since it first appeared 
 as a clinical syndrome in central africa 
 in the late 1970s , ebola hemorrhagic fever , now termed ebola virus 
 disease , has intrigued infectious disease physicians , virologists , 
 and epidemiologists because of the striking clinical presentation 
 associated with the end stage of the disease , its high case fatality 
 rate , and the ease with which it is transmitted among close contacts , 
 including caregivers . isolation and identification 
 of the zaire ebola virus soon after the first outbreak gave clinicians , 
 scientists , and public health representatives tangible evidence of 
 the pathogen responsible for severe illness in 318 people which resulted 
 in 280 deaths and fueled four decades of research on the biology of 
 the highly lethal pathogen . this virus , 
 currently referred to as ebola virus , is rapidly disseminated to lymph 
 nodes by monocytes , macrophages , and dendritic cells where it quickly 
 spreads to the liver and the spleen . this 
 process is extremely efficient , making development of a therapeutic 
 regimen to treat ebola virus disease a race against the clock due 
 to the narrow window between the time when viremia and/or the onset 
 of fever and other clinical symptoms can be detected and death . several experimental candidates have shown promise 
 for treating ebola virus disease in animal models of infection , however , there are currently no therapeutic or 
 preventative agents approved for human use . basic supportive care 
 ( fluid and electrolyte replacement ; administration of antibiotics 
 and antimalarials for concurrent infections and antiemetics for gastrointestinal 
 symptoms ) continues to be the cornerstone of therapy for ebola virus 
 disease and can notably improve outcomes when administered early in 
 the course of the disease . the current outbreak in 
 west africa not only emphasizes the important 
 relationship between early detection of infection and supportive treatment , 
 it also highlights a critical need for a well tolerated , highly effective 
 ebola vaccine that can rapidly elicit protection with a single dose . 
 according to the world health organization ( who ) at the time of this 
 writing ( october 30 , 2014 ) there have been 13,703 cases of ebola infection , 
 4,922 of which have resulted in death . it is estimated that more than 521 of the reported cases have been 
 healthcare workers , the majority of which ( 99% ) reside in countries 
 with widespread and intense transmission , and that more than half 
 ( 52% ) of these people did not recover from infection . even though this might be an underestimate of the actual 
 situation due to delays in reporting of data and the rapid evolution 
 of the outbreak , this represents a significant loss to an already 
 understaffed and resource poor healthcare system in a region of extreme 
 poverty and civil unrest . considering 
 that the who and other modeling experts have predicted that more than 
 20,000 new cases of ebola infection will occur by the end of november 
 2014 and that in the worst case scenario 1.4 million cases will be 
 seen before the current outbreak ends , an effective 
 needle - free vaccine would bolster the medical response and health 
 care infrastructure of affected nations by allowing a large number 
 of medical personnel to provide aid and immunizations to those under 
 outbreak conditions without concern for their personal health . the overall goal of these studies was to identify an immunization 
 platform that is easy to administer and capable of eliciting long - term 
 protection from ebola infection . using results generated in mouse 
 and guinea pig models , two studies were designed to evaluate the clinical 
 profile of a recombinant adenovirus serotype 5-based vaccine given 
 as a single dose by respiratory or sublingual ( sl ) administration 
 to non - human primates ( nhp ) . the first study , conducted with 9 male 
 cynomolgus macaques , was designed to identify a subtherapeutic dose 
 for each route of administration to assist in identifying formulations 
 that improved vaccine performance . the second study , conducted with 
 11 macaques , was designed to evaluate a novel formulation that improved 
 the immunological profile of the vaccine after intranasal administration 
 in rodents and to evaluate the longevity of the immune response conferred 
 by each platform . a comprehensive assessment 
 of the immune response elicited by each vaccine platform after exposure 
 to ebola virus was performed in this study . full toxicological profiles 
 were generated for each animal after immunization in both studies . to our knowledge , this is the first evaluation of a needle - free ebola 
 vaccine in primates and one of the first to demonstrate long - term 
 protection from lethal infection . the e1/e3 deleted recombinant 
 adenovirus serotype 5 vector expressing a codon optimized full - length 
 ebola glycoprotein sequence under the control of the chicken -actin 
 promoter ( ad - cagoptzgp ) and a host range mutant adenovirus serotype 
 5 ( ad5mut ) that can replicate in non - human primates were amplified 
 in hek 293 cells and purified as described . concentration of each virus preparation was determined by uv spectrophotometric 
 analysis at 260 nm and with the adeno - x rapid titer kit ( clontech , 
 mountain view , ca ) according to the manufacturer s instructions . preparations with infectious to physical particle ratios of 1:37 were 
 used in these studies . buffers and reagents used in the production 
 and purification of each virus preparation were of the highest quality 
 available and were tested for the presence of endotoxin using a qcl-1000 
 chromogenic lal end point assay ( cambrex bioscience , walkersville , 
 md ) . all reagents contained less than 0.1 e.u./ml , and each virus 
 preparation contained less than 0.2 e.u./ml . sterility of each preparation 
 was confirmed employing the methods outlined in the united states 
 pharmacopeia for parenteral products . a two cell line bioassay was performed on each preparation to determine 
 the presence of rca as described . less 
 than one rca was detected for every 3  10 virus 
 particles tested . non - human primate studies 
 were conducted 
 under a contract at bioqual inc . , the animal management 
 program of this institution is accredited by the american association 
 for the accreditation of laboratory animal care and meets nih standards 
 as outlined in the guide for the care and use of laboratory animals . this institution also accepts as mandatory phs policy on humane care 
 of vertebrate animals used in testing , research , and training . twenty 
 male cynomolgus macaques ( macaca fascicularis ) of 
 chinese origin were allowed to acclimate for 30 days in quarantine 
 prior to immunization . animals received standard monkey chow , treats , 
 vegetables , and fruits throughout the study . husbandry enrichment 
 consisted of commercial toys and visual stimulation . specific details about the 
 primates used in each of these studies are summarized in tables 1 and 2 . animals were screened 
 for signs of prior exposure to adenovirus ( anti - ad5 nab , ad5 dna , 
 t cell responses ) 4 days prior to immunization . samples were taken for evaluation 
 of blood chemistry and adenovirus shedding ( nasal and oral swabs , 
 urine , feces ) 6 h after immunization and on days 1 , 2 , and 7 . on day 
 20 , serum and bal were collected for assessment of shedding and anti - ad5 
 nab and anti - ebola gp antibody levels . bal , pbmcs , and ilns were also 
 screened for ebola gp - specific cd8 and cd4 t cells at this time point . on day 38 , additional samples were taken 
 for assessment of anti - ebola gp and anti - ad5 antibodies and antigen - specific 
 t cell proliferation ( ebola gp and ad5 ) . 42 days after immunization , 
 nhps were shipped to the national microbiology laboratory in winnipeg , 
 canada , for challenge . after an acclimation period , primates were 
 challenged with 1,000 pfu of ebola ( 1995 , kikwit ) by intramuscular 
 ( i m ) injection . two animals were given the vaccine by intramuscular injection in a 
 total volume of 1 ml of potassium phosphate buffered saline ( kpbs ) 
 divided equally between the left and right deltoid muscles . three 
 animals were given the vaccine by the sublingual route by placing 
 50 l of the preparation under each side of the tongue and waiting 
 for 15 min between doses to allow for absorption . this was achieved by slowly 
 dispensing two 250 l volumes of the preparation into each nostril 
 and waiting for 15 min between doses to allow for absorption . the 
 remaining dose of the vaccine ( 5 ml volume ) was instilled into the 
 lungs via an endotracheal tube . this route of administration will 
 be referred to as respiratory immunization or as intranasal / intratracheal 
 ( in / it ) throughout the manuscript to illustrate that the vaccine was 
 administered to the respiratory mucosa by two different routes . one 
 primate was given 1 ml of kpbs divided equally between the left and 
 right deltoid muscles . blood 
 was collected 6 h after immunization and on days 1 , 2 , and 7 . full 
 blood chemistry panels and complete blood counts were performed by 
 idexx bioresearch ( west sacramento , ca ) . two animals ( negative controls ) were given 1 ml each of kpbs divided 
 between the left and right deltoid muscles . the respiratory formulation 
 was prepared at five times the working concentration [ 10 mg / ml poly(maleic 
 anhydride - alt-1-octadecene ) substituted with 3-(dimethylamino)propylamine 
 ( anatrace , maumee , oh ) ] , sterilized by filtration , and diluted with 
 freshly purified virus in kpbs ( ph 7.4 ) prior to use . three animals 
 were given the vaccine in this formulation in the respiratory tract 
 as described for study 1 . three animals were given an adenovirus serotype 
 5 host range mutant virus to establish pre - existing immunity ( pei ) 
 by i m injection 28 days prior to immunization with the vaccine by 
 the sublingual route as described above . three animals with no prior 
 exposure to adenovirus were given the vaccine by the sublingual route 
 for comparison . animals were transported 
 to the national 
 microbiology laboratory in winnipeg and , after an acclimation period , 
 transferred to the biosafety level 4 ( bsl-4 ) laboratory there for 
 challenge . challenge studies were approved by the canadian science 
 centre for human and animal health ( cschah ) animal care committee 
 following the guidelines of the canadian council on animal care . for 
 challenge , animals were infected by intramuscular injection at two 
 sites with a total volume of 1 ml of freshly prepared ebola virus 
 ( strain kikwit 95 , passage 3 on veroe6 cells ) of an inoculum containing 
 1,000 times the 50% tissue culture infectious dose ( tcid50 ) in diluent ( minimal essential medium containing 0.3% bovine serum 
 albumin ) . ebola virus titers were confirmed ( 1.21  10 tcid50/ml ) by back - titration of the challenge preparation 
 following administration of the virus . animals were monitored daily 
 and scored for disease progression using an internal filovirus scoring 
 protocol approved by the cschah animal care committee . the scoring 
 system graded changes from normal in the subject 's posture , attitude , 
 activity level , feces / urine output , food / water intake , weight , temperature , 
 and respiration and ranked disease manifestations such as a visible 
 rash , hemorrhage , cyanosis , or flushed skin . samples were taken for 
 assessment of anti - ebola gp antibodies and full blood panels on days 
 3 , 7 , 14 , 21 , and 28 postchallenge and upon death . hematological analysis 
 of samples was performed in the bsl-4 lab with a horiba abx scil abc 
 vet animal blood counter , and blood chemistries were analyzed with 
 a vetscan vs1 ( abraxis ) . ifn- elispot assays were performed 
 in triplicate according to the manufacturer s protocol ( bd 
 biosciences , san diego , ca ) with 5  10 peripheral 
 blood mononuclear cells ( pbmcs ) per well in crpmi media ( rpmi 1640 , 
 1 mm l - glutamine , 50 m -mercaptoethanol , 10% 
 fbs and 1% penicillin / streptomycin ) . cells were stimulated with three 
 peptide pools for the ebola glycoprotein ( 2.5 g / ml ) for 18 
 h. spots were visualized with the aec substrate ( bd biosciences ) and 
 quantified with the elispot plate reader ( aid cell technology , strassberg , 
 germany ) . the frequency of cd8 and cd4 t cells producing ifn- , il-2 , il-4 , and cd107a were 
 assessed by flow cytometry with the following antibodies : cd3 alexa 
 fluor 700 ( clone sp34 - 2 ) and cd4 peridinin chlorophyll protein ( percp)-cy5.5 
 ( clone l200 ) from bd biosciences ( san jose , ca ) ; cd8 phycoerythrin 
 ( pe)-cy7 ( clone rpa - t8 ) , cd107a brilliant violet 421 ( clone h4a3 ) , 
 il-2 alexa fluor 488 ( clone mq1 - 17h12 ) , il-4 pe ( clone 8d4 - 8 ) , and 
 ifn- allophycocyanin ( apc , clone b27 ) from biolegend ( san diego , 
 ca ) . one million pbmcs were stimulated overnight with peptides ( 5 
 g / ml ) using golgiplug ( 0.5 l / ml ) and golgistop ( 0.6 
 l / ml ) in the presence of the anti - cd107a antibody . after surface 
 staining for cd3 , cd4 , and cd8 , samples were incubated two times ( 30 
 min each ) in cytofix / cytoperm ( bd biosciences ) for permeabilization . intracellular staining was performed , and the samples were kept overnight 
 in pbs/1% paraformaldehyde . approximately 250,000500,000 events 
 were captured on a bd lsr ii flow cytometer and data analyzed with 
 flowjo vx0.6 software ( tree star , ashland , or ) . blood was collected 
 from each primate in edta tubes , shipped same 
 day and pbmcs isolated as described previously . cells were resuspended in r10 medium ( rpmi 1640 , 2 mm l - glutamine , 50 m -mercaptoethanol , 10% fbs , and 
 100 iu / ml penicillin and streptomycin ) and stimulated using either 
 an ebola glycoprotein - specific peptide library ( 2.5 g / ml ) , 
 a first generation adenovirus that is genetically identical to the 
 vaccine but does not contain a transgene cassette ( adnull , 1,000 moi ) , or 5 g / ml cona ( sigma , st . after 3 days , cells 
 were fed by removing 50 l of spent medium and replacing it 
 with 100 l of fresh r10 medium . on day 5 , cells were washed 
 with phosphate buffered saline ( pbs ) for subsequent immunostaining 
 for cell surface markers and for ki-67 , an intracellular marker for 
 proliferation as described . proliferation 
 was calculated by subtraction of values obtained from cells cultured 
 in medium alone . flat bottom , 
 immulon 2hb plates ( fisher scientific , pittsburgh , pa ) were coated 
 with purified ebola virus gp33637tm - ha ( 3 
 g / well ) in pbs ( ph 7.4 ) overnight at 4 c . one hundred microliters of each dilution were added 
 to antigen - coated plates for 2 h at room temperature . plates were 
 washed 4 times and incubated with a hrp - conjugated goat anti - monkey 
 igg antibody ( 1:2,000 , kpl , inc . , gaithersburg , md ) for 1 h at room 
 temperature . optical densities were read at 450 nm on a microplate reader 
 ( tecan usa , research triangle park , nc ) . primate sera were heat 
 inactivated at 56 
 c for 45 min and then serially diluted in 2-fold increments 
 in dulbecco s modified eagle s medium ( dmem ) in triplicate 
 prior to incubation at 37 c for 1 h with an equal volume of 
 medium containing ebov - egfp ( 100 pfu per well ) as described . serum mixtures were then added 
 to vero e6 cells and placed at 37 c for 2 days and then fixed 
 in 10% phosphate buffered formalin . gfp levels were quantified by 
 a fluorescent plate reader ( aid cell technology ) . these assays were 
 performed under bsl-4 conditions at the national microbiology laboratory 
 in winnipeg . primate sera were heat inactivated and serially 
 diluted as described for the ebola virus assay . samples were incubated 
 with a first generation adenovirus serotype 5 expressing beta - galactosidase 
 for 1 h before they were added to hela cell monolayers . an equal volume 
 of medium containing 20% fbs was then added to each well , and infections 
 continued for 24 h. cells were then histochemically stained for beta - galactosidase 
 expression as described . positive cells 
 were quantified by visual inspection with a lecia dm lb microscope 
 ( leica microsystems inc . , bannockburn , il ) . for both assays , the serum 
 dilution that corresponded to a 50% reduction in transgene expression 
 was calculated by the method of reed and muench and reported as the 
 reciprocal of this dilution . total rna was extracted 
 from whole blood 
 using a qiamp viral rna mini kit ( qiagen ) . ebola virus rna was detected 
 by a qrt - pcr assay targeting the rna polymerase ( nucleotides 16472 
 to 16538 , af086833 ) and lightcycler 480 rna master hydrolysis probes 
 ( roche diagnostics gmbh , mannheim , germany ) . the reaction conditions 
 were as follows : 63 c for 3 min , 95 c for 30 s , and cycling 
 of 95 c for 15 s , 60 c for 30 s for 45 cycles with a lightcycler 
 480 ii ( roche ) . primer sequences for this assay were as follows : ebovlf2 
 cagccagcaatttcttccat , ebovlr2 tttcggttgctgtttctgtg , 
 and ebovlp2fam fam - atcattggcgtactggaggagcag - bhq1 . urine and bal 
 fluid were concentrated using 
 amicon ultra 100k centrifugal filter devices ( millipore , billerica , 
 ma ) . dna was isolated from blood , concentrated bal , and oral and nasal 
 swabs using a qiamp dna mini kit according to the manufacturer s 
 instructions ( qiagen , valencia , ca ) . dna was isolated from rectal 
 swabs using a modified protocol and the qiamp dna mini kit . dna was 
 extracted from the urine concentrate using a qiaamp viral rna mini 
 kit ( qiagen ) according to the manufacturer s instructions . dna was isolated from stool samples using a qiaamp fast dna stool 
 mini kit ( qiagen ) . quantification of viral dna was determined by real 
 time pcr according to a published protocol . dna amplifications were carried out using a viia 7 real - time pcr 
 system ( life technologies , carlsbad , ca ) with the following cycling 
 conditions : 50 c for 2 min , 95 c for 10 min , 95 c 
 for 15 s , and 62 c for 1 min for a total of 41 cycles . primer 
 sequences , used to amplify a region of the adenovirus serotype 5 hexon 
 protein , were 5-act ata tgg aca acg tca acc cat t-3 
 ( forward ) and 5-acc ttc tga ggc acc tgg atg t-3 ( reverse ) . the internal probe sequence , tagged with 6fam fluorescence dye at 
 the 5 end and tamra quencher at the 3 end , was 5-acc 
 acc gca atg ctg gcc tgc-3. each sample was run in triplicate 
 in a given pcr assay . two primate 
 studies are summarized here . the first , referred to 
 as study 1 , involved 9 male cynomolgus macaques and served to identify 
 suitable doses of vaccine that were semiprotective for further evaluation 
 of test formulations to improve survival in the nhp model . the second 
 study , referred to as study 2 , evaluated a novel formulation for the 
 respiratory platform and involved refinement of the sublingual platform 
 in naive animals and those with prior exposure to adenovirus . the 
 workflow and treatment schedules for each study are depicted in figures 1 and 7 . administration of the vaccine at a dose of 1.4 
  10 infectious virus particles ( ivp)/kg to the respiratory 
 and the sublingual mucosa was well tolerated with no adverse reactions 
 noted . of particular note is that all animals experienced a transient 
 increase in serum phosphate levels 6 h after immunization with a primate 
 from each treatment group falling outside normal values ( 22473 , i m , 
 1.4 times normal , 50459 , in / it , 1.2 times normal , 62125 , sl , 1.3 times 
 normal , figure 2a ) . phosphate levels for all 
 animals reached their nadir at the 24 h time point and were within 
 the normal range for the remainder of the study . blood urea nitrogen 
 ( bun ) levels peaked for all animals 24 h after immunization . two of 
 these animals , one given the vaccine by i m injection ( 40347 , 29 mg / dl ) 
 and another given the vaccine by the in / it route ( 52945 , 33 mg / dl ) , 
 had levels that were notably outside of the normal range ( figure 2b ) . these values returned to normal by 48 h and 
 remained so throughout the course of the study . serum aspartate aminotransferase 
 ( ast ) , a standard indicator of adenovirus toxicity , was significantly elevated above normal values in all animals 
 24 h after immunization except for one animal given the vaccine by 
 the sl route ( 62125 ) and another given the vaccine by the i m route 
 ( 40347 ) . ast levels fell 48 h after immunization with only a few animals 
 remaining above normal limits ( figure 2c ) . ast values for all animals were within normal limits by the 7 day 
 time point . serum alkaline phosphatase ( alp ) of two animals fell outside 
 the normal range during the study . samples from one animal given the 
 vaccine by i m injection were only mildly over the normal acceptable 
 limit ( 22473 , figure 2d ) while those of an 
 animal immunized by the in / it route ( 52945 ) were 2 times the normal 
 acceptable limit . in both cases , this parameter was high throughout 
 the study and this elevation was not in response to the vaccine . other 
 serological parameters evaluated during the first week after immunization 
 ( calcium , creatinine , albumin , globulin , total protein , total bilirubin , 
 alanine aminotransferase ( alt ) , glucose , sodium , potassium , chloride , 
 and cholesterol ) all fell within normal limits during the course of 
 the study . study 1 : clinical parameters evaluated over time in non - human primates 
 immunized by various routes . cynomolgus macaques were given a single 
 dose of vaccine by i m injection or by the respiratory or the sl route . each line represents alterations for each parameter during the course 
 of therapy for one primate . in each panel : red lines / squares : saline 
 control . adenovirus shedding was also evaluated 
 using a standard real time 
 pcr assay to detect adenovirus genomes in serum , nasal swabs , bal fluid , oral swabs , urine , and feces ( figure 3 ) . a significant number of adenovirus genomes were 
 found in the serum of one animal immunized by the respiratory route 
 2 days after immunization ( 50459 , 1,452 genomes / ml serum , figure 3a ) and another immunized by i m injection 7 days 
 after treatment ( 22473 , 7,296 genomes / ml serum ) . as expected , substantial 
 amounts of adenovirus serotype 5 genomes were found in nasal swabs 
 obtained from primates immunized by the in / it route ( 50459 , 4.2  
 10 , 52483 , 1.4  10 , 59245 , 7.5  
 10 ) 24 h after immunization ( figure 3b ) . swabs from one primate immunized by the sl route also contained 
 a notable amount of ad5 genomes ( 62361 , 8,090 ) at the 24 h time point . swabs from one animal immunized by the in / it route contained a significant 
 amount of adenovirus genomes 2 days after immunization ( 52945 , 6,333 ) . samples taken at days 7 and 20 fell below detection limits of the 
 assay . very low amounts of ad5 genomes were found in the bal fluid 
 of animals immunized by the in / it route 20 days after immunization 
 ( figure 3c ) . oral swabs taken 24 h after treatment 
 from one nhp immunized by the in / it route ( 52483 , 4.5  10 , figure 3d ) and two animals immunized 
 by the sl route ( 62125 , 4.9  10 , and 62361 , 9.3 
  10 ) contained significant numbers of adenovirus 
 genomes . swabs collected from animals at the 2 day time point did 
 not contain any adenovirus genomes . a significant number of virus 
 genomes were detected in the urine of 2 animals within 6 h after treatment 
 ( 52945 , 621 copies / ml , and 62361 , 1,228 copies / ml ) . adenovirus dna 
 was also found 24 h after treatment in the urine of 3 animals ( 50459 , 
 1,163 , 62361 , 801 , and 62125 , 116 copies / ml , figure 3e ) . samples from all other animals throughout the time course 
 of this study fell below detection limits of the assay . interestingly , 
 adenovirus genomes were only detected in the feces of animals immunized 
 by the in / it route ( figure 3f ) . as early as 
 6 h after immunization , 2,362 and 7,302 adenovirus genomes were found 
 in fecal samples from animals 52483 and 52945 respectively . feces 
 collected from animal 50459 24 h after vaccination contained 5,919 
 adenovirus genomes . this increased to 7,405 in samples taken from 
 the same animal at the 48 h time point . samples from animal 52945 
 also taken 48 h after treatment contained 2,772 virus genomes . study 1 : adenovirus 
 genomes are released predominantly in the nasal 
 mucosa and feces after respiratory immunization and in the oral and 
 nasal mucosa after sublingual immunization . male cynomolgus macaques 
 were given either 1  10 ivp by i m injection or 1 
  10 ivp by the respiratory or the sl route . dna 
 was isolated from each sample , and viral genomes were determined by 
 real time pcr . twenty days after immunization , 
 pbmcs were isolated from whole blood and incubated with peptides specific 
 for ebola glycoprotein ( gp ) . cells were then subjected to intracellular 
 cytokine staining for cd8 and cd4 surface 
 antigens and ifn- and sorted by flow cytometry . at this time 
 point , few cells responsive to ebola glycoprotein could be detected 
 in pbmcs obtained from any of the animals ( data not shown ) . a similar 
 trend was observed in samples taken from iliac lymph nodes ( ilns ) 
 of animals . profound responses were seen in samples obtained from 
 the bal fluid of animals given the vaccine by the in / it route . the 
 strongest response was seen in cd4 cells with 12.5% of 
 the population obtained from primate 52945 and 3.03% of the population 
 from primate 50459 responding ( figure 4a ) . although the response from the third primate in this treatment group 
 ( 52483 ) was small in comparison ( 0.71% ) , it was significantly higher 
 than that observed in animals given the vaccine by i m injection . study 1 : respiratory immunization induces strong antigen - specific 
 t cell responses after administration of a single dose of a formulated 
 adenovirus - based ebola vaccine . ( a ) quantitative analysis of ebola 
 glycoprotein - specific cd4 t cells in bal 
 fluid . cells were isolated from whole blood 20 days after immunization 
 and stimulated with a peptide library for ebola glycoprotein or peptides 
 specific for the mhc class ii associated invariant chain peptide that 
 binds the mhc class ii groove of cells ( h - clip , negative control ) . each 
 cell population was stimulated for 5 h , stained for phenotypic markers , 
 and analyzed by flow cytometry . ( b ) quantitative analysis of ebola 
 glycoprotein - specific cd8 t cells in bal 
 fluid . cells were treated as described for panel a. ( c ) magnitude 
 of the antigen - specific response of mononuclear cells isolated from 
 whole blood of macaques . pbmcs were isolated 20 days after immunization 
 from whole blood and evaluated for ifn- secretion after stimulation 
 with an ebola gp - specific peptide library by elispot . ( d ) magnitude 
 of the antigen - specific response in mononuclear cells isolated from 
 iliac lymph nodes ( ilns ) of primates . mncs were isolated 20 days after 
 immunization from ilns and evaluated for ifn- secretion after 
 stimulation with an ebola gp - specific peptide library by elispot . ( e ) proliferative capacity of ebola gp - specific t cells collected 
 38 days after immunization of naive primates by various routes . the 
 proliferative capacity of cd4 ( white bars ) and cd8 ( black bars ) t cells isolated from whole blood was evaluated 
 for each animal by stimulation for 5 days with an ebola gp - specific 
 peptide library and subsequent staining for ki-67 , an intracellular 
 marker for proliferation . ( f ) proliferative 
 capacity of adenovirus serotype 5-specific t cells after immunization 
 by various routes . cells were isolated from whole blood 38 days after 
 immunization and stimulated for 5 days with a first generation adenovirus 
 that does not contain a transgene cassette ( adnull , moi 1:1,000 ) . the proliferative capacity of cd4 ( white bars ) and cd8 ( black bars ) t cells was determined by intracellular staining 
 for ki-67 . animal numbers displayed in each panel and their corresponding 
 treatments are summarized in table 1 . pbmc and iln populations were 
 further analyzed for ifn- 
 production in response to ebola gp by elispot . samples from animals 
 immunized by the i m route ( 22473 and 40347 ) both had significant numbers 
 of ifn- producing cells ( 255 and 642 spot forming cells ( sfcs)/million 
 mononuclear cells ( mncs ) respectively , figure 4c ) . pbmc samples from two nhps immunized by the sl route ( 52165 , 
 62361 ) also had measurable numbers of ifn- producing cells 
 ( 257 and 98 sfcs / million mncs ) . samples from nhps immunized by the 
 in / it route contained the highest numbers of ifn- producing 
 cells ( 1,100 , 607 , and 2,055 sfcs / million mncs ) . samples from the 
 ilns of 2 nhps given the vaccine by the in / it route ( 50459 and 52945 ) 
 contained approximately 7 and 18 times the number of ifn- producing 
 cells found in the saline control ( animal 22457 ) respectively ( figure 4d ) . 38 days after immunization , the proliferative 
 capacity of cd4 and cd8 cells in response 
 to ebola gp and adenovirus 
 serotype 5 was assessed by a ki-67 staining assay . two samples , each obtained from animals immunized by the 
 respiratory route , contained significant numbers of proliferative 
 ebola gp - specific cd4 t cells ( 50459 , 11.9% , and 52945 , 
 6.5% , white bars , figure 4e ) . the sample obtained 
 from nhp 50459 also contained the most ebola gp - specific cd8 t cells ( 8.8% , black bars , figure 4e ) . the 
 sample from nhp 62125 immunized by the sl route contained the second 
 highest amount of cd8 t cells ( 4.9% ) . all remaining samples 
 contained approximately 34% cd8 t cells that could 
 proliferate in response to ebola gp except for that from animal 52483 
 ( 1.1% ) . only one sample obtained from a primate immunized by the in / it 
 route , 52165 , contained a significant population of proliferative 
 adenovirus 5-specific cd4 t cells ( 8.1% , white bars , figure 4f ) . one sample from a primate in the in / it group 
 ( 50459 ) and another from the sl group ( 62125 ) contained notable populations 
 of cd8 cells that proliferated in response to ad5 ( 9.4 
 and 9.3% respectively , black bars , figure 4f ) . all remaining samples contained approximately 4% cd8 t cells that could proliferate in response to adenovirus except 
 for animal 40347 ( 2.2% ) . anti - ebola 
 gp and anti - adenovirus antibody levels were assessed in serum and 
 bal fluid 20 and 38 days after immunization ( figure 5 ) . marked levels of anti - ebola gp igg antibodies were found 
 in serum from animals immunized by the i m and the in / it routes 20 
 days after treatment ( figure 5a ) . anti - ebola gp antibodies 
 were found in the serum of only one of the animals immunized by the 
 sl route ( 52165 ) . this animal also had ebola gp - specific igg antibodies 
 in bal fluid 20 days after treatment ( figure 5b ) that were similar to those found in samples from animals immunized 
 by the respiratory route . bal from animals immunized by the i m route 
 did not contain any detectable levels of anti - ebola gp antibodies . one sample from a primate immunized by the i m route ( 40347 ) contained 
 a significant amount of circulating anti - adenovirus neutralizing antibodies 
 ( nabs , 1,007 reciprocal dilution , figure 5c ) . the sample from the remaining animal in the i m group and 2 others 
 from the in / it group contained anti - adenovirus nab titers of 200 
 reciprocal dilution . serum from animals immunized by the sl route 
 did not contain measurable levels of anti - adenovirus 5 nabs . study 1 : respiratory 
 immunization induces strong anti - ebola gp 
 and minimal anti - adenovirus antibody responses in serum and bal fluid . these samples 
 were screened for the presence of anti - ebola gp antibodies by elisa . serum collected on day 20 was also screened for anti - adenovirus 5 
 nabs using an infectious titer assay ( panel c ) . data in panel c is 
 reported as the dilution at which the infectious titer of a first 
 generation adenovirus expressing the beta - galactosidase transgene 
 was reduced by 50% . in each panel , error bars represent the standard 
 error of samples assayed in triplicate from each primate for each 
 time point . 62 days 
 after immunization , nhps were challenged with 1,000 pfu of ebola virus 
 ( 1995 , kikwit ) . one primate immunized by i m injection ( 40347 ) and 
 one animal immunized by the sl route ( 62125 ) succumbed to infection 
 6 days after challenge ( figure 6a ) . at this 
 time animal 62125 had a clinical score of 23 , and substantial petechiae 
 were noted upon necropsy . primate 40347 had a temperature of 40.3 
 c and a clinical score of 25 and experienced notable bleeding . one primate immunized by the in / it route ( 52483 ) and one primate immunized 
 by the sl route ( 62361 ) died the following day . each of these animals 
 had clinical scores above 25 and significantly decreased food intake 
 the previous day . the remaining primate immunized by the sl route 
 ( 52165 ) expired 8 days after challenge . one of the primates vaccinated 
 by i m injection ( 22473 ) and two of the animals immunized by the in / it 
 route ( 50459 , 52945 ) survived challenge ( 50 and 67% survival i m and 
 in / it respectively , figure 6a ) . a slight increase in weight of one animal immunized by 
 the in / it route ( 50459 ) was noted during the study period . changes 
 in body temperature ( figure 6c ) and clinical 
 scores ( figure 6d ) for each primate were in 
 line with survival results . the most striking changes in hematology 
 and blood chemistry values were observed around day 5 postchallenge 
 in the animals that did not survive . these include significantly elevated 
 liver enzymes with alt ( figure 6e ) and alp 
 ( figure 6f ) values rising to levels 27 and 
 16 times baseline respectively and blood urea nitrogen levels rising 
 to 7.5 times normal values before the animals expired ( figure 6 g ) . platelet counts , however , dropped to half the 
 baseline values in these animals ( figure 6h ) . in contrast , a sharp increase in platelets was noted in samples obtained 
 from animals that survived challenge . other hematology and blood chemistry 
 values in these animals remained largely unchanged ( data not shown ) . naive male cynomolgus macaques ( see table 1 for characteristics ) were challenged 62 days after immunization 
 with a lethal dose of 1,000 pfu ( 1,000 tcid50 ) of ebola 
 virus ( 1995 , kikwit ) . ( d ) daily clinical scores for each primate using 
 a standard , approved scoring methodology throughout the challenge . 
 variations in serum ( e ) alanine aminotransferase ( alt ) , ( f ) alkaline 
 phosphatase ( alp ) , ( g ) blood urea nitrogen ( bun ) , and ( h ) platelets 
 ( plt ) were noted in animals that did not survive challenge . the most exciting finding extracted from study 1 was that the combined 
 in / it administration of the vaccine was able to confer long - term immunity 
 to ebola . since it was not known if immunity induced by adenovirus - based 
 vaccines for ebola is persistent over time , we decided to extend the length of time between respiratory administration 
 of a formulated version of the vaccine and challenge . a secondary 
 goal was to increase the dose of vaccine given by the sublingual route 
 and to evaluate the ability of the sublingual vaccine to confer protection 
 in animals with prior exposure to adenovirus since improved responses 
 in this population were observed in studies with rodents . the long - term immune response of surviving animals 
 postchallenge was also a major point of interest in this study especially 
 in animals receiving vaccine containing a novel formulation and in those given the sublingual vaccine to 
 identify parameters to target during additional refinement of each 
 immunization platform . three male cynomolgus macaques were given 
 the vaccine in a potassium 
 phosphate buffer ( ph 7.4 ) containing an amphiphilic polymer ( formula 
 weight ( fw ) 39,000 ) formulation that improved the antigen - specific 
 immune response in rodent models of infection . the goal was to immunize this group as early in the study 
 as possible so that there would be a significant amount of time between 
 immunization and challenge ( figure 7 ) . 42 days after these animals were immunized , 3 
 macaques were given 1  10 particles of a host range 
 mutant adenovirus serotype 5 that can replicate in non - human primates by intramuscular injection to establish pre - existing immunity . 42 
 days later , animals were then given the vaccine by the sublingual 
 route . at this time the animals had an average circulating anti - adenovirus 
 antibody titer of 320  160 reciprocal dilution . three naive 
 animals were also given the same dose of vaccine by the sublingual 
 route at the same time for comparison . eleven male cynomolgus macaques 
 of chinese origin were immunized according to the schedule depicted 
 in the figure . animals were shipped to the national microbiology laboratory 
 ( nml ) in winnipeg 126 days after immunization for challenge on day 
 150 of the study . animals 
 were screened for signs of prior exposure to adenovirus and ebola 
 ( anti - ad5 nab , ad5 dna , anti - ebola gp antibodies ) 1 week prior to 
 the initiation of the study . samples were taken for evaluation of blood chemistry 
 and adenovirus shedding ( nasal , oral , rectal swabs , urine , feces ) 
 6 h after immunization as well as on days 1 , 2 , and 7 . on day 20 , 
 serum and bal were collected for assessment of shedding , anti - ad5 
 nabs , and anti - ebola gp antibodies . on day 42 , additional samples 
 were taken for assessment of anti - ebola gp and anti - ad5 antibodies 
 and antigen - specific t cell proliferation ( ebola gp and ad5 ) . 150 
 days after immunization , nhps were shipped to the national microbiology 
 laboratory in winnipeg for challenge . in / it : intranasal / intratracheal . in contrast 
 to the first study , a notable spike in creatine phosphokinase ( cpk ) 
 was detected in the serum of all animals 24 h after immunization ( figure 8a ) . this enzyme increased to 8 times baseline in 
 one animal immunized by the in / it route ( 810003 , 8,209 iu / l ) and to 
 10 times baseline in a primate with pre - existing immunity to adenovirus 
 immunized by the sublingual route ( 804819 , 4,483 ) . a notable spike 
 in serum lactate dehydrogenase ( ldh ) was also noted at the 24 h time 
 point . this was not as sharp as that seen with cpk with the highest 
 elevations found to be approximately 3 times baseline ( 804317 , 849 as seen in the first study , 
 serum ast increased in all primates after immunization . this occurred 
 at the 24 h time point for animals immunized by the respiratory and 
 sublingual routes but was not observed in primates with pre - existing 
 immunity to adenovirus until 48 h ( figure 8c ) . as in the first study , serum alkaline phosphatase ( alp ) levels 
 varied between primates , however , in this trial a distinct drop in 
 this parameter was noted in samples collected from most animals between 
 the 6 and 24 h time points , after which values remained constant ( figure 8d ) . other serological parameters evaluated during 
 the first week after immunization ( calcium , creatinine , albumin , globulin , 
 total protein , gamma glutamyl transferase ( ggt ) , total bilirubin , 
 alanine aminotransferase ( alt ) , bun , glucose , sodium , potassium , phosphate , 
 chloride , and cholesterol ) all fell within normal limits throughout 
 the course of the study ( data not shown ) . study 2 : clinical parameters 
 demonstrating transient changes after 
 immunization of naive non - human primates and those with pre - existing 
 immunity to adenovirus immunized by various routes . naive cynomolgus 
 macaques were given a single dose of vaccine by the respiratory ( in / it ) 
 or the sl routes . a separate group of animals first received a dose 
 of an adenovirus serotype 5 host range mutant virus 42 days prior 
 to immunization . black lines / squares : sl immunization ( primates 
 with pre - existing immunity to adenovirus ) . orange lines / diamonds : 
 sl immunization ( naive primates ) . adenovirus genomes were only found in serum samples collected 
 from 
 animals immunized by the respiratory route ( figure 9a ) . the most significant numbers of virus genomes detected 
 in any of the biological samples collected throughout the second study 
 were found in nasal swabs collected from primates 6 h after in / it 
 immunization [ 810003 ( 8.18  10 genome copies ( gc ) ) , 
 809077 ( 1.44  10 gc ) , and 802197 ( 1.36  10 gc , figure 9b ) ] and in oral swabs 
 collected from primates 6 h after sublingual immunization : [ 804317 
 ( 9.06  10 gc ) , 805257 ( 1.74  10 gc ) , and 808233 ( 7.92  10 gc , figure 9d ) ] . as seen in the first study , adenovirus genomes 
 were only found in the bal fluid of animals immunized by the in / it 
 route ( figure 9c ) . urine collected from one 
 naive animal immunized by the sl route and another with pre - existing 
 immunity also immunized by the sl route 6 h after treatment contained 
 notable amounts of adenovirus ( 808233 , 9,821 gc ; 807243 , 2,363 gc , 
 figure 9e ) . adenovirus genomes were found in 
 feces collected from one primate with pre - existing immunity to adenovirus 
 24 h after immunization by the sl route ( 804819 , 2.71  10 gc ) and in another primate 2 days after it was immunized 
 by the in / it route ( 802197 , 6.51  10 gc , figure 9f ) . virus continued to be shed in feces of this 
 animal 1 week after immunization ( 802197 , 2.52  10 gc ) . adenovirus dna was found on rectal swabs collected from each 
 animal throughout the course of the study ( table 3 ) . study 2 : adenovirus genomes are released in the serum and nasal 
 mucosa after in / it administration of formulated vaccine and in the 
 oral mucosa after sublingual immunization . male cynomolgus macaques 
 were given either 1.6  10 ivp / kg of vaccine in a 
 formulation of 10 mg / ml poly(maleic anhydride - alt-1-octadecene ) substituted with 3-(dimethylamino)propylamine by the 
 respiratory route or 2  10 ivp / kg of vaccine in 
 potassium phosphate buffered saline by the sl route . dna was isolated 
 from each sample , and viral genomes were determined by real time pcr . 
 animal numbers and corresponding treatments are outlined in table 2 data were obtained by real - time 
 taqman pcr on dna isolated from samples as described . none detected . sample fell below 
 the detection limit of the assay ( 10 viral genomes/100 ng of dna ) . the ebola virus 
 glycoprotein - specific t cell response was examined in pbmcs isolated 
 from whole blood immediately prior to challenge , 150 days postimmunization . multiparameter flow cytometry provided a comprehensive analysis of 
 the types of antigen - specific t cells elicited by each treatment ( figure 10 ) . the cd4 t cell population present 
 in animals immunized by the in / it route was much more diverse than 
 the cd8 t cell population ( figure 10a , b ) . six specific cd4 t cell subpopulations were found 
 in animal 802197 with the most predominate phenotype being cd4 cd107a il-2 ( 39% of the cd4 population , figure 10a ) . this animal also 
 had the most diverse antigen - specific cd8 t cell population 
 with 4 different subpopulations detected by intracellular staining 
 ( figure 10b ) . cells that were cd4 il-2 were most prevalent ( 45% ) in this primate . the cd8 population in this animal was composed of 3 specific 
 subtypes with relatively equal distribution ( cd8 cd107a il-2 , cd8 ifn- , and cd8 il-2 ) . the cd4 t cell 
 population was less diverse in primate 810003 with the majority of 
 antigen - specific cells also having the cd4 il-2 phenotype ( 85% ) . the cd8 il-2 subpopulation 
 was the most prominent of two types of antigen - specific cd8 t cells found in this primate . study 2 : 
 mucosal immunization elicits diverse populations of t 
 cells capable of responding to ebola glycoprotein 150 days after treatment . 
 quantitative analysis of cd4 t cell populations secreting 
 individual and combinations of cytokines in response to antigen stimulation 
 after in / it administration ( panel a ) , sl administration to naive animals 
 ( panel c ) , and sl administration to those with pre - existing immunity 
 to adenovirus ( panel d ) . panel b reflects the quantitative analysis 
 of cd8 t cell populations after immunization by the in / it 
 route . each positively responding cell was assigned to one of 8 possible 
 categories reflecting the production of ifn- , il-2 , and il-4 
 alone or in combination . pie charts depict the variety of t cell populations 
 found in each individual animal . cd4 t cells were not 
 found in samples obtained from primate 808233 ( sl immunization ) . a 
 single cd8 il-2 population was detected in 
 samples from primate 804819 ( pei - sl ) and is not illustrated as a pie 
 chart . cd4 and cd8 t cell populations were noticeably 
 less diverse in animals immunized by the sl route ( figure 10c ) . antigen - specific cd4 t cells were 
 not detected in samples collected from primate 808233 . cd4 ifn- il-2 cells were present to a 
 lesser degree than cd4 ifn- cells in 
 samples collected from animal 805257 ( 25% and 75% of the population 
 respectively ) . the most diverse cd4 population elicited 
 by sl immunization was found in primate 804317 with cd4 il-2 cells being the most prominent of 5 different subtypes 
 identified in this population . antigen - specific cd8 t 
 cells were only found in samples collected from this animal with the 
 majority being of the cd8 cd107a phenotype 
 ( 92.6% ) and the remaining cells of the cd8 il-2 phenotype ( 7.4% , data not shown ) . pre - existing immunity to 
 adenovirus did not noticeably alter the 
 diversity of t cells elicited by sublingual immunization ( figure 10d ) . five distinct subpopulations of cd4 t cells were found in primate 809227 with those of the cd4 il-2 being the most prominent ( 63.1% ) . a single population 
 of cd8 cd107a cells was also found in samples 
 collected from this animal ( data not shown ) . cd4 il-4 cells were the most prominent of the two antigen - specific 
 cd4 t cell populations found in samples collected from 
 primate 807243 . antigen - specific cd8 t cells were not 
 detected in samples collected from this animal . sl immunization induced 
 a single population of cd4 il-2 cells and 
 a single population of cd8 cd107a cells in 
 primate 804819 . anti - ebola 
 gp and anti - adenovirus antibody levels were assessed in serum and 
 bal fluid at various time points after immunization ( figure 11 ) . antigen - specific antibody levels mildly increased 
 between day 20 and day 104 in serum collected from two animals immunized 
 by the in / it route ( 0810003 , 1.5-fold increase , 0809077 , 1.3-fold 
 increase , 0802197 , no change , figure 11a ) . antibody levels remained high at the 142 day time point and were comparable 
 to those found in animals immunized by the respiratory route in the 
 first primate study . significant anti - ebola gp antibody levels were 
 detected in the bal fluid of only one primate immunized by the in / it 
 route ( 0802197 , figure 11b ) . samples obtained 
 from one of the animals immunized by the sublingual route ( 0808233 ) 
 contained the highest level of anti - ebola gp antibodies than any of 
 the other animals given a single dose of vaccine ( figure 11c ) . it is also important to note that a significant 
 change in anti - ebola gp antibody levels between day 20 and day 57 
 postimmunization was detected in samples obtained from only one animal 
 in this treatment group ( 0805257 , 2.4-fold increase ) . samples from 
 only one of the animals with prior exposure to adenovirus immunized 
 by the sublingual route contained anti - ebola gp antibodies above the 
 detection limit of the assay ( 809227 , figure 11d ) . while a notable amount of anti - adenovirus neutralizing antibody 
 ( nab ) was detected in the serum of one primate 20 days after immunization 
 by the in / it route ( 802197 , 1:640 reciprocal dilution ) , circulating 
 anti - adenovirus nabs were low in samples obtained from other primates 
 immunized in the same manner ( figure 11e ) . anti - adenovirus nabs were not found in the bal of any of the primates 
 immunized by the in / it route during the course of the study ( data 
 not shown ) . while anti - adenovirus nabs were quite high in the serum 
 of one animal with pre - existing immunity 20 days after immunization 
 by the sl route ( 809227 , 1:2,560 reciprocal dilution ) , they were not 
 detected in samples collected from two naive primates immunized in 
 the same manner ( 805257 , 804317 , figure 11f ) . study 
 2 : respiratory immunization induces production of antigen - specific 
 antibodies that are sustained over time . serum was collected from 
 cynomolgus macaques immunized by the in route ( panel a ) on days 20 , 
 104 , and 142 after immunization and analyzed for anti - ebola gp igg 
 by elisa as described . serum was also 
 collected from naive primates ( panel c ) and those with pre - existing 
 immunity to adenovirus ( panel d ) on days 20 and 57 after immunization . these samples along with bal fluid ( panel b ) collected from all primates 
 were screened for anti - ebola gp antibodies in the same manner . serum 
 from animals immunized by the in / it route ( panel e ) and from animals 
 immunized by the sl route ( panel f ) was also screened for anti - adenovirus 
 neutralizing antibodies . in each panel , error bars represent the standard 
 error of samples assayed in triplicate from each primate for each 
 time point . 150 days 
 after immunization , animals were challenged with 1,000 pfu of ebola 
 virus ( 1995 , kikwit ) . six days after challenge , both primates given 
 saline , two animals immunized by the sl route ( 804317 , 808233 ) , and 
 one animal with pre - existing immunity to adenovirus immunized by the 
 sl route ( 809227 ) expired from infection ( figure 12a ) . the remaining primates with pre - existing immunity succumbed 
 to infection on days 7 ( 804819 ) and 8 ( 807243 ) respectively . the remaining 
 animal given the vaccine by the sl route ( 805257 ) expired on day 9 . these animals experienced minimal changes in body weight ( figure 12b ) and temperature ( figure 12c ) during the course of infection with their clinical scores peaking 
 at about 47 days after challenge ( figure 12d ) . study 2 : a single dose of a formulated adenovirus - based 
 vaccine 
 protects from lethal challenge 150 days after immunization . cynomolgus macaques given a single dose of 1.4  
 10 ivp of ad - cagoptzgp formulated with 10 mg / ml poly(maleic 
 anhydride - alt-1-octadecene ) substituted with 3-(dimethylamino)propylamine 
 in phosphate buffered saline survived lethal challenge . animals succumbing 
 to infection experienced a change of 10% of body weight during 
 the active infection period . body temperature declined in each animal during challenge 
 with the most dramatic drops observed in animals that were not protected 
 from infection . clinical scores were recorded for 
 each primate by a blinded technician using a standard , approved scoring 
 methodology . lymphocytes of surviving animals 
 recovered from an initial drop 3 days after challenge and remained 
 stable throughout the remainder of the study . ( f ) elispot analysis 
 of the cellular immune response in surviving animals 14 days after 
 challenge . pbmcs were isolated from whole blood and stimulated with 
 a peptide pool spanning the ebola glycoprotein . samples were collected from animals 
 on day 3 and day 14 and at the time of death . ( i ) blood urea nitrogen 
 ( bun ) profile of immunized animals during challenge . blue lines / triangles : in / it immunization . 
 orange lines / diamonds : sl immunization . black lines / squares : animals 
 with pre - existing immunity to adenovirus immunized by the sl route . a notable drop in lymphocyte levels 
 of all animals lymphocytes 
 abruptly spiked in one animal immunized by the sl route ( 808233 ) and 
 another with pre - existing immunity to adenovirus ( 804819 ) 6 days after 
 challenge . lymphocyte levels of primates immunized by the in / it route 
 slowly increased to day 14 where they remained constant . elispot 
 analysis revealed that a significant amount of mncs capable of producing 
 ifn- in response to stimulation with ebola gp peptides were 
 present in pbmcs isolated from whole blood of surviving animals 14 
 days after challenge ( figure 12f ) . a sharp 
 drop in platelet counts was noted in all animals that did not survive 
 challenge ( figure 12 g ) . mild drops in platelet 
 counts were observed in animals immunized by the in / it route 3 days 
 after challenge . alt 
 ( figure 12h ) and bun ( figure 12i ) sharply rose to values as high as 24 and 6 times baseline 
 respectively in animals that succumbed to ebola infection . assessment of sera taken during challenge revealed that primates 
 immunized by the in / it route had very high levels of circulating anti - ebola 
 gp antibodies ( figure 13a ) . these were neutralizing 
 since very low levels of infectious ebola were found in samples taken 
 from two primates 3 days postchallenge ( figure 13b ) . infectious ebola virus was not detected in any samples collected 
 from the third animal in this treatment group ( 809077 ) . ebola virus 
 genomes were also not detected in samples taken from any of the animals 
 immunized by the respiratory route ( table 4 ) . although samples from two animals immunized by the sublingual 
 route also contained high levels of anti - ebola neutralizing antibody 
 ( 804317 , 808233 , 1,280 reciprocal dilution , figure 13c ) , they were only partially neutralizing since a concentration 
 of 316 tcid50/ml was found in samples collected from both 
 primates at the 3 day time point that escalated to 1.47  10 and 6.81  10 tcid50/ml respectively 
 by the 6 day time point ( figure 13d ) . the number 
 of circulating virus genomes in these animals followed a similar trend 
 ( table 4 ) . one animal that was exposed to the 
 adenovirus serotype 5 host range mutant prior to immunization by the 
 sl route ( 804819 ) also had high levels of anti - ebola gp circulating 
 antibodies ( 1,280 reciprocal dilution , figure 13e ) , however , ebola virus rna was detected in samples collected from 
 this animal at a concentration of 8.19  10 genome 
 copies / ml ( table 4 ) . this animal expired before 
 any infectious virus could be detected in its serum ( figure 13f ) . anti - ebola gp antibodies generated by a formulated 
 adenovirus - based 
 respiratory vaccine are neutralizing while those produced by an unformulated 
 sublingual vaccine are partially neutralizing . the neutralizing capacity 
 of antibodies in serum collected from each primate was assessed using 
 a fluorescence neutralization assay ( panels a , c , and e ) . the amount 
 of ebola virus present in the serum of animals during challenge was 
 determined using a standard infectious titer assay ( panels b , d , and 
 f ) . in each panel , data obtained from animals given saline prior to 
 challenge with ebola are included as red symbols and lines for reference . tcid50 = median tissue culture infectious dose 50 or the 
 amount of virus that will produce pathological change in 50% of cells 
 that are infected in culture . these assays were performed under bsl-4 
 conditions at the national microbiology laboratory in winnipeg . data were 
 obtained by quantitative 
 rt - pcr on rna isolated from whole blood as described . sample fell below 
 the detection limit of the assay ( 86 viral genomes / ml ) . units are genome copies per milliliter 
 of whole blood ( gc / ml ) . the ongoing epidemic in west africa is the largest 
 ebola outbreak 
 ever recorded and is rapidly crossing borders . in response to this 
 public health crisis , the who has supported a movement to initiate 
 small phase i clinical trials of vaccine candidates that have successfully 
 prevented non - human primates from developing ebola virus disease after 
 exposure to a lethal dose of the virus . one candidate , an attenuated vesicular stomatitis virus ( vsv ) that 
 expresses the ebola glycoprotein in place of its own envelope protein , 
 has shown efficacy in inducing both prophylactic and postexposure 
 protection from infection . while this 
 fast - acting platform clearly holds promise for people in high - risk 
 settings who may have already been exposed to ebola , the longevity 
 of the immune response elicited by the vaccine has only just been 
 evaluated in rodent models of infection . a second vaccine candidate , a bivalent recombinant chimpanzee adenovirus 
 serotype 3 virus expressing the glycoproteins of both ebola and sudan 
 species , the most lethal of the known ebola viruses , is also undergoing clinical testing . this platform , which does not generate a protective immune response 
 as rapidly as the vsv - based vaccine , requires supplementation with 
 a modified vaccinia ankara virus also expressing the ebola and sudan 
 glycoproteins to generate long - term protective immunity . both of these vaccine candidates have been developed 
 and are entering the clinic as injectable products . the ideal 
 characteristics for an effective ebola vaccine should 
 be greatly influenced by the population where infections are endemic . long - lasting protection from ebola is necessary for at - risk populations 
 ( medical personnel ) and for rural villagers where repeated prime - boost 
 regimens are not feasible . development of easy to administer , noninvasive 
 immunization platforms eliminates the potential for transmission and 
 spread of other blood - borne pathogens like hepatitis and hiv due to 
 needle stick injuries that occur from unsafe practices during large 
 immunization campaigns and from improper handling of biomedical waste . establishment of mucosal as well as systemic immunity to ebola is 
 also important since transmission of the virus occurs through direct 
 contact of mucosal areas with body fluids of infected individuals . while the manner by which we delivered the vaccine 
 to the respiratory mucosa may seem complex by traditional standards , 
 the in / it dual route method was previously found to stimulate a significant 
 igg response as early as 14 days after immunization , which is much 
 earlier than that observed after intramuscular injection . thus , we envision this approach to be quite 
 attractive when rapid immune protection is desired especially in the 
 case of first responders to an outbreak . another important consideration 
 is that of the prevalence of pre - existing immunity ( pei ) to adenovirus 
 serotype 5 in the global population and the negative impact it might 
 have on the potency of our vaccine . we have previously 
 found that the performance of the unformulated vaccine when given 
 by in / it administration to primates at a dose equivalent to that used 
 in the studies outlined in this manuscript was not affected by pre - existing 
 immunity to adenovirus 5 . however , survival 
 was not complete as 25% of the naive animals and those with pre - existing 
 immunity to adenovirus succumbed to infection . it is also important 
 to realize that , in this case , the vaccine was not given alone but 
 in combination with another adenovirus vector expressing interferon 
 alpha , which may play a significant role in development of the antigen - specific 
 immune response necessary for survival from ebola infection in animals 
 with prior exposure to adenovirus . in contrast , respiratory administration 
 of our formulated vaccine alone afforded full protection to all animals 
 challenged at a much later time after immunization ( figure 12 ) . while this improvement in the absence of an 
 immunostimulatory cytokine may suggest that our formulated vaccine 
 may improve the potency of the vaccine in those with prior exposure 
 to adenovirus , additional dose ranging studies in animals with pei 
 to adenovirus are greatly warranted . both the unformulated and 
 the formulated vaccines given by the 
 respiratory route were well tolerated by each primate with only mild , 
 transient changes in a few blood chemistry parameters noted ( figures 2 and 8) . shedding of the 
 vaccine was also minimal as the adenovirus was fully cleared from 
 the nasal passages , urine , and feces within 48 h after immunization 
 ( figures 3 and 9 ) . administration 
 of the unformulated vaccine by the respiratory route induced notable 
 systemic and mucosal ebola gp - specific t cell responses ( figure 4 ) as well as anti - ebola gp - specific antibodies in 
 the circulation and bal fluid ( figure 5 ) . to 
 date , a considerable body of evidence indicates that , regardless of 
 vaccine platform , a robust antibody response to ebola glycoprotein 
 is essential for protection from lethal infection . additional studies have shown that a strong 
 antigen - specific t cell response is required to prevent the dysregulation 
 of host protective immune responses during ebola infection and is supportive when the antibody - mediated 
 response is suboptimal . these principles were illustrated 
 by the primate given the respiratory vaccine that did not survive 
 challenge in study 1 as the t cell response and the antibody mediated 
 response were significantly lower in samples collected from this animal 
 than in samples collected from other primates immunized in the same 
 manner . for the past 20 years , there has been solid evidence 
 in the scientific 
 literature that heterologous prime - boost regimens are required for 
 induction of long - lived protective cd8 t cells against 
 a variety of microbial infections . as 
 a result , there are very little if any data delineating the durability 
 of the immune response generated by a single dose of a recombinant 
 adenovirus - based vaccine since most of these platforms involve a priming 
 dose of a recombinant dna plasmid containing an antigenic sequence 
 similar to that encoded in a recombinant adenovirus used as a boost 
 or the use of two different adenovirus serotypes expressing the same 
 antigen . detailed study 
 of prime - boost regimens revealed that this approach elicited a diverse 
 cd8 t cell population with a variety of immunological 
 functions as defined by the production of two or more cytokines at 
 one time and cell surface mobilization of the degranulation marker 
 cd107a . while the presence of polyfunctional 
 t cells has been shown to be important for protection in some infectious 
 disease models , the role of polyfunctional t 
 cells is not yet clear in the context of ebola infection . previous 
 studies in rodent models of ebola infection revealed that pre - existing 
 immunity to adenovirus compromised the production of polyfunctional 
 cd8 t cells , which was indicative 
 of poor survival upon challenge . in the non - human primate model , it 
 has recently been found that the presence of cd8 ifn-tnf- t cells correlated with survival while other studies suggest that polyfunctional 
 cd4 t cells , especially those producing ifn-  and 
 il-2 in response to stimulation with ebola gp peptides , are important 
 for survival . data generated in our study best 
 correlate with these latter studies as samples obtained from two of 
 the primates receiving the respiratory vaccine that survived challenge 
 contained highly diverse cd4 t cell populations and the 
 most diverse cd8 t cell populations of all immunized animals 
 ( figure 10 ) . however , one common cell surface 
 marker / cytokine profile could not be detected among the surviving 
 animals . to our knowledge , this is the first time that durable protection 
 from a single dose respiratory recombinant adenovirus - based ebola 
 vaccine has been demonstrated in non - human primates . the step 
 and hvtn studies , which utilized an adenovirus serotype 
 5-based hiv vaccine , and some non - human primate data generated with 
 recombinant adenoviruses have raised concerns that induced antigen - specific , 
 vector - specific , or total cd4 lymphocytes at mucosal surfaces 
 may lead to enhanced hiv-1 infection . here , we show that respiratory 
 administration of a recombinant adenovirus 5-based vaccine does induce 
 strong cd4 t cell responses in bal fluid and in the periphery 
 that are long lasting . in contrast , sl immunization did not foster 
 production of antigen - specific cd4 t cells in bal but 
 did support antigen - specific t cell responses in the periphery , which 
 were not hampered by prior exposure to adenovirus . while it is not 
 clear if these observations would have adverse impact in populations 
 where there is currently a heightened need for an ebola vaccine and 
 where hiv is quite prevalent , the fact that adenovirus genomes were 
 detected from rectal swabs taken from animals at 7 and 20 days should 
 be further evaluated . because these samples were contaminated with 
 residual fecal matter , screening of rectal biopsies taken 104 days 
 after immunization for signs of inflammation and cellular activation one very important point illustrated in the second primate study 
 is that the antibody response generated by a given vaccine platform 
 may not be fully neutralizing . this was somewhat evident in the first 
 primate study as two animals with what appeared to be strong ebola 
 gp - specific antibody responses as determined by an elisa assay did 
 not survive challenge ( figure 5 and 6 ) . tcid50 and quantitative rt - pcr assays 
 performed in the second study provided solid evidence that antibodies 
 generated after immunization by the sl route could not effectively 
 neutralize the virus ( figure 13 ) . the highly 
 diverse ebola gp - specific cd4 t cell populations found 
 in one naive animal immunized by the sl route and another with pre - existing 
 immunity could not compensate for the poor antibody - mediated immune 
 response generated by this immunization strategy . while these results 
 were disappointing , it is important to realize that the primary goal 
 of this project was to develop an adenovirus - based vaccine that could 
 be given by either the respiratory or the oral route . during development 
 of the oral platform , we discovered that sublingual administration 
 of the vaccine could elicit protective responses to rodent - adapted 
 ebola . recently , studies evaluating this route 
 of immunization have gained presence in the scientific literature 
 with promising results for a variety of pathogens . the respiratory 
 platform is attractive for use in areas where there is a dire need 
 for ebola vaccines and therapeutics because it allows for self - immunization 
 in a needle free capacity . however , devices required for instillation 
 into the nose and lung can be bulky , require some skill for proper 
 use , and may require refrigeration for storage . many of the dosage forms used for sublingual delivery are 
 compact and can stabilize compounds at ambient temperature . thus , refinement of this platform for adenovirus - based 
 vaccines and other ebola vaccine candidates is warranted .
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  109 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 ( i m ) 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  1010 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 .