Bolstering Components of the Immune Response Compromised by Prior Exposure to Adenovirus: Guided Formulation Development for a Nasal Ebola Vaccine
Identifieur interne : 000056 ( Pmc/Corpus ); précédent : 000055; suivant : 000057Bolstering Components of the Immune Response Compromised by Prior Exposure to Adenovirus: Guided Formulation Development for a Nasal Ebola Vaccine
Auteurs : Jin Huk Choi ; Stephen X0a C. Schafer ; Alexander N. Freiberg ; Maria A. CroyleSource :
- Molecular Pharmaceutics [ 1543-8384 ] ; 2014.
Abstract
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
Url:
DOI: 10.1021/mp5006454
PubMed: 25549696
PubMed Central: 4525322
Links to Exploration step
PMC:4525322Le document en format XML
<record><TEI><teiHeader><fileDesc><titleStmt><title xml:lang="en">Bolstering Components of the Immune Response Compromised
by Prior Exposure to Adenovirus: Guided Formulation Development for
a Nasal Ebola Vaccine</title><author><name sortKey="Choi, Jin Huk" sort="Choi, Jin Huk" uniqKey="Choi J" first="Jin Huk" last="Choi">Jin Huk Choi</name><affiliation><nlm:aff id="aff1">Division of Pharmaceutics, College of Pharmacy,<institution>The University of Texas at Austin</institution>, Austin, Texas 78712,<country>United States</country></nlm:aff></affiliation></author><author><name sortKey="Schafer, Stephen X0a C" sort="Schafer, Stephen X0a C" uniqKey="Schafer S" first="Stephen X0a C." last="Schafer">Stephen X0a C. Schafer</name><affiliation><nlm:aff id="aff1">Division of Pharmaceutics, College of Pharmacy,<institution>The University of Texas at Austin</institution>, Austin, Texas 78712,<country>United States</country></nlm:aff></affiliation></author><author><name sortKey="Freiberg, Alexander N" sort="Freiberg, Alexander N" uniqKey="Freiberg A" first="Alexander N." last="Freiberg">Alexander N. Freiberg</name><affiliation><nlm:aff id="aff2">Department of Pathology,<institution>The University of Texas Medical Branch</institution>, Galveston, Texas 77555,<country>United States</country></nlm:aff></affiliation></author><author><name sortKey="Croyle, Maria A" sort="Croyle, Maria A" uniqKey="Croyle M" first="Maria A." last="Croyle">Maria A. Croyle</name><affiliation><nlm:aff id="aff1">Division of Pharmaceutics, College of Pharmacy,<institution>The University of Texas at Austin</institution>, Austin, Texas 78712,<country>United States</country></nlm:aff></affiliation><affiliation><nlm:aff id="aff3">Center for Infectious Disease,<institution>The University of Texas at Austin</institution>, Austin, Texas 78712,<country>United States</country></nlm:aff></affiliation></author></titleStmt><publicationStmt><idno type="wicri:source">PMC</idno><idno type="pmid">25549696</idno><idno type="pmc">4525322</idno><idno type="url">http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4525322</idno><idno type="RBID">PMC:4525322</idno><idno type="doi">10.1021/mp5006454</idno><date when="2014">2014</date><idno type="wicri:Area/Pmc/Corpus">000056</idno><idno type="wicri:explorRef" wicri:stream="Pmc" wicri:step="Corpus" wicri:corpus="PMC">000056</idno></publicationStmt><sourceDesc><biblStruct><analytic><title xml:lang="en" level="a" type="main">Bolstering Components of the Immune Response Compromised
by Prior Exposure to Adenovirus: Guided Formulation Development for
a Nasal Ebola Vaccine</title><author><name sortKey="Choi, Jin Huk" sort="Choi, Jin Huk" uniqKey="Choi J" first="Jin Huk" last="Choi">Jin Huk Choi</name><affiliation><nlm:aff id="aff1">Division of Pharmaceutics, College of Pharmacy,<institution>The University of Texas at Austin</institution>, Austin, Texas 78712,<country>United States</country></nlm:aff></affiliation></author><author><name sortKey="Schafer, Stephen X0a C" sort="Schafer, Stephen X0a C" uniqKey="Schafer S" first="Stephen X0a C." last="Schafer">Stephen X0a C. Schafer</name><affiliation><nlm:aff id="aff1">Division of Pharmaceutics, College of Pharmacy,<institution>The University of Texas at Austin</institution>, Austin, Texas 78712,<country>United States</country></nlm:aff></affiliation></author><author><name sortKey="Freiberg, Alexander N" sort="Freiberg, Alexander N" uniqKey="Freiberg A" first="Alexander N." last="Freiberg">Alexander N. Freiberg</name><affiliation><nlm:aff id="aff2">Department of Pathology,<institution>The University of Texas Medical Branch</institution>, Galveston, Texas 77555,<country>United States</country></nlm:aff></affiliation></author><author><name sortKey="Croyle, Maria A" sort="Croyle, Maria A" uniqKey="Croyle M" first="Maria A." last="Croyle">Maria A. Croyle</name><affiliation><nlm:aff id="aff1">Division of Pharmaceutics, College of Pharmacy,<institution>The University of Texas at Austin</institution>, Austin, Texas 78712,<country>United States</country></nlm:aff></affiliation><affiliation><nlm:aff id="aff3">Center for Infectious Disease,<institution>The University of Texas at Austin</institution>, Austin, Texas 78712,<country>United States</country></nlm:aff></affiliation></author></analytic><series><title level="j">Molecular Pharmaceutics</title><idno type="ISSN">1543-8384</idno><idno type="eISSN">1543-8392</idno><imprint><date when="2014">2014</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p content-type="toc-graphic"><graphic xlink:href="mp-2014-006454_0010" id="ab-tgr1"></graphic></p><p>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 <italic>in vitro</italic> 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 (ND<sub>50</sub>). <italic>In vivo</italic> performance of rAd in F16 was compared
with unformulated virus, virus modified with poly(ethylene) glycol
(PEG), and virus incorporated into poly(lactic-<italic>co</italic>-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<sup>+</sup> 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
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<front><journal-meta><journal-id journal-id-type="nlm-ta">Mol Pharm</journal-id><journal-id journal-id-type="iso-abbrev">Mol. Pharm</journal-id><journal-id journal-id-type="publisher-id">mp</journal-id><journal-id journal-id-type="coden">mpohbp</journal-id><journal-title-group><journal-title>Molecular Pharmaceutics</journal-title></journal-title-group><issn pub-type="ppub">1543-8384</issn><issn pub-type="epub">1543-8392</issn><publisher><publisher-name>American Chemical
Society</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="pmid">25549696</article-id><article-id pub-id-type="pmc">4525322</article-id><article-id pub-id-type="doi">10.1021/mp5006454</article-id><article-categories><subj-group><subject>Article</subject></subj-group></article-categories><title-group><article-title>Bolstering Components of the Immune Response Compromised
by Prior Exposure to Adenovirus: Guided Formulation Development for
a Nasal Ebola Vaccine</article-title></title-group><contrib-group><contrib contrib-type="author" id="ath1"><name><surname>Choi</surname><given-names>Jin Huk</given-names></name><xref rid="aff1" ref-type="aff">†</xref></contrib><contrib contrib-type="author" id="ath2"><name><surname>Schafer</surname><given-names>Stephen
C.</given-names></name><xref rid="aff1" ref-type="aff">†</xref></contrib><contrib contrib-type="author" id="ath3"><name><surname>Freiberg</surname><given-names>Alexander N.</given-names></name><xref rid="aff2" ref-type="aff">‡</xref></contrib><contrib contrib-type="author" corresp="yes" id="ath4"><name><surname>Croyle</surname><given-names>Maria A.</given-names></name><xref rid="cor1" ref-type="other">*</xref><xref rid="aff1" ref-type="aff">†</xref><xref rid="aff3" ref-type="aff">§</xref></contrib><aff id="aff1"><label>†</label>Division of Pharmaceutics, College of Pharmacy,<institution>The University of Texas at Austin</institution>, Austin, Texas 78712,<country>United States</country></aff><aff id="aff2"><label>‡</label>Department of Pathology,<institution>The University of Texas Medical Branch</institution>, Galveston, Texas 77555,<country>United States</country></aff><aff id="aff3"><label>§</label>Center for Infectious Disease,<institution>The University of Texas at Austin</institution>, Austin, Texas 78712,<country>United States</country></aff></contrib-group><author-notes><corresp id="cor1"><label>*</label>The University of Texas at Austin,
College of Pharmacy, PHR 4.214D, 2409 W. University Ave., Austin,
TX 78712-1074. Tel: <phone>512-471-1972</phone>. Fax: <fax>512-471-7474</fax>. E-mail: <email>macroyle@austin.utexas.edu</email>.</corresp></author-notes><pub-date pub-type="epub"><day>30</day><month>12</month><year>2014</year></pub-date><pub-date pub-type="ppub"><day>03</day><month>08</month><year>2015</year></pub-date><volume>12</volume><issue>8</issue><issue-title>Advances
in Respiratory and Nasal Drug Delivery</issue-title><fpage>2697</fpage><lpage>2711</lpage><history><date date-type="received"><day>24</day><month>09</month><year>2014</year></date><date date-type="accepted"><day>30</day><month>12</month><year>2014</year></date><date date-type="rev-recd"><day>21</day><month>12</month><year>2014</year></date></history><permissions><copyright-statement>Copyright © 2014 American Chemical Society</copyright-statement><copyright-year>2014</copyright-year><copyright-holder>American Chemical Society</copyright-holder><license license-type="editor-choice"><license-p>This is an open access article published under an ACS AuthorChoice <ext-link ext-link-type="uri" xlink:href="http://pubs.acs.org/page/policy/authorchoice_termsofuse.html">License</ext-link>, which permits copying and redistribution of the article or any adaptations for non-commercial purposes.</license-p></license></permissions><abstract><p content-type="toc-graphic"><graphic xlink:href="mp-2014-006454_0010" id="ab-tgr1"></graphic></p><p>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 <italic>in vitro</italic> 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 (ND<sub>50</sub>). <italic>In vivo</italic> performance of rAd in F16 was compared
with unformulated virus, virus modified with poly(ethylene) glycol
(PEG), and virus incorporated into poly(lactic-<italic>co</italic>-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<sup>+</sup> 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.</p></abstract><kwd-group><kwd>formulation</kwd><kwd>pre-existing immunity</kwd><kwd>adenovirus
serotype 5</kwd><kwd>Ebola virus</kwd><kwd>nasal vaccine</kwd><kwd>multifunctional CD8<sup>+</sup> T cell response</kwd></kwd-group><funding-group><funding-statement><funding-source>National Institutes of Health, United States</funding-source></funding-statement></funding-group><custom-meta-group><custom-meta><meta-name>document-id-old-9</meta-name><meta-value>mp5006454</meta-value></custom-meta><custom-meta><meta-name>document-id-new-14</meta-name><meta-value>mp-2014-006454</meta-value></custom-meta><custom-meta><meta-name>ccc-price</meta-name><meta-value></meta-value></custom-meta></custom-meta-group></article-meta></front><body><sec sec-type="intro" id="sec1"><title>Introduction</title><p>Since the resolution of the Plague of
Athens in 426 B.C., it has
been understood that exposure to microorganisms that cause disease
is protective against reinfection.<sup><xref ref-type="bibr" rid="ref1">1</xref></sup> This
concept of immunity through exposure to pathogens provided the foundation
for early vaccine development, which has led to the control of several
major diseases including smallpox, diphtheria, tetanus, yellow fever,
pertussis, and poliomyelitis.<sup><xref ref-type="bibr" rid="ref2">2</xref></sup> Within the
past 20 years, advances in synthetic and systems biology have significantly
compressed the time between identification of a pathogen responsible
for a pandemic and global dispersement of an effective vaccine.<sup><xref ref-type="bibr" rid="ref3">3</xref></sup> This technological revolution has also fostered
production of highly sophisticated vaccine platforms consisting of
recombinant viruses, virus like particles, synthetic protein antigens,
polysaccharide conjugates, and human cell isolates.<sup><xref ref-type="bibr" rid="ref4">4</xref></sup> Despite this progress, vaccines continue to be produced
primarily as injectable products.</p><p>Administration of drugs within
the nasal cavity was first exploited
to alleviate respiratory conditions such as allergic and infectious
rhinitis, nasal polyposis, and sinusitis.<sup><xref ref-type="bibr" rid="ref5">5</xref></sup> Further understanding of the rich vascularization and active transport
regions along the nasal mucosa has made it an attractive site of delivery
for small molecule compounds that are subject to extensive first pass
metabolism when given orally.<sup><xref ref-type="bibr" rid="ref6">6</xref></sup> The presence
of specialized mucosal lymphoid follicles throughout the nasal mucosa,
coupled with the fact it is often the place at which most bacterial
and viral infections are initiated,<sup><xref ref-type="bibr" rid="ref7">7</xref></sup> suggests
that intranasal immunization is a logical approach for protection
against many pathogens. However, reported cases of anaphylaxis and
development of neurological side effects after nasal administration
of vaccines have prevented intranasal delivery technology from being
exploited to its full potential.<sup><xref ref-type="bibr" rid="ref8">8</xref></sup> To date,
only three intranasal vaccines have been approved for human use: FluMist
in the United States,<sup><xref ref-type="bibr" rid="ref9">9</xref></sup> Fluenz in the European
Union,<sup><xref ref-type="bibr" rid="ref10">10</xref></sup> and Nasovac, a pandemic H1N1 vaccine
approved by the Drug Controller General of India.<sup><xref ref-type="bibr" rid="ref11">11</xref></sup></p><p>We have utilized nasal delivery of a recombinant
adenovirus serotype
5-based vaccine against the Ebola virus (formerly Zaire ebolavirus)
as a method to improve vaccine performance in the presence of pre-existing
immunity (PEI) to the adenovirus vector. This delivery method fosters
very high, localized, and notable systemic immune responses to an
encoded antigen and, more specifically, has fully protected animals
with PEI to adenovirus from lethal challenge with Ebola.<sup><xref ref-type="bibr" rid="ref12">12</xref>,<xref ref-type="bibr" rid="ref13">13</xref></sup> However, we have found that, if prior exposure to adenovirus occurs
in the respiratory tract in a manner similar to natural infection,
multifunctional CD8<sup>+</sup> T cells and antigen-specific antibody
responses are compromised in mouse and guinea pig models of Ebola
infection.<sup><xref ref-type="bibr" rid="ref14">14</xref></sup> Some of these responses are
starting to be observed in the clinic.<sup><xref ref-type="bibr" rid="ref15">15</xref></sup> Both of these responses have been found to support protection from
Ebola in rodent and primate models of disease.<sup><xref ref-type="bibr" rid="ref14">14</xref>,<xref ref-type="bibr" rid="ref16">16</xref>,<xref ref-type="bibr" rid="ref17">17</xref></sup> This finding prompted us to adopt the hypothesis
that reinvigoration of these responses through the use of relatively
nontoxic compounds that increase residence time in the nasal cavity
and improve bioavailability of the vaccine beyond the epithelial barrier
would significantly improve vaccine potency in those with prior exposure
to adenovirus. Reagents selected for this purpose fell into four major
categories: sugars and sugar derivatives (sucrose, melezitose, raffinose),
surfactants (Pluronic F68, <italic>N</italic>-dodecyl-β-<sc>d</sc>-maltopyranoside (nDMPS)), polymers (poly(ethylene) glycol,
poly(lactide-<italic>co</italic>-glycolide), poly(maleic anhydrides)),
and permeability enhancers (sorbitol, mannitol). Formulation candidates
were evaluated in a graded, stepwise manner. Preparations that improved
transduction efficiency with minimal cytotoxic effects in an <italic>in vitro</italic> model of the airway epithelium were selected for
further testing <italic>in vivo</italic> in rodent models of Ebola
virus infection.</p></sec><sec id="sec2"><title>Experimental Section</title><sec sec-type="materials" id="sec2.1"><title>Materials</title><p>Acepromazine
was purchased from Fort Dodge
Laboratories (Atlanta, GA). Ketamine was purchased from Wyeth, Fort
Dodge, Animal Health (Overland Park, KS). Dulbecco’s phosphate-buffered
saline (DPBS), xylazine, tresyl chloride activated monomethoxypoly(ethylene)glycol, <sc>l</sc>-lysine, poly(ethylene) glycol 3000, ethyl acetate, poly(lactide-<italic>co</italic>-glycolide) copolymers (PLGA, 50:50 lactide:glycolide),
poly(vinyl alcohol) (PVA), glutaraldehyde (grade I, 25% in water), <italic>o</italic>-phenylenediamine, sucrose (USP grade), <sc>d</sc>-mannitol
(USP grade), <sc>d</sc>-sorbitol (USP grade), bovine serum albumin
(RIA grade), brefeldin A, potassium ferricyanide, and potassium ferrocyanide
were purchased from Sigma-Aldrich (St. Louis, MO). Eosin Y and Tween
20 were purchased from Fisher Scientific (Kalamazoo, MI, and Pittsburgh,
PA respectively). Melezitose monohydride was purchased from MP Biomedicals
(Solon, OH) and raffinose pentahydrate from Alfa Aesar (Ward Hill,
MA). Sodium hydroxide, potassium phosphate monobasic, potassium phosphate
dibasic, and sodium dodecyl sulfate were purchased from Mallinckrodt
Baker (Phillipsburg, NJ). Pluronic F68 was purchased from BASF (Mount
Olive, NJ). Dulbecco’s modified Eagle’s medium (DMEM),
RPMI-1640, minimal essential medium (MEM), and <sc>l</sc>-glutamine
were purchased from Mediatech (Manassas, VA). Fetal bovine serum (qualified,
US origin), penicillin, and streptomycin were purchased from Gibco
Life Technologies (Grand Island, NY). Sodium pyruvate and nonessential
amino acids were purchased from Lonza (Walkersville, MD). 5-Bromo-4-chloro-3-indolyl-β-<sc>d</sc>-galactoside (X-gal) was purchased from Gold Biotechnology
(St. Louis, MO). <italic>N</italic>-Dodecyl-β-<sc>d</sc>-maltopyranoside
(nDMPS), poly(maleic anhydride-<italic>alt</italic>-1-decene) substituted
with 3-(dimethylamino)propylamine (PMAL C8, formula weight (FW) 8,500),
poly(maleic anhydride-<italic>alt</italic>-1-tetradecene) substituted
with 3-(dimethylamino)propylamine (PMAL C12, FW 12,000), and poly(maleic
anhydride-<italic>alt</italic>-1-octadecene) substituted with 3-(dimethylamino)propylamine
(PMAL C16, FW 39,000) were purchased from Anatrace (Maumee, OH). The
TELRTFSI peptide was purchased from New England Peptide (Gardner,
MA). The negative control peptide (YPYDVPDYA) was purchased
from GenScript (Piscataway, NJ). Antibodies used for ELISPOT and flow
cytometry, Cytofix/Cytoperm and Perm/Wash reagents were purchased
from BD Pharmingen (San Diego, CA).</p></sec><sec id="sec2.2"><title>Adenovirus Production</title><p>Four different recombinant adenoviruses
were used in the studies outlined in this manuscript. All were first
generation E1/E3 deleted recombinant adenovirus serotype 5 vectors
that differed only by transgene expression cassettes. Two vectors,
AdlacZ, expressing <italic>Escherichia coli</italic> beta-galactosidase,
and AdGFP, expressing green fluorescent protein, were used for rapid
screening of the transduction efficiency of formulations <italic>in
vitro</italic> and <italic>in vivo</italic> due to the ease by which
their transgene products could be visualized and quantitated. AdNull,
an E1/E3 deleted adenovirus 5 vector with a similar genetic backbone
as the other viruses used in this study except that it does not contain
a marker transgene expression cassette, was used to induce pre-existing
immunity to adenovirus 5 in mice prior to immunization. These viruses
were each amplified in HEK 293 cells (ATCC CRL-1573 Manassas, VA).
They were purified from cell lysates by banding twice on cesium chloride
gradients and desalted over Econo-Pac 10 DG disposable chromatography
columns (BioRad, Hercules, CA) equilibrated with potassium phosphate
buffer (KPBS, pH 7.4). The concentration of each preparation was determined
by UV spectrophotometric analysis at 260 nm and by an infectious titer
assay as described.<sup><xref ref-type="bibr" rid="ref18">18</xref></sup> Preparations with
a ratio of infectious to physical particles of 1:100 were used for
these studies. For immunization and challenge studies, an E1/E3 deleted
recombinant adenovirus serotype 5 vector expressing a codon optimized
full-length Ebola glycoprotein sequence under the control of the chicken
β-actin promoter (Ad-CAGoptZGP) was amplified in HEK 293 cells
and purified as described.<sup><xref ref-type="bibr" rid="ref14">14</xref></sup> Concentration
of this and AdNull 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 1:200 of each of these
viruses were used in this study.</p></sec><sec id="sec2.3"><title>PEGylation of Adenovirus</title><p>PEGylation was performed according
to established protocols.<sup><xref ref-type="bibr" rid="ref19">19</xref>,<xref ref-type="bibr" rid="ref20">20</xref></sup> Characterization of
these preparations revealed significant changes in the biophysical
properties of the virus such as the PEG-dextran partition coefficient
and peak elution times during capillary electrophoresis.<sup><xref ref-type="bibr" rid="ref20">20</xref></sup> Approximately 18,245 ± 546 PEG molecules
were associated with each virus particle in the studies outlined here
as determined by a PEG-biotin assay.<sup><xref ref-type="bibr" rid="ref21">21</xref></sup></p></sec><sec id="sec2.4"><title>PLGA Microspheres</title><p>PLGA microspheres were prepared using
a standard water-in-oil-in-water (W/O/W) double emulsion and solvent
evaporation method.<sup><xref ref-type="bibr" rid="ref22">22</xref></sup> One milliliter of
virus (5 × 10<sup>12</sup> virus particles) was added to ethyl
acetate containing 100 mg of PLGA. The primary water-in-oil emulsion
was prepared by homogenization for 30 s and was then added to 10 mL
of an aqueous solution containing 5% (w/v) PVA. The secondary W/O/W
emulsion was prepared by homogenization for 60 s and further agitated
with a magnetic stirring rod for 2 h at 4 °C to evaporate the
cosolvent. Microspheres were collected by centrifugation at 2,000
rpm for 3 min and washed five times with sterile KPBS. The diameter
of the microspheres fell between 0.3 and 5 μm with an average
particle size of 2.06 ± 1.4 μm as determined by dynamic
light scattering using a DynaPro LSR laser light scattering device
and detection system (Wyatt Technology, Santa Barbara, CA). Regularization
histograms and assignment of hydrodynamic radius values to various
subpopulations within the sample were calculated using DynaLS software
(Wyatt). The amount of virus embedded in the microspheres was determined
by digesting a portion of each preparation with 1 N NaOH for 24 h.
The average encapsulation efficiency of this process was 21.6 ±
4.4% (<italic>n</italic> = 6). Aliquots of each preparation were dried,
placed in sterile, light resistant containers, and stored at room
temperature for evaluation of stability over time. Release profiles
of each preparation were determined by placing 10 mg of microspheres
in 0.5 mL of sterile KPBS on a magnetic stir plate (Corning, Tewksbury,
MA) in a 37 °C incubator. Each day, microspheres were collected
by centrifugation, and the supernatant was collected and replaced
with KPBS prewarmed to 37 °C. The number of infectious virus
particles released from microspheres was determined by serial dilution
of collected samples and subsequent infection of Calu-3 cells (ATCC,
HTB-55), an established model of the airway epithelia.<sup><xref ref-type="bibr" rid="ref23">23</xref></sup></p></sec><sec id="sec2.5"><title><italic>In Vitro</italic> Screening of Formulations</title><p>Two vectors, AdlacZ, expressing <italic>E. coli</italic> beta-galactosidase,
and AdGFP, expressing green fluorescent protein, were used for rapid
screening of the transduction efficiency of virus in a variety of
formulations due to the ease by which their transgene products could
be visualized and quantitated. Formulations were prepared at five
times the working concentration, sterilized by filtration and diluted
with freshly purified virus in KPBS (pH 7.4) prior to use. Two hundred
microliters of formulation containing virus (MOI 100) in the absence
or presence of anti-adenovirus antibodies was added to differentiated
Calu-3 cells seeded at a density of 1.25 × 10<sup>5</sup> cells/well
in 12 well plates. Formulations remained in contact with cell monolayers
for 2 h at 37 °C in 5% CO<sub>2</sub>. Cytotoxicity was assessed
by measuring lactate dehydrogenase (LDH) release into the formulation
with a standard cytotoxicity kit (Roche Applied Science, Indianapolis,
IN) according to the manufacturer’s instructions. Complete
lysis was achieved by adding 1% sodium dodecyl sulfate to cells not
exposed to formulations (positive control). Transduction efficiency
was measured 48 h later either by histochemical staining and quantitation
of cells expressing beta-galactosidase by visual inspection or by
flow cytometry to quantitate cells expressing GFP.</p></sec><sec id="sec2.6"><title>Animal Studies</title><p>All procedures were approved by the
Institutional Animal Care and Use Committees at The University of
Texas at Austin and the University of Texas Medical Branch in Galveston
and are in accordance with the guidelines established by the National
Institutes of Health for the humane treatment of animals. Two different
strains of mice were used in these studies. Male B10.Br mice (MHC
H-2<sup>k</sup>) were used to characterize the immune response to
Ebola glycoprotein after immunization with the Ad-CAGoptZGP vector
as described previously.<sup><xref ref-type="bibr" rid="ref13">13</xref>,<xref ref-type="bibr" rid="ref14">14</xref>,<xref ref-type="bibr" rid="ref24">24</xref></sup> Because this strain is difficult to breed,<sup><xref ref-type="bibr" rid="ref25">25</xref>,<xref ref-type="bibr" rid="ref26">26</xref></sup> and is often not readily available in quantities sufficient from
the supplier to perform the studies outlined in this manuscript, male
C57/BL6 (MHC H-2<sup>d</sup>) mice were used for initial screening
of formulations that improved transgene expression <italic>in vitro</italic> with minimal cytotoxic effects. Both strains were obtained from
the Jackson Laboratory (age 4–6 weeks, Bar Harbor, ME).</p></sec><sec id="sec2.7"><title>Nasal
Administration of Virus/Immunization</title><p>Animals
were housed in a temperature-controlled, 12 h light-cycled facility
at the Animal Research Center of The University of Texas at Austin
with free access to standard rodent chow (Harlan Teklad, Indianapolis,
IN) and tap water. Animals were anesthetized by a single intraperitoneal
injection of a 3.9:1 mixture of ketamine (100 mg/mL) and xylazine
(100 mg/mL). Once deep plane anesthesia was achieved, animals were
placed on their stomach. The sedated animal’s head was rested
upon an empty tuberculin syringe to keep the head in an upright position
and to minimize choking or accidental swallowing of vaccine (as illustrated
in Table of Contents graphic). Each mouse received a dose of 1 ×
10<sup>8</sup> infectious particles of unformulated or formulated
vaccine by direct application in the nasal cavity. The inhalation
pressure from the animal’s natural breathing was sufficient
to allow small droplets from a standard micropipette (Gilson, Middleton,
WI) to gently enter the nasal cavity without the need to forcefully
inject the solution. The right nostril received 10 μL and was
allowed to dry for up to 5 min before adding an additional 10 μL
to the left nostril, for a total volume of 20 μL per animal.
The animal was observed in the relaxed position for an additional
10 min to guarantee comfortable breathing and ensure that the vaccine
was not lost via sneezing (a rare occurrence that can result from
touching the animal’s nose with the micropipette tip instead
of allowing the tiny droplet to be gently pulled into the nose through
natural inhalation pressure).</p></sec><sec id="sec2.8"><title>Establishment of Pre-Existing
Immunity to Adenovirus</title><p>A first generation adenovirus that
that does not contain a transgene
cassette (AdNull) was used to establish pre-existing immunity to adenovirus
serotype 5.<sup><xref ref-type="bibr" rid="ref27">27</xref></sup> Twenty-eight days prior to
vaccination, mucosal PEI was induced by placing 5 × 10<sup>10</sup> particles of AdNull in the nasal cavity as described above under
the immunization protocol. Twenty-four days later, blood was collected
via the saphenous vein and serum screened for anti-Ad neutralizing
antibodies (NABs) as described below. At the time of vaccination,
animals had an average anti-Ad circulating NAB titer of 315 ±
112 reciprocal dilution, which falls within the range of average values
reported in humans after natural infection.<sup><xref ref-type="bibr" rid="ref28">28</xref></sup></p></sec><sec id="sec2.9"><title>Challenge with Mouse-Adapted Ebola Virus</title><p>Challenge
experiments were performed under biosafety level 4 (BSL-4) conditions
in an AAALAC accredited animal facility at the Robert E. Shope BSL-4
Laboratory at the University of Texas Medical Branch in Galveston,
Texas. Twenty-one days postimmunization, vaccinated mice were transported
to the BSL-4 lab, where they were challenged on day 28 by intraperitoneal
injection with 1,000 pfu of mouse-adapted (30,000 × LD<sub>50</sub>) Ebola.<sup><xref ref-type="bibr" rid="ref29">29</xref></sup> After challenge, animals were
monitored for clinical signs of disease and weighed daily for 14 days.
Serum alanine aminotransferase (ALT) and aspartate aminotransferase
(AST) levels were determined using AST/SGOT and ALT/SGPT DT slides
on a Vitros DTSC autoanalyzer (Ortho Clinical Diagnostics, Rochester,
NY).</p></sec><sec id="sec2.10"><title>ELISPOT</title><p>ELISpot assays were performed using the ELISpot
Mouse Set (BD Pharmingen) according to the manufacturer’s instructions.
Mononuclear cells were isolated from the spleen and bronchoalveolar
lavage fluid as described previously,<sup><xref ref-type="bibr" rid="ref14">14</xref></sup> washed twice with complete DMEM, and added to wells of a 96 well
ELISpot plate (5 × 10<sup>5</sup> cells/well) with the TELRTFSI
peptide (0.5 μg/well) that carries the Ebola virus glycoprotein
immunodominant MHC class I epitope for mice with the H-2<sup>k</sup> haplotype (B10.Br).<sup><xref ref-type="bibr" rid="ref30">30</xref></sup> Negative control
cells were stimulated with an irrelevant peptide, which carries a
binding sequence for influenza hemagglutinin (YPYDVPDYA,
0.5 μg/well). Spots were counted using an automated ELISpot
reader (CTL-ImmunoSpot S5Micro Analyzer, Cellular Technology Ltd.,
Shaker Heights, OH).</p></sec><sec id="sec2.11"><title>Multiparameter Flow Cytometry</title><p>Splenocytes
(2 ×
10<sup>6</sup>) isolated from immunized mice were cultured with TELRTFSI
peptide (0.5 μg/well) and 1 μg/mL brefeldin A for 5 h
at 37 °C in 5% CO<sub>2</sub>. Negative control cells were incubated
with the YPYDVPDYA peptide (0.5 μg/well). Following
stimulation, cells were surface stained with anti-mouse CD8α
antibodies (1:150 in DPBS) and followed by intracellular staining
with anti-mouse IFN-γ, TNF-α, and IL-2 antibodies as described.<sup><xref ref-type="bibr" rid="ref14">14</xref></sup> Positive cells were counted using four-color
flow cytometry (FACS Fortessa, BD Biosciences, Palo Alto, CA). Over
500,000 events were captured per sample. Data were analyzed using
FlowJo software (Tree Star, Inc., Ashland, OR).</p></sec><sec id="sec2.12"><title>CFSE Assay</title><p>Splenocytes were isolated 42 days postvaccination,
stained using the Vybrant CFDA SE Cell Tracer kit (Invitrogen, Carlsbad,
CA), seeded at a concentration of 5 × 10<sup>5</sup> cells/well
in 96 well plates, and cultured for 5 days at 37 °C with 5% CO<sub>2</sub> in the presence of the TELRTFSI or YPYDVPDYA
peptides (0.5 μg/well) as described previously.<sup><xref ref-type="bibr" rid="ref24">24</xref></sup> Cells were incubated with a cocktail of antibodies (perCPCy5.5
labeled-anti-mouse-CD8, PE labeled-anti-mouse-CD44, and allophycocyanin
(APC) labeled-anti-mouse-CD62L, 1:150) and analyzed by flow cytometry
with over 1,000,000 events captured per sample.</p></sec><sec id="sec2.13"><title>Characterization
of Ebola Glycoprotein-Specific Antibodies</title><p>Flat bottom, Immulon
2HB plates (Fisher Scientific, Pittsburgh,
PA) were coated with purified Ebola virus GP<sub>33-637</sub>ΔTM-HA (3 μg/well) in PBS (pH 7.4) overnight at 4 °C.<sup><xref ref-type="bibr" rid="ref31">31</xref></sup> Heat-inactivated serum samples were diluted
(1:20) in PBS. One hundred microliters of each dilution was added
to antigen-coated plates for 2 h at room temperature. Plates were
washed 4 times and incubated with HRP-conjugated goat anti-mouse IgG,
IgG1, IgG2a, IgG2b, and IgM (1:2,000, Southern Biotechnology Associates,
Birmingham, AL) antibodies in separate wells for 1 h at room temperature.
Plates were washed, and substrate solution was added to each well.
Optical densities were read at 450 nm on a microplate reader (Tecan
USA, Research Triangle Park, NC).</p></sec><sec id="sec2.14"><title>Adenovirus Neutralizing
Assay</title><p>Heat-inactivated serum
was diluted in 2-fold increments starting from a 1:20 dilution. Each
dilution was incubated with AdlacZ (1 × 10<sup>6</sup> pfu) for
1 h at 37 °C and applied to HeLa cells (ATCC# CCL-2) seeded in
96 well plates (1 × 10<sup>4</sup> cells/well). After this time,
100 μL of DMEM supplemented with 20% FBS was added to each well.
Twenty-four hours later, beta-galactosidase expression was measured
by histochemical staining. Dilutions that reduced transgene expression
by 50% were calculated using the method of Reed and Muench.<sup><xref ref-type="bibr" rid="ref32">32</xref></sup> The absence of neutralization in samples containing
medium only (negative control) and FBS (serum control) and an average
titer of 1:1,280 ± 210 read from an internal positive control
stock serum were the criteria for qualification of each assay.</p></sec><sec id="sec2.15"><title>Statistical
Analysis</title><p>Data were analyzed for statistical
significance using SigmaStat (Systat Software Inc., San Jose, CA)
by performing a one-way analysis of variance (ANOVA) between control
and experimental groups, followed by a Bonferoni/Dunn post hoc test
when appropriate. Differences in the raw values among treatment groups
were considered statistically significant when <italic>p</italic> <
0.05.</p></sec></sec><sec sec-type="results" id="sec3"><title>Results</title><p>One of the primary objectives of our laboratory
is to develop a
needle-free adenovirus based vaccine against Ebola. To achieve this
goal, a variety of novel formulations were identified, prepared, and
evaluated for their ability to maintain or improve transduction efficiency
of the adenovirus with minimal cytotoxicity in Calu-3 cells, an <italic>in vitro</italic> model of the human respiratory epithelium.<sup><xref ref-type="bibr" rid="ref23">23</xref></sup> Over 400 formulations were assessed using a
recombinant adenovirus expressing <italic>E. coli</italic> beta-galactosidase
that differed from our vaccine construct only by the transgene cassette.
This virus was chosen for these studies because it was available in
sufficient quantity to support our high-throughput screening approach
and for the ease by which the beta-galactosidase transgene product
could be visualized and quantitated <italic>in vitro</italic> and <italic>in vivo</italic>. Data summarized here illustrate our heuristic approach
where the number of formulation candidates tested <italic>in vitro</italic> is significantly reduced prior to the first <italic>in vivo</italic> screen for transduction efficiency and safety and further reduced
to a select few for characterization of the immune response and subsequent
evaluation of protection from lethal exposure to rodent-adapted Ebola.</p><sec id="sec3.1"><title><italic>In Vitro</italic> Characterization of Formulated Adenovirus
Preparations</title><p>While many of the formulations included in the
initial screen could maintain the stability of the adenovirus at ambient
temperatures (data not shown), very few improved the <italic>in vitro</italic> transduction efficiency of the virus above that seen from virus
formulated in saline. However, a preparation of 5% w/v sucrose increased
transduction efficiency by a factor of 1.96 with respect to virus
formulated in saline (pH 7.4, Figure <xref rid="fig1" ref-type="fig">1</xref>A). Pluronic
F68 (0.005% w/v), mannitol (1.25% w/v), and melezitose (1.25% w/v)
alone each increased transduction by a factor of 1.78, 1.61, and 1.51,
respectively. Formulations consisting of either raffinose or sorbitol
alone at a 1.25% (w/v) concentration did not significantly enhance
transduction (<italic>p</italic> = 0.06). A multicomponent formulation,
F3, consisting of sucrose (10 mg/mL), mannitol (40 mg/mL), and 1%
v/v poly(ethylene) glycol 3,000, previously found to stabilize the
virus at ambient temperatures for extended periods of time,<sup><xref ref-type="bibr" rid="ref33">33</xref></sup> increased transduction 5-fold (<italic>p</italic> = 0.03). A formulation of 100 μM nDMPS was the second most
efficacious formulation, however, significant cytoxicity (>80%
cell
lysis) was observed in cultures treated with this formulation. Less
than 1% lysis was observed in cells treated with F3, making it sutiable
for further evaluation <italic>in vivo</italic> (data not shown).</p><fig id="fig1" position="float"><label>Figure 1</label><caption><p>Multicomponent
formulations improve adenovirus transduction efficiency
and stabilize virus in PLGA microspheres. (A) Transduction efficiency
of excipients and formulations in differentiated Calu-3 cells. Cell
monolayers were exposed to formulations containing a model recombinant
adenovirus serotype 5 vector expressing beta-galactosidase (AdlacZ)
for 2 h at 37 °C. Transduction efficiency was determined by comparison
of the number of cells expressing the beta-galactosidase transgene
after treatment with formulated virus to the number of beta-galactosidase
positive cells after treatment with virus in saline. Results are reported
as the mean ± standard error of the mean of data generated from
triplicate samples over three separate experiments (<italic>n</italic> = 9 each formulation). PF68, Pluronic F68; nDMPS, <italic>N</italic>-dodecyl-β-<sc>d</sc>-maltopyranoside; F3, formulation containing
sucrose (10 mg/mL), mannitol (40 mg/mL), and 1% (v/v) poly(ethylene)
glycol 3,000. * indicates a significant difference with respect to
unformulated virus. (B) Adenovirus concentration versus time profiles
of supernatants collected from PLGA microspheres stored at 37 °C.
Ten milligrams of microspheres containing AdlacZ was suspended in
0.5 mL of sterile saline immediately after preparation (Immediate
Release) or after storage at room temperature (25 °C) for 7 or
30 days. The number of infectious particles released at each time
point was determined by serial dilution of collected supernatants
and subsequent infection of Calu-3 cells. (C) <italic>In vitro</italic> release profiles of adenovirus from PLGA microspheres stored at
room temperature over time. Release rates for freshly prepared beads
did not significantly differ from those of beads stored at 25 °C
for 7 days. The release rate increased 3-fold after storage for one
month under the same conditions. Results depicted in panels B and
C are reported as the mean ± standard error of the mean of data
generated from triplicate samples collected from six separate experiments.</p></caption><graphic xlink:href="mp-2014-006454_0002" id="gr1" position="float"></graphic></fig><p>In an effort to improve transduction
efficiency in the presence
of anti-adenovirus neutralizing antibodies, a method for encapsulation
of adenovirus with the biocompatible polymer poly(lactic-<italic>co</italic>-glycolic acid) was also developed.<sup><xref ref-type="bibr" rid="ref34">34</xref></sup> Only
preparations that were capable of releasing active virus at concentrations
relevant for immunization after storage at ambient temperatures would
be considered for further <italic>in vivo</italic> testing. Approximately
10 ± 0.43% of the virus particles embedded in the polymer matrix
were rendered uninfectious during processing, leaving the average
virus concentration to be 1.08 ± 0.5 × 10<sup>9</sup> infectious
virus particles (ivp) per milligram of microspheres. Infectious virus
particles were released for 14 days, after which virus could no longer
be detected (Figure <xref rid="fig1" ref-type="fig">1</xref>B). Approximately 50%
of the total amount of infectious virus embedded in freshly made polymer
beads and in beads stored at 25 °C for 7 days was released within
24 h (Figure <xref rid="fig1" ref-type="fig">1</xref>C). Storage of the beads at room
temperature for 7 days did not significantly impact release rate as
1.34 × 10<sup>8</sup> ivp were released per day from freshly
prepared beads and 1.76 × 10<sup>8</sup> ivp released per day
after this time. Storage at room temperature for one month increased
the rate of release to 3.5 × 10<sup>8</sup> ivp/day and promoted
release of the entire dose (97.95%) within 48 h (Figure <xref rid="fig1" ref-type="fig">1</xref>C).</p></sec><sec id="sec3.2"><title><italic>In Vivo</italic> Transduction Efficiency
of Formulated
Adenovirus Preparations in Naive Mice and Those with Pre-Existing
Immunity</title><p>Based upon their ability to improve transduction
efficiency and maintain virus stability, the F3 formulation and PLGA
microspheres were selected for further evaluation of their effect
on the transduction efficiency of adenovirus in naive mice and those
with pre-existing immunity (PEI) to adenovirus. PEGylated virus was
also selected for evaluation as a vaccine platform since we have previously
shown that covalent attachment of poly(ethylene) glycol to the virus
capsid improves the transduction efficiency in animals that have been
exposed to adenovirus.<sup><xref ref-type="bibr" rid="ref35">35</xref></sup> Intranasal administration
of AdlacZ, the model virus used for <italic>in vitro</italic> screening
studies summarized in Figure <xref rid="fig1" ref-type="fig">1</xref>, in each respective
formulation resulted in high levels of transgene expression in epithelial
cells of the conducting airways of naive mice 4 days after treatment
(Figure <xref rid="fig2" ref-type="fig">2</xref>, panels A–D). More importantly,
each formulation significantly improved transgene expression in mice
with pre-existing immunity to adenovirus (Figure <xref rid="fig2" ref-type="fig">2</xref>, panels F–H) with respect to unformulated virus (Figure <xref rid="fig2" ref-type="fig">2</xref>, panel E).</p><fig id="fig2" position="float"><label>Figure 2</label><caption><p>Formulations improve adenovirus transduction
efficiency in the
lungs of naive mice and those with prior exposure to adenovirus. Naive
C57BL/6 mice were given 5 × 10<sup>10</sup> particles of the
model recombinant virus used for <italic>in vitro</italic> screening
of formulations (AdlacZ) suspended in potassium phosphate buffered
saline (panel A), in formulation F3 (panel B), PEGylated virus (panel
C), or 4.6 mg of PLGA microspheres containing the same dose of virus
(panel D) by the intranasal route. A second set of mice were divided
into the same treatment groups 28 days after receiving a dose of 5
× 10<sup>10</sup> particles of AdNull, an E1/E3 deleted recombinant
adenovirus serotype 5 virus similar to the AdlacZ vector which does
not contain a transgene cassette (panels E–H). Mice in each
group were sacrificed 4 days after administration of the AdlacZ vector.
Images display representative gene expression patterns for 6 mice
per treatment group. Magnification in each panel: 200×.</p></caption><graphic xlink:href="mp-2014-006454_0003" id="gr2" position="float"></graphic></fig></sec><sec id="sec3.3"><title>The T Cell Response: Magnitude</title><p>Since the transduction
efficiency data in mice with pre-existing immunity to adenovirus looked
promising for each formulation, the immune response elicited by each
formulation was evaluated in B10.Br mice with the Ad-CAGoptZGP vector
as described previously.<sup><xref ref-type="bibr" rid="ref13">13</xref>,<xref ref-type="bibr" rid="ref14">14</xref>,<xref ref-type="bibr" rid="ref24">24</xref></sup> The systemic antigen-specific T cell response generated by each
formulation candidate was evaluated by quantitation of IFN-γ
secreting mononuclear cells (MNCs) in the spleen by ELISpot. There
was no significant difference in the amount of antigen-specific cells
present in samples obtained from naive animals immunized with PEGylated,
PLGA encapsulated, or unformulated virus (<italic>p</italic> >
0.05,
Figure <xref rid="fig3" ref-type="fig">3</xref>A). Samples from mice immunized with
the F3 formulation contained slightly more antigen-specific cells
than those from mice given unformulated vaccine (486.7 ± 4.8
spot-forming cells (SFCs)/million MNCs, F3, vs 414.7 ± 27.6 SFCs/million
MNCs, unformulated). In contrast to what was observed in naive animals,
PEI significantly decreased the number of activated IFN-γ secreting
MNCs in the spleens of animals given each preparation except in those
given the PEGylated vaccine (317.3 ± 58.2 spot-forming cells
(SFCs)/million mononuclear cells (MNCs), naive, vs 234.7 ± 54.3
SFCs/million MNCs, PEI, Figure <xref rid="fig3" ref-type="fig">3</xref>A). The most
significant reduction in IFN-γ secreting MNCs was observed in
animals given the microsphere preparation (426.7 ± 33.8 SFCs/million
MNCs, naive, vs 57.3 ± 7.1 SFCs/million MNCs, PEI). Pre-existing
immunity also significantly reduced the number of IFN-γ secreting
cells recovered from bronchoalveolar lavage (BAL) fluid in mice given
unformulated vaccine (1,513.3 ± 63.6 SFCs/million MNCs, naive,
vs 526.7 ± 98.2 SFCs/million MNCs, PEI, <italic>p</italic> <
0.01, Figure <xref rid="fig3" ref-type="fig">3</xref>B). This response was not compromised
in animals given the PEGylated and PLGA encapsulated vaccines (PEG,
2,666.7 ± 54.6 SFCs/million MNCs, naive, vs 580 ± 61.1 SFCs/million
MNCs, PEI; PLGA, 1,280 ± 90.2 SFCs/million MNCs, naive, vs 1,360
± 231.8 SFCs/million MNCs, PEI).</p><fig id="fig3" position="float"><label>Figure 3</label><caption><p>Formulated preparations maintain antigen
specific polyfunctional
T cell responses in naive mice and those with prior exposure to adenovirus.
Characterization of the immune response to Ebola glycoprotein was
performed in B10.Br mice as described previously.<sup><xref ref-type="bibr" rid="ref12">12</xref>−<xref ref-type="bibr" rid="ref14">14</xref>,<xref ref-type="bibr" rid="ref24">24</xref></sup> (A) Magnitude of the systemic CD8<sup>+</sup> T cell
response against Ebola glycoprotein. The number of IFN-γ secreting
mononuclear cells was quantitated in isolates taken 10 days after
immunization from the spleen of naive B10.Br mice and those with prior-exposure
to adenovirus by ELISpot. (B) Magnitude of the mucosal CD8<sup>+</sup> T cell response against Ebola glycoprotein. The number of IFN-γ
secreting mononuclear cells was quantitated 10 days after immunization
in bronchoalveolar lavage (BAL) fluid of naive mice and those with
prior-exposure to adenovirus by ELISpot. (C) Polyfunctionality of
the Ebola glycoprotein-specific T cell response in naive mice. Ten
days after immunization, splenocytes from 5 mice per treatment group
were pooled and stimulated with an Ebola glycoprotein-specific peptide.
Bar graphs illustrate the percentage of CD8<sup>+</sup> tumor necrosis
factor α (TNF-α)-, interleukin 2 (IL-2)-, and interferon
γ (IFN-γ)-producing cells detected after 5 h of antigen
stimulation. Distribution of single-, double-, and triple-cytokine-producing
CD8<sup>+</sup> T cells is shown as various colors in pie chart diagrams.
The relative frequency of cells that produce all three cytokines defines
the quality of the vaccine-induced CD8<sup>+</sup> T cell response.
The proportion of these cells (IFN-γ<sup>+</sup>IL-2<sup>+</sup>TNF-α<sup>+</sup>) generated in response to each treatment
is written in the red section of each pie chart while the proportion
of cells producing a single cytokine are represented by the light
blue, purple, and yellow sections of each pie chart. (D) Polyfunctionality
of the Ebola glycoprotein-specific T cell response in mice with prior
exposure to adenovirus. Pre-existing immunity to adenovirus 5 was
induced by instilling 5 × 10<sup>10</sup> virus particles of
AdNull, an E1/E3 deleted virus that does not contain a transgene cassette,
in the nasal cavity of mice 28 days prior to immunization. Ten days
after immunization, splenocytes were harvested and pooled as described
in panel C. An increase in the number of polyfunctional cells, as
indicated by an increase in the size of the red section of each pie
graph, was fostered by several of the test formulations with respect
to that produced by unformulated vaccine. (E) Quantitative analysis
of the effector memory T cell response. Splenocytes were harvested
42 days after immunization, stained with CFSE, and stimulated with
the TELRTFSI peptide for 5 days. Cells positive for CD8<sup>+</sup>, CD44<sup>HI</sup>, and CD62L<sup>LOW</sup> markers were then evaluated
for CFSE by four-color flow cytometry. Data represent the average
values obtained from three separate experiments each containing 5
mice per treatment. Error bars reflect the standard error of the data.
*<italic>p</italic> < 0.05, **<italic>p</italic> < 0.01, ***<italic>p</italic> < 0.001, one-way ANOVA, Bonferroni/Dunn post hoc analysis.</p></caption><graphic xlink:href="mp-2014-006454_0004" id="gr3" position="float"></graphic></fig></sec><sec id="sec3.4"><title>The T Cell Response: Quality</title><p>Both the quantity and
quality of antigen-specific CD8<sup>+</sup> T cells induced by a vaccine
platform significantly contribute to protection from a variety of
infectious diseases such as AIDS, malaria, and hepatitis C.<sup><xref ref-type="bibr" rid="ref36">36</xref></sup> In animal models of infection, the quality of
the antigen-specific T cell response can be assessed by stimulation
of splenocytes, intracellular staining, and multiparameter flow cytometry
to characterize the diversity of CD8<sup>+</sup> T cell populations
induced after immunization.<sup><xref ref-type="bibr" rid="ref37">37</xref></sup> The presence
of polyfunctional CD8<sup>+</sup> T cells, capable of producing several
cytokines (IFN-γ, IL-2, and TNF-α) in response to the
antigen, has been found to correlate with a reduction in circulating
antigen and viral load since they are known to be the most responsive
cells early in the infection process.<sup><xref ref-type="bibr" rid="ref38">38</xref></sup> Thus, strategies to increase the presence of cells capable of producing
variety of cytokines and chemokines in response to a pathogen are
part of many immunization strategies.<sup><xref ref-type="bibr" rid="ref39">39</xref>,<xref ref-type="bibr" rid="ref40">40</xref></sup> In this context,
functional analysis of cytokine producing CD8<sup>+</sup> T cells
at the single-cell level was performed to determine the ability of
our formulations to improve the quality of the antigen-specific CD8<sup>+</sup> T cell response. As part of this analysis, we were able to
delineate seven distinct cytokine-producing cell populations based
upon IFN-γ, IL-2, and TNF-α secretion patterns.</p><p>As stated above, the relative frequency of cells that produce all
three cytokines defines the quality of the vaccine-induced CD8<sup>+</sup> T cell response. In naive mice, each formulation increased
the number of polyfunctional CD8<sup>+</sup> T cells, with the PLGA
encapsulated vaccine producing the highest amount of these cells (IFN-γ<sup>+</sup>IL-2<sup>+</sup>TNF-α<sup>+</sup>, 37.1 ± 5.04%,
Figure <xref rid="fig3" ref-type="fig">3</xref>C). Prior exposure to adenovirus in
the nasal mucosa reduced the quality of the response generated by
the unformulated vaccine (23.9 ± 3.24%, naive, vs 19.1 ±
6.76%, PEI) and F3 (27.2 ± 4.60%, naive, vs 14.8 ± 2.85%,
PEI) while the response induced by the PEGylated vaccine was not compromised
(24.8 ± 3.69%, naive, vs 26.9 ± 4.74%, PEI, Figure <xref rid="fig3" ref-type="fig">3</xref>D). The polyfunctional response was somewhat strengthened
in mice with prior exposure to adenovirus given the PLGA microspheres
(37.1 ± 5.04%, naive, vs 41.7 ± 7.88%, PEI). This effect
was also seen 42 days after immunization.</p></sec><sec id="sec3.5"><title>The T Cell Response: Memory</title><p>Antigen-specific CD8<sup>+</sup> memory T cells are crucial components
of long-term protection
against viral infections. In order to predict the long-term efficacy
of our formulated vaccines, we evaluated the effector memory CD8<sup>+</sup> T cell response with a CFSE proliferation assay. Forty-two
days after immunization, splenocytes isolated from naive mice given
the unformulated vaccine contained 8.8 ± 1.02% effector memory
CD8<sup>+</sup> T cells capable of proliferating in response to an
Ebola virus glycoprotein-specific MHC I-restricted peptide (Figure <xref rid="fig3" ref-type="fig">3</xref>E). The number of effector memory CD8<sup>+</sup> T cells was lower in samples harvested from animals immunized with
the other formulations. Prior exposure to adenovirus significantly
reduced the memory response in mice given the F3 formulation, PEGylated
vaccine, and unformulated vaccine. The response elicited by PLGA encapsulated
vaccine was suppressed by pre-existing immunity to a lesser degree
than that observed in the other treatment groups (2.32 ± 0.09%
vs 0.85 ± 0.08, F3, vs 0.72 ± 0.04, PEG).</p></sec><sec id="sec3.6"><title>The Anti-Ebola
Virus Antibody Response</title><p>We have previously
found that prior exposure to adenovirus significantly reduced antibody-mediated
immune response to Ebola glycoprotein in mice and guinea pigs.<sup><xref ref-type="bibr" rid="ref14">14</xref></sup> More specifically, we also found that a reduction
in glycoprotein-specific IgG1 antibodies correlated with poor survival
after challenge with rodent-adapted Ebola. Thus, we evaluated total
anti-Ebola glycoprotein-specific immunoglobulin (IgG) and IgG isotypes
in serum to determine if each formulation could counterbalance the
effect of prior mucosal exposure to adenovirus on B cell mediated
immune responses (Figure <xref rid="fig4" ref-type="fig">4</xref>). Each formulation
significantly increased the amount of each antibody isotype specific
for Ebola glycoprotein (GP) in naive mice (Figure <xref rid="fig4" ref-type="fig">4</xref>A). The IgG2a level in mice given PLGA microspheres was the
only deviation from this trend as it was reduced by 29.9% with respect
to unformulated virus (Figure <xref rid="fig4" ref-type="fig">4</xref>A). Prior exposure
to adenovirus significantly reduced each anti-Ebola GP-specific IgG
isotype evaluated (Figure <xref rid="fig4" ref-type="fig">4</xref>B). Although IgG2b
levels in samples collected from mice immunized with the F3 formulation
doubled, IgG1 and IgG2a levels were not significantly different from
those seen in animals given unformulated virus. IgG1 and IgG2b levels
in mice immunized with PLGA microspheres were 9.5 and 1.3 times those
found in samples from mice given unformulated vaccine (Figure <xref rid="fig4" ref-type="fig">4</xref>B). PEI to adenovirus reduced IgG2b levels by 45.9%
in mice given PEGylated vaccine. IgG1 and IgG2a could not be detected
in serum of mice immunized with this preparation. Trace levels of
Ebola GP-specific IgM antibodies were found in serum from mice given
the PLGA and PEGylated preparations.</p><fig id="fig4" position="float"><label>Figure 4</label><caption><p>Formulated vaccines improve the anti-Ebola
glycoprotein antibody
response. Serum collected from individual mice 42 days after immunization
was screened for total IgG and IgG isotypes by ELISA. (A) Antibody
profile for naive mice. Naive B10.Br mice were given 1 × 10<sup>8</sup> particles of Ad-CAGoptZGP suspended in formulation or 4.6
mg of PLGA microspheres containing the virus in KPBS by the intranasal
route. (B) Antibody profile for mice with pre-existing immunity to
adenovirus. Pre-existing immunity was established by instillation
of a dose of 5 × 10<sup>10</sup> particles of AdNull in the nasal
passages of B10.Br mice 28 days prior to immunization with formulated
vaccines. In both panels, the average optical density read from samples
obtained from each treatment group are presented to serve as a measure
of relative antibody concentration and data reported as average values
± the standard error of the mean obtained from three separate
experiments each containing 5 mice per treatment. In each panel, the
asterisk indicates a significant difference with respect to naive,
immunized animals. *<italic>p</italic> < 0.05, **<italic>p</italic> < 0.01, ***<italic>p</italic> < 0.001, one-way ANOVA, Bonferroni/Dunn
post hoc analysis.</p></caption><graphic xlink:href="mp-2014-006454_0005" id="gr4" position="float"></graphic></fig></sec><sec id="sec3.7"><title>Survival From Lethal Challenge</title><p>A marked reduction in
the quality of the T cell response and in Th2 type antibody responses
were found to be indicative of poor protection against lethal infection
with Ebola virus in animals with PEI to adenovirus.<sup><xref ref-type="bibr" rid="ref14">14</xref></sup> Using this criteria, we decided that mice immunized with
vaccine in formulation F3 would not be subject to challenge with a
lethal dose of a mouse-adapted variant of Ebola (MA-EBOV) since neither
facet of the immune response was notably improved by the formulation
in mice with PEI to adenovirus. All of the naive mice given unformulated
vaccine and the PLGA microsphere preparation survived lethal challenge
with MA-EBOV (1,000 pfu ≃ 30,000 × LD<sub>50</sub>, Figure <xref rid="fig5" ref-type="fig">5</xref>A). Twenty-five percent of naive mice given the
PEGylated vaccine succumbed to infection. Sixty percent of the animals
with PEI to adenovirus that were immunized with unformulated vaccine
survived challenge. Eighty percent of mice with prior exposure to
adenovirus that were immunized with the PEGylated preparation did
not survive challenge. This group also demonstrated the most notable
drop in body weight during the course of infection (Figure <xref rid="fig5" ref-type="fig">5</xref>B). Samples taken from this group also revealed
sharp elevations in ALT (842 ± 342 U/L) and AST (602 ± 298
U/L), indicative of severe liver damage from infection (Figure <xref rid="fig5" ref-type="fig">5</xref>C). The PLGA microsphere preparation protected 80%
of the mice with PEI to adenovirus from challenge. Serum ALT (195
± 7.25 U/L) and AST (232 ± 10.1 U/L) levels were significantly
lower in this treatment group with respect to those from animals given
only saline (ALT, 1,913.6 ± 228.6 U/L; AST, 2,152 ± 394.77
U/L) for which the challenge was uniformly lethal and from mice with
PEI given unformulated vaccine (ALT, 879 ± 197 U/L; AST, 898
± 241 U/L, <italic>p</italic> < 0.01).</p><fig id="fig5" position="float"><label>Figure 5</label><caption><p>Formulations that augment
both the polyfunctional T cell response
and antigen-specific IgG1 antibody levels in mice with prior exposure
to adenovirus improve survival from lethal challenge. Naive B10.Br
mice were given 1 × 10<sup>8</sup> particles of Ad-CAGoptZGP
suspended in formulation or 4.6 mg of PLGA microspheres containing
the virus in KPBS by the intranasal route. Pre-existing immunity (PEI)
was established by instillation of a dose of 5 × 10<sup>10</sup> particles of AdNull in the nasal passages of B10.Br mice 28 days
prior to immunization. Twenty-eight days after immunization, mice
(<italic>n</italic> = 10/group) were challenged with a lethal dose
of 1,000 pfu mouse-adapted Ebola (30,000 × LD<sub>50</sub>) by
intraperitoneal injection. (A) Kaplan–Meier survival curve.
* indicates a significant difference with respect to the PEI/unformulated
treatment group. (B) Body weight profile after challenge. No significant
changes in body weight were noted in animals that survived challenge.
The most significant drop in weight (∼15% reduction) was observed
in animals with prior exposure to adenovirus immunized with the PEGylated
preparation. (C) Serum alanine (ALT) and aspartate (AST) aminotransferase
levels postchallenge. Samples from nonsurvivors were taken at time
of death. Samples from survivors were taken 14 days postchallenge.
In all panels, *<italic>p</italic> < 0.05, **<italic>p</italic> < 0.01, ***<italic>p</italic> < 0.001, one-way ANOVA, Bonferroni/Dunn
post hoc analysis.</p></caption><graphic xlink:href="mp-2014-006454_0006" id="gr5" position="float"></graphic></fig></sec><sec id="sec3.8"><title>An <italic>In Vitro</italic> Assay for Quantitative Evaluation
of Transduction Efficiency of Formulated Virus in the Presence of
Neutralizing Antibodies</title><p>Because the PLGA and PEGylated preparations
did not fully protect mice with PEI to adenovirus from lethal challenge,
a secondary effort to identify formulations to improve survival was
initiated. Based upon our initial results with the maltoside, nDMPS,
we sought to identify compounds with similar properties but reduced
toxicity profiles for further testing. Evaluation of transduction
efficiency in the presence of neutralizing antibody was also included
as a more stringent test to predict <italic>in vivo</italic> performance
of formulation candidates. Three different amphiphols, differing only
in the length of carbon chain in the hydrophobic region of the molecule,
were first evaluated for their ability to preserve the transduction
efficiency of the model AdlacZ vector in Calu-3 cells in the presence
of neutralizing antibodies. Transduction efficiency of the virus in
a formulation of 10 mg/mL of poly(maleic anhydride-<italic>alt</italic>-1-decene) substituted with 3-(dimethylamino)propylamine (referred
to as F8) was reduced from 2.58 ± 0.03 × 10<sup>7</sup> to
1.94 ± 0.14 × 10<sup>7</sup> ivp/mL when the anti-adenovirus
5 antibody concentration in the infection media increased from 0.5
ND<sub>50</sub> to 5 ND<sub>50</sub> (Figure <xref rid="fig6" ref-type="fig">6</xref>A). Virus formulated with 10 mg/mL poly(maleic anhydride-<italic>alt</italic>-1-tetradecene) substituted with 3-(dimethylamino)propylamine
(F12) experienced the most significant drop in transduction efficiency
when antibody concentration was increased from 0.5 ND<sub>50</sub> to 5 ND<sub>50</sub> (74% reduction, 1.64 ± 0.18 × 10<sup>7</sup> (0.5 ND<sub>50</sub>), to 4.28 ± 0.48 × 10<sup>6</sup> (5 ND<sub>50</sub>) lfu/mL). Transduction efficiency of the
virus formulated with 10 mg/mL poly(maleic anhydride-<italic>alt</italic>-1-octadecene) substituted with 3-(dimethylamino)propylamine (F16)
in the presence of 5 ND<sub>50</sub> neutralizing antibody was not
significantly different from that in the presence of the 0.5 ND<sub>50</sub> concentration (<italic>p</italic> = 0.08, Figure <xref rid="fig6" ref-type="fig">6</xref>A). This compound also had a very favorable toxicity
profile as formulations of 1 and 10 mg/mL were cytotoxic to only 1.9
± 0.47 and 1.8 ± 0.61% of the Calu-3 cell population respectively
(Figure <xref rid="fig6" ref-type="fig">6</xref>B). Increasing the concentration to
five times that of the effective concentration (50 mg/mL) was still
well tolerated by the Calu-3 cell monolayer with 3.63 ± 0.35%
lysis noted.</p><fig id="fig6" position="float"><label>Figure 6</label><caption><p>Poly(maleic anhydrides): amphiphilic compounds that improve
adenovirus
transduction efficiency with minimal toxicity. A series of zwitterionic
polymers of varying size were screened for their ability to improve
the transduction efficiency of recombinant adenoviruses in lung epithelial
cells. Initial screening of formulations <italic>in vitro</italic> and <italic>in vivo</italic> was performed with AdlacZ containing
the beta-galactosidase transgene (panels A and D). Use of an E1/E3
deleted recombinant adenovirus expressing green fluorescent protein
(AdGFP) and quantitation of infected cells by flow cytometry enhanced
sensitivity of the screening assay so that subtle differences in transduction
efficiency in the presence of anti-adenovirus neutralizing antibodies
could be detected (panel C). (A) Transduction efficiency of formulated
AdlacZ in the presence of neutralizing antibody. Formulations containing
1 × 10<sup>8</sup> infectious particles of AdlacZ were incubated
with aliquots of a highly characterized neutralizing antibody stock
for 1 h prior to infection of Calu-3 cells. Forty-eight hours later,
beta-galactosidase positive cells were identified by histochemical
staining. The number of infectious virus particles was tallied and
calculated as described previously.<sup><xref ref-type="bibr" rid="ref18">18</xref></sup> (B)
Toxicity profile of F16. Formulations were placed on differentiated
Calu-3 cell monolayers for a period of 2 h. Culture medium was then
assessed for LDH activity. Lysis buffer served as a positive control
(100% lysis) and KPBS as a negative control. (C) Quantitative assessment
of transduction efficiency of formulated AdGFP over a range of neutralizing
antibody concentrations. In this experiment, 1 × 10<sup>8</sup> infectious particles of AdGFP were incubated in solution containing
concentrations of anti-adenovirus antibody reflective of that found
in the global population<sup><xref ref-type="bibr" rid="ref28">28</xref>,<xref ref-type="bibr" rid="ref41">41</xref></sup> as described in panel
A. Twenty-four hours after infection, infected cells, positive for
GFP, were counted by flow cytometery. (D) Histological evaluation
of transgene expression in the lung 4 days after intranasal administration
of formulated virus. A single dose of 5 × 10<sup>10</sup> infectious
particles of AdlacZ was given to naive mice or mice with PEI to adenovirus
induced by the intranasal route. Four days later, mice were sacrificed,
and tissue was harvested and stained for transgene expression. Sections
illustrate representative transgene expression patterns found in tissue
collected from 6 animals per treatment. Magnification for unformulated
panels: 200×. Magnification for F16 panels: 400×. Results
in panels A–C are reported as the mean ± standard error
of the mean of data generated from triplicate samples collected from
four separate experiments.</p></caption><graphic xlink:href="mp-2014-006454_0007" id="gr6" position="float"></graphic></fig><p>Before the vaccine formulated with the F16 preparation was
tested <italic>in vivo</italic>, it underwent an additional round
of screening to
confirm that transduction efficiency was adequately improved in the
presence of neutralizing antibody. In order to increase the sensitivity
of this assay and make it a better predictor of <italic>in vivo</italic> performance, we decided to incorporate a model a recombinant adenovirus
5 vector expressing green fluorescent protein (AdGFP) into our test
formulations and evaluate transduction efficiency by fluorescence-activated
cell sorting (FACS). In order to generate data that was clinically
relevant, the virus was incubated with five different solutions containing
a series of neutralizing antibody concentrations spanning those found
in the general population.<sup><xref ref-type="bibr" rid="ref28">28</xref>,<xref ref-type="bibr" rid="ref41">41</xref></sup> Cells infected with
the virus were quantitated by flow cytometry 48 h after infection.
While 8.58% of the monolayer infected with unformulated virus expressed
the transgene, the F16 formulation increased transduction efficiency
to 38.8% (Figure <xref rid="fig6" ref-type="fig">6</xref>C). This assay allowed us
to see significant differences in transduction efficiency of the formulated
virus in the presence of the 0.5 ND<sub>50</sub> and 5 ND<sub>50</sub> antibody concentrations that were not detected by the infectious
titer assay (3.44%, 0.5 ND<sub>50</sub>, vs 34.4%, 5 ND<sub>50</sub>). Although the formulation improved transduction efficiency of the
virus over a wide range of antibody concentrations, the limit of this
improvement was reached at the 50 ND<sub>50</sub> concentration, where
the formulation could no longer protect the virus from neutralization.</p><p>Adenovirus formulated with the F16 compound was well tolerated
in naive animals and those with PEI to adenovirus. Almost every epithelial
cell in both large and small airways was transduced by virus formulated
with F16 (Figure <xref rid="fig6" ref-type="fig">6</xref>D). Highly concentrated areas
of transgene expression were also found in small airways of mice with
circulating neutralizing antibody levels of 262 ± 43 reciprocal
dilution given virus in this formulation. In contrast, PEI to adenovirus
prevented transgene expression in both large and small airways of
mice given unformulated virus. Serum transaminases, standard indicators
of adenovirus toxicity,<sup><xref ref-type="bibr" rid="ref42">42</xref></sup> in both naive
animals and those with PEI to adenovirus immunized with the F16 formulation
were reduced by 40% with respect to similar treatment groups given
unformulated vaccine (data not shown).</p></sec><sec id="sec3.9"><title>The Immune Response Generated
by Formulation F16 in Mice with
Pre-Existing Immunity</title><p>Because prior formulation candidates
did not fully confer protection in mice in which PEI was established
through the nasal mucosa, evaluation of the F16 formulation <italic>in vivo</italic> focused solely on the ability of this formulation
to improve the immune response to the encoded Ebola glycoprotein under
these specific conditions. As seen in prior studies, PEI significantly
compromised the production of GP-specific IFN-γ-secreting mononuclear
cells isolated from spleen (Figure <xref rid="fig7" ref-type="fig">7</xref>A) and
BAL fluid (Figure <xref rid="fig7" ref-type="fig">7</xref>B) in animals given unformulated
vaccine (<italic>p</italic> < 0.01). PEI induced by the mucosal
route also significantly reduced the frequency of GP-specific multifunctional
CD8<sup>+</sup> T cells elicited by the unformulated vaccine (naive,
64.9 ± 4.88%, vs IN PEI/unformulated, 48.6 ± 3.66%, <italic>p</italic> < 0.05; Figure <xref rid="fig7" ref-type="fig">7</xref>C).</p><fig id="fig7" position="float"><label>Figure 7</label><caption><p>Formulation
F16 improves quantitative and qualitative Ebola glycoprotein-specific
CD8<sup><bold>+</bold></sup> T cell responses in mice with prior exposure
to adenovirus. PEI to adenovirus 5 was induced by instilling 5 ×
10<sup>10</sup> virus particles of AdNull, an E1/E3 deleted virus
that does not contain a transgene cassette, in the nasal cavity of
mice 28 days prior to immunization. (A) The systemic effector CD8<sup><bold>+</bold></sup> T cell response. Ten days after vaccination,
mononuclear cells from the spleen were harvested and stimulated with
an Ebola GP-specific peptide and responsive cells were quantitated
by ELISpot. (B) The mucosal effector CD8<sup><bold>+</bold></sup> T
cell response. Ten days after vaccination, mononuclear cells collected
from BAL fluid were harvested, pooled according to treatment, and
stimulated with an Ebola GP-specific peptide and responsive cells
were quantitated by ELISpot. (C) The polyfunctional CD8<sup><bold>+</bold></sup> T cell response. Ten days after immunization, splenocytes
from 5 mice per treatment group were pooled and stimulated with an
Ebola glycoprotein-specific peptide. Each positively responding cell
was assigned to one of 7 possible combinations of IFN-γ, IL-2,
and TNF-α production and quantitated as shown in the bar graph.
The most potent responders, those producing all 3 cytokines in response
to stimulation, are depicted by the red arcs in the pie charts. The
proportion of cells in samples from each treatment group that produce
IFN-γ is depicted by the blue arc. The number in each pie chart
denotes the percentage of triple producers found in samples from a
given treatment group. Data reflect average values ± the standard
error of the mean for six mice per group. * indicates a significant
difference with respect to the naive/unformulated group, *<italic>p</italic> < 0.05, **<italic>p</italic> < 0.01, one-way ANOVA,
Bonferroni/Dunn post hoc analysis.</p></caption><graphic xlink:href="mp-2014-006454_0008" id="gr7" position="float"></graphic></fig><p>The F16 formulation improved the immune response in animals
with
PEI to adenovirus as the number of GP-specific, IFN-γ-secreting
mononuclear cells isolated from the spleen of these animals was notably
higher than that from naive animals given unformulated vaccine (2,290
± 51 SFCs/million MNCs, naive, vs 2,840 ± 110 SFCs/million
MNCs, PEI/F16, <italic>p</italic> < 0.05, Figure <xref rid="fig7" ref-type="fig">7</xref>A). The number of antigen-specific IFN-γ-secreting mononuclear
cells isolated from the BAL fluid of animals given the F16 formulation
was not statistically different from that found in naive animals given
unformulated vaccine (<italic>p</italic> = 0.07, Figure <xref rid="fig7" ref-type="fig">7</xref>B). This trend was also observed with respect to the multifunctional
CD8<sup>+</sup> T cell response as it also did not change with respect
to that found in naive animals given unformulated vaccine (naive/unformulated,
64.9 ± 4.88%, vs IN PEI/F16 (10 mg/mL), 60.0 ± 9.1%, <italic>p</italic> = 0.055; Figure <xref rid="fig7" ref-type="fig">7</xref>C). Forty-two
days after immunization, the effector memory CD8<sup>+</sup> T cell
response was also evaluated in mice immunized with unformulated vaccine
or the F16 preparation. The F16 formulation increased the memory response
by a factor of 3.3 from 0.28 ± 0.15% (unformulated) to 0.93 ±
0.25% (F16, data not shown). Serum from animals with pre-existing
immunity to adenovirus that were immunized with the F16 preparation
contained 4 times more anti-Ebola glycoprotein antibodies than that
from animals given unformulated vaccine (Figure <xref rid="fig8" ref-type="fig">8</xref>). Samples from these animals also contained 5 times more of the
IgG1 isotype and notable levels of antigen-specific IgM antibodies.</p><fig id="fig8" position="float"><label>Figure 8</label><caption><p>Formulation
F16 improves the antigen specific antibody response
in mice with prior exposure to adenovirus. The average optical density
read from individual samples obtained from each treatment group are
presented to serve as a measure of relative antibody concentration
and data reported as average values ± the standard error of the
mean obtained from two separate experiments each containing 6 mice
per treatment. The limit of detection for the assay is 0.01 absorbance
unit. **<italic>p</italic> < 0.01, one-way ANOVA, Bonferroni/Dunn
post hoc analysis.</p></caption><graphic xlink:href="mp-2014-006454_0009" id="gr8" position="float"></graphic></fig></sec></sec><sec sec-type="discussion" id="sec4"><title>Discussion</title><p>Development
of effective vaccines against Ebola is a difficult
task due to the lethality of the virus, which makes testing of any
vaccine on humans impossible. In addition, outbreaks remain quite
unpredictable, making identification of a specific high-risk population
to be targeted for a clinical efficacy trial problematic.<sup><xref ref-type="bibr" rid="ref43">43</xref></sup> Therefore, potential vaccine candidates must
be evaluated in animal models of Ebola infection prior to clinical
testing. Data from these models must provide a clear indication of
measurable, predictive markers that strongly correlate with survival,
which can then be evaluated in the absence of exposure in a clinical
setting.<sup><xref ref-type="bibr" rid="ref44">44</xref></sup> To date, a single dose of several
different vaccines derived from recombinant adenovirus serotype 5
that express the Ebola virus glycoprotein has been highly efficacious
in naive animals.<sup><xref ref-type="bibr" rid="ref12">12</xref>,<xref ref-type="bibr" rid="ref45">45</xref>,<xref ref-type="bibr" rid="ref46">46</xref></sup> However, none of these platforms have been completely effective
in animals with PEI to adenovirus.<sup><xref ref-type="bibr" rid="ref47">47</xref>−<xref ref-type="bibr" rid="ref49">49</xref></sup></p><p>We have recently
shown that immunization with an adenovirus 5-based
Ebola vaccine by the same route in which pre-existing immunity is
induced significantly diminishes the antigen-specific multifunctional
CD8<sup>+</sup> T cell response and antibody production and that both
these parameters contribute the protective efficacy of the vaccine.<sup><xref ref-type="bibr" rid="ref14">14</xref></sup> Therefore, the primary objective of these studies
was to develop novel formulations for a nasal vaccine against Ebola
that would be effective in those with PEI to adenovirus by two distinct
mechanisms: physical protection of the virus from neutralization by
anti-adenovirus antibodies and enhancement of antigen expression from
virus that escaped neutralization. Prior work in our laboratory identified
formulations consisting of pharmaceutically acceptable excipients
that enhanced the physical stability of the virus and increased transduction
efficiency to the lung epithelium with minimal toxicity.<sup><xref ref-type="bibr" rid="ref33">33</xref>,<xref ref-type="bibr" rid="ref50">50</xref></sup> One of these formulations, referred to in this manuscript as F3,
significantly improved transduction efficiency of the virus in an <italic>in vitro</italic> model of the airway epithelium and in naive animals
(Figures <xref rid="fig1" ref-type="fig">1</xref> and <xref rid="fig2" ref-type="fig">2</xref>). However,
this effect alone was not sufficient to fortify the antigen-specific
immune response in mice exposed to adenovirus 5 28 days prior to immunization
(Figures <xref rid="fig3" ref-type="fig">3</xref> and <xref rid="fig4" ref-type="fig">4</xref>).</p><p>Nanoparticles made of poly(lactide-<italic>co</italic>-glycolide)
copolymer blends have been evaluated and developed for controlled
release of a variety of therapeutic drugs since the 1980s.<sup><xref ref-type="bibr" rid="ref51">51</xref></sup> While this delivery platform has been utilized
in several licensed products to improve the bioavailability of hydrophobic
drugs,<sup><xref ref-type="bibr" rid="ref52">52</xref></sup> it has yet to be successfully
utilized in a vaccine product.<sup><xref ref-type="bibr" rid="ref53">53</xref></sup> However,
improvements in manufacturing and sterilization processes for PLGA
nanoparticles have allowed several vaccine candidates using this system
to progress to phase I/II and II/III clinical testing.<sup><xref ref-type="bibr" rid="ref54">54</xref></sup> Particle size is one of the most influential
characteristics of the potency of a vaccine preparation as it can
dictate how antigens are processed.<sup><xref ref-type="bibr" rid="ref55">55</xref></sup> Recently,
it was found that immunization with PLGA particles within the 200–600
nm size range stimulated strong Th1 responses with high levels of
circulating IFN-γ while particles within the 2–8 μm
range fostered IL-4 production and associated Th2 responses.<sup><xref ref-type="bibr" rid="ref56">56</xref></sup> This paradigm is supported by our studies as
the average particle size of the encapsulated vaccine preparation
fell within the 2–8 μm range and Th2 responses were predominant
in naive animals and those with PEI to adenovirus (Figure <xref rid="fig4" ref-type="fig">4</xref>). Surprisingly, the encapsulated vaccine was only
partially protective in animals with PEI. This most likely is due
to the significant reduction in the systemic CD8<sup>+</sup> T cell
response resulting from poor distribution of particles from the respiratory
compartment. It is also important to realize that the local antigen-specific
immune response was not compromised by PEI to adenovirus. Thus, full
protection may have been observed if the immunized mice were exposed
to the challenge virus via the respiratory tract. Additional studies
to tailor the size and surface chemistry of the microparticles to
facilitate uptake and processing by migrating antigen presenting cells
are warranted.</p><p>PEGylation has been successfully used to reduce
aggregation, improve
solubility, and dampen the immunogenicity of a variety of licensed
protein-based therapeutics.<sup><xref ref-type="bibr" rid="ref57">57</xref></sup> We developed
a process for PEGylation of recombinant adenoviruses that improved
transgene expression in several animal models of PEI.<sup><xref ref-type="bibr" rid="ref20">20</xref>,<xref ref-type="bibr" rid="ref35">35</xref></sup> Although the immune response against the transgene was not evaluated
in those studies, data summarized in this manuscript suggest that
PEGylation significantly alters the immune response to an encoded
transgene in naive animals and those with PEI to the virus by potentially
different mechanisms. In naive animals, the PEGylated vector generated
qualitative antigen-specific T cell responses that were similar to
those generated by unmodified virus (Figure <xref rid="fig3" ref-type="fig">3</xref>C). The antibody-mediated response was also not impacted by PEGylation
(Figure <xref rid="fig4" ref-type="fig">4</xref>). The quantitative T cell response,
however, was reduced in these animals (Figure <xref rid="fig3" ref-type="fig">3</xref>A). While the PEGylated vector improved the polyfunctional T cell
response in mice with PEI to adenovirus (Figure <xref rid="fig3" ref-type="fig">3</xref>C), IgG1 and IgG2a antibodies against the Ebola virus glycoprotein
were not detected in serum collected from these animals (Figure <xref rid="fig4" ref-type="fig">4</xref>B). Taken together, these findings suggest that
PEG alters the manner by which the virus and the transgene product
are processed by antigen presenting cells.<sup><xref ref-type="bibr" rid="ref58">58</xref>,<xref ref-type="bibr" rid="ref59">59</xref></sup> They also suggest, especially in naive animals, that conjugation
of PEG to the virus capsid delays processing of an encoded antigenic
transgene. This has been observed by others who have found that priming
and boosting doses of a PEGylated vector were necessary to induce
antigen-specific T cell responses that were greater than that achieved
by a single dose of the PEGylated vector or the unmodified virus alone.<sup><xref ref-type="bibr" rid="ref60">60</xref></sup></p><p>Immune responses correlating with protection
from Ebola virus infection
have long been the subject of fervid debate.<sup><xref ref-type="bibr" rid="ref61">61</xref>−<xref ref-type="bibr" rid="ref63">63</xref></sup> Some reports
suggest that the humoral arm of the adaptive immune response is required
for protection,<sup><xref ref-type="bibr" rid="ref48">48</xref>,<xref ref-type="bibr" rid="ref64">64</xref>,<xref ref-type="bibr" rid="ref65">65</xref></sup> while others point to cell mediated immunity as the predominant
protective mechanism.<sup><xref ref-type="bibr" rid="ref17">17</xref>,<xref ref-type="bibr" rid="ref66">66</xref></sup> In the studies summarized here,
we found that when specific IgG antibody subtypes were not produced
against the Ebola glycoprotein in response to the PEGylated vaccine,
survival was poor, with 20% of mice with PEI surviving challenge.
This finding may be useful in refining strategies to utilize combinations
of antibodies to treat Ebola infection.<sup><xref ref-type="bibr" rid="ref67">67</xref></sup> When antibody levels were high and the antigen-specific quantitative
T cell response was suppressed, significant yet only partial protection
from challenge was achieved in naive animals given the PEGylated preparation
(75% survival) and in mice with PEI given the PLGA preparation (80%
survival). Taken together, these results suggest that antigen-specific
antibodies are critical for protection and that the T cell response
supports protection if a certain level of neutralizing antibody is
present. These observations are in line with prior observations in
rodent models of infection<sup><xref ref-type="bibr" rid="ref14">14</xref>,<xref ref-type="bibr" rid="ref63">63</xref></sup> and should be evaluated
in larger models of Ebola hemorrhagic fever that are more reflective
of the human disease.<sup><xref ref-type="bibr" rid="ref44">44</xref></sup></p><p>The immune
response generated by the preparation delineated as
F16 restored the antigen-specific multifunctional CD8<sup>+</sup> T
cell and antibody responses compromised by PEI to adenovirus serotype
5. The primary component in this formulation is an amphiphilic polymer
with alternating hydrophilic and hydrophobic side chains that was
originally developed as an alternative to surfactants for stabilizing
membrane proteins in aqueous solution and delivering biomolecules
across lipid bilayers of the cell membrane.<sup><xref ref-type="bibr" rid="ref68">68</xref>,<xref ref-type="bibr" rid="ref69">69</xref></sup> Unlike detergent-based micelles, this compound assumes a belt-like
structure around the trans-membrane domain of proteins in the cell
membrane. At neutral pH, the overall surface charge of the polymer
is highly positive, allowing it to immobilize negatively charged biomolecules
and promote interaction with the anionic cell surface.<sup><xref ref-type="bibr" rid="ref70">70</xref></sup> Since the adenovirus capsid bears a net negative
charge at neutral pH,<sup><xref ref-type="bibr" rid="ref71">71</xref></sup> we believe that
this compound enhances transduction efficiency of the virus in this
manner. The observation that transduction efficiency of the virus
in the presence of neutralizing antibody is directly influenced by
the length of the hydrophobic region of the compound suggests that
the F16 compound covered the virus in a manner that most effectively
prevented antibody binding. Additional studies to characterize the
interaction between this compound and the virus and how it impacts
uptake and processing by antigen presenting cells are warranted. Evaluation
of this compound in a primate challenge model has recently been described.<sup><xref ref-type="bibr" rid="ref72">72</xref></sup></p><p>Since many pathogens, much like Ebola,
have mastered the art of
outwitting host immune responses,<sup><xref ref-type="bibr" rid="ref73">73</xref>−<xref ref-type="bibr" rid="ref75">75</xref></sup> vaccine candidates must
elicit strong and broad immune responses that are long lasting and
difficult for the pathogen to counteract. While advances in reverse
vaccinology and proteomics have accelerated discovery and production
of antigenic candidates that do not bear the safety concerns associated
with live and attenuated organisms, they cannot, on their own, generate
fully protective immune responses. Thus, identification of delivery
systems that protect the antigen and direct it to specific components
of the immune system and discovery of compounds that stimulate antigen-specific
immune responses are critical to the development of any vaccine program.
This is even more important for preparations that are to be delivered
to the nasal mucosa since many of the known conventional adjuvants
are either toxic or not active when given by the intranasal route.<sup><xref ref-type="bibr" rid="ref7">7</xref>,<xref ref-type="bibr" rid="ref76">76</xref></sup> In the studies summarized here, we have identified a novel, nontoxic
excipient with unique physicochemical properties that substantially
augments the transduction efficiency and immunogenicity of a recombinant
adenovirus serotype 5-based Ebola vaccine in the presence of pre-existing
immunity to the adenovirus vector. Graded, stepwise evaluation of
each formulation <italic>in vitro</italic> and <italic>in vivo</italic> allowed us to confirm that specific components of the immune response
play a role in protection from Ebola. While some of these results
may only apply specifically to the adenovirus vector and Ebola, they
may be informative for improving other vaccine platforms based upon
microbes also routinely encountered in the environment like <italic>Salmonella</italic>, vaccinia, herpes simplex, and human respiratory
syncytial virus.<sup><xref ref-type="bibr" rid="ref77">77</xref>−<xref ref-type="bibr" rid="ref80">80</xref></sup></p></sec></body><back><notes id="notes-1" notes-type="conflict-of-interest"><p>The authors
declare the following competing financial interest(s): As Director
of the Robert E. Shope BSL-4 Laboratory, A.N.F. has participated in
the development of a variety of vaccines and therapeutics for Ebola
virus disease including the vaccine described in this manuscript.
J.H.C., S.C.S., and M.A.C. have no competing interests to declare.</p></notes><ack><title>Acknowledgments</title><p>The authors thank Lihong Zhang, Terry Juelich, and
Jennifer
Smith of the UTMB BSL-4 facility for their help with Ebola challenge
studies. We acknowledge technical assistance of Michael Boquet and
Joe Dekker in early formulation development. We would also like to
extend deep appreciation to Dr. Erica Ollmann Saphire and Marnie Fucso
of The Scripps Research Institute for providing the Ebola GP<sub>33-637</sub>ΔTM-HA plasmid and assistance with glycoprotein purification.
This work was funded by the National Institutes of Health NIAID Grant
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