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* Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215;
Crucell Holland, Leiden, The Netherlands;
Therion Biologics, Cambridge, MA 02142; and
Vaccine Research Center, National Institutes of Health, Bethesda, MD 20892
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The development of adenovirus (Ad) vaccine vectors that elicit potent Ag-specific immune responses, but are not inhibited by pre-existing anti-Ad5 immunity, is therefore an important research priority. One strategy involves the development of Ad vectors from species other than humans (8, 9). For example, chimpanzee Ads are currently being developed as candidate vaccine vectors that are only marginally affected by anti-Ad5 immunity (10, 11). However, nonhuman Ads may pose significant regulatory challenges as a result of their unknown clinical disease associations in humans.
Another strategy involves the development of vaccine vectors from rare human Ad serotypes (12, 13, 14, 15), but cross-reactive antivector immune responses between heterologous serotypes have been reported and could prove a major limitation of this approach (16). Ad35 is one of the rarest of the 51 known human serotypes, with a seroprevalence of <7%, and thus may offer a significant advantage over Ad5 as a candidate vaccine vector (15). However, the immunogenicity of rAd35 vaccines and the extent of immunologic cross-reactivity between Ad5 and Ad35 have not previously been determined. We therefore investigated the impact of anti-Ad5 immunity on the immunogenicity of rAd5 and rAd35 vaccines expressing SIV Gag in mice.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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E1/E3-deleted, replication-incompetent Ad5 or Ad35 vectors were generated in PER.C6/55K cells using pBR322-based adaptor plasmid pAdAptor pAdApt535 together with cosmid pWE.Ad.AfllII-rITR
E3 orpWE.Ad35.pIX-rITRE3 essentially as previously described (17, 18). The adaptor plasmids contained the left portion of the Ad genomes (nt 1–454 in Ad5 or nt 1–464 in Ad35), followed by transcriptional control elements and the adaptor Ad DNA region (nt 3511–6095 in Ad5 or nt 3401–4669 in Ad35). The SIVmac239 gag gene optimized for high levels of expression in mammalian cells (GeneART, Regensburg, Germany) was cloned into the expression cassette in the adaptor plasmids. The resulting pAdApt-Gag and pAdApt535-Gag plasmids expressed the SIVmac239 gag gene under transcriptional control of the human, full-length, immediate-early CMV promoter and the SV40 polyadenylation signal. These plasmids were linearized and transfected into PER.C6/55K cells together with the cosmid pWE.Ad.AfllII-rITRE3 or pWE.Ad35.pIX-rITRE3 containing the right portion of the Ad genomes using Lipofectamine (Invitrogen, Breda, The Netherlands). Homologous recombination led to the generation of rAd5-Gag or rAd35-Gag virus. Ad vectors in crude lysates were plaque-purified using limiting dilutions and agar overlays, and Ad vector clones were analyzed for presence and expression of the transgene. Positive clones were amplified for large-scale production using PER.C6/55K cells in 24–48 triple-layer 3 x 175-cm2 flasks. Stock viruses were purified by standard two-step CsCl gradient ultracentrifugation and dialyzed three times into PBS containing 5% sucrose. Purified Ad vectors were aliquoted and stored at –80°C. Virus particle (vp) titers were determined by HPLC. Infectivity was assessed by plaque assays using PER.C6/55K cells. SIV Gag expression was assessed by infection of A549 cells, followed by analysis of culture lysates using a commercial Gag ELISA kit (Murex Biotech, Dartford, U.K.). Purified rAd5-Gag and rAd35-Gag vectors were negative in replication-competent Ad assays. Replication-incompetent rAd5-luciferase, rAd35-luciferase, rAd5-empty, and rAd35-empty vectors were produced using similar methods.
Mice and immunizations
Six- to 8-wk-old C57/BL6 or BALB/c mice were purchased from Charles River Laboratories (Wilmington, MA). For rAd5 or rAd35 immunizations, mice were injected i.m. with 106–1010 vp replication-incompetent E1/E3-deleted rAd5 or rAd35 expressing SIVmac239 Gag in 100 µl of sterile PBS in the quadriceps muscles. For DNA immunizations, mice were injected i.m. with 50 µg of plasmid VRC-4307 expressing SIVmac239 Gag-Pol-Nef (Vaccine Research Center, National Institutes of Health, Bethesda, MD) in 100 µl of sterile saline. For rMVA immunizations, mice were injected i.p. with 108 PFU of rMVA-T338 expressing SIVmac239 Gag in 100 µl of sterile PBS (Therion Biologics, Cambridge, MA). To induce active anti-Ad5 immunity, mice were preimmunized once or twice, separated by a 4-wk interval, i.m. with 1010 vp of rAd5-empty containing no insert in 100 µl of sterile PBS.
Gag-specific ELISPOT
Gag-specific cellular immune responses were assessed by IFN-
ELISPOT assays using murine splenocytes in response to individual Gag epitope peptides or a pool of overlapping 15-aa peptides covering the entire SIVmac239 Gag protein. Ninety-six-well multiscreen plates (Millipore, Bedford, MA) coated overnight with 100 µl/well of 10 µg/ml rat anti-mouse IFN- (PharMingen, San Diego, CA) in PBS were washed three times with endotoxin-free Dulbecco’s PBS (Life Technologies, Gaithersburg, MD) containing 0.25% Tween 20 and blocked with PBS containing 5% FBS for 2 h at 37°C. The plates were washed three times with Dulbecco’s PBS containing 0.25% Tween 20, rinsed with RPMI 1640 containing 10% FBS, and incubated in triplicate with 2 x 105 or 5 x 105 splenocytes/well in a 100-µl reaction volume containing 1 µg/ml peptide. For studies using the Gag peptide pool, each peptide in the pool was present at 1 µg/ml. After an 18-h incubation, the plates were washed nine times with Dulbecco’s PBS containing 0.25% Tween 20 and once with distilled water. The plates were then incubated for 2 h with 75 µl/well of 5 µg/ml biotinylated rat anti-mouse IFN- (BD PharMingen), washed six times with Coulter Wash (Coulter, Miami, FL), and incubated for 2 h with a 1/500 dilution of streptavidin-alkaline phosphatase (Southern Biotechnology Associates, Birmingham, AL). After five washes with Coulter Wash and one wash with PBS, the plates were developed with nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl-phosphate chromogen (Pierce, Rockford, IL), development was stopped by washing with tap water, and plates were air-dried and read using an ELISPOT reader (Hitech Instruments, Edgement, PA). For depletion studies, splenocytes were incubated with magnetic microbeads coated with anti-CD4 (L3T4) or anti-CD8 (Ly-2) mAbs (Miltenyi Biotec, Auburn, CA) and separated using MiniMACS columns before performing the ELISPOT assay. Cell depletions were >95% efficient.
Gag-specific ELISA
Serum anti-Gag Ab titers from immunized mice were measured by a direct ELISA. Ninety-six-well plates coated overnight with 100 µl/well of 1 µg/ml recombinant SIV Gag protein (Intracel, Cambridge, MA) in PBS were blocked for 2 h with PBS containing 2% BSA and 0.05% Tween 20. Sera were then added in serial dilutions and incubated for 1 h. The plates were washed three times with PBS containing 0.05% Tween 20 and incubated for 1 h with a 1/2000 dilution of a peroxidase-conjugated, affinity-purified, rabbit anti-mouse secondary Ab (The Jackson Laboratory, Bar Harbor, ME). The plates were then washed three times, developed with tetramethylbenzidine (Kirkegaard & Perry Laboratories, Gaithersburg, MD), stopped with 1% HCl, and analyzed at 450 nm with an MR5000 ELISA plate reader (Dynatech Laboratories, Chantilly, VA).
AL11-specific tetramer binding assays
Tetrameric H-2Db complexes folded around the immunodominant SIV Gag AL11 epitope (AAVKNWMTQTL) were prepared and used to stain peptide-specific CD8+ T lymphocytes essentially as previously described (4, 19). Mouse blood was collected in RPMI 1640 containing 40 U/ml heparin. After lysis of the RBCs, 0.1 µg of PE-labeled Db/AL11 tetramer in conjunction with allophycocyanin-labeled anti-CD8
mAb (Ly-2; Caltag Laboratories, San Francisco, CA) was used to stain AL11-specific CD8+ T lymphocytes. The cells were washed in PBS containing 2% FBS and fixed in 0.5 ml of PBS containing 1.5% paraformaldehyde. Samples were analyzed by two-color flow cytometry on a FACSCalibur (BD Biosciences, Mountain View, CA). Gated CD8+ T lymphocytes were examined for staining with the Db/AL11 tetramer. CD8+ T lymphocytes from naive mice were used as negative controls and exhibited <0.1% tetramer staining.
Ad-specific ELISPOT
Ad5- or Ad35-specific cellular immune responses were assessed by IFN- ELISPOT assays using murine splenocytes from C57/BL6 mice in response to Ad5- or Ad35-infected syngeneic BLK CL4 stimulator cells (ATCC TIB-81; American Type Culture Collection, Manassas, VA) essentially as previously described (15). BLK CL4 cells were plated at a density of 1 x 106 cells/well in a six-well plate and infected with E1/E3-deleted rAd5-empty or rAd35-empty at a multiplicity of infection of 2 x 104 for 3 days. ELISPOT assays using splenocytes from immunized C57/BL6 mice were then performed as described above, using 5 x 105 splenocytes and 1 x 105 Ad-infected BLK CL4 stimulator cells/well in place of peptide Ags. For negative controls, splenocytes were incubated with uninfected BLK CL4 cells or medium alone.
Ad-specific neutralizing Ab assay
Ad5- or Ad35-specific neutralizing Ab (NAb) responses were assessed by luciferase-based virus neutralization assays essentially as previously described (20). A549 human lung carcinoma cells were plated at a density of 1 x 104 cells/well in 96-well plates. E1/E3-deleted rAd5-luciferase or rAd35-luciferase reporter constructs were then added at a multiplicity of infection of 500 with 2-fold serial dilutions of serum in 200-µl reaction volumes. After a 24-h incubation, luciferase activity in the cells was measured using the Steady-Glo Luciferase Reagent System (Promega, Madison, WI). Ninety percent neutralization titers were defined as the maximum serum dilution that neutralized 90% of luciferase activity.
Ad-specific cellular proliferation assay
Ad5- or Ad35-specific CD4+ cellular proliferative responses were assessed by [3H]thymidine incorporation assays. Splenocytes from immunized mice were depleted of CD8+ T cells using anti-CD8 (Ly-2)-coated magnetic microbeads (Miltenyi Biotec, Auburn, CA). Cell depletions were >95% efficient. CD8-depleted splenocytes were then resuspended at 4 x 106 cells/ml in RPMI 1640 containing 5% FBS. One hundred microliters was added to each well in 96-well plates with 108 vp of rAd5-empty, 108 vp of rAd35-empty, or medium alone. After 4 days of culture, 1 µCi of [3H]thymidine (ICN Biochemicals, Costa Mesa, CA) was added to each well. After a 16-h incubation, cells were harvested on glass filter paper, and radioactivity was measured in a 1450 Microbeta liquid scintillation counter (Wallac, Gaithersburg, MD). The stimulation index was calculated as: (cpm with Ag stimulation)/(background cpm without Ag).
Statistical analyses
ELISPOT and tetramer data are presented as the mean and SE. Statistical analyses were performed with PRISM version 2.01 (GraphPad, San Diego, CA). Comparisons of mean ELISPOT responses among groups of mice were performed by two-tailed t tests for two groups of animals or by ANOVA for more than two groups. Bonferroni adjustments were included when appropriate to account for multiple comparisons. In all cases, p < 0.05 was considered significant.
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Results |
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We initiated studies to determine the immunogenicity of E1/E3-deleted, replication-incompetent rAd5-Gag and rAd35-Gag vaccines in naive mice. Groups of BALB/c and C57/BL6 mice (n = 4/group) were immunized once i.m. with 1010 or 108 vp of each vector. Vaccine-elicited cellular immune responses were assessed by ELISPOT assays using a pool of 15-aa peptides overlapping by 11 aa covering the entire SIV Gag protein. Vaccine-elicited humoral immune responses were assessed by Gag-specific ELISAs.
As shown in Fig. 1, both rAd5-Gag and rAd35-Gag elicited only marginal SIV Gag-specific cellular immune responses in BALB/c mice. In contrast, both vectors at a dose of 1010 vp elicited rapid and potent ELISPOT responses by 2 wk after vaccination in C57/BL6 mice. At the dose of 108 vp, SIV Gag-specific cellular immune responses elicited by rAd35-Gag were 
2-fold lower than those elicited by rAd5-Gag. High titer anti-Gag Ab responses were elicited by rAd5-Gag in both BALB/c and C57/BL6 mice. However, no anti-Gag Ab responses were detected after immunization with rAd35-Gag. These data demonstrate that rAd35-Gag elicited slightly lower cellular immune responses, but markedly lower humoral immune responses, than rAd5-Gag.
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The rapid emergence of high frequency, Gag-specific, cellular immune responses in C57/BL6 mice (Fig. 1C) suggested the presence of immunodominant Db- or Kb-restricted CD8+ T lymphocyte epitopes. We therefore used a matrix-based ELISPOT approach to identify candidate epitopes within SIV Gag to facilitate the development of quantitative peptide-specific cellular immune assays for rAd5-Gag and rAd35-Gag vectors. As depicted in Fig. 2, A–C, C57/BL6 mice immunized with 1010 vp of rAd5, 1010 vp of rAd35, or 50 µg of plasmid DNA expressing SIV Gag developed an immunodominant cellular immune response to the 15-aa P78 peptide (QTDAAVKNWMTQTLL) and a subdominant response to the P19 peptide (ENLKSLYNTVCVIWC). ELISPOT assays using splenocytes depleted of CD4+ or CD8+ T lymphocytes demonstrated that both P78 and P19 were, in fact, CD8+ T lymphocyte epitopes.
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Impact of low levels of anti-Ad5 immunity on the immunogenicity of rAd5-Gag and rAd35-Gag
We next determined the impact of low levels of anti-Ad5 immunity on cellular immune responses elicited by rAd5-Gag and rAd35-Gag. To model pre-existing anti-Ad5 immunity, C57/BL6 mice were preimmunized once with 1010 vp rAd5-empty 4 wk before immunization. As shown in Fig. 3C, mice preimmunized with rAd5-empty developed mean Ad5-specific NAb titers of 128, but no detectable Ad35-specific NAb titers (<16). These Ad5-specific NAb responses represent average titers found in individuals in the United States and Western Europe (15, 21). Ad-specific T lymphocyte responses in these mice were assessed by virus-specific ELISPOT assays using splenocytes stimulated with Ad5- or Ad35-infected syngeneic BLK CL4 cells. As shown in Fig. 3D, mice preimmunized with rAd5-empty developed mean Ad5-specific ELISPOT responses of 250 spot-forming cells (SFC)/106 splenocytes, but no detectable Ad35-specific ELISPOT responses (<25 SFC/106 splenocytes).
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Vector-specific humoral and cellular immune responses were also assessed in these groups of mice. As shown in Fig. 3, C and D, naive mice immunized with rAd5-Gag or rAd35-Gag developed Ad serotype-specific immune responses. No detectable cross-reactive Ad5/Ad35-specific NAb or ELISPOT responses were observed in these mice, suggesting that these two vectors were largely immunologically distinct. As expected, mice preimmunized with rAd5-empty generated potent, anamnestic Ad5-specific NAb and ELISPOT responses after rAd5-Gag immunization. Interestingly, mice preimmunized with rAd5-empty generated unexpectedly potent Ad35-specific NAb responses after rAd35-Gag immunization (Fig. 3C). In fact, these responses were 10-fold higher than the Ad35-specific NAb responses generated in naive mice after the same rAd35-Gag immunization. These data suggest that preimmunization with rAd5-empty may have primed low levels of cross-reactive antivector immune responses that became apparent only after administration of the heterologous rAd35-Gag vector. However, these cross-reactive responses did not appear to suppress the immunogenicity of rAd35-Gag.
Impact of high levels of anti-Ad5 immunity on the immunogenicity of rAd5-Gag and rAd35-Gag
As the majority of individuals in the developing world have high titers of Ad5-specific NAbs, we assessed whether high levels of anti-Ad5 immunity would suppress cellular immune responses elicited by rAd35-Gag. To investigate this, mice were preimmunized twice with 1010 vp of rAd5-empty 8 and 4 wk before immunization. As shown in Fig. 4C, these mice developed mean Ad5-specific NAb titers of 16,384, but no detectable Ad35-specific NAb titers (<16). These NAb titers represent the upper limit of those typically found in sub-Saharan Africa (21). As shown in Fig. 4D, these mice also developed high frequency, Ad5-specific ELISPOT responses of 560 SFC/106 splenocytes, but no detectable Ad35-specific ELISPOT responses (<25 SFC/106 splenocytes). Thus, preimmunization of mice with two doses of rAd5-empty generated potent anti-Ad5 immunity.
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Tetramer binding assays
We next used tetramer binding assays to assess both the kinetics and the magnitudes of CD8+ T lymphocyte responses specific for the immunodominant AL11 epitope. Tetrameric Db/AL11 complexes were constructed essentially as previously described (4, 19) and were used to quantitate the emergence of AL11-specific CD8+ T lymphocyte responses elicited by rAd5-Gag and rAd35-Gag in mice with or without anti-Ad5 immunity. C57/BL6 mice were preimmunized once with 1010 vp rAd5-empty 4 wk before immunization and developed Ad5-specific NAb titers of 128–256. Groups of naive mice or mice with anti-Ad5 immunity (n = 4/group) were then immunized with 1010 or 108 vp of rAd5-Gag or rAd35-Gag. Db/AL11 tetramer binding to gated CD8+ T lymphocytes was assessed at multiple time points after immunization. As shown in Fig. 5, A and B, anti-Ad5 immunity markedly blunted the emergence of tetramer+CD8+ T lymphocyte responses elicited by rAd5-Gag, but did not substantially affect the responses elicited by rAd35-Gag. These data confirm the absence of functionally suppressive, cross-reactive, antivector immune responses between Ad5 and Ad35. We also assessed the immunologic potencies of rAd5-Gag and rAd35-Gag administered at doses of 1010, 109, 108, 107, and 106 vp in naive mice. As shown in Fig. 5C, the threshold dose required to elicit detectable tetramer+ CD8+ T lymphocyte responses in naive mice was 108 vp for rAd35-Gag compared with 107 vp for rAd5-Gag.
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We next investigated the immunogenicity of homologous and heterologous prime-boost vaccine regimens in naive C57/BL6 mice. Groups of mice (n = 4/group) were primed at wk 0 with 1010 vp rAd5-Gag or rAd35-Gag and boosted at wk 4 with homologous or heterologous vectors. As shown in Fig. 6A, heterologous rAd5 prime-rAd35 boost and rAd35 prime-rAd5 boost regimens were more immunogenic than homologous prime-boost regimens. Readministration of homologous vectors did not substantially boost Gag-specific cellular immune responses under these conditions, presumably as a result of the generation of potent antivector immunity. Readministration of homologous vectors similarly failed to boost Gag-specific responses in mice with pre-existing anti-Ad5 immunity (data not shown).
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Immunogenicity of heterologous prime-boost regimens in mice with anti-Ad5 immunity
Heterologous DNA prime-rAd5 boost vaccine regimens have been shown to elicit particularly potent immune responses in animal models and are therefore being investigated as candidate vaccine strategies in clinical trials (1, 2, 3). As the majority of humans have pre-existing anti-Ad5 immunity, we sought to assess the immunogenicity of various prime-boost regimens in animals with anti-Ad5 immunity. We therefore compared the immunogenicities of four heterologous prime-boost regimens in C57/BL6 mice that were preimmunized once with 1010 vp of rAd5-empty 4 wk before primary immunization. These mice had low Ad5-specific NAb titers of 128–256 (Fig. 7C). Groups of mice (n = 4/group) were primed at wk 0 with 50 µg of DNA or 1010 vp of rAd5 expressing SIV Gag and then boosted at wk 4 with 1010 vp of rAd5, 1010 vp of rAd35, or 108 PFU of rMVA expressing SIV Gag. Mice were sacrificed at wk 8 for immunologic assays.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We did detect low levels of cross-reactive Ad5/Ad35-specific CD4+ T lymphocyte responses. Cross-reactive, Ad-specific, Th lymphocytes have also been observed in humans (16, 22), suggesting that Ads from heterologous serotypes are not completely immunologically distinct. Moreover, these cross-reactive CD4+ T lymphocyte responses probably contributed to the potent Ad-specific NAbs that developed after heterologous vector administration in mice (Fig. 6B). However, despite these cross-reactive immune responses, Gag-specific cellular immune responses elicited by rAd35-Gag were not detectably suppressed by pre-existing anti-Ad5 immunity. These data suggest that antivector NAb responses that are present at the time of immunization are more important than those that develop after immunization in determining their suppressive effects on vaccine immunogenicity.
In mice with anti-Ad5 immunity at levels typically found in humans, rAd35-Gag elicited significantly higher cellular immune responses than rAd5-Gag. These data demonstrate the potential utility of Ad35 as a vaccine vector in the presence of pre-existing anti-Ad5 immunity. In naive mice, however, rAd35-Gag elicited slightly lower cellular immune responses and substantially lower humoral immune responses compared with rAd5-Gag. These differences in immunogenicity are consistent with our previous finding that rAd35-mediated transgene expression was several-fold lower than rAd5-mediated transgene expression in mouse muscle (15). Ad5 interacts with the coxsackievirus and Ad receptor on the surface of cells with its long and flexible fiber protein (23, 24, 25, 26). In contrast, the Ad35 fiber protein is shorter and more rigid than the Ad5 fiber (26, 27), and CD46 has recently been identified as an Ad35 receptor (28). As a result, Ad5 and Ad35 have different cellular tropisms (29, 30, 31, 32) and intracellular trafficking pathways (33). These differences may explain in part the differences in immunogenicity between rAd5-Gag and rAd35-Gag in this animal model. At present, it is not clear why we observed large differences between rAd5-Gag and rAd35-Gag in their ability to elicit Gag-specific antibodies. It is possible that a higher threshold of Ag is needed to generate Ab responses compared with T lymphocyte responses in this system.
Heterologous prime-boost vaccine strategies are commonly used to elicit high frequency, Ag-specific immune responses. We confirmed that boosting with heterologous vectors elicited higher immune responses than boosting with homologous vectors (Fig. 6A). However, regimens that include rAd5 will probably have substantially reduced immunogenicity and clinical utility in the presence of anti-Ad5 immunity. In mice with anti-Ad5 immunity, a DNA prime-rAd35 boost regimen was significantly more immunogenic than a DNA prime-rAd5 boost regimen (Fig. 7). Thus, DNA prime-rAd35 boost as well as rAd5 prime-rAd35 boost regimens warrant further investigation as candidate HIV-1 prime-boost vaccine strategies.
These data demonstrate the potential utility of Ad vaccine vectors derived from rare human serotypes to elicit immune responses in the presence of pre-existing anti-Ad5 immunity. It is possible that cross-reactive cellular immune responses between serologically distinct Ads may limit the utility of certain vector combinations, although this problem appears to be minimal for Ad5 and Ad35. Therefore, this strategy may prove an effective and general approach for developing vectors that avoid the suppressive effects of pre-existing anti-Ad5 immunity. However, the ultimate utility of this approach in humans will require data from clinical trials.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Dan H. Barouch, Research East Room 113, Division of Viral Pathogenesis, Beth Israel Deaconess Medical Center, 330 Brookline Avenue, Boston, MA 02215. E-mail address: dbarouch{at}bidmc.harvard.edu
3 Abbreviations used in this paper: rAd5, adenovirus serotype 5; Ad, adenovirus; NAb, neutralizing Ab; SFC, spot-forming cell; vp, virus particle.
Received for publication December 19, 2003. Accepted for publication March 4, 2004.
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S. Worgall, A. Krause, J. Qiu, J. Joh, N. R. Hackett, and R. G. Crystal Protective Immunity to Pseudomonas aeruginosa Induced with a Capsid-Modified Adenovirus Expressing P. aeruginosa OprF J. Virol., December 15, 2007; 81(24): 13801 - 13808. [Abstract] [Full Text] [PDF]
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C. Stahl-Hennig, S. Kuate, M. Franz, Y. S. Suh, H. Stoiber, U. Sauermann, K. Tenner-Racz, S. Norley, K. S. Park, Y. C. Sung, et al. Atraumatic Oral Spray Immunization with Replication-Deficient Viral Vector Vaccines J. Virol., December 1, 2007; 81(23): 13180 - 13190. [Abstract] [Full Text] [PDF]
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L. J. White, M. M. Parsons, A. C. Whitmore, B. M. Williams, A. de Silva, and R. E. Johnston An Immunogenic and Protective Alphavirus Replicon Particle-Based Dengue Vaccine Overcomes Maternal Antibody Interference in Weanling Mice J. Virol., October 1, 2007; 81(19): 10329 - 10339. [Abstract] [Full Text] [PDF]
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S. Santra, Y. Sun, J. G. Parvani, V. Philippon, M. S. Wyand, K. Manson, A. Gomez-Yafal, G. Mazzara, D. Panicali, P. D. Markham, et al. Heterologous Prime/Boost Immunization of Rhesus Monkeys by Using Diverse Poxvirus Vectors J. Virol., August 15, 2007; 81(16): 8563 - 8570. [Abstract] [Full Text] [PDF]
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K. Radosevic, C. W. Wieland, A. Rodriguez, G. J. Weverling, R. Mintardjo, G. Gillissen, R. Vogels, Y. A. W. Skeiky, D. M. Hone, J. C. Sadoff, et al. Protective Immune Responses to a Recombinant Adenovirus Type 35 Tuberculosis Vaccine in Two Mouse Strains: CD4 and CD8 T-Cell Epitope Mapping and Role of Gamma Interferon Infect. Immun., August 1, 2007; 75(8): 4105 - 4115. [Abstract] [Full Text] [PDF]
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K. Lore, W. C. Adams, M. Havenga, M. L. Precopio, L. Holterman, J. Goudsmit, and R. A. Koup Myeloid and Plasmacytoid Dendritic Cells Are Susceptible to Recombinant Adenovirus Vectors and Stimulate Polyfunctional Memory T Cell Responses J. Immunol., August 1, 2007; 179(3): 1721 - 1729. [Abstract] [Full Text] [PDF]
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P. Abbink, A. A. C. Lemckert, B. A. Ewald, D. M. Lynch, M. Denholtz, S. Smits, L. Holterman, I. Damen, R. Vogels, A. R. Thorner, et al. Comparative Seroprevalence and Immunogenicity of Six Rare Serotype Recombinant Adenovirus Vaccine Vectors from Subgroups B and D J. Virol., May 1, 2007; 81(9): 4654 - 4663. [Abstract] [Full Text] [PDF]
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J. R. Greenland, R. Geiben, S. Ghosh, W. A. Pastor, and N. L. Letvin Plasmid DNA Vaccine-Elicited Cellular Immune Responses Limit In Vivo Vaccine Antigen Expression through Fas-Mediated Apoptosis J. Immunol., May 1, 2007; 178(9): 5652 - 5658. [Abstract] [Full Text] [PDF]
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J. Liu, B. A. Ewald, D. M. Lynch, A. Nanda, S. M. Sumida, and D. H. Barouch Modulation of DNA Vaccine-Elicited CD8+ T-Lymphocyte Epitope Immunodominance Hierarchies J. Virol., December 15, 2006; 80(24): 11991 - 11997. [Abstract] [Full Text] [PDF]
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A. R. Thorner, A. A. C. Lemckert, J. Goudsmit, D. M. Lynch, B. A. Ewald, M. Denholtz, M. J. E. Havenga, and D. H. Barouch Immunogenicity of Heterologous Recombinant Adenovirus Prime-Boost Vaccine Regimens Is Enhanced by Circumventing Vector Cross-Reactivity J. Virol., December 15, 2006; 80(24): 12009 - 12016. [Abstract] [Full Text] [PDF]
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A. A. C. Lemckert, J. Grimbergen, S. Smits, E. Hartkoorn, L. Holterman, B. Berkhout, D. H. Barouch, R. Vogels, P. Quax, J. Goudsmit, et al. Generation of a novel replication-incompetent adenoviral vector derived from human adenovirus type 49: manufacture on PER.C6 cells, tropism and immunogenicity. J. Gen. Virol., October 1, 2006; 87(Pt 10): 2891 - 2899. [Abstract] [Full Text] [PDF]
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P. L. Leopold, R. L. Wendland, T. Vincent, and R. G. Crystal Neutralized Adenovirus-Immune Complexes Can Mediate Effective Gene Transfer via an Fc Receptor-Dependent Infection Pathway. J. Virol., October 1, 2006; 80(20): 10237 - 10247. [Abstract] [Full Text] [PDF]
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M. Havenga, R. Vogels, D. Zuijdgeest, K. Radosevic, S. Mueller, M. Sieuwerts, F. Weichold, I. Damen, J. Kaspers, A. Lemckert, et al. Novel replication-incompetent adenoviral B-group vectors: high vector stability and yield in PER.C6 cells. J. Gen. Virol., August 1, 2006; 87(Pt 8): 2135 - 2143. [Abstract] [Full Text] [PDF]
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M. J. Cayabyab, A.-H. Hovav, T. Hsu, G. R. Krivulka, M. A. Lifton, D. A. Gorgone, G. J. Fennelly, B. F. Haynes, W. R. Jacobs Jr., and N. L. Letvin Generation of CD8+ T-Cell Responses by a Recombinant Nonpathogenic Mycobacterium smegmatis Vaccine Vector Expressing Human Immunodeficiency Virus Type 1 Env J. Virol., February 15, 2006; 80(4): 1645 - 1652. [Abstract] [Full Text] [PDF]
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S. Capone, A. Meola, B. B. Ercole, A. Vitelli, M. Pezzanera, L. Ruggeri, M. E. Davies, R. Tafi, C. Santini, A. Luzzago, et al. A Novel Adenovirus Type 6 (Ad6)-Based Hepatitis C Virus Vector That Overcomes Preexisting Anti-Ad5 Immunity and Induces Potent and Broad Cellular Immune Responses in Rhesus Macaques J. Virol., February 15, 2006; 80(4): 1688 - 1699. [Abstract] [Full Text] [PDF]
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S. Verhaagh, E. de Jong, J. Goudsmit, S. Lecollinet, G. Gillissen, M. de Vries, K. van Leuven, I. Que, K. Ouwehand, R. Mintardjo, et al. Human CD46-transgenic mice in studies involving replication-incompetent adenoviral type 35 vectors J. Gen. Virol., February 1, 2006; 87(2): 255 - 265. [Abstract] [Full Text] [PDF]
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O. J. A. E. Ophorst, K. Radosevic, M. J. E. Havenga, M. G. Pau, L. Holterman, B. Berkhout, J. Goudsmit, and M. Tsuji Immunogenicity and Protection of a Recombinant Human Adenovirus Serotype 35-Based Malaria Vaccine against Plasmodium yoelii in Mice Infect. Immun., January 1, 2006; 74(1): 313 - 320. [Abstract] [Full Text] [PDF]
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S. M. Barratt-Boyes, A. C. Soloff, W. Gao, E. Nwanegbo, X. Liu, P. A. Rajakumar, K. N. Brown, P. D. Robbins, M. Murphey-Corb, R. D. Day, et al. Broad cellular immunity with robust memory responses to simian immunodeficiency virus following serial vaccination with adenovirus 5- and 35-based vectors J. Gen. Virol., January 1, 2006; 87(1): 139 - 149. [Abstract] [Full Text] [PDF]
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T.-C. Yang, J. Millar, T. Groves, N. Grinshtein, R. Parsons, S. Takenaka, Y. Wan, and J. L. Bramson The CD8+ T Cell Population Elicited by Recombinant Adenovirus Displays a Novel Partially Exhausted Phenotype Associated with Prolonged Antigen Presentation That Nonetheless Provides Long-Term Immunity J. Immunol., January 1, 2006; 176(1): 200 - 210. [Abstract] [Full Text] [PDF]
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A. B. McDermott, D. H. O'Connor, S. Fuenger, S. Piaskowski, S. Martin, J. Loffredo, M. Reynolds, J. Reed, J. Furlott, T. Jacoby, et al. Cytotoxic T-Lymphocyte Escape Does Not Always Explain the Transient Control of Simian Immunodeficiency Virus SIVmac239 Viremia in Adenovirus-Boosted and DNA-Primed Mamu-A*01-Positive Rhesus Macaques J. Virol., December 15, 2005; 79(24): 15556 - 15566. [Abstract] [Full Text] [PDF]
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A. Nanda, D. M. Lynch, J. Goudsmit, A. A. C. Lemckert, B. A. Ewald, S. M. Sumida, D. M. Truitt, P. Abbink, M. G. Kishko, D. A. Gorgone, et al. Immunogenicity of Recombinant Fiber-Chimeric Adenovirus Serotype 35 Vector-Based Vaccines in Mice and Rhesus Monkeys J. Virol., November 15, 2005; 79(22): 14161 - 14168. [Abstract] [Full Text] [PDF]
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M. Hashimoto, J. L. Boyer, N. R. Hackett, J. M. Wilson, and R. G. Crystal Induction of Protective Immunity to Anthrax Lethal Toxin with a Nonhuman Primate Adenovirus-Based Vaccine in the Presence of Preexisting Anti-Human Adenovirus Immunity Infect. Immun., October 1, 2005; 73(10): 6885 - 6891. [Abstract] [Full Text] [PDF]
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B. Peng, L. R. Wang, V. R. Gomez-Roman, A. Davis-Warren, D. C. Montefiori, V. S. Kalyanaraman, D. Venzon, J. Zhao, E. Kan, T. J. Rowell, et al. Replicating Rather than Nonreplicating Adenovirus-Human Immunodeficiency Virus Recombinant Vaccines Are Better at Eliciting Potent Cellular Immunity and Priming High-Titer Antibodies J. Virol., August 15, 2005; 79(16): 10200 - 10209. [Abstract] [Full Text] [PDF]
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A. A. C. Lemckert, S. M. Sumida, L. Holterman, R. Vogels, D. M. Truitt, D. M. Lynch, A. Nanda, B. A. Ewald, D. A. Gorgone, M. A. Lifton, et al. Immunogenicity of Heterologous Prime-Boost Regimens Involving Recombinant Adenovirus Serotype 11 (Ad11) and Ad35 Vaccine Vectors in the Presence of Anti-Ad5 Immunity J. Virol., August 1, 2005; 79(15): 9694 - 9701. [Abstract] [Full Text] [PDF]
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L. Wu, W.-p. Kong, and G. J. Nabel Enhanced Breadth of CD4 T-Cell Immunity by DNA Prime and Adenovirus Boost Immunization to Human Immunodeficiency Virus Env and Gag Immunogens J. Virol., July 1, 2005; 79(13): 8024 - 8031. [Abstract] [Full Text] [PDF]
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J. Vellinga, S. Van der Heijdt, and R. C. Hoeben The adenovirus capsid: major progress in minor proteins J. Gen. Virol., June 1, 2005; 86(6): 1581 - 1588. [Abstract] [Full Text] [PDF]
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S. M. Sumida, D. M. Truitt, A. A. C. Lemckert, R. Vogels, J. H. H. V. Custers, M. M. Addo, S. Lockman, T. Peter, F. W. Peyerl, M. G. Kishko, et al. Neutralizing Antibodies to Adenovirus Serotype 5 Vaccine Vectors Are Directed Primarily against the Adenovirus Hexon Protein J. Immunol., June 1, 2005; 174(11): 7179 - 7185. [Abstract] [Full Text] [PDF]
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S. Santra, M. S. Seaman, L. Xu, D. H. Barouch, C. I. Lord, M. A. Lifton, D. A. Gorgone, K. R. Beaudry, K. Svehla, B. Welcher, et al. Replication-Defective Adenovirus Serotype 5 Vectors Elicit Durable Cellular and Humoral Immune Responses in Nonhuman Primates J. Virol., May 15, 2005; 79(10): 6516 - 6522. [Abstract] [Full Text] [PDF]
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) or mice with anti-Ad5 immunity (
and 
) were immunized with 1010 or 108 vp of rAd5-Gag (A) or rAd35-Gag (B). Db/AL11 tetramer binding to CD8+ T lymphocytes is shown at various time points after immunization. C, Groups of naive mice were immunized with 1010, 109, 108, 107, or 106 vp of rAd5-Gag or rAd35-Gag, and Db/AL11 tetramer binding to CD8+ T lymphocytes was determined on day 14 after immunization.
