A Simian Replication-Defective Adenoviral Recombinant to HIV-1 Gag Julie C. Fitzgerald, Guang-Ping Gao, Arturo Reyes-Sandoval, George N. Pavlakis, Zhi Q. Xiang, This information is current as Anthony P. Wlazlo, Wynetta Giles-Davis, James M. Wilson of September 25, 2021. and Hildegund C. J. Ertl J Immunol 2003; 170:1416-1422; ; doi: 10.4049/jimmunol.170.3.1416

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2003 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

A Simian Replication-Defective Adenoviral Recombinant Vaccine to HIV-1 Gag1

Julie C. Fitzgerald,* Guang-Ping Gao,† Arturo Reyes-Sandoval,2*‡ George N. Pavlakis,§ Zhi Q. Xiang,* Anthony P. Wlazlo,* Wynetta Giles-Davis,* James M. Wilson,† and Hildegund C. J. Ertl3*

In animal models, E1-deleted human adenoviral recombinants of the serotype 5 (AdHu5) have shown high efficacy as vaccine carriers for different Ags including those of HIV-1. Humans are infected by common serotypes of human adenovirus such as AdHu5 early in life and a significant percentage has high levels of neutralizing Abs to these serotypes, which will very likely impair the efficacy of recombinant based on the homologous . To circumvent this problem, a novel replication-defective adenoviral vaccine carrier based on an E1-deleted recombinant of the chimpanzee adenovirus 68 (AdC68) was developed. An AdC68 construct expressing a codon-optimized, truncated form of gag of HIV-1 induces CD8؉ T cells to gag in mice which at the Downloaded from height of the immune response encompass nearly 20% of the entire splenic CD8؉ T cell population. The vaccine-induced immune response provides protection to challenge with a vaccinia gag recombinant virus. Induction of transgene-specific CD8؉ T cells and protection against viral challenge elicited by the AdC68 vaccines is not strongly inhibited in animals preimmune to AdHu5 virus. However, the response elicited by the AdHu5 vaccine is greatly attenuated in AdHu5 preimmune animals. The Journal of Immunology, 2003, 170: 1416–1422. http://www.jimmunol.org/

espite recent progress in antiviral strategies, the pan- fere with the efficacy of such vaccines. To circumvent this inter- demic of HIV-1 continues to expand. Correlates of pro- ference while preserving the known advantages of adenoviral D tection to HIV-1 remain ill-defined, confounding the de- recombinants, we developed a novel vector system. The vaccine is velopment of vaccines which have thus far failed to show efficacy based on E1-deleted chimpanzee adenovirus 68 (AdC68) (11), in clinical trials. Studies indicate that both neutralizing Abs to the which does not circulate in the human population and fails to carry envelope protein and cytolytic T cells to internal proteins, most neutralizing B cell epitopes that cross-react with the common se- notably gag, are required to limit infections with HIV-1 or SIV rotypes of human adenoviruses (12). Lack of preexisting virus-

(1Ð5). Replication-defective, E1-deleted human adenoviral recom- neutralizing Abs in the human population suggests that this novel by guest on September 25, 2021 binant vaccines of the serotype 5 (AdHu5)4 induce both types of adenoviral recombinant may provide an improved vaccine carrier immune responses with high efficiency in experimental animals for use in humans. In this study, we present preclinical efficacy (6Ð9). Additionally, an AdHu5 SIV gag vaccine protected nonhu- data comparing the CD8ϩ T cell response in mice to gag of HIV-1 man primates from challenge with a pathogenic chimeric simian elicited by AdHu5 and AdC68 recombinant vaccines expressing a HIV and provided better control of and maintenance of truncated form of gag. CD4ϩ counts than vaccination with adjuvanted DNA or poxvirus recombinants (9), demonstrating a favorable outlook for the use of Materials and Methods an adenoviral recombinant as an HIV vaccine. Nevertheless, Mice nearly all humans repeatedly encounter AdHu5 virus and a signif- Female 6- to 8-wk-old BALB/c mice were purchased from Jackson Lab- icant proportion has neutralizing Abs (10) that are likely to inter- oratory (Bar Harbor, ME). Cell lines *The Wistar Institute, and †University of Pennsylvania, Philadelphia, PA 19104; Ϫ ¤ Mammalian cells, i.e., E1-transfected 293 cells, TK 143B human osteo- Center for Cancer Research, National Cancer Institute-Frederick, Frederick, MD sarcoma cells (The Wistar Institute, Philadelphia, PA), and P815 mouse 21702; ‡Centro de Investigacion en Ciencia Aplicada y Tecnologia Avanzada del Instituto Politecnico Nacional, Mexico City, Mexico mastecytoma cells were propagated in DMEM supplemented with glu- tamine, sodium pyruvate, nonessential amino acids, HEPES buffer, antibi- Received for publication July 9, 2002. Accepted for publication November 22, 2002. otic, and 10% FBS. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance Generation, propagation, and titration of viral recombinants with 18 U.S.C. Section 1734 solely to indicate this fact. These methods have been described in detail for AdHu5 recombinants (8). 1 This work was funded by grants from National Institutes of Health/National Institute The same technology was applied for generation of the AdC68 recombi- of Allergy and Infectious Diseases and by the W.W. Smith Foundation and Genovo. nant (12). A pC68-CMV shuttle vector carrying the gag sequence was J.M.W. owns equity in Targeted Genomics (formerly Genovo). cotransfected with SspI-digested AdC68 genomic DNA into 293 cells and 2 Current address: Programa Institucional de Biomedicina Molecular Escuela Nacio- plaques were selected. Both E1-deleted AdHu5 and AdC68 recombinants nal de Medicina y Homeopatia-Instituto Politecnico Nacional, Mexico City, Mexico. were propagated on 293 cells transfected with the E1 gene of AdHu5 virus. 3 Address correspondence and reprint requests to Dr. Hildegund C. J. Ertl, The Wistar Virus was purified by CsCl gradient centrifugation and titrated on 293 cells Institute, 3601 Spruce Street, Philadelphia, PA 19104. E-mail address: ertl@wistar. to determine PFUs. Vaccinia gag recombinant virus (VVgag) (vDK1; con- upenn.edu tributed by Dr. D. Kuritzkes, National Institutes of Health AIDS Research 4 Abbreviations used in this paper: AdHu5, human adenoviral recombinants of the and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, serotype 5; AdC68, chimpanzee adenovirus 68; rab.gp, glycoprotein of rabies virus; Ϫ HPV, human papillomavirus; GFP, green fluorescent protein; VVgag, vaccinia gag MD) was propagated and titrated on TK 143B cells. Adenoviral recom- recombinant virus. binants expressing unrelated viral proteins, such as the glycoprotein of

Copyright © 2003 by The American Association of Immunologists, Inc. 0022-1767/03/$02.00 The Journal of Immunology 1417 rabies virus (rab.gp) or the L1 protein of human papillomavirus (HPV)-16, described previously (8, 13), were used as controls. Construction of AdHu5 and AdC68 recombinant expressing the green fluorescent protein (GFP) has been described previously (14). Peptides The AMQMLKETI (15) peptide which carries the immunodominant MHC class I epitope of gag for mice of the H-2d haplotype and the control peptide 31D delineated from the nucleoprotein of rabies virus (16) were synthesized by the Peptide Facility of The Wistar Institute. The peptides were purified by high pressure liquid chromatography and sequence veri- fied by mass spectrometry. Peptides were diluted in DMSO to a concen- tration of 1 mg/ml and stored at Ϫ20¡C. Immunization of mice Groups of 4Ð5 BALB/c mice were immunized at 6Ð8 wk of age with recombinant vaccines given i.m. (adenoviral recombinants), s.c., or i.p. (vaccinia virus recombinants). Western blot Gag protein was identified in supernatants of infected TKϪ143B by West- Ϫ 6 ern blotting using a mouse mAb to gag. TK 143B cells (1 ϫ 10 ) were Downloaded from infected for 48 h with AdHu5gag37 or AdC68gag37 virus (10 PFU/cell). Additional TKϪ143B cells were infected with constructs expressing the rab.gp (AdHu5rab.gp, AdC68rab.gp). Proteins in the culture supernatant were separated on a 12% denaturing polyacrylamide gel and transferred by electroblotting to a polyvinylidene fluoride membrane. The blot was stained with the mAb 183-H12-5C to HIV-1 p24 (provided by Dr. B. Chesebro and K. Wehrly, National Institutes of Health AIDS Reference and Reagents Center, Division of AIDS, National Institute of Allergy and http://www.jimmunol.org/ Infectious Diseases, National Institutes of Health). Intracellular cytokine staining Splenocytes (1 ϫ 106/sample) were cultured for5hat37¡C in 96-well round bottom microtiter plate wells in DMEM supplemented with 2% FBS and 10Ϫ6 M 2-ME. Brefeldin A (GolgiPlug; BD PharMingen, San Diego, CA) was added at 1 ␮l/ml. The AMQMLKETI peptide was used for pep- tide stimulation at a concentration of 3 ␮g/ml. Control cells were incubated with an unrelated peptide or without peptide. After washing, cells were incubated for 30 min at 4¡C with 25 ␮l of a 1/100 dilution of a FITC- by guest on September 25, 2021 labeled Ab to mouse CD8 (BD PharMingen). They were washed again and permeabilized in 1ϫ Cytofix/Cytoperm (BD PharMingen) for 20 min at 4¡C, washed three times with Perm/Wash (BD PharMingen), and incubated in the same buffer for 30 min at 4¡C with 25 ␮l of a 1/100 dilution of a PE-labeled Ab to mouse IFN-␥ (BD PharMingen). After washing, cells were examined by two-color flow cytometry using an EPICS Elite XL (Beckman Coulter, Miami, FL), and data were analyzed by WinMDi soft- ware. The number in the right hand corner of the graphs in the results section shows the percentage of CD8ϩ cells that stained positive for IFN-␥ over all CD8ϩ T cells. Cells incubated without the peptide to gag showed Ͻ0.5% PE staining of CD8ϩ T cells (data not shown). 51Cr-release assay Splenocytes were tested in a 5-h 51Cr-release assay at varied E:T cell ratios on 1 ϫ 104 P815 cells treated for 16Ð24 h at room temperature with either the gag peptide or the control peptide 31D. The graph shows the mean percentage of specific lysis of triplicate wells Ϯ SDs. MHC class I tetramer staining A biotin-labeled H-2Kd AMQMLKETI peptide tetramer was obtained from the National Institutes of Health Tetramer Facility (Emory University,

FIGURE 2. CD8ϩ T cell responses to gag in immunized mice. Freshly isolated splenocytes of BALB/c mice immunized i.m. with 2 ϫ 105,2ϫ 106, or 2 ϫ 107 PFU of AdC68gag37 virus; 2 ϫ 106 PFU of an AdHu5 recom- binant expressing the L1 protein of HPV-16 (AdHu5L1); 2 ϫ 106 PFU of AdHu5gag37 virus; 2 ϫ 107 PFU of VVgag virus; or 2 ϫ 107 PFU of VVgag virus followed by 4 ϫ 106 PFU of VVgag virus were tested for CD8ϩ T cell FIGURE 1. Gag secretion by TKϪ143B cells infected with adenoviral response to gag 10 days later. A, Intracellular staining for IFN-␥: x-axis anti- recombinants. The figure shows a Western Blot with supernatants from CD8, y-axis anti-IFN-␥. B, 51Cr-relrease assay: lysis of target cells coated with TKϪ143B cells infected for 48 h with 10 PFU/cell of adenoviral recom- the peptide to gag (f), lysis of target cells coated with a control peptide (X) binants expressing gag or a control protein. The blot was stained with a (i.e., peptide 31D delineated from the sequence of the rabies virus nucleopro- mouse mAb to gag. Lane 1, AdHu5rab.gp; lane 2, AdC68rab.gp; lane 3, tein). C, Tetramer staining: stained with streptavidin-PE (left panels), stained AdHu5gag37; lane 4, AdC68gag37. with PE-labeled gag tetramer (right panels). 1418 SIMIAN ADENOVIRUS VACCINE TO HIV-1 GAG

Atlanta, GA). Lymphocytes (106/sample) were incubated for 45 min on ice constructed (12). To circumvent rev dependency (17) of transgene with 200 ␮l of a mixture of a 1/100 dilution of a FITC-labeled Ab to CD8 expression, we inserted a codon-modified sequence of gag from and a 1/400 dilution of the tetramer or as a control with a comparable which genetic instability elements had been removed (18). The amount of PE-labeled streptavidin. Cells were washed and analyzed by two-color flow cytometry using an EPICS Elite XL. introduced gene encodes the truncated, partially secreted p37 gag protein (p17 and p24) (19). Both recombinants, termed Vaccinia virus challenge AdHu5gag37 and AdC68gag37, were generated and propagated on Immunized mice were injected with 106 PFU of the VVgag i.p. Paired 293 cells transfected with the E1 gene of AdHu5, which can ovaries from individual mice harvested 5 days later were homogenized, transcomplement the E1-deleted AdC68 virus recombinants freeze-thawn three times, and titrated on confluent monolayers of Ϫ thereby reducing the risk of recombination associated with the use TK 143B cells. Titers were read 48 h later. of a homologous E1 sequence. Both recombinants expressed the Ϫ Adoptive transfer of AdHu5 preimmune lymphocytes and sera transgene product in infected TK 143B cells (Fig. 1).

8 Mice were immunized with 10 PFU of the AdHu5rab.gp recombinant ϩ virus and sacrificed 2 wk later. Blood samples were incubated for1hat Induction of CD8 T cell responses to gag room temperature then centrifuged to separate the serum and cellular com- To assess the immunogenicity of the two adenoviral recombinants ponents and the serum was collected. Splenocytes were also isolated and to gag, naive BALB/c mice were immunized once with 2 ϫ 106 lymphocytes purified on a Ficoll cushion. The lymphocytes were then 5 stained with a 1/100 dilution of an FITC-labeled Ab to CD8 for 30 min at PFU of the AdHu5gag37 vaccine or various amounts (2 ϫ 10 , 4¡C and sorted using a Cytomation MoFlo (Cytomation, Fort Collins, CO) 2 ϫ 106, and 2 ϫ 107 PFU/mouse) of the AdC68gag37 vaccine. ϩ Ϫ into CD8 and CD8 populations. Naive mice were injected i.v. with 5 ϫ Control mice were immunized with an AdHu5 vaccine to L1 of 6 ϩ ϫ 6 Ϫ ϫ 7 Downloaded from 10 CD8 splenocytes, 5 10 CD8 splenocytes, 5 10 unsorted HPV-16 or once or twice with the VVgag construct. Splenocytes splenocytes, or 250 ␮l of preimmune serum given i.p. and then vaccinated ϩ the following day with AdHu5gag37 or AdC68gag37 virus. were tested 10 days later for CD8 T cells specific for the immu- nodominant epitope of gag either by an intracellular cytokine Sorting and cell surface marker analysis of adenovirus-infected (IFN-␥) assay or by a 51Cr-release assay conducted with freshly APCs isolated splenocytes that had not been expanded in vitro. As shown ϩ AdHu5 preimmune or naive mice were immunized with 2.5 ϫ 1011 par- in Fig. 2A, both recombinants induced CD8 T cells that produced ticles of AdHu5GFP or AdC68GFP recombinant virus i.m. in the lower IFN-␥ in response to the immunodominant epitope of gag. These http://www.jimmunol.org/ hind limbs. Mice were sacrificed 48 h later and cells from the popliteal ϩ Ϫ cells mediated lysis of gag-expressing H-2 compatible target cells lymph nodes were isolated and then sorted into GFP and GFP popula- ϩ ϩ (Fig. 2B). Gag-specific CD8 T cell activity was superior upon tions using a Cytomation MoFlo. GFP cells were then stained with a 1/100 dilution of a PE-labeled Ab to MHC class II of H-2d (BD Phar- immunization with the AdC68gag37 construct. Depending on the Mingen) and a CyChrome C-labeled Ab to CD11b (eBioscience, San Di- dose, the AdC68gag37 vaccine induced frequencies of gag-specific ego, CA) for 30 min at 4¡C, washed, and analyzed by three-color flow CD8ϩ T cells that encompassed nearly 20% of the entire splenic cytometry using an EPICS Elite XL. CD8ϩ cell population (Fig. 2A). The AdHu5gag37 recombinant induced optimal frequencies of ϳ9% at 2 ϫ 106 PFU (Fig. 2A) Results

which were not significantly enhanced upon increasing the dose of by guest on September 25, 2021 Transgene product expression by E1-deleted adenoviral this vaccine (data not shown). A vaccinia virus recombinant ex- recombinants pressing full-length gag (VVgag) stimulated far lower CD8ϩ T cell E1-deleted adenoviral recombinants derived from the AdC68 and responses to gag (Fig. 2, A and B). The control construct failed to the AdHu5 virus carrying the gag gene of HIV-1 clade B were induce CD8ϩ T cells that responded with IFN-␥ production or

FIGURE 3. Kinetics of the CD8ϩ T cell response to gag. Groups of BALB/c mice were immunized with 5 ϫ 106 PFU of AdHu5gag37 virus (upper panels) or AdC68gag37 virus (lower panels). Splenocytes were harvested 6Ð12 days later and tested for IFN-␥ production. The Journal of Immunology 1419 Downloaded from http://www.jimmunol.org/ by guest on September 25, 2021

FIGURE 4. The effect of preexisting immunity to AdHu5 virus on CD8ϩ T cell responses to gag. Mice were immunized i.m. with 108 PFU of the AdHu5L1 vaccine. Two weeks later AdHu5L1-immune (a and c), as well as naive (b and d) mice were injected with 2 ϫ 107 PFU of AdHu5gag37 (a and b) or AdC68gag37 (c and d) recombinants. Mice were sacrificed 9 days after immunization with the adenoviral recombinants and splenocytes were tested by intracellular staining for IFN-␥ production to the gag peptide (A) or target cell lysis (B).

target cell lysis to the gag epitope. Intracellular cytokine assays have delayed transgene expression and thus shifted the develop- with mouse splenocytes commonly show some cytokine produc- ment of a transgene-specific CD8ϩ T cell response. tion by CD8Ϫ cells. To further ensure that the high frequencies of ϩ ␥ CD8 IFN- -producing T cells were indeed specific for gag, we ϩ tested splenocytes from mice immunized with the highest dose of The gag-specific CD8 T cell response in mice pre-exposed to the AdC68gag37 vaccine for CD8ϩ T cells that stained with a AdHu5 virus tetramer specific for the immunodominant epitope of gag. As To study the impact of previous exposure to AdHu5 Ags, mice ϩ shown in Fig. 2C, frequencies of gag-specific CD8 T cells de- were immunized with a single dose of 108 PFU of an E1-deleted tectable by this technique were comparable to those observed with AdHu5 recombinant expressing an irrelevant Ag. This dose in- intracellular cytokine staining. duces virus neutralizing Ab titers to AdHu5 virus of ϳ1/100Ð1/

ϩ 200 (data not shown). Two weeks later, mice were vaccinated Kinetics of the gag-specific CD8 T cell response either with the AdHu5gag37 or the AdC68gag37 construct. Mice The kinetics of the CD8ϩ T cell response to gag elicited by the two preimmune to AdHu5 virus failed to develop a gag-specific CD8ϩ adenoviral recombinants differed (Fig. 3). The response to gag T cell response after vaccination with the AdHu5gag37 vaccine presented by the AdHu5gag37 virus peaked 2Ð4 days earlier than (Fig. 4, A and B, a and b). The CD8ϩ T cell response to gag was the CD8ϩ T cell response to the AdC68gag37 recombinant. We only slightly decreased in mice pre-exposed to an AdHu5 construct reported previously that the AdC68 construct induces production before immunization with the AdC68gag37 vaccine (Fig. 4, A and of high levels of type 1 IFN upon infection of splenocytes (20). B, c and d). IFNs reduce in part by down-regulating the ac- Both the AdHu5gag37 and the AdC68gag37 vaccine induced tivity of viral promoters such as the CMV promoter, which drives protective immunity against a subsequent i.p. challenge with the transgene expression in both types of adenoviral recombinants. We VVgag recombinant that replicated to high titers in the ovaries of assume, and this remains to be proven, that dampening of the pro- mice immunized with adenoviral recombinants to an unrelated Ag. moter activity upon inoculation of the AdC68 recombinant may Pre-exposure of mice to AdHu5 virus abolished protection induced 1420 SIMIAN ADENOVIRUS VACCINE TO HIV-1 GAG

rum harvested. The splenocytes were sorted into CD8ϩ and CD8Ϫ populations and injected i.v. into naive syngeneic animals. Serum from the preimmune animals was injected i.p. into naive animals. These animals, as well as groups of naive and preimmune controls, were subsequently vaccinated with AdHu5gag37 or AdC68gag37 virus. Finally, the percentage of gag-specific IFN-␥-producing CD8ϩ splenocytes was determined for each group. This experi- ment was performed twice; in both experiments, the results were consistent and the results are summarized in Table I. As expected, AdHu5 preimmune serum caused the greatest re- duction in AdHu5gag37 efficacy (denoted by the percentage of reduction in frequency of IFN-␥-producing CD8ϩ T cells in trans- fused as compared with untreated AdHu5gag37-vaccinated control mice). In contrast, AdHu5 preimmune sera caused no reduction in the induction of IFN-␥-producing CD8ϩ T cells by the AdC68gag37 vaccine. This shows that nonneutralizing Abs that cross-react between the two serotypes of adenovirus do not impact the induction of transgene product-specific CD8ϩ T cells by the heterologous vaccine construct, implicating a T cell-mediated Downloaded from mechanism in AdC68gag37 inhibition. The induction of a gag- specific immune response to the simian origin vaccine was de- creased in animals that received AdHu5 preimmune CD8ϩ spleno- FIGURE 5. The effect of preexisting immunity to AdHu5 on the effi- cytes (Table I). This reduction in Ag-specific CD8ϩ T cell cacy of the vaccines in inducing protection to viral challenge. Groups of 8 frequency was comparable to the reduction in AdHu5 preimmune eight mice were injected i.m. with 10 PFU of AdHu5L1 virus. Two weeks http://www.jimmunol.org/ control animals vaccinated with AdC68gag37, suggesting that later, they as well as additional groups of naive mice were immunized with ϩ 2 ϫ 107 PFU of AdHu5gag37 (left panel) or AdC68gag37 (right panel) cross-reactive adenovirus-specific CD8 T cells are a main con- virus. Eight mice each were vaccinated with 2 ϫ 107 PFU of AdHu5rab.gp tributor to this phenomenon. Cell sorting does not remove all ϩ or AdC68rab.gp constructs as nonprotected controls. Nine days later, mice CD8 cells from the population and this contamination most likely were injected i.p. with 106 PFU of the VVgag recombinant. Ovaries were caused the small reduction (13%) seen in animals that received harvested 5 days later and titers of vaccinia virus in paired ovaries from AdHu5 preimmune CD8Ϫ splenocytes. individual mice were determined and are denoted by individual circles or We next tested if this decrease in gag-specific CD8ϩ T cell squares. X ϭ mean titers. ϩ frequency by AdHu5 preimmune CD8 T cells can be explained

by a reduction in adenovirus-infected APCs. For this experiment, by guest on September 25, 2021 by the AdHu5gag37 vaccine, but only slightly reduced that elicited groups of AdHu5 preimmune and naive mice were injected i.m. by the AdC68gag37 vaccine (Fig. 5). into the lower hind legs with GFP-expressing adenoviral recom- binants. This results in transduction of APCs, presumably dendritic ϩ Impact of AdHu5-specific CD8 lymphocytes on AdC68gag37 cells, which upon maturation migrate from the injection site to efficacy draining lymph nodes and express GFP (H. C. J. Ertl, manuscript Adenoviruses of different human- and chimpanzee-derived sero- in preparation). Draining popliteal lymph nodes were isolated 48 h types show a high degree of amino acid sequence homology which after i.m. injection of AdGFP constructs and the percentage of ϩ is expected to be recognized by cross-reactive T cells. We hypoth- GFP-expressing (adenovirus transduced), CD11b , MHC class ϩ esized that the presence of such cross-reactive adenovirus-specific II cells in the lymph nodes was determined; these cell surface CD8ϩ T cells caused the reduction in gag-specific CD8ϩ T cell markers were chosen to further characterize the transduced cells as responses seen in AdHu5 preimmune animals vaccinated with APCs. The result of this experiment is shown in Fig. 6 and Table AdC68gag37 by selectively killing adenovirus-infected APCs. To II. The overall percentage of GFP-expressing cells is lower for test this hypothesis, we performed an adoptive transfer experiment AdC68 than for AdHu5-immunized animals; this result is highly to study the effects of different components of the AdHu5 immune reproducible (H. C. J. Ertl, manuscript in preparation) and we nev- response on subsequent vaccination with either adenoviral recom- ertheless see a better CD8ϩ response in the AdC68-vaccinated binant. Preimmune mice were sacrificed and splenocytes and se- mice. AdHu5 preimmune mice showed an ϳ50-fold reduction in

Table I. Effect of passive transfer of AdHu5 preimmune cells or sera on CD8ϩ T cell responses to gaga

Percentage of Reduction of IFN-␥-Producing CD8ϩ T Cells

AdHu5 AdHu5 Preimmune Splenocyte AdHu5 CD8ϩ AdHu5 CD8Ϫ AdHu5 Sera Vaccine (No Transfer) Transfer Transfer Transfer Transfer

AdHu5gag37 92 NRb NR NR 53 AdC68gag37 55 28 42 13 NR

a Groups of naive mice were passively transferred with CD8ϩ, CD8Ϫ, or unsorted splenocytes, or sera from AdHu5 preimmune animals. They were then vaccinated with AdHu5gag37 or AdC68gag37 virus. Naive and AdHu5 preimmune animals were also vaccinated as controls. The CD8ϩ immune response was measured by intracellular cytokine staining of CD8ϩ T cells for IFN-␥. Data are expressed as percentage of reduction of frequencies of IFN-␥-producing CD8ϩ T cells of the transfused mice compared to frequencies of IFN-␥-producing CD8ϩ T cells by AdHu5 naive controls vaccinated with the gag constructs. Data are averaged from two experiments. b NR, No reduction. The Journal of Immunology 1421

Discussion E1-deleted AdHu5 recombinants induce potent B and T cell re- sponses to both the adenoviral Ags and the transgene product (21), which far surpass those elicited by other types of vaccines. The high immunogenicity of adenoviral recombinants relates in part to the noncytopathic nature of such E1-deleted viruses resulting in sustained Ag expression (8) and to their ability to efficiently trans- duce professional APCs (22). When tested in nonhuman primates, an AdHu5 vaccine to gag of HIV-1 showed superior efficacy in both preventing CD4 loss and in controlling acute and set point viral load upon challenge in comparison to adjuvanted DNA and modified vaccinia Ankara vaccines expressing the same transgene product (9). Nevertheless, in animals pre-exposed to AdHu5 virus to mimic the status of many adult humans, the interference due to neutralizing Abs to the vaccine carrier could only be overcome by increasing the vaccine dose 1000-fold (23). To forestall problems with preexisting immunity to common serotypes of human adeno- viruses, we developed an alternative vaccine system based on a simian serotype of adenovirus, which does not circulate in the Downloaded from human population (12). Our data show that adenoviral recombi- nants to gag based on the human or chimpanzee serotypes induce CD8ϩ T cells to gag at frequencies surpassing those elicited by previously described vaccines or by chronic infections (24Ð27). FIGURE 6. The effect of AdHu5 preimmunity on adenovirus-infected Pre-exposure to AdHu5 resulting in serotype-specific virus-neu- APCs. Naive and AdHu5 preimmune mice were inoculated with tralizing Abs titers comparable to those detectable in a sizable http://www.jimmunol.org/ AdHu5GFP or AdC68GFP virus. Forty-eight hours later, cells from the ϩ fraction of the human population severely reduced the efficacy of draining lymph nodes were sorted to isolate GFP cells and stained for ϩ CD11b and MHCII. The graphs are gated on GFPϩ cells. the AdHu5gag37 vaccine, but only slightly impaired the CD8 T cell response to the AdC68gag37 virus. Reduction of the trans- gene-specific CD8ϩ T cell response to AdC68gag37 vaccination in

ϩ ϩ AdHu5 pre-exposed mice is most likely due to the activity of in- infected (i.e., GFP-expressing) CD11b , MHC class II cells terserotype cross-reactive CD8ϩ T cells which lyse adenovirus- upon injection of the AdHu5GFP recombinant (0.001% in preim- infected dendritic cells, diminishing the capacity of the Ag pre- mune mice vs 0.055% in naive controls); this is most likely due to sentation machinery. We believe that this effect is a byproduct of the presence of neutralizing Abs to AdHu5 which prevent infection our experimental system in which we vaccinated mice early, i.e., 2 by guest on September 25, 2021 of APCs by the AdHu5GFP construct. AdC68GFP-vaccinated wk after exposure to AdHu5. It is possible that a longer time in- mice showed a far more modest reduction of ϳ2-fold of GFPϩ, ϩ ϩ terval between pre-exposure and vaccination would diminish the CD11b , MHC class II cells in draining lymph nodes (0.014 vs impact of AdHu5 pre-exposure on the CD8ϩ T cell response to the 0.025% in naive controls). We hypothesize that this reduction is ϩ AdC68 vaccine. A longer interval between pre-exposure and vac- due to the presence of adenovirus-specific CD8 T cells, which cination is unlikely to rescue the CD8ϩ T cell response to AdHu5 could lyse AdC68GFP-infected APCs, and this reduction from the vaccination that was dampened by Abs rather than CD8ϩ T cells. normal level of Ag presentation partially diminishes the magnitude The more dramatic impact of CD8ϩ T cells on the efficacy of the of the immune response to the transgene product of the AdC68 AdC68 vaccine than on the homologous vaccine presumably re- vaccine. Indeed, the 2-fold reduction of AdC68GFP-infected flects differences in kinetics of CD8ϩ T cell induction by the two APCs in AdHu5 preimmune mice fits with the 55% (ϳ2-fold) ϩ vaccine constructs. The AdC68 vaccine shows induction of trans- ␥ ϩ reduction in gag-specific IFN- -producing CD8 T cells seen in gene product-specific CD8 T cells with a 3- to 4-day delay com- AdHu5 preimmune mice vaccinated with AdC68gag37 (Table I). pared with AdHu5 recombinants which may allow for better re- cruitment and reactivation of CD8ϩ T cells to cross-reactive epitopes of adenovirus. Table II. Percentage of adenovirus infected cells in draining lymph In summary, data presented in this study demonstrate that E1- ϩ node after AdGFP immunization in AdHu5 preimmune vs naive micea deleted AdC68 recombinants induce superior CD8 T cell re- sponses to the transgene product, even in the presence of neutral- % GFPϩ, izing Abs to a common strain of a human adenovirus, than ϩ ϩ % GFP , MHCII , previously described AdHu5 recombinants. Thus, they are ex- % GFPϩ MHCIIϩ CD11bϩ pected to be more suitable as vaccine carriers for use in humans, AdHu5/AdHu5GFP 0.002 0.001 0.001 compared with vaccines based on human serotypes. Naive/AdHu5GFP 0.956 0.176 0.055 AdHu5/AdC68GFP 0.064 0.036 0.014 Acknowledgments Naive/AdC68GFP 0.167 0.054 0.025 We thank the Vector and Immunology Cores of the Institute for Human a AdHu5 preimmune or naive mice were immunized with AdHu5- or AdC68GFP for technical support. i.m. in the lower leg. Two days later cells were isolated from the popliteal lymph nodes and sorted by flow cytometry into GFPϩ populations to enrich the population of adenovirus transduced cells. The GFPϩ populations were subsequently stained for References MHCII and CD11b, and finally analyzed again by flow cytometry. The data in Fig. 6 1. Amara, R. R., F. Villinger, J. D. Altman, S. L. Lydy, S. P. O’Neil, S. I. Staprans, (i.e., percentage of MHCIIϩ and percentage of CD11bϩ) were used to calculate the D. C. Montefiori, Y. Xu, J. G. Herndon, L. S. Wyatt, et al. 2001. Control of a percentage of positive cells in each column out of all isolated and sorted cells from mucosal challenge and prevention of AIDS by a multiprotein DNA/MVA vac- the draining lymph nodes. cine. Science 292:69. 1422 SIMIAN ADENOVIRUS VACCINE TO HIV-1 GAG

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