations that increase susceptibility to dextran sodium sulfate (DSS)-induced colitis in mice, we identified two nonsense mutations in Lrba Although Treg cells have been a main focus in LRBA research to date, we found that dendritic cells (DCs) contribute significantly to DSS-induced intestinal inflammation in LRBA-deficient mice. Lrba-/- DCs exhibited excessive IRF3/7- and PI3K/mTORC1-dependent signaling and type I IFN production in response to the stimulation of the Toll-like receptors (TLRs) 3, TLR7, and TLR9. Substantial reductions in cytokine expression and sensitivity to DSS in LRBA-deficient mice were caused by knockout of Unc93b1, a chaperone necessary for trafficking of TLR3, TLR7, and TLR9 to endosomes. Our data support a function for LRBA in limiting endosomal TLR signaling and consequent intestinal inflammation.

Biallelic loss of function variant in the unfolded protein response gene PDIA6 is associated with asphyxiating thoracic dystrophy and neonatal-onset diabetes.

Protein disulfide isomerase A6 (PDIA6) is an unfolded protein response (UPR)-regulating protein. PDIA6 regulates the UPR sensing proteins, Inositol requiring enzyme 1, and EIF2AK3. Biallelic inactivation of the two genes in mice and humans resulted in embryonic lethality, diabetes, skeletal defects, and renal insufficiency. We recently showed that PDIA6 inactivation in mice caused embryonic and early lethality, diabetes and immunodeficiency. Here, we present a case with asphyxiating thoracic dystrophy (ATD) syndrome and infantile-onset diabetes. Whole exome sequencing revealed a homozygous frameshift variant in the PDIA6 gene. RNA expression was reduced in a gene dosage-dependent manner, supporting a loss-of-function effect of this variant. Phenotypic correlation with the mouse model recapitulated the growth defect and delay, early lethality, coagulation, diabetes, immunological, and polycystic kidney disease phenotypes. In general, the phenotype of the current patient is consistent with phenotypes associated with the disruption of PDIA6 and the sensors of UPR in mice and humans. This is the first study to associate ATD to the UPR gene, PDIA6. We recommend screening ATD cases with or without insulin-dependent diabetes for variants in PDIA6.

Dominant atopy risk mutations identified by mouse forward genetic analysis.

BACKGROUND: Atopy, the overall tendency to become sensitized to an allergen, is heritable but seldom ascribed to mutations within specific genes. Atopic individuals develop abnormally elevated IgE responses to immunization with potential allergens. To gain insight into the genetic causes of atopy, we carried out a forward genetic screen for atopy in mice. METHODS: We screened mice carrying homozygous and heterozygous N-ethyl-N-nitrosourea (ENU)-induced germline mutations for aberrant antigen-specific IgE and IgG1 production in response to immunization with the model allergen papain. Candidate genes were validated by independent gene mutation. RESULTS: Of 31 candidate genes selected for investigation, the effects of mutations in 23 genes on papain-specific IgE or IgG1 were verified. Among the 20 verified genes influencing the IgE response, eight were necessary for the response, while 12 repressed IgE. Nine genes were not previously implicated in the IgE response. Fifteen genes encoded proteins contributing to IgE class switch recombination or B-cell receptor signaling. The precise roles of the five remaining genes (Flcn, Map1lc3b, Me2, Prkd2, and Scarb2) remain to be determined. Loss-of-function mutations in nine of the 12 genes limiting the IgE response were dominant or semi-dominant for the IgE phenotype but did not cause immunodeficiency in the heterozygous state. Using damaging allele frequencies for the corresponding human genes and in silico simulations (Monte Carlo) of undiscovered atopy mutations, we estimated the percentage of humans with heterozygous atopy risk mutations. CONCLUSIONS: Up to 37% of individuals may be heterozygous carriers for at least one dominant atopy risk mutation.

Excessive endosomal TLR signaling causes inflammatory disease in mice with defective SMCR8-WDR41-C9ORF72 complex function.

The SMCR8-WDR41-C9ORF72 complex is a regulator of autophagy and lysosomal function. Autoimmunity and inflammatory disease have been ascribed to loss-of-function mutations of Smcr8 or C9orf72 in mice. In humans, autoimmunity has been reported to precede amyotrophic lateral sclerosis caused by mutations of C9ORF72 However, the cellular and molecular mechanisms underlying autoimmunity and inflammation caused by C9ORF72 or SMCR8 deficiencies remain unknown. Here, we show that splenomegaly, lymphadenopathy, and activated circulating T cells observed in Smcr8-/- mice were rescued by triple knockout of the endosomal Toll-like receptors (TLRs) TLR3, TLR7, and TLR9. Myeloid cells from Smcr8-/- mice produced excessive inflammatory cytokines in response to endocytosed TLR3, TLR7, or TLR9 ligands administered in the growth medium and in response to TLR2 or TLR4 ligands internalized by phagocytosis. These defects likely stem from prolonged TLR signaling caused by accumulation of LysoTracker-positive vesicles and by delayed phagosome maturation, both of which were observed in Smcr8-/- macrophages. Smcr8-/- mice also showed elevated susceptibility to dextran sodium sulfate-induced colitis, which was not associated with increased TLR3, TLR7, or TLR9 signaling. Deficiency of WDR41 phenocopied loss of SMCR8. Our findings provide evidence that excessive endosomal TLR signaling resulting from prolonged ligand-receptor contact causes inflammatory disease in SMCR8-deficient mice.

Altered Marginal Zone B Cell Selection in the Absence of IκBNS.

Marginal zone (MZ) B cells reside in the splenic MZ and play important roles in T cell-independent humoral immune responses against blood-borne pathogens. IκBNS-deficient bumble mice exhibit a severe reduction in the MZ B compartment but regain an MZ B population with age and, thus, represent a valuable model to examine the biology of MZ B cells. In this article, we characterized the MZ B cell defect in further detail and investigated the nature of the B cells that appear in the MZ of aged bumble mice. Flow cytometry analysis of the splenic transitional B cell subsets demonstrated that MZ B cell development was blocked at the transitional-1 to transitional-2-MZ precursor stage in the absence of functional IκBNS. Immunohistochemical analysis of spleen sections from wild-type and bumble mice revealed no alteration in the cellular MZ microenvironment, and analysis of bone marrow chimeras indicated that the MZ B cell development defect in bumble mice was B cell intrinsic. Further, we demonstrate that the B cells that repopulate the MZ in aged bumble mice were distinct from age-matched wild-type MZ B cells. Specifically, the expression of surface markers characteristic for MZ B cells was altered and the L chain Igλ+ repertoire was reduced in bumble mice. Finally, plasma cell differentiation of sorted LPS-stimulated MZ B cells was impaired, and aged bumble mice were unable to respond to NP-Ficoll immunization. These results demonstrate that IκBNS is required for an intact MZ B cell compartment in C57BL/6 mice.

Skin-specific regulation of SREBP processing and lipid biosynthesis by glycerol kinase 5.

The recessive N-ethyl-N-nitrosourea-induced phenotype toku is characterized by delayed hair growth, progressive hair loss, and excessive accumulation of dermal cholesterol, triglycerides, and ceramides. The toku phenotype was attributed to a null allele of Gk5, encoding glycerol kinase 5 (GK5), a skin-specific kinase expressed predominantly in sebaceous glands. GK5 formed a complex with the sterol regulatory element-binding proteins (SREBPs) through their C-terminal regulatory domains, inhibiting SREBP processing and activation. In Gk5toku/toku mice, transcriptionally active SREBPs accumulated in the skin, but not in the liver; they were localized to the nucleus and led to elevated lipid synthesis and subsequent hair growth defects. Similar defective hair growth was observed in kinase-inactive GK5 mutant mice. Hair growth defects of homozygous toku mice were partially rescued by treatment with the HMG-CoA reductase inhibitor simvastatin. GK5 exists as part of a skin-specific regulatory mechanism for cholesterol biosynthesis, independent of cholesterol regulation elsewhere in the body.

IgD class switching is initiated by microbiota and limited to mucosa-associated lymphoid tissue in mice.

Class-switch recombination (CSR) alters the Ig isotype to diversify antibody effector functions. IgD CSR is a rare event, and its regulation is poorly understood. We report that deficiency of 53BP1, a DNA damage-response protein, caused age-dependent overproduction of secreted IgD resulting from increased IgD CSR exclusively within B cells of mucosa-associated lymphoid tissues. IgD overproduction was dependent on activation-induced cytidine deaminase, hematopoietic MyD88 expression, and an intact microbiome, against which circulating IgD, but not IgM, was reactive. IgD CSR occurred via both alternative nonhomologous end-joining and homologous recombination pathways. Microbiota-dependent IgD CSR also was detected in nasal-associated lymphoid tissue of WT mice. These results identify a pathway, present in WT mice and hyperactivated in 53BP1-deficient mice, by which microbiota signal via Toll-like receptors to elicit IgD CSR.

TLR4/MD-2 activation by a synthetic agonist with no similarity to LPS.

Structurally disparate molecules reportedly engage and activate Toll-like receptor (TLR) 4 and other TLRs, yet the interactions that mediate binding and activation by dissimilar ligands remain unknown. We describe Neoseptins, chemically synthesized peptidomimetics that bear no structural similarity to the established TLR4 ligand, lipopolysaccharide (LPS), but productively engage the mouse TLR4 (mTLR4)/myeloid differentiation factor 2 (MD-2) complex. Neoseptin-3 activates mTLR4/MD-2 independently of CD14 and triggers canonical myeloid differentiation primary response gene 88 (MyD88)- and Toll-interleukin 1 receptor (TIR) domain-containing adaptor inducing IFN-beta (TRIF)-dependent signaling. The crystal structure mTLR4/MD-2/Neoseptin-3 at 2.57-Å resolution reveals that Neoseptin-3 binds as an asymmetrical dimer within the hydrophobic pocket of MD-2, inducing an active receptor complex similar to that induced by lipid A. However, Neoseptin-3 and lipid A form dissimilar molecular contacts to achieve receptor activation; hence strong TLR4/MD-2 agonists need not mimic LPS.

Real-time resolution of point mutations that cause phenovariance in mice.

With the wide availability of massively parallel sequencing technologies, genetic mapping has become the rate limiting step in mammalian forward genetics. Here we introduce a method for real-time identification of N-ethyl-N-nitrosourea-induced mutations that cause phenotypes in mice. All mutations are identified by whole exome G1 progenitor sequencing and their zygosity is established in G2/G3 mice before phenotypic assessment. Quantitative and qualitative traits, including lethal effects, in single or multiple combined pedigrees are then analyzed with Linkage Analyzer, a software program that detects significant linkage between individual mutations and aberrant phenotypic scores and presents processed data as Manhattan plots. As multiple alleles of genes are acquired through mutagenesis, pooled "superpedigrees" are created to analyze the effects. Our method is distinguished from conventional forward genetic methods because it permits (1) unbiased declaration of mappable phenotypes, including those that are incompletely penetrant (2), automated identification of causative mutations concurrent with phenotypic screening, without the need to outcross mutant mice to another strain and backcross them, and (3) exclusion of genes not involved in phenotypes of interest. We validated our approach and Linkage Analyzer for the identification of 47 mutations in 45 previously known genes causative for adaptive immune phenotypes; our analysis also implicated 474 genes not previously associated with immune function. The method described here permits forward genetic analysis in mice, limited only by the rates of mutant production and screening.

Integrin Receptors Play a Key Role in the Regulation of Hepatic CYP3A.

Landmark studies describing the effect of microbial infection on the expression and activity of hepatic CYP3A used bacterial lipopolysaccharide as a model antigen. Our efforts to determine whether these findings were translatable to viral infections led us to observations suggesting that engagement of integrin receptors is key in the initiation of processes responsible for changes in hepatic CYP3A4 during infection and inflammation. Studies outlined in this article were designed to evaluate whether engagement of integrins, receptors commonly used by a variety of microbes to enter cellular targets, is vital in the regulation of CYP3A in the presence and absence of virus infection. Mice infected with a recombinant adenovirus (AdlacZ) experienced a 70% reduction in hepatic CYP3A catalytic activity. Infection with a mutant virus with integrin-binding arginine-glycine-aspartic acid (RGD) sequences deleted from the penton base protein of the virus capsid (AdΔRGD) did not alter CYP3A activity. CYP3A mRNA and protein levels in AdlacZ-treated animals were also suppressed, whereas those of mice given AdΔRGD were not significantly different from uninfected control mice. Silencing of the integrinß-subunit reverted adenovirus-mediated CYP3A4 suppression in vitro. Silencing of theα-subunit did not. Suppression of integrin subunits had a profound effect on nuclear receptors pregnane X receptor and constitutive androstane receptor, whereas retinoid X receptorαwas largely unaffected. To our knowledge, this is the first time that extracellular receptors, like integrins, have been indicated in the regulation of CYP3A. This finding has several implications owing to the important role of integrins in normal physiologic process and in many disease states.

Bolstering Components of the Immune Response Compromised by Prior Exposure to Adenovirus: Guided Formulation Development for a Nasal Ebola Vaccine.

The severity and longevity of the current Ebola outbreak highlight the need for a fast-acting yet long-lasting vaccine for at-risk populations (medical personnel and rural villagers) where repeated prime-boost regimens are not feasible. While recombinant adenovirus (rAd)-based vaccines have conferred full protection against multiple strains of Ebola after a single immunization, their efficacy is impaired by pre-existing immunity (PEI) to adenovirus. To address this important issue, a panel of formulations was evaluated by an in vitro assay for their ability to protect rAd from neutralization. An amphiphilic polymer (F16, FW ∼39,000) significantly improved transgene expression in the presence of anti-Ad neutralizing antibodies (NAB) at concentrations of 5 times the 50% neutralizing dose (ND50). In vivo performance of rAd in F16 was compared with unformulated virus, virus modified with poly(ethylene) glycol (PEG), and virus incorporated into poly(lactic-co-glycolic) acid (PLGA) polymeric beads. Histochemical analysis of lung tissue revealed that F16 promoted strong levels of transgene expression in naive mice and those that were exposed to adenovirus in the nasal cavity 28 days prior to immunization. Multiparameter flow cytometry revealed that F16 induced significantly more polyfunctional antigen-specific CD8+ T cells simultaneously producing IFN-γ, IL-2, and TNF-α than other test formulations. These effects were not compromised by PEI. Data from formulations that provided partial protection from challenge consistently identified specific immunological requirements necessary for protection. This approach may be useful for development of formulations for other vaccine platforms that also employ ubiquitous pathogens as carriers like the influenza virus.

A Single Dose Respiratory Recombinant Adenovirus-Based Vaccine Provides Long-Term Protection for Non-Human Primates from Lethal Ebola Infection.

As the Ebola outbreak in West Africa continues and cases appear in the United States and other countries, the need for long-lasting vaccines to preserve global health is imminent. Here, we evaluate the long-term efficacy of a respiratory and sublingual (SL) adenovirus-based vaccine in non-human primates in two phases. In the first, a single respiratory dose of 1.4×10(9) infectious virus particles (ivp)/kg of Ad-CAGoptZGP induced strong Ebola glycoprotein (GP) specific CD8+ and CD4+ T cell responses and Ebola GP-specific antibodies in systemic and mucosal compartments and was partially (67%) protective from challenge 62 days after immunization. The same dose given by the SL route induced Ebola GP-specific CD8+ T cell responses similar to that of intramuscular (IM) injection, however, the Ebola GP-specific antibody response was low. All primates succumbed to infection. Three primates were then given the vaccine in a formulation that improved the immune response to Ebola in rodents. Three primates were immunized with 2.0×10(10) ivp/kg of vaccine by the SL route. Diverse populations of polyfunctional Ebola GP-specific CD4+ and CD8+ T cells and significant anti-Ebola GP antibodies were present in samples collected 150 days after respiratory immunization. The formulated vaccine was fully protective against challenge 21 weeks after immunization. While diverse populations of Ebola GP-specific CD4+ T cells were produced after SL immunization, antibodies were not neutralizing and the vaccine was unprotective. To our knowledge, this is the first time that durable protection from a single dose respiratory adenovirus-based Ebola vaccine has been demonstrated in primates.