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  • Hexon Hypervariable Region-Modified Adenovirus Type 5 (Ad5)Vectors Display Reduced Hepatotoxicity but Induce T LymphocytePhenotypes Similar to Ad5 Vectors

    Jeffrey E. Teigler,a Pablo Penaloza-MacMaster,a Rebecca Obeng,a Nicholas M. Provine,a Rafael A. Larocca,a Erica N. Borducchi,a

    Dan H. Baroucha,b

    Center for Virology and Vaccine Research, Beth Israel Deaconess Medical Center, Boston, Massachusetts, USAa; Ragon Institute of MGH, MIT, and Harvard, Boston,Massachusetts, USAb

    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.

    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).

    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

    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.

    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

    Received 9 April 2014 Returned for modification 30 April 2014Accepted 4 June 2014

    Published ahead of print 18 June 2014

    Editor: S. A. Plotkin

    Address correspondence to Dan H. Barouch, dbarouch@bidmc.harvard.edu.

    J.E.T. and P.P.-M. contributed equally to this work.

    Copyright 2014, American Society for Microbiology. All Rights Reserved.

    doi:10.1128/CVI.00207-14

    The authors have paid a fee to allow immediate free access to this article.

    August 2014 Volume 21 Number 8 Clinical and Vaccine Immunology p. 11371144 cvi.asm.org 1137

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  • 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.

    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).

    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-

    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).

    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.

    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

    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.

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  • 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)

    (31). The fixed cells were acquired using an LSR II flow cytometer (BDBiosciences) and analyzed using FlowJo (TreeStar).

    Data analysis and statistical methods. The concentrations of cyto-kines and che