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<ul><li><p>Hexon Hypervariable Region-Modified Adenovirus Type 5 (Ad5)Vectors Display Reduced Hepatotoxicity but Induce T LymphocytePhenotypes Similar to Ad5 Vectors</p><p>Jeffrey E. Teigler,a Pablo Penaloza-MacMaster,a Rebecca Obeng,a Nicholas M. Provine,a Rafael A. Larocca,a Erica N. Borducchi,a</p><p>Dan H. Baroucha,b</p><p>Center for Virology and Vaccine Research, Beth Israel Deaconess Medical Center, Boston, Massachusetts, USAa; Ragon Institute of MGH, MIT, and Harvard, Boston,Massachusetts, USAb</p><p>Hexon modification of adenovirus type 5 (Ad5) vectors with the hypervariable regions (HVRs) of Ad48 has been shown to allowAd5HVR48 vectors to circumvent the majority of the preexisting Ad5-neutralizing antibodies. However, it remains unclearwhether modifying hexon HVRs impacts innate or adaptive immune responses elicited by this vector. In this study, we investi-gated the influence of the HVR substitution of Ad5 on innate and adaptive immune responses following vaccination. Ad5HVR48displayed an intermediate level of innate immune cytokines and chemokines relative to those of Ad5 and Ad48, consistent withits chimeric nature. Hepatotoxicity was observed after Ad5 immunization but not after Ad5HVR48 or Ad48 immunization.However, the CD8 T-cell responses elicited by Ad5HVR48 vectors displayed a partially exhausted phenotype, as evidenced bythe sustained expression of programmed death 1 (PD-1), decreased effector-to-central memory conversion, and reduced mem-ory recall responses, similar to those elicited by Ad5 vectors and in contrast to those induced by Ad48 vectors. Taken together,these results indicate that although Ad5HVR48 largely bypasses preexisting Ad5 neutralizing antibodies and shows reduced hep-atotoxicity compared to that of Ad5, it induces adaptive immune phenotypes that are functionally exhausted similar to thoseelicited by Ad5.</p><p>Adenovirus (Ad) vectors are widely utilized as platforms forvaccines and gene therapy (13). The utility of certain com-mon Ad serotypes, however, is hampered by prevalent preexistingvector-specific neutralizing antibodies in the global population (4,5). In particular, adenovirus type 5 (Ad5)-based vectors have beenshown to exhibit decreased immunogenicity in the presence ofbaseline Ad5 antibodies (6). Ad5 vectors at high doses also exhibithepatotoxicity as a result of liver tropism due to the binding ofblood coagulation factor X (FX) in the systemic circulation (710). Both of these properties appear to be mediated primarily bythe hexon hypervariable regions (HVRs) (8, 1116). HVRs arehighly variable among Ad serotypes and represent the primarydeterminant of neutralization specificity (1720).</p><p>The role of the HVRs in influencing the immunogenicity andbiological properties of adenoviral vectors has led to the develop-ment of strategies to either circumvent preexisting immunity ordecrease liver tropism and associated hepatotoxicity. These strat-egies include HVR chemical modifications, such as PEGylation,HVR sequence modification by poly-His sequence insertion, orreplacement of HVRs with those from less prevalent Ad serotypes(12, 2126). Our laboratory has described an Ad5 vector with itssurface HVRs replaced with those of Ad48, a chimeric vector re-ferred to as Ad5HVR48 (1921). This vector evades the majorityof preexisting Ad5 neutralizing antibodies and has been evaluatedas a human immunodeficiency virus (HIV) vaccine vector inmice, nonhuman primates, and humans in a recent phase I clinicaltrial (Integrated Preclinical/Clinical AIDS Vaccine Developmentprogram [IPCAVD] 002; D. H. Barouch, unpublished data) (19,21). Furthermore, studies from our laboratory and others havedescribed marked differences in vaccine-elicited innate and adap-tive immune responses following Ad5 or Ad48 immunization (4,27, 28). While Ad5HVR48 has shown a lack of vector-induced</p><p>toxicity in both nonhuman primates (NHP) and humans, a pre-vious report suggested that Ad5HVR48 may exhibit unexpectedhepatotoxic and inflammatory responses in mice that are greaterthan those observed with Ad5 or Ad48 vectors (29). Understand-ing the impact of HVR modifications on the toxicity and innateand adaptive immune phenotypes elicited by Ad vectors is impor-tant for future clinical development of HVR-chimeric Ad vectors.</p><p>In this study, we investigated the innate and adaptive immuneresponses elicited by Ad5HVR48 vectors compared with those ofthe parental vectors Ad5 and Ad48 in mice. We found that theserum cytokine and chemokine responses elicited by Ad5HVR48were higher than those elicited by Ad5 but lower than those elic-ited by Ad48, consistent with its chimeric nature. NeitherAd5HVR48 nor Ad48 vectors induced hepatotoxicity, as mea-sured by liver enzymes and histopathology. The CD8 T lympho-cytes elicited by Ad5HVR48 exhibited a partially exhausted phe-notype similar to those induced by Ad5 vectors and characterizedby a high surface expression of programmed death 1 (PD-1) andlow production of gamma interferon (IFN-) and interleukin 2(IL-2). Taken together, these data show that hexon modification</p><p>Received 9 April 2014 Returned for modification 30 April 2014Accepted 4 June 2014</p><p>Published ahead of print 18 June 2014</p><p>Editor: S. A. Plotkin</p><p>Address correspondence to Dan H. Barouch, dbarouch@bidmc.harvard.edu.</p><p>J.E.T. and P.P.-M. contributed equally to this work.</p><p>Copyright 2014, American Society for Microbiology. All Rights Reserved.</p><p>doi:10.1128/CVI.00207-14</p><p>The authors have paid a fee to allow immediate free access to this article.</p><p>August 2014 Volume 21 Number 8 Clinical and Vaccine Immunology p. 11371144 cvi.asm.org 1137</p><p> on Septem</p><p>ber 17, 2018 by guesthttp://cvi.asm</p><p>.org/D</p><p>ownloaded from</p><p>http://dx.doi.org/10.1128/CVI.00207-14http://cvi.asm.orghttp://cvi.asm.org/</p></li><li><p>of Ad5, while enabling the chimeric Ad5HVR48 vector to circum-vent the majority of preexisting neutralizing antibodies, resultedin innate immune profiles intermediate between Ad5 and Ad48vectors but did not detectably improve the cellular adaptive im-mune phenotype compared with those from Ad5 vectors.</p><p>MATERIALS AND METHODSAnimals and viruses. Replication-incompetent E1/E3-deleted Ad5,Ad5HVR48, and Ad48 were generated as previously described (4, 21).Viruses were grown in E1-complementing PER.55K cells and purified ona CsCl gradient, and they exhibited similar viral particle-to-PFU (vp/PFU) ratios. C57BL/6 and MF1 mice were purchased from Charles RiverLaboratories (Wilmington, MA). For measuring the adaptive immune re-sponses vectors expressing a simian immunodeficiency virus (SIVmac239),the Gag transgene were used. The mice were injected intravenously with3 1010 or 1 1011 viral particles of the vectors indicated. All studiesinvolving animals were approved by the Beth Israel Deaconess MedicalCenter Institutional Animal Care and Use Committee (IACUC).</p><p>Serum cytokine and transaminase level determination. Serum sam-ples were collected 6 h and 48 h after vaccination. For the cytokine andchemokine assays, the sera were thawed on ice prior to inactivation with0.05% Tween for 15 min at room temperature and then utilized on aMilliplex MAP mouse magnetic cytokine and chemokine panel, accord-</p><p>ing to the manufacturers instructions (Millipore, Billerica, MA) (27). TheLuminex data were analyzed on the Bio-Plex 200 instrument runningBio-Plex Manager 4.1 (Bio-Rad, Hercules, CA) with an 80% to 120%standard acceptance range. For serum aminotransferase level determina-tion, the sera were thawed on ice and then measured for aspartate amino-transferase (AST) and alanine aminotransferase (ALT) activity by stan-dard methods at the Beth Israel Deaconess Medical Center Small AnimalImaging Facility (SAIF).</p><p>Microscopy. The livers were harvested from mice 48 h following vac-cination and processed for paraffin embedding and hematoxylin and eo-sin staining according to standard methods developed by the Dana-Far-ber/Harvard Cancer Center Rodent Histopathology Core at HarvardMedical School. The slides were imaged on a Zeiss AxioImager M1 run-ning AxioVision version 4.6.3.0 (Carl Zeiss GmbH, Jena, Germany) at theBeth Israel Deaconess Medical Center Imaging Core.</p><p>Antibodies and flow cytometry. Single-cell suspensions were stainedat 4C for 30 min. The following antibody clones were used: CD8 (53-6.7), CD127 (A7R34), CD62L (MEL-14), PD-1 (RMP1-30), gamma in-terferon (IFN-) (XMG1.2), and interleukin 2 (IL-2) (JES6-5H4). Allantibodies were purchased from BD Pharmingen, except for PD-1 (Bio-Legend). Biotinylated major histocompatibility complex I (MHC-I)H-2Db monomers loaded with the previously described immunodomi-nant AL11 peptide (AAVKNWMTQTL) (30) were obtained from the</p><p>FIG 1 Serum cytokines and chemokines of mice after Ad5, Ad5HVR48, or Ad48 vector administration. Mice (C57BL/6 or MF1) were injected intravenously with3 1010 (n 4/group) or 1 1011 (n 3/group) vp Ad vectors. Serum samples were collected 6 h postvaccination, and systemic cytokine and chemokine levelswere measured by Luminex analyses. Data are shown as means standard errors of the mean (SEM). The horizontal lines at the top indicate P values of 0.05using Students t tests.</p><p>Teigler et al.</p><p>1138 cvi.asm.org Clinical and Vaccine Immunology</p><p> on Septem</p><p>ber 17, 2018 by guesthttp://cvi.asm</p><p>.org/D</p><p>ownloaded from</p><p>http://cvi.asm.orghttp://cvi.asm.org/</p></li><li><p>NIH Tetramer Core Facility from Emory University. Intracellular cyto-kine staining was used for measuring the functionality of Gag-specificCD8 T-cell responses using the AL11 peptide. Intracellular cytokinestaining was performed with the Cytofix/Cytoperm kit (BD Biosciences)</p><p>(31). The fixed cells were acquired using an LSR II flow cytometer (BDBiosciences) and analyzed using FlowJo (TreeStar).</p><p>Data analysis and statistical methods. The concentrations of cyto-kines and chemokines were obtained from Luminex assays using a 5-pa-rameter logistic model. For all statistics shown, P values were calculatedusing unpaired two-tailed Students t tests with no multiple-comparisoncorrections. All statistics were calculated in GraphPad Prism v5.0 (Graph-Pad Software, Inc., La Jolla, CA).</p><p>RESULTSAd5HVR48 displays an intermediate innate stimulatory pheno-type compared to those of parental Ad5 and Ad48 vectors. Weinitiated studies by assessing the systemic cytokine and chemokineresponses in two strains of mice after vaccination, inbred C57BL/6mice that had been extensively utilized for characterizing Ad vec-tor immunology, and outbred MF1 mice that had been used inprevious studies assessing Ad vector toxicity (4, 8, 12, 14, 19, 21).The mice (C57BL/6 or MF1; n 3 to 4/group) were inoculatedintravenously (i.v.) with 3 1010 or 1 1011 viral particles (vp) ofAd5, Ad5HVR48, or Ad48 vectors, consistent with procedures inprior studies (29). Serum samples were collected 6 h after vacci-nation, and systemic levels of cytokines and chemokines were as-sessed by Luminex analyses. A comparison of the systemic cyto-kine and chemokine levels revealed a greater induction of manycytokines and chemokines in mice vaccinated with Ad48 than inthose vaccinated with Ad5, including gamma interferon-inducedprotein 10 (IP-10), interleukin 1 beta (IL-1), IL-6, IL-12(p70),tumor necrosis factor alpha (TNF-), and monokine induced bygamma interferon (MIG) (Fig. 1). These differences were ob-served in both C57BL/6 strain and MF1 mice, although an overalllower cytokine induction was observed in MF1 mice. Ad5HVR48displayed an intermediate cytokine and chemokine elicitation</p><p>FIG 2 Serum AST and ALT levels in mice after Ad5, Ad5HVR48, or Ad48vector administration. Mice (C57BL/6 or MF1) were injected intravenouslywith 3 1010 (n 4/group) or 1 1011 (n 3/group) vp Ad vectors. Serumsamples were collected 48 h postvaccination, and systemic AST and ALT levelswere measured. Data are shown as means SEM.</p><p>FIG 3 Ad5 and Ad5HVR48 are highly immunogenic but result in generation of partially exhausted CD8 T cells. Mice (C57BL/6; n 8/group) were injectedintramuscularly with 3 1010 vp of Ad5, Ad5HVR48, or Ad48 expressing SIV Gag. Each mouse was sacrificed 60 days postimmunization, and memory CD8</p><p>T-cell responses were assessed by flow cytometry. (A) Representative fluorescence-activated cell sorter (FACS) plots showing the percentage of PD-1 expressingAL11-specific CD8 T cells in tissues. (B) Absolute numbers of Gag-specific CD8 T cells (DbAL11) in tissues. (C) Mean fluorescence intensity indicating PD-1expression by AL11-specific CD8 T cells in tissues. Data are shown as means SEM. The horizontal lines at the top indicate P values of 0.05 using Studentst tests.</p><p>Hexon-Modified Adenovirus Type 5 Vectors</p><p>August 2014 Volume 21 Number 8 cvi.asm.org 1139</p><p> on Septem</p><p>ber 17, 2018 by guesthttp://cvi.asm</p><p>.org/D</p><p>ownloaded from</p><p>http://cvi.asm.orghttp://cvi.asm.org/</p></li><li><p>profile (e.g., IP-10, IL-1, IL-13, IL-12[p70], and TNF-) com-pared with those of Ad5 and Ad48 vectors in both strains of mice(Fig. 1), although it more closely resembled the profile elicited byAd5 vectors.</p><p>Ad5HVR48 vectors display reduced hepatotoxicity relativeto those of Ad5 vectors. We next sought to assess the potentialimpact of HVR modification on Ad5-induced hepatotoxicity.Mice (C57BL/6 or MF1; n 3 to 4/group) were injected i.v. with3 1010 or 1 1011 vp Ad5, Ad5HVR48, or Ad48 vectors, andserum samples and livers were harvested 48 h after immunization.Serum aspartate aminotransferase (AST) and alanine aminotrans-ferase (ALT) levels were determined, and liver sections were as-sessed by histopathology. The C57BL/6 mice exhibited negligiblehepatotoxicity with all vectors, but MF1 mice immunized withAd5 vectors demonstrated a dramatic increase in systemic levels ofAST and ALT (Fig. 2). Ad48 and Ad5HVR48 vectors did not resultin increased AST or ALT levels in either mouse strain, indicatingthat Ad5HVR48 was less hepatotoxic than Ad5. None of the vec-tors at the doses utilized led to histopathological changes (data notshown).</p><p>Both Ad5 and Ad5HVR48 induce partially exhausted CD8</p><p>T-cell responses. We next assessed the potential influence of HVRmodification on the phenotype and function of Ad-elicited CD8</p><p>T-cell responses. Mice (C57BL/6; n 8/group) were vaccinatedintramuscularly with 3 1010 vp Ad5, Ad5HVR48, or Ad48 ex-pressing SIV Gag. Each mouse was sacrificed 60 days after vacci-</p><p>nation, and Gag-specific CD8 T-cell responses were assessed inthe liver, spleen, and lymph nodes. Analysis of Gag-specific CD8</p><p>T-cell responses by Db/AL11 tetramer staining showed that bothAd5 and Ad5HVR48 vectors elicited potent and comparable anti-gen-specific immune responses in the spleen and liver (Fig. 3A). Incontrast, the magnitude of antigen-specific CD8 T cells elicitedby Ad48 vectors was lower than that elicited by Ad5 andAd5HVR48 in the spleen (3.7- and 3.5-fold-lower induction andP 0.0045 and 0.0031, respectively; Students t tests) and liver(3.2- and 2.5-fold-lower induction and P 0.0003 and 0.0273,respectively) (Fig. 3B). However, while Ad5 and Ad5HVR48 elic-ited greater numbers of antigen-specific CD8 T cells than thoseelicited by Ad48, the CD8 T cel...</p></li></ul>