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* The Wistar Institute, and
University of Pennsylvania, Philadelphia, PA 19104;
Center for Cancer Research, National Cancer Institute-Frederick, Frederick, MD 21702;
Centro de Investigacion en Ciencia Aplicada y Tecnologia Avanzada del Instituto Politecnico Nacional, Mexico City, Mexico
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Introduction |
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Materials and Methods |
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Female 6- to 8-wk-old BALB/c mice were purchased from Jackson Laboratory (Bar Harbor, ME).
Cell lines
Mammalian cells, i.e., E1-transfected 293 cells, TK-143B human osteosarcoma cells (The Wistar Institute, Philadelphia, PA), and P815 mouse mastecytoma cells were propagated in DMEM supplemented with glutamine, sodium pyruvate, nonessential amino acids, HEPES buffer, antibiotic, and 10% FBS.
Generation, propagation, and titration of viral recombinants
These methods have been described in detail for AdHu5 recombinants (8). The same technology was applied for generation of the AdC68 recombinant (12). A pC68-CMV shuttle vector carrying the gag sequence was cotransfected with SspI-digested AdC68 genomic DNA into 293 cells and plaques were selected. Both E1-deleted AdHu5 and AdC68 recombinants were propagated on 293 cells transfected with the E1 gene of AdHu5 virus. Virus was purified by CsCl gradient centrifugation and titrated on 293 cells to determine PFUs. Vaccinia gag recombinant virus (VVgag) (vDK1; contributed by Dr. D. Kuritzkes, National Institutes of Health AIDS Research and Reference Reagent Program, Division of AIDS, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD) was propagated and titrated on TK-143B cells. Adenoviral recombinants expressing unrelated viral proteins, such as the glycoprotein of 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 viruses 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 verified by mass spectrometry. Peptides were diluted in DMSO to a concentration of 1 mg/ml and stored at -20°C.
Immunization of mice
Groups of 4–5 BALB/c mice were immunized at 6–8 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 TK-143B by Western blotting using a mouse mAb to gag. TK-143B cells (1 x 106) were infected for 48 h with AdHu5gag37 or AdC68gag37 virus (10 PFU/cell). Additional TK-143B 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 Infectious Diseases, National Institutes of Health).
Intracellular cytokine staining
Splenocytes (1 x 106/sample) were cultured for 5 h at 37°C in 96-well round bottom microtiter plate wells in DMEM supplemented with 2% FBS and 10-6 M 2-ME. Brefeldin A (GolgiPlug; BD PharMingen, San Diego, CA) was added at 1 µl/ml. The AMQMLKETI peptide was used for peptide stimulation at a concentration of 3 µg/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 µl of a 1/100 dilution of a FITC-labeled Ab to mouse CD8 (BD PharMingen). They were washed again and permeabilized in 1x 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 µl 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 software. The number in the right hand corner of the graphs in the results section shows the percentage of CD8+ cells that stained positive for IFN- over all CD8+ T cells. Cells incubated without the peptide to gag showed <0.5% PE staining of CD8+ 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 x 104 P815 cells treated for 16–24 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 ± 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, Atlanta, GA). Lymphocytes (106/sample) were incubated for 45 min on ice with 200 µl of a mixture of a 1/100 dilution of a FITC-labeled Ab to CD8 and a 1/400 dilution of the tetramer or as a control with a comparable amount of PE-labeled streptavidin. Cells were washed and analyzed by two-color flow cytometry using an EPICS Elite XL.
Vaccinia virus challenge
Immunized mice were injected with 106 PFU of the VVgag i.p. Paired ovaries from individual mice harvested 5 days later were homogenized, freeze-thawn three times, and titrated on confluent monolayers of TK-143B cells. Titers were read 48 h later.
Adoptive transfer of AdHu5 preimmune lymphocytes and sera
Mice were immunized with 108 PFU of the AdHu5rab.gp recombinant virus and sacrificed 2 wk later. Blood samples were incubated for 1 h at room temperature then centrifuged to separate the serum and cellular components and the serum was collected. Splenocytes were also isolated and lymphocytes purified on a Ficoll cushion. The lymphocytes were then stained with a 1/100 dilution of an FITC-labeled Ab to CD8 for 30 min at 4°C and sorted using a Cytomation MoFlo (Cytomation, Fort Collins, CO) into CD8+ and CD8- populations. Naive mice were injected i.v. with 5 x 106 CD8+ splenocytes, 5 x 106 CD8- splenocytes, 5 x 107 unsorted splenocytes, or 250 µl of preimmune serum given i.p. and then vaccinated the following day with AdHu5gag37 or AdC68gag37 virus.
Sorting and cell surface marker analysis of adenovirus-infected APCs
AdHu5 preimmune or naive mice were immunized with 2.5 x 1011 particles of AdHu5GFP or AdC68GFP recombinant virus i.m. in the lower hind limbs. Mice were sacrificed 48 h later and cells from the popliteal lymph nodes were isolated and then sorted into GFP+ and GFP- populations 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 PharMingen) and a CyChrome C-labeled Ab to CD11b (eBioscience, San Diego, CA) for 30 min at 4°C, washed, and analyzed by three-color flow cytometry using an EPICS Elite XL.
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Results |
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E1-deleted adenoviral recombinants derived from the AdC68 and the AdHu5 virus carrying the gag gene of HIV-1 clade B were constructed (12). To circumvent rev dependency (17) of transgene expression, we inserted a codon-modified sequence of gag from which genetic instability elements had been removed (18). The introduced gene encodes the truncated, partially secreted p37 gag protein (p17 and p24) (19). Both recombinants, termed AdHu5gag37 and AdC68gag37, were generated and propagated on 293 cells transfected with the E1 gene of AdHu5, which can transcomplement the E1-deleted AdC68 virus recombinants thereby reducing the risk of recombination associated with the use of a homologous E1 sequence. Both recombinants expressed the transgene product in infected TK-143B cells (Fig. 1).
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To assess the immunogenicity of the two adenoviral recombinants to gag, naive BALB/c mice were immunized once with 2 x 106 PFU of the AdHu5gag37 vaccine or various amounts (2 x 105, 2 x 106, and 2 x 107 PFU/mouse) of the AdC68gag37 vaccine. Control mice were immunized with an AdHu5 vaccine to L1 of HPV-16 or once or twice with the VVgag construct. Splenocytes were tested 10 days later for CD8+ T cells specific for the immunodominant epitope of gag either by an intracellular cytokine (IFN-) assay or by a 51Cr-release assay conducted with freshly isolated splenocytes that had not been expanded in vitro. As shown in Fig. 2A, both recombinants induced CD8+ T cells that produced IFN- in response to the immunodominant epitope of gag. These cells mediated lysis of gag-expressing H-2 compatible target cells (Fig. 2B). Gag-specific CD8+ T cell activity was superior upon immunization with the AdC68gag37 construct. Depending on the dose, the AdC68gag37 vaccine induced frequencies of gag-specific CD8+ T cells that encompassed nearly 20% of the entire splenic CD8+ cell population (Fig. 2A). The AdHu5gag37 recombinant induced optimal frequencies of 
9% at 2 x 106 PFU (Fig. 2A) which were not significantly enhanced upon increasing the dose of this vaccine (data not shown). A vaccinia virus recombinant expressing full-length gag (VVgag) stimulated far lower CD8+ T cell responses to gag (Fig. 2, A and B). The control construct failed to induce CD8+ T cells that responded with IFN- production or target cell lysis to the gag epitope. Intracellular cytokine assays with mouse splenocytes commonly show some cytokine production by CD8- cells. To further ensure that the high frequencies of CD8+ IFN--producing T cells were indeed specific for gag, we tested splenocytes from mice immunized with the highest dose of the AdC68gag37 vaccine for CD8+ T cells that stained with a tetramer specific for the immunodominant epitope of gag. As shown in Fig. 2C, frequencies of gag-specific CD8+ T cells detectable by this technique were comparable to those observed with intracellular cytokine staining.
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The kinetics of the CD8+ T cell response to gag elicited by the two adenoviral recombinants differed (Fig. 3). The response to gag presented by the AdHu5gag37 virus peaked 2–4 days earlier than the CD8+ T cell response to the AdC68gag37 recombinant. We reported previously that the AdC68 construct induces production of high levels of type 1 IFN upon infection of splenocytes (20). IFNs reduce viral replication in part by down-regulating the activity of viral promoters such as the CMV promoter, which drives transgene expression in both types of adenoviral recombinants. We assume, and this remains to be proven, that dampening of the promoter activity upon inoculation of the AdC68 recombinant may have delayed transgene expression and thus shifted the development of a transgene-specific CD8+ T cell response.
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To study the impact of previous exposure to AdHu5 Ags, mice were immunized with a single dose of 108 PFU of an E1-deleted AdHu5 recombinant expressing an irrelevant Ag. This dose induces virus neutralizing Ab titers to AdHu5 virus of 1/100–1/200 (data not shown). Two weeks later, mice were vaccinated either with the AdHu5gag37 or the AdC68gag37 construct. Mice preimmune to AdHu5 virus failed to develop a gag-specific CD8+ T cell response after vaccination with the AdHu5gag37 vaccine (Fig. 4, A and B, a and b). The CD8+ T cell response to gag was only slightly decreased in mice pre-exposed to an AdHu5 construct before immunization with the AdC68gag37 vaccine (Fig. 4, A and B, c and d).
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Adenoviruses of different human- and chimpanzee-derived serotypes show a high degree of amino acid sequence homology which is expected to be recognized by cross-reactive T cells. We hypothesized that the presence of such cross-reactive adenovirus-specific CD8+ T cells caused the reduction in gag-specific CD8+ T cell responses seen in AdHu5 preimmune animals vaccinated with AdC68gag37 by selectively killing adenovirus-infected APCs. To test this hypothesis, we performed an adoptive transfer experiment to study the effects of different components of the AdHu5 immune response on subsequent vaccination with either adenoviral recombinant. Preimmune mice were sacrificed and splenocytes and serum harvested. The splenocytes were sorted into CD8+ and CD8- 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 CD8+ splenocytes was determined for each group. This experiment was performed twice; in both experiments, the results were consistent and the results are summarized in Table I.
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-producing CD8+ T cells in transfused as compared with untreated AdHu5gag37-vaccinated control mice). In contrast, AdHu5 preimmune sera caused no reduction in the induction of IFN--producing CD8+ 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 CD8+ T cells by the heterologous vaccine construct, implicating a T cell-mediated mechanism in AdC68gag37 inhibition. The induction of a gag-specific immune response to the simian origin vaccine was decreased in animals that received AdHu5 preimmune CD8+ splenocytes (Table I). This reduction in Ag-specific CD8+ T cell frequency was comparable to the reduction in AdHu5 preimmune control animals vaccinated with AdC68gag37, suggesting that cross-reactive adenovirus-specific CD8+ T cells are a main contributor to this phenomenon. Cell sorting does not remove all CD8+ cells from the population and this contamination most likely caused the small reduction (13%) seen in animals that received AdHu5 preimmune CD8- splenocytes.
We next tested if this decrease in gag-specific CD8+ T cell frequency by AdHu5 preimmune CD8+ T cells can be explained by a reduction in adenovirus-infected APCs. For this experiment, groups of AdHu5 preimmune and naive mice were injected i.m. into the lower hind legs with GFP-expressing adenoviral recombinants. This results in transduction of APCs, presumably dendritic cells, which upon maturation migrate from the injection site to draining lymph nodes and express GFP (H. C. J. Ertl, manuscript in preparation). Draining popliteal lymph nodes were isolated 48 h after i.m. injection of AdGFP constructs and the percentage of GFP-expressing (adenovirus transduced), CD11b+, MHC class II+ cells in the lymph nodes was determined; these cell surface markers were chosen to further characterize the transduced cells as APCs. The result of this experiment is shown in Fig. 6 and Table II. The overall percentage of GFP-expressing cells is lower for AdC68 than for AdHu5-immunized animals; this result is highly reproducible (H. C. J. Ertl, manuscript in preparation) and we nevertheless see a better CD8+ response in the AdC68-vaccinated mice. AdHu5 preimmune mice showed an 50-fold reduction in infected (i.e., GFP-expressing) CD11b+, MHC class II+ cells upon injection of the AdHu5GFP recombinant (0.001% in preimmune mice vs 0.055% in naive controls); this is most likely due to the presence of neutralizing Abs to AdHu5 which prevent infection of APCs by the AdHu5GFP construct. AdC68GFP-vaccinated mice showed a far more modest reduction of 2-fold of GFP+, CD11b+, MHC class II+ cells in draining lymph nodes (0.014 vs 0.025% in naive controls). We hypothesize that this reduction is due to the presence of adenovirus-specific CD8+ T cells, which could lyse AdC68GFP-infected APCs, and this reduction from the normal level of Ag presentation partially diminishes the magnitude of the immune response to the transgene product of the AdC68 vaccine. Indeed, the 2-fold reduction of AdC68GFP-infected APCs in AdHu5 preimmune mice fits with the 55% (2-fold) reduction in gag-specific IFN--producing CD8+ T cells seen in AdHu5 preimmune mice vaccinated with AdC68gag37 (Table I).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In summary, data presented in this study demonstrate that E1-deleted AdC68 recombinants induce superior CD8+ T cell responses to the transgene product, even in the presence of neutralizing Abs to a common strain of a human adenovirus, than previously described AdHu5 recombinants. Thus, they are expected to be more suitable as vaccine carriers for use in humans, compared with vaccines based on human serotypes.
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Acknowledgments |
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Footnotes |
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2 Current address: Programa Institucional de Biomedicina Molecular Escuela Nacional de Medicina y Homeopatia-Instituto Politecnico Nacional, Mexico City, Mexico.
3 Address correspondence and reprint requests to Dr. Hildegund C. J. Ertl, The Wistar Institute, 3601 Spruce Street, Philadelphia, PA 19104. E-mail address: ertl{at}wistar.upenn.edu
4 Abbreviations used in this paper: AdHu5, human adenoviral recombinants of the serotype 5; AdC68, chimpanzee adenovirus 68; rab.gp, glycoprotein of rabies virus; HPV, human papillomavirus; GFP, green fluorescent protein; VVgag, vaccinia gag recombinant virus.
Received for publication July 9, 2002. Accepted for publication November 22, 2002.
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S. M. Sumida, D. M. Truitt, M. G. Kishko, J. C. Arthur, S. S. Jackson, D. A. Gorgone, M. A. Lifton, W. Koudstaal, M. G. Pau, S. Kostense, et al. Neutralizing Antibodies and CD8+ T Lymphocytes both Contribute to Immunity to Adenovirus Serotype 5 Vaccine Vectors J. Virol., March 15, 2004; 78(6): 2666 - 2673. [Abstract] [Full Text] [PDF]
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A. R. Pinto, J. C. Fitzgerald, W. Giles-Davis, G. P. Gao, J. M. Wilson, and H. C. J. Ertl Induction of CD8+ T Cells to an HIV-1 Antigen through a Prime Boost Regimen with Heterologous E1-Deleted Adenoviral Vaccine Carriers J. Immunol., December 15, 2003; 171(12): 6774 - 6779. [Abstract] [Full Text] [PDF]
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), lysis of target cells coated with a control peptide (X) (i.e., peptide 31D delineated from the sequence of the rabies virus nucleoprotein). C, Tetramer staining: stained with streptavidin-PE (left panels), stained with PE-labeled gag tetramer (right panels).
